Organisatrice : Prof. Christelle Breton

26es Journées du Groupe

Français des Glycosciences

Aussois, 23-27 mai 2016

Livre des résumés

Le GFG 2016 remercie les organismes suivants pour leur soutien financier ou matériel

partenaires académiques

partenaires industriels

Sommaire

BIENVENUE À AUSSOIS ............................................................... 1

COMITÉ D’ORGANISATION ....................................................... 1

COMITÉ SCIENTIFIQUE NATIONAL ......................................... 1

COMITÉ SCIENTIFIQUE LOCAL .................................................. 1

PROGRAMME ................................................................................. 2

COMMUNICATIONS INVITÉES (CI) ........................................ 5

COMMUNICATIONS ORALES (CO + DUO) ......................... 19

POSTERS ......................................................................................... 31

LISTE DES POSTERS ..................................................................... 52

LISTE DES PARTICIPANTS ......................................................... 54

1

Bienvenue à Aussois

Chers participants, chers membres du GFG, chers amis,

Vous m’avez fait l’honneur, il y a 4 ans, en me nommant Présidente du GFG pour la période 2015-2016, de me confier l’organisation des 26es Journées du GFG. Nous y sommes. Je suis particulièrement heureuse de vous accueillir, pour cette nouvelle édition, au Centre Paul Langevin à Aussois, village savoyard aux portes du Parc National de la Vanoise en Maurienne.

Comme les précédentes éditions, ces Journées seront l’occasion de rassembler chimistes, polyméristes, structuralistes, biochimistes et biologistes du domaine riche et fascinant des Glycosciences. Ces rencontres bisannuelles sont un moment fort du GFG : elles permettent de présenter les dernières avancées, favorisent les échanges scientifiques et enrichissent nos connaissances. Elles sont aussi l’occasion de retrouver des collègues, de nouer de nouvelles amitiés et collaborations et de partager des moments de convivialité.

Ces journées seront aussi l’occasion d’attribuer le prix du GFG2016 et le prix Bernard Fournet- André Verbert et de tenir l’Assemblée Générale des membres du GFG. Au cours de cette AG, les membres du bureau actuel présenteront le bilan des actions de ces deux dernières années. Vous aurez droit, en avant-première, à la présentation du nouveau site web du GFG ainsi qu’à une proposition de logo. Nous élirons également le prochain vice-président Biologiste qui aura en charge l’organisation de la 28e édition de ces journées en 2020. L’Assemblée générale des membres est un moment unique pour faire entendre votre point de vue. Vous êtes tous invités à prendre part à la réflexion sur les actions futures à mener pour accroitre la visibilité des Glycosciences en France et dynamiser la vie du GFG.

L’organisation d’un congrès est avant tout un travail d’équipe. Je tiens à remercier très sincèrement les membres du comité scientifique local et du comité scientifique national du GFG qui ont participé à l’élaboration du programme. Nous espérons que ce programme sera à la hauteur de vos attentes. J’adresse une mention spéciale à Sylvie Armand (secrétaire du GFG) et Michèle Carret pour leur aide très précieuse dans l’organisation pratique de ce congrès et à Martine Morales et Sandrine Coindet pour les aspects administratifs. Merci à Kawthar Bouchemal pour le logo du GFG2016 et à Michèle Carret pour le design du site web du colloque. Arnaud, Emeline, Emilie, Milène et Valérie vous accueilleront et vous guideront pendant cette semaine. Merci à eux. Merci également aux partenaires académiques et privés pour leur soutien financier.

Je n’oublie pas non plus Vincent Ferrières, notre trésorier du GFG, et Arnaud Tatibouët, notre Vice-Président chimiste, pour leurs actions au sein de l’association et Philippe Delannoy pour son aide dans l’attribution des prix du GFG.

Tous les membres du comité d’organisation vous souhaitent un très bon congrès et un séjour agréable à Aussois. Nous espérons que le cadre sera propice aux échanges et que vous apprécierez la beauté et la quiétude du site.

Bien amicalement à tous,

Christelle

Comité D’organisation

Dr Sylvie Armand, Pr Christelle Breton, Michèle Carret, Sandrine Coindet & Martine Morales, Cermav

Comité scientifique national

Pr Christelle Breton Cermav, Grenoble

Pr Arnaud Tatibouët ICOA, Orléans Pr Vincent Ferrières ENSC, Rennes

Pr David Bonnaffé ICMMO, Orsay Pr Philippe Delannoy UGSF, Lille

Pr. Florence Djedaïni-Pilard LG, Amiens Pr Pierre Monsan INSA, Toulouse

Dr Serge Pérez DPM, Grenoble Dr Catherine Ronin SiaMed’Xpress

Comité scientifique local

Pr Christelle Breton Cermav, Grenoble

Dr Sylvie Armand Cermav, Grenoble Pr Rachel Auzély Cermav, Grenoble Dr Sébastien Fort Cermav, Grenoble

Dr Sami Halila Cermav, Grenoble Dr William Helbert Cermav, Grenoble

Dr Anne Imberty Cermav, Grenoble Dr Hugues Lortat Jacob IBS, Grenoble

Dr Serge Pérez DPM, Grenole Dr Jean-Luc Putaux Cermav, Grenoble

2

PROGRAMME

lundi 23 mai

17h30 CEREMONIE D’OUVERTURE

Session 1 modérateur

Ph. Delannoy

17h45 CI-01 Jacques Le Pendu Glycans in enteric virus infection

18h15 DUO-01 Solange Morera & Yves Queneau A key pyranose-2-phosphate motif is responsible for both antibiotic import and quorum-sensing regulation in Agrobacterium tumefaciens

mardi 24 mai

Session 2 modérateurs J. Le Pendu V. Ferrières

08h45 CI-02 François Foulquier Congenital disorders of glycosylation and Golgi homeostasis: an unexpected link !

09h15 CO-01 Steffi Baldini Regulation of hepatic Fatty Acid Synthase properties by O-GlcNAcylation in vivo and ex vivo 09h30 CO-02 Giuliano Cutolo The MG system as a ligation tool in biological chemistry 09h45 CI-03 Frédéric Friscourt Novel cyclooctyne-based probes with exciting physical properties for the bioorthogonal labeling of glycoconjugates 10h15 - pause

Session 3 modérateurs J. Bouckaert A. Tatibouët

10h45 CI-04 Cyrille Grandjean Galectins, key players of homeostasis

11h15 CO-03 Annabelle Varrot Tectonin2 from Laccaria bicolor is designed for methylated glycans recognition 11h30 CO-04 Sébastien Gouin Modulateurs multimériques de l’activité des glycosidases 11h45 CI-05 Olivier Renaudet Chemoselective ligations: highly efficient strategies for the construction of biologically active multivalent glycoconjugates 12h15 - déjeuner

Session 4 modérateurs

R. Auzély C. Tellier

14h30 CI-06 Claire Moulis Discovery and applications of new sucrose-active enzymes from GH70 family

15h00 CO-05 Claire Dumon Exploration of the lignocellulolytic potential of invertebrate microbiome 15h15 CO-06 Mehdi Omri Selective oxidation of free carbohydrates to corresponding aldonates using gold supported catalysts under microwave-irradiation 15h30 CI-07 Françoise Quignard Polysaccharides: from hydrocolloids to textured materials 16h00 : pause

Session 5 modérateurs C. Boisset

Y. Queneau

16h30 CI-08 Nathalie Bourgougnon Conventional and sustainable bioprocesses for the extraction of antiherpetic oligo and polysaccharides from the invasive Solieria chordalis (Rhodophyta, Gigartinales)

17h00 CO-07 Agata Zykwinska Assembly of a marine exopolysaccharide into microgels for protein delivery applications 17h15 CO-08 Véronique Bonnet Preparation of new nanovectors by synthesis of glycerolipidyl and phosphoramidyl-cyclodextrins

17h30 SESSION POSTERS 1 (n° impairs)

3

mercredi 25 mai

Session 6 modérateurs T. Lefebvre J-B. Behr

08h45 CI-09 Jérôme Nigou Molecular bases of Mycobacterium tuberculosis recognition by C-type lectins: from the modulation of innate immune response to the design of therapeutic molecules

09h15 CO-09 Emeline Richard Bacterial synthesis of polysialic acid lactosides in recombinant Escherichia coli K-12

09h30 CO-10 Joanne Xie Synthesis and property of N-oxyamide-linked glycoconjugates

09h45 CI-10 Laurence Mulard Synthetic carbohydrate-based vaccines against shigellosis: from concept to clinic … and more 10h15 : pause

Session 7 modérateurs N. Aghajari

S. Fort

10h45 CI-11 Marcelo E. Guerin Membrane enzymes: the structural basis of phosphatidylinositol mannosides biosynthesis in mycobacteria

11h15 CO-11 Thomas Hurtaux Activity and structural characterization of Candida albicans β-1,2 mannosyltransferase CaBmt3 involved in the elongation of the cell-wall phosphopeptidomannan

11h30 CO-12 Régis Fauré How to tip the balance from hydrolysis toward transglycosylation: molecular basis in retaining GHs

11h45 CI-12 Yves Blériot The glycosyl cation: from observation to exploitation 12h15 : déjeuner - après-midi libre

jeudi 26 mai

Session 8 modérateurs

A. Varrot R. Vivès

08h45 CI-13 Muriel Bardor Microalgae could help deciphering the evolution of N-glycosylation pathways

09h15 CO-13 Elizabeth Ficko-Blean Unraveling the multivalent binding of a marine family 6 carbohydrate-binding module with its native laminarin ligand

09h30 CO-14 Corinne Pau-Roblot Polygalacturonase from Arabidopsis thaliana: to new enzymes for industrial applications

09h45 CI-14 Jérôme Pelloux Roles of pectin methylesterases (PMEs) in plant development: how to fine-tune the degree of methylesterification of pectins ? 10h15 : pause

Session 9 modérateurs D. Bonnaffé

S. Pérez

10h45 CI-15 Caroline Rémond Glycoside hydrolases as enzymatic tools for the functionalization of carbohydrates

11h15 CO-15 Cédric Peyrot Chemo-enzymatic synthesis of innovant glycolipids for cosmetic formulation

11h30 CO-16 Isabelle Compagnon Infrared Multiple Photon Dissociation Spectroscopy : a new powerful technique for structural characterization of carbohydrates

11h45 CI-16 Etienne Fleury Preparation, characterization and properties of bio-hybrid materials from guar gum, ionic liquid and poly(ionic liquid) 12h15 : déjeuner

14h30 SESSION POSTERS 2 (n° pairs)16h00 : pause

Session 10 modérateurs P. Lerouge

J-C. Michalski

16h30 CI-17 Richard Daniellou Enzymatic synthesis of thioglycoconjugates: our recent progresses

17h00 CI-18 Catherine Ronin Glycoengineering therapeutic biologics : optimization of next generation antibodies

17h30 CI-19 Serge Pérez Popular glycosciences: building, seeing and playing with complex carbohydrates

18h00 ASSEMBLEE GENERALE DU GFG

vendredi 27 mai

Session 11 modérateurs

F. Allain C. Lopin-Bon

08h45 CI-20 Cédric Przybylski Interaction of glycosaminoglycans with cytokine biochips probed by Surface Plasmon Resonance Imaging coupled with Mass Spectrometry (SPRi-MS)

09h15 DUO-02 Samir Dahbi & Isabelle Bertin-Jung

Chemical synthesis and development of modified xylosides as potential inhibitors targeting β4GalT7, a key enzyme in glycosaminoglycan biosynthesis initiation

09h30 CI-21 David Bonnaffé 1,2-cis-glycosylation: the 2-azido-2-deoxy-D-gluco case in heparan sulfate fragment synthesis

10h00 CEREMONIE DE CLOTURE

5

COMMUNICATIONS INVITéES (CI)

7

CI-01

CI-02

Glycans in enteric virus infection

Jacques Le Pendu

CRCNA, Inserm UMR892, CNRS UMR 6299, Université de Nantes Noroviruses (NoVs) and rotaviruses (RVs) represent the most common causes of gastroenteritis. Despite their complete lack of phylogenetic relationship, human strains of these 2 families of viruses share similar carbohydrate-binding properties. Thus, human NoVs have been known for some time to attach to histo-blood group antigens (HBGAs) and recent data indicate that some strains additionally bind to gangliosides [1, 4]. Likewise, recent works showed that human RV strains appear to recognize both gangliosides and HBGAs [5]. These common glycan-binding properties within the 2 virus families, suggests shared molecular mechanisms of infection. Moreover, either volunteers’ studies and/or analyses of outbreaks demonstrated that for both NoVs and RVs, the HBGA polymorphism restricts infection to individuals presenting the correct HBGAs [2, 4]. A given strain appears to infect a subgroup of the population only, suggesting a past co-evolution of humans and both NoVs and RVs that led to a trade-off where the human population is partly protected whilst the virus circulation is maintained [3]. The partial protection of the population afforded by the HBGAs polymorphism, termed herd innate protection, can be complemented by herd immunity [4]. This can have important implications for the development of vaccines. In addition, blocking glycan-binding could provide a common preventive or therapeutic approach. 1. Han L, Tan M, Xia M, Kitova EN, Jiang X, Klassen JS (2014) Gangliosides are ligands for

human noroviruses. JACS 136:12631-12637 2. Imbert-Marcille B-M, Barbé L, Dupé M, Le Moullac-Vaidye B, Besse B, Peltier C, Ruvoën-

Clouet N, Le Pendu J (2013) A FUT2 gene common polymorphism determines resistance to rotavirus A of the P[8] genotype. J Infect Dis 209:1227-1230

3. Le Pendu J, Nystrom K, Ruvoen-Clouet N (2014) Host-pathogen co-evolution and glycan interactions. Curr Opin Virol 7:88-94

4. Ruvöen-Clouet N, Belliot G, Le Pendu J (2013) Noroviruses and histo-blood groups: the impact of common host genetic polymorphisms on virus transmission and evolution. Rev Med Virol 23:355-366

5. Tan M, Jiang X (2014) Histo-blood gorup antigens: a common nich for norovirus and rotavirus. Expert Rev Mol Med 16:e5

Congenital Disorders of Glycosylation and Golgi homeostasis: an unexpected link!

François Foulquier, Sven Potelle, Eudoxie Dulary, Sandrine Duvet, Dorothée Vicogne, Marie-

Ange Krzewinski-Recchi, Willy Morelle & Geoffroy de Bettignies

1 Univ. Lille, CNRS, UMR 8576 – UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F-59000

Lille, France.

Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. As GDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165 deficient cells, Mn2+ supplementation could restore a normal glycosylation. This suggests that TMEM165 is a key determinant for the regulation of Golgi Mn2+ homeostasis.

8

CI-03

CI-04

Novel cyclooctyne-based probes with exciting physical properties for the bioorthogonal labeling of glycoconjugates

Frédéric Friscourt 1, Petr Ledin 2, Richard Steet 2, Geert-Jan Boons 2 & Christoph Fahrni 3

1 Institut Européen de Chimie et Biologie, Université de Bordeaux, INCIA, CNRS UMR5287, Pessac, France, f.friscourt@iecb.u-bordeaux.fr

2 Complex Carbohydrate Research Center, Athens, University of Georgia, GA USA 3 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA USA

The bioorthogonal chemical reporter strategy, which elegantly combines the use of metabolically labeled azido sugars and highly reactive cyclooctyne probes, is emerging as a versatile technology for labeling and visualizing glycans.1

Although, the first generation of cyclooctynes exhibited relatively slow kinetics,2 efforts to increase reaction rates by tailoring the cyclooctyne structure have led to the identification of the dibenzocyclooctyne framework as key scaffold for high reactivity.3 However, increasing the aromatic nature of the cyclooctyne probes also augments their hydrophobicity, which can promote their sequestration by membranes or nonspecific binding to serum proteins, thereby increasing background signal.

To address these difficulties, we have developed two novel dibenzocyclooctynes (Fig 1): 1. A highly polar O-sulphated-dibenzocyclooctyne (S-DIBO),4 which does not penetrate the

cellular membrane, resulting in the selective labeling of extracellular glycoconjugates in living cells;

2. A fluorogenic cyclooctyne (Fl-DIBO)5 that undergoes fast cycloadditions with azides to yield�strongly fluorescent triazoles.

Figure 1 : Novel cyclooctynes with enhanced physical properties

References : [1] J.A. Prescher, C.R. Bertozzi, Nat. Chem. Biol., 2005, 1, 13-21. [2] N.J. Agard, J.M. Baskin, J.A. Prescher, A. Lo, C.R. Bertozzi, ACS Chem. Biol., 2006, 1, 644-648. [3] (a) X. Ning, J. Guo, M. A. Wolfert, G-J. Boons, Angew. Chem. Int. Ed., 2008, 47, 2253-2255; (b)

J.C. Jewett, E.M. Sletten, C.R. Bertozzi, J. Am. Chem. Soc., 2010, 132, 3688-3690; (c) M.F. Debets, S.S. van Berkel, S. Schoffelen, F.P.J. Rutjes, J.C.M. van Hest, F.L. van Delft, Chem. Commun., 2010, 46, 97-99.

[4] F. Friscourt, P.A. Ledin, N. E. Mbua, H.R. Flanagan-Steet, M.A. Wolfert, R. Steet, G-J. Boons, J. Am. Chem. Soc., 2012, 134, 5381-5389.

[5] F. Friscourt, C.J. Fahrni, G-J. Boons, J. Am. Chem. Soc., 2012, 134, 18809-18815.

Galectins, key players of homeostasis

Johann Dion, Christophe Dussouy, Samir Dahbi, Annie Lambert, Nataliya Storozhylova, Claude Solleux, Charles Tellier, Stéphane Téletchéa & Cyrille Grandjean

Unité Fonctionnalité & Ingénierie des Protéines, UMR CNRS 6286, Université des Sciences et Techniques de Nantes

The galectins form a ubiquitous family of lectins which bind to β-galactoside motifs through a conserved carbohydrate recognition domain (CRD). Galectins play a major role in cell development, homeostasis as well as in immune and inflammatory response. The deregulation of their expression/function is directly or indirectly associated to more than 100 pathologies such as cancer, arthritis, fibrosis, polycystic kidney disease… Evidence of their mode of action is, however, often indirect and their study made difficult due to their spatiotemporal localization and possible co-expression of several galectins within the same cell/tissue. [1] Focusing on Galectin-3, we aim at developing inhibitors of high affinity and specificity to shed light on biological processes Gal-3 is involved in and, at term, to propose novel therapeutic strategies. Lactosamine of type I (Galb1-3GlcNAc] and type II (Galb1-4GlcNAc) are the minimal natural motifs recognized by Gal-3. While presence of OH groups at positions C4’, C6’ and C3 (or C4) is mandatory for the binding, modifications at other positions is tolerated. We have developed access to either type I or type II lactosamine-based inhibitors according to chemo-enzymatic or chemical strategy, respectively. Pharmacophores have been introduced at key positions of these sugar motifs so as to optimize the recognition by the galectin-3 CRD (Figure). Selected inhibitors have been further modified for studying the role of galectin-3 in cell division and migration as well as in inflammation. Some of these biological results will also been discussed.

Figure: Access to type I or type II lactosamine and their recognition by Galectin-3 CRD

References: [1] Galectins, eds. A.A. Klyosov, Z.J. Witczak and D. Platt, Wiley, Hoboken (2008). [2] S. André, C. Grandjean, F.-M. Gautier, S. Bernardi, F. Sansone, H .-J. Gabius, R. Ungaro,

Chem. Commun., 2011, 47, 6126-6128

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CI-05

CI-06

CI-05 Chemoselective ligations: highly efficient strategies for the construction of biologically active multivalent glycoconjugates

Olivier Renaudet1,2 1 Univ. Grenoble Alpes, DCM, 38000 Grenoble, France; CNRS, DCM, 38000 Grenoble, France

2 Institut Universitaire de France, 103 boulevard Saint-Michel, 75005 Paris, France Synthetic glycoclusters and glycodendrimers have stimulated increasing interests over the past decade [1]. Among the large variety of multivalent scaffolds reported so far, our group is focusing on cyclopeptide-based glycoconjugates for diverse biological applications [2]. In this context, well-defined structures with various size, sugar density and combination (Figure 1) have been prepared in a controlled manner using either single or orthogonal chemoselective procedures (i.e. oxime ligation, Huisgen 1,3-dipolar cycloaddition, thiol-ene coupling, thiol-chloroacetyl coupling). Here we present the synthesis of several 4-, 16- and 64-valent compounds [3] and their biological properties as nanomolar lectin ligands [4] and antitumoral vaccines [5].

Figure 1: Molecular model of 4-, 16- and 64-valent glycoconjugates.

References : [1] O. Renaudet, R. Roy, Chem. Soc. Rev., 2013, 42, 4515. [2] M. C. Galan, P. Dumy, O. Renaudet, Chem. Soc. Rev., 2013, 42, 4599. [3] a) B. Thomas, C. Pifferi, G. C. Daskhan, M. Fiore, N. Berthet, O. Renaudet. Org. Biomol. Chem.,

2015, 13, 11529; b) B. Thomas, M. Fiore, G. C. Daskhan, N. Spinelli, O. Renaudet, Chem. Commun., 2015, 51, 5436; b) B. Thomas, N. Berthet, J. Garcia, P. Dumy, O. Renaudet, Chem. Commun., 2013, 49, 10796.

[4] a) N. Berthet, B. Thomas, I. Bossu, E. Dufour, E. Gillon, J. Garcia, N. Spinelli, A. Imberty, P. Dumy, O. Renaudet, Bioconjugate Chem., 2013, 24, 1598; b) M. Fiore, N. Berthet, A. Marra, E. Gillon, P. Dumy, A. Dondoni, A. Imberty, O. Renaudet, Org. Biomol. Chem., 2013, 11, 7113.

[5] a) B. Richichi, B. Thomas, M. Fiore, R. Bosco, H. Qureshi, C. Nativi, O. Renaudet, L. BenMohamed. Angew. Chem. Int. Ed., 2014, 53, 11917; b) O. Renaudet, L. BenMohamed, G. Dasgupta, I. Bettahi, P. Dumy, ChemMedChem, 2008, 3, 737.

Discovery and applications of new sucrose-active enzymes from GH70 family

Marlène Vuillemin, Marion Claverie, Florent Grimaud, Etienne Severac,

Sandrine Morel, Magali Remaud-Simeon & Claire Moulis

LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 avenue de Rangueil, 31077 Toulouse, France

Polysaccharide-based materials are now recognized as attractive alternatives to polymers derived from carbon fossil fuels, as revealed by their broad range of applications in food & feed, agriculture, health, or in chemical industries. In this context, some α-transglucosylases produced by lactic acid bacteria can be of interest, as they catalyze the synthesis of high molar mass α-glucans, glucooligosaccharides or gluco-conjugates from sucrose[1], a low-cost and abundant renewable resource.

These α-transglucosylases are classified in GH70 family[2], which comprises today around 300 sequences for only about sixty enzymes biochemically characterized, that remains low. To accelerate the development of enzymatic glucosylation tools with desired properties, our work is focused on their structure-activity relationship studies and engineering. However, the natural diversity of GH70 enzymes is far from being fully explored, and the repertoire of our enzymatic tool-box enzymes could be expand by exploring the numerous lactic acid bacterial genomes sequences available in databases. This presentation will describe our recent findings on several very original GH70 enzymes isolated thanks to data mining or genome sequencing campaigns. Distinctive specificities in term of glucan molar masses and/or structure (degree of α-1,2 or α-1,3 branching onto linear α-1,6 backbones) will be reported, as their impact on the physico-chemical properties of the final products. References : [1] Leemhuis H et al. (2013) J. Biotechnol. 163, 250–272 [2] Lombard Vet al. (2013) Nucleic Acids Res. 42, 490–495

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CI-07

CI-08

Polysaccharides: from hydrocolloids to textured materials

Françoise Quignard

ICGM, UMR 5253 CNRS-UM2-ENSCM-UM1, Matériaux Avancés pour la Catalyse et la Santé, 8 Rue de l'Ecole Normale, 34296 Montpellier Cedex 5, France.

The introduction of renewable resources in the production of catalyst supports and adsorbent is only possible if the materials intended to replace oil-derived or energy-intensive solids comply with strict requirements, like as high surface area, appropriate surface chemistry and porosity, thermal and chemical stability, and low cost. Hydrocolloid-forming polysaccharides are natural polyelectrolytes able to gelify water when added in tiny amounts. Hydrogels containing 1-2 % polymer and 98-99 % water can be shaped as self-standing spheres or films with good mechanical stability. Natural polysaccharides, albeit known for many years as supports for enzymatic catalysts and gelling agents in aqueous phase, suffer from diffusional limitations, due to the low surface area of the dried materials generally used, xerogels or lyophilised solids. This lecture deals with the proper methods to prepare dry materials which retain the dispersion of the polymer hydrogel, namely polysaccharide aerogels [1]. In one hand, the aerogel formulation opens the way to the exploitation of the surface properties and the high dispersion of the large family of polysaccharides for reactions at the interface between the polymer and a gas or an organic solvent. Applications in catalysis [2, 3], adsorption and chemical sensing can take advantage of the reactivity of the functional groups of the polymers or of catalytic sites in electrostatic or covalent interaction with the polysaccharide. In the other hand, aerogel formulation allows the extension to hydrocolloid derivatives of the techniques classically used for the characterization of inorganic solids, in particular those implying a high vacuum environment [4]. References :

[1] Quignard, F; Valentin, R; Di Renzo, New . J. Chem. 2008, 32, 1300. DOI: 10.1039/b808218a [2] Chtchigrovsky, M; Lin, Y; Ouchaou, K; Chaumontet, M; Robitzer, M ; Quignard, F; Taran, F. Chem. Mater. 2012, 24, 1505. DOI: 10.1021/cm3003595 -Chtchigrovsky, M; Primo, A; Gonzalez, P; Molvinger, K; Robitzer, M; Taran, F; Quignard, F.Angew Chem, 2009, 32, 5916. DOI: 10.1002/anie.200901309 [3] Pettignano, A; Bernardi, L; Fochi, M; Geraci, L; Robitzer, M; Tanchoux, N; Quignard, F. New . J. Chem. 2015, 39, 4222. DOI: 10.1039/c5nj00349k. [4] Robitzer, M; Di Renzo, F; Quignard, F. Microp. Mesop. Mat., 2011, 140, 9-16 DOI: 10.1016/j.micromeso.2010.10.006.

Conventional and sustainable bioprocesses for the extraction of antiherpetic oligo- and polysaccharides from the invasive Solieria chordalis (Rhodophyta, Gigartinales). Nathalie Bourgougnon1, Anne-Sophie Burlot1, Romain Boulho1, Yolanda Freile-Pelegrin2,

Christel Marty1, Gilles Bedoux1, Daniel Robledo2. 1Univ. Bretagne-Sud, EA 3884, LBCM, IUEM, F-56000 Vannes, France.

2Marine Resources Department, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV), Mérida, Mexico

Carrageenan is a generic name for a family of natural, water-soluble, sulphated galactans that are isolated from Rhodophyta and exploited on commercial scale. These phycocolloids exhibit high viscosity, and stabilizing, emulsifying and unique gelling properties used in the pharmaceutical, chemical and food industries. They were also shown to be potent and selective inhibitors of several enveloped viruses replication in vitro. Their modes of action have been attributed to the blockage of some early stages of the virus replication cycle. The carrageenophyte Solieria chordalis (C. Agardh) J.Agardh (Gigartinales, Solieriaceae) has been observed in the Gulf of Morbihan (France) since 2005 and in the Sarzeau peninsula (Morbihan, France) where strandings have become more abundant between July and October. S. chordalis is a real economic and environmental burden due to its littoral anarchic proliferation. The processing of this raw material is little developed and provides little added value whereas it constitutes a biomass potentially rich in highly bioactive polysaccharides that could represent useful avenues for the development of new functional ingredients in pharmaceutical industries. The aim of this conference is then to compare and discuss the use of sustainable bioprocesses for extracting and purifying antiviral polysaccharides from Solieria chordalis. To improve the extraction conditions of polysaccharides, we propose to use Microwave Assisted extraction (MAE) and Enzyme-Assisted Extraction (EAE) techniques in comparison with the conventional Hot Water Extraction (HWE). Comparison of yields and chemical composition analysis of extracts were performed for each processes. Antiviral activity from oligo- and polysaccharides was evaluated in mammalian cell lines infected by Herpes simplex virus type 1 (HSV-1; family Herpesviridae) in vitro.

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CI-09

CI-10

Molecular bases of Mycobacterium tuberculosis

recognition by C-type lectins: from the modulation of innate immune response

to the design of therapeutic molecules

Jérôme Nigou

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, France

Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens. It has evolved multiple molecular mechanisms to alter immune responses, including inflammation, thereby securing its colonization and survival inside the infected host. In particular, M. tuberculosis exposes specific glycolipids and lipoglycans at its cell envelope surface to target C-type lectin receptors (CLRs), DC-SIGN, Mannose Receptor or Mincle, expressed by innate immune cells, such macrophages and dendritic cells.

The strategies used by M. tuberculosis to modulate the host inflammatory response prompted us to design synthetic molecules that mimic the bioactive structure of natural mycobacterial glycoconjugates, with the objective of developing innovative immunomodulatory compounds. To achieve this goal, we used a combination of approaches, including identification of the natural CLR agonist molecules present in the mycobacterial cell envelope, deciphering the molecular mechanisms of ligand-receptor interaction and bio-guided chemical synthesis.

During my talk, I will present the example of two fully synthetic families of molecules that display powerful activities in vitro and in vivo in mouse models: i) anti-inflammatory mannodendrimers, ligands of DC-SIGN, that prevent acute lung inflammation; ii) adjuvant glycolipids, ligands of Mincle, that induce strong Th1 and Th17 immune responses.

These immunomodulatory compounds are currently tested in different pathologically models to determine the broader applicability of their therapeutic use.

Synthetic carbohydrate-based vaccines against shigellosis: from concept to clinic … and more

Laurence Mulard 1,2

1 Institut Pasteur, Unité de Chimie des Biomolécules, 28 rue du Dr Roux, 75724 Paris Cedex 15, France

2 CNRS UMR 3523, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France

Shigellosis, or bacillary dysentery, caused by the non capsulated enteroinvasive bacteria Shigella, is a major burden especially in developing countries. It remains one of the top four diarrheal diseases in children under five.1 Species and serotype diversity, added to their geographical distribution, strongly support the need for a multivalent vaccine.2 With regard to endemic shigellosis, special attention is paid to Shigella flexneri and Shigella sonnei. Interestingly, protection against re-infection is thought to be achieved, to a large extent, by antibodies specific for the O-antigen moiety of the lipopolysaccharide. In this context, a multidisciplinary strategy toward vaccine candidates encompassing synthetic oligosaccharides mimicking the “protective” determinants carried by the O-antigen of selected serotypes was undertaken in the laboratory. The two-step process under development aims first at identifying sets of “protective” epitopes, and second at designing conjugates thereof acting as strong immunogens.

This presentation first highlights the pre-clinical development of a monovalent S. flexneri 2a glycovaccine candidate now entering the clinical stage.3 Second, it addresses our strategy for broadening vaccine coverage. Interestingly, with the exception of serotypes 6 and 6a, all known repeating units from S. flexneri O-antigens comprise a common tetrasaccharide backbone. Diversity and serotype specificity are related to the occurrence of α-D-glucosylation and/or acetylation at specific hydroxyl groups of the basic tetrasaccharide, itself a linear combination of three L-rhamnose residues and a N-acetyl-D-glucosamine.4 The possible impact of these substitutions on vaccine development is discussed, while their influence on hapten synthesis is exemplified. In particular, we illustrate the multidisciplinary strategy that we have implemented to identify promising well-defined mimics of the O-antigen from S. flexneri 3a, another prevalent serotype. We report a detailed investigation of the immunodominant role of O-antigen stoichiometric O-acetylation as revealed by chemical synthesis, immunochemistry, physical chemistry, NMR, and X-ray crystallography studies. Next, we describe the rational design, synthesis, and immunogenicity data of the first synthetic carbohydrate-based vaccine candidate against S. flexneri 3a. Finally, we discuss preliminary immunogenicity data for a set of S. flexneri 2a / S. flexneri 3a glycoconjugate combinations and extension to additional serotypes prevalent in the field. References : [1] K. L. Kotloff et al., The Lancet 2013, 382, 209. [2] M. M. Levine et al., Nat. Rev. Microbiol., 2007, 5, 540. [3] R. van der Put et al., Bioconjugate Chem., 2016, DOI: 10.1021/acs.bioconjchem.5b00617. [4] A. V. Perepelov et al., FEMS Immunol. Med. Microbiol., 2012, 66, 201.

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CI-12

Membrane Enzymes: the Structural Basis of Phosphatidylinositol Mannosides Biosynthesis

in Mycobacteria

Marcelo E. Guerin 1,2

1 Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain

2 IKERBASQUE, Basque Foundation for Science, 48013, Bilbao, Spain. Membrane enzymes constitute a large class of proteins with critical roles in a variety of cellular processes in all living organisms. They generate a significant amount of structural diversity in biological systems, which are particularly apparent not only in the maintenance of the structural integrity of the cell, but also in cell signaling and metabolism, and cell-pathogen interactions. Many of these cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of cytoplasm and organelles. Thus, enzymes performing this type of reaction are required to access a lipophilic substrate located in the membranes and to catalyze its reaction with a polar, water-soluble compound.[1] Here we focus on the membrane enzymes involved in the early steps of the phosphatidylinositol mannosides (PIMs) biosynthetic pathway, unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidylinositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Of particular relevance, we demonstrate the occurrence of a conformational switch during the catalytic cycle of the retaining glycosyltransferase PimA, the enzyme that start the pathway, involving both β-strand–to–α-helix and α-helix–to–β-strand transitions.[2] These structural changes seem to modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane surface. Although scant structural information is currently available on protein catalysis at the lipid-water interface, our studies demonstrate that protein-membrane interactions might entail unanticipated structural changes in otherwise well conserved protein architectures, and suggests that similar changes may also play a functional role in other membrane-associated enzymes. Finally, we report the crystal structures of PatA, an essential membrane associated acyltransferase that transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring added by PimA, in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl–CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design. References : [1] Forneris, F.; Mattevi, A. Science 2008, 321, 213-216. [2] Giganti et al., Nat. Chem. Biol. 2015, 11,

16-18. Highlighted in the News and Views Section: Brodhun F, Tittmann K. Nat. Chem. Biol. 2015, 11, 102-103. [3] Albesa-Jove et al., Angew. Chem. Int. Ed. Engl. 2015, 54, 9898-9902.

[4] Albesa-Jove et al., Nat. Commun. 2016, 7, 10906.

The glycosyl cation: from observation to exploitation

Yves Blériot 1, Amélie Martin 1, Ana Arda 2, Jérôme Désiré 1, Agnès Mingot 1, Nicolas

Probst1, Jesus Jimenez-Barbero 2 & Sébastien Thibaudeau 1 1 IC2MP, UMR CNRS 7285, Equipe “Synthèse organique” Université de Poitiers, France

2 CIC bioGUNE, Bizkaia Technological Park, 48160 Derio-Bizkaia, Spain The central reaction in glycosciences is arguably glycosylation, the formation of the glycosidic bond that connects a sugar to another molecule. It can be performed enzymatically through the use of glycosyl transferases or chemically using glycosyl donors and acceptors. Surprisingly, while the enzymatic mechanism has gained a high level of knowledge and sophistication, some of the details of the chemical glycosylation mechanism are still poorly understood (Figure 1).1 Both mechanisms probably involve transient glycosyl cations to some degree. Observation, characterisation and further exploitation of these key ionic species could have a strong impact on applied and fundamental aspects of glycosciences. Our recent contribution to this field will be presented.2

Figure 1 : Prototype of the chemical glycosylation mechanism References : [1] L. Bohé, D. Crich, C.R. Chimie, 2011, 14, 3-16. [2] A. Martin, A. Arda, J. Désiré, N. Probst, A. Mingot, P. Sinaÿ, J. Jimenez-Barbero, S. Thibaudeau,Y. Blériot, Nat. Chem. 2016, 8, 186-191.

carbon) and 2.57 ppm (methyl protons)) in the reaction mixture sup-ports a pathway involving departure of the protonated anomericacetate, which is further dissociated into a methyl acylium ion andwater23. Application of the methodology to sugar pyranoses wasthen studied, and required suitable protective groups for the sugarsand benign nucleophilic characteristics for the superacid medium.A rapid survey of sugar protecting groups including esters, ethersand acetals led to the selection of the acetyl as the group of choice.This can withstand the harsh acidic conditions, and can be recoveredafter superacid quenching. The easily available peracetylated gluco-pyranoses were tested first to check the compatibility of HF/SbF5with monosaccharides. Fortunately, the 1H NMR spectrum of thecrude reaction mixture resulting from the treatment of the peracety-lated α-D-glucopyranose with HF/SbF5 was immaculate, allowing itsfull assignment. A polycationic pyranosidic structure 2 with aregular 4C1 chair conformation, and in which the carbonyl function

of each of the five ester groups is protonated (presence of fiveprotons as singlets at 12 ppm < δ < 14 ppm by 1H NMR), wasdeduced from this spectrum (Fig. 1c). At this stage, the presence ofpartially protonated species that undergo a rapid dynamic equili-brium that is not detectable on the NMR timescale cannot be ruledout24. It is remarkable that, under these severe conditions, no elimin-ation reaction takes place to yield unsaturated heterocycles. Similarly,peracetylated α-D-glucosamine furnished the corresponding polypro-tonated species 3, also with a 4C1 chair conformation. Taking advan-tage of the neighbouring group participation of the acetyl protectinggroup at C2 (ref. 25), the peracetylated β-D-glucopyranose yielded theknown dioxalenium ion 4 (ref. 26), as confirmed by the appearance ofthe dioxonium signal (δ = 193.6 ppm), the strong deshielding of theC1 carbon (δ = 108.6 ppm) and the shielding of the methyl group(δ = 13.8 ppm). A 4H5 conformation was attributed to ion 4 by com-paring the experimental and GIAO-DFT (gauge including atomicorbitals–density functional theory)-calculated 13C chemical shiftsand coupling constants for its optimized structure. In parallel, theknown oxazolinium ion 5 (ref. 27) was generated from peracetylatedN-acetyl-β-D-glucosamine, and was found to adopt a major OS2 con-formation according to NMR analysis and DFT calculations (Fig. 1c).

These results indicate that, even in superacid, transient glycosylcations can be trapped and stabilized by a vicinal acetate or amidegroup at C2, further supporting an equilibrium between the proto-nated and unprotonated forms of these functions allowing anchi-meric assistance by the carbonyl group at C2. This preliminarystudy, targeting stabilized glycosyl cationic intermediates and com-bining superacid chemistry, NMR analysis and DFT calculations,confirmed the potential of our approach, which was next exploitedfor the study of highly challenging non-stabilized glycosyl cations.To avoid potential problems associated with the presence of a sub-stituent at C2, we focused our first efforts on the peracetylated2-deoxy-β-D-glucopyranose. Extensive NMR analysis of thecrude reaction mixture resulting from the treatment of the sugarderivative with HF/SbF5 at −40 °C provided strong evidence forthe formation of the 2-deoxyglucosyl oxocarbenium ion 6 as themain species (anomeric proton at δ = 8.89 ppm and anomericcarbon at δ = 229.1 ppm) (Fig. 2a–e).

The quality of the recorded NMR spectra, assisted by compu-tational tools, enabled access to the conformation of oxocarbeniumion 6 for the first time in a condensed phase. Evidence for flatteningof the sugar ring was provided by J-coupling analysis and a 4E con-formation was assigned according to the perfect match between theexperimental and theoretical NMR parameters (Fig. 3a). This posi-tive result prompted us to pursue glycosyl cations displaying a sub-stituent at C2, as is classically found and used in carbohydratechemistry. We initially turned our attention to the peracetylated2-azido-β-D-glucopyranose that did not provide the correspondingglycosyl cation under HF/SbF5 treatment. Instead, a species inwhich the azido28 and acetate groups were protonated was produced(Supplementary Section 7). To explain the stability of this monosac-charide, we postulated that the generation of a positive charge inclose vicinity to the anomeric centre precludes glycosyl cation for-mation. We thus moved to known peracetylated 2-bromo-29 and2-fluoroglucopyranoses30, which are extensively used as glycosyldonors. Although the 2-fluoro derivative gave complex NMRspectra, the 2-bromo sugar furnished a species that, from NMRdata (anomeric proton at δ = 8.36 ppm and anomeric carbon atδ = 198.1 ppm), corresponded to glucosyl cation 7. Application ofa similar set of NMR and computational experiments as for ion 6led to the attribution of a 4H5 conformation for 7 (Fig. 3b). Bothions 6 and 7 display half-chair or close conformations in whichthe positively charged acetate substituents adopt pseudoequatorialorientations, probably to minimize charge repulsion, notably withthe cationic anomeric carbon. In contrast, the bromine atom in 7adopts a pseudoaxial orientation that might be imposed by a

O

LGPO

OPO

E X

OPO

Glycosyl donor A

X

O

LGPO

EB

X

OPO

F

X

DE

OPO

C

Conformation?Stereochemical outcome?

OAc*

O

1

O13C: 228.5 ppm

1H: 8.89 ppm

13C: 3.8 ppm1H: 2.57 ppm

13C: 150.7 ppm

O

OAc*

*AcO*AcO

*AcOOAc*

2 (4C1)

O

OAc*

*AcO*AcO

*AcHNOAc*

3 (4C1) 4 (4H5)

5 (OS2)

O

OAc*

*AcO

HNO

O*AcO

*AcO

OAc*O O

*AcO =

O

OH

a

c

b

Figure 1 | Prototype of the glycosylation mechanism and ionic speciesobserved by NMR. a, Generally accepted mechanism for glycosylationstarting from glycosyl donor A and involving associated species B, glycosyloxocarbenium ion C, covalent intermediate D, contact ion pair E or solventseparated ion pair F, depending on the glycosylation conditions and thestructure of the glycosyl donor. P, protecting group; LG, leaving group;E+, electrophile; X−, electrophile counterion. b, Key NMR signalsrecorded for tetrahydropyranosyl cation 1 (obtained by treatment of2-acetoxytetrahydropyran with HF/SbF5) and methyl acylium ion insuperacid. c, Structure and major conformation adopted by sugar-derivedions 2–5 (obtained by treatment of peracetylated α-D-glucopyranose,α-D-glucosamine, β-D-glucopyranose and β-D-glucosamine, respectively,with HF/SbF5), as deduced from the analysis of the experimental vicinalJH,H coupling constants and by comparison between the experimental andGIAO-DFT calculated 13C chemical shifts and coupling constants for theiroptimized structure.

ARTICLES NATURE CHEMISTRY DOI: 10.1038/NCHEM.2399

NATURE CHEMISTRY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemistry2

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CI-14

Microalgae could help deciphering the evolution of N-glycosylation pathways

Clément Ovide1*, Gaëtan Vanier1*, Elodie Mathieu-Rivet1, Carole Burel1,

Patrice Lerouge1, Marie-Christine Kiefer-Meyer1 & Muriel Bardor1, 2

1 Normandie Univ, UNIROUEN, Laboratoire Glycobiologie et Matrice Extracellulaire végétale 76000 Rouen, France.

2 Institut Universitaire de France (IUF), Paris, France. *Equal contribution of the two authors

Despite the biological and physiological significance, along with knowledge regarding the N-glycosylation processing in Eukaryotes, little attention has been paid so far to this biosynthetic pathway in microalgae even if they are interesting organisms spread in different phyla of the tree of life. Moreover, microalgae emerged recently as potential cell bio-factories for the production of biopharmaceuticals [1] for which glycosylation represent a critical quality attribute [2].

In order to characterize the N-glycosylation pathways in microalgae, we took advantage of the recent genomic sequencing of several microalgae models belonging to different phyla of the tree of life and identify a set of putative orthologs involved in the different key steps of the N-glycan biosynthesis and maturation. For some of the microalgae like the green microalgae Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, detailed structural analyses of the N-glycans bearing by their endogenous proteins have already been performed, thus reflecting their capabilities in term of N-glycan biosynthesis [3-5]. Moreover, we started to clone and functionally characterize some of the microalgae glycosyltransferases and glycosidases. We already demonstrated that a Phaeodactylum tricornutum gene is encoding for a N-acetylglucosaminyltransferase I which is functional. Indeed, this gene encodes for an active N-acetylglucosaminyltransferase I which is able to restore complex-type N-glycans maturation in the Chinese Hamster Ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I [3]. This piece of work represented the first functional characterisation of N-glycan glycosyltransferase from microalgae. Further functional characterizations and localisation of putative glycosyltransferases are currently under investigation to shed the light about the specific Golgi maturations and organisation occurring in microalgae N-glycosylation pathway. References: [1] Vanier G., Hempel F., Chan P., Rodamer M., Vaudry D., Maier U., Lerouge P. and Bardor M.

(2015) Plos ONE, DOI:10.1371/journal.pone.0139282. [2] Lingg N., Zhang P., Song Z. and Bardor M. (2012) Biotechnology Journal, 12, 1462-1472. [3] Baiet B., Burel C., Saint-Jean B., Louvet R., Menu-Bouaouiche L., Kiefer-Meyer M.-C., Mathieu-

Rivet E., Lefebvre T., Castel H., Carlier A., Cadoret J.-P., Lerouge P. and Bardor M. (2011) Journal of Biological Chemistry, 286, 6152-64.

[4] Mathieu-Rivet E., Scholz M., Arias C., Dardelle F., Schulze S., Le Mauff F., Teo G., Hochmal A.K., Blanco-Rivero A., Loutelier-Bourhis C., Kiefer-Meyer M.-C., Fufezan C., Burel C., Lerouge P., Martinez F., Bardor M.* and Hippler M.* (2013) Mol cell Proteomics. 12(11):3160-83.

[5] Mathieu-Rivet E., Kiefer-Meyer M.C., Vanier G., Ovide C., Burel C., Lerouge P. and Bardor M. (2014) Frontiers in Plant Science, section Plant physiology, Jul 28; 5:359.

Roles of pectin methylesterases (PMEs) in plant development: How to fine-tune the degree of

methylesterification of pectins?

Ludivine Hocq1, Fabien Sénéchal1, Olivier Habrylo1, Françoise Fournet1, Jean-Marc Domon1, Paulo Marcelo2, Alexis Peaucelle3, Françoise Guérineau1, Katra Kolšek4, Davide

Mercadante4, Valérie Lefebvre1, Jérôme Pelloux1

1 EA3900-BIOPI Biologie des Plantes et Innovation SFR Condorcet FR CNRS 3417, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, France.

2 Plateforme d’Ingénierie Cellulaire & Analyses des Protéines ICAP Université de Picardie Jules Verne, 80039 Amiens, France.

3 UMR1318-IJPB, INRA Centre de Versailles-Grignon, Versailles, France. 4 HITS gGmbH - Heidelberg Institute for Theoretical Studies, Schloß-Wolfsbrunnenweg 35,

69118 Heidelberg, Germany. The fine-tuning of the degree of methylesterification of cell wall pectin is a key to regulate cell elongation and ultimately the shape of plant body. Pectin methylesterification is spatio-temporally controlled by pectin methylesterases (PMEs, 66 members in Arabidopsis). The comparably large number of proteinaceous pectin methylesterase inhibitors (PMEIs) questions the specificity of the PME-PMEI interaction and the functional role of such abundance. We first characterized the role of PMEs in regulating cell expansion during dark-grown hypocotyl. In this simple model, developmental and cell biology, genomics, biochemistry, and biophysics can be integrated at a cellular level. Using mutant plants impaired for the expression of PME2 and PME32, we show how PMEs can mediate changes in pectin chemistry and cell wall mechanics, with consequent effects on elongation. To gain more insights into the fine tuning of PME activity, we characterized PME-PMEI interactions. For this purpose, we combined biochemistry and Molecular Dynamic (MD) simulations approaches to assess the determinants of the pH-dependence of the interaction. Using site-directed mutagenesis, we confirmed the role of specific amino acids in modulating the interaction. MD simulation have proven to be powerful to predict the differences between PMEI, allowing the discovery of a strategy that may be used by PMEIs to inhibit PMEs in different micro-environmental conditions and paving the way to identify the specific role of distinct PMEIs in muro.

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CI-16

Glycoside hydrolases as enzymatic tools for the functionalization of carbohydrates

Caroline Rémond

UMR FARE 614 - Chaire AFERE, Université de Reims Champagne Ardenne - INRA

Glycoside corresponds to sugar moiety (monosaccharide to polysaccharide) covalently linked to an aglycon part (small aglycon to protein and lipid). Numerous glycosides exist in nature and some can be synthesized for various applications (surfactants, food, biological activities, antimicrobial activities, …). The attachment of the sugar moiety modifies the properties of the aglycon notably by improving its solubility in aqueous media. Glycosylation of aglycons can be obtained by conventional chemical synthesis which allow obtaining reasonable yields but suffer from numerous drawbacks such as protection and deprotection steps of the substrates, use of solvents and toxic catalysts [1, 2]. Enzymatic glycosylation represents an interesting alternative and can be achieved by glycosyl transferases, glycoside phosphorylases, transglycosidases and glycoside hydrolases [3]. In vivo, glycoside hydrolases catalyze the hydrolysis of glycosidic linkages. In presence of acceptor molecules different from water, some glycoside hydrolases can catalyze reverse hydrolysis (thermodynamically controlled) or transglycosylation (kinetically controlled) reactions which conduct to the synthesis of glycosides [4].

Figure 1 : Reactions catalyzed by glycoside hydrolases

Some examples of glycosides synthesis (alkyl glycosides, vitamin glycosides, …) with glycoside hydrolases will be presented as well as strategies developed to improve synthesis which can be achieved by protein engineering and/or by optimization of reactional conditions. References : [1] Brusa, C., et al., beta-xylopyranosides: synthesis and applications. RSC Advances, 2015. 5(110):

91026-91055. [2] de Roode, B.M., et al., Perspectives for the industrial enzymatic production of glycosides.

Biotechnology Progress, 2003. 19(5): 1391-1402. [3] Thuan, N.H. and J.K. Sohng, Recent biotechnological progress in enzymatic synthesis of

glycosides. Journal of Industrial Microbiology & Biotechnology, 2013. 40(12): 1329-1356. [4] van Rantwijk, F., M. Woudenberg-van Oosterom, and R.A. Sheldon, Glycosidase-catalysed

synthesis of alkyl glycosides. Journal of Molecular Catalysis B: Enzymatic, 1999. 6: 511-532.

Preparation, characterization and Properties of Bio-Hybrid materials from Guar Gum,

Ionic Liquid and Poly(Ionic Liquid)

Biao Zhang, Anatoli Serghei, Guillaume Sudre, Julien Bernard, Aurélia Charlot & Etienne Fleury

Université de Lyon, Lyon, F-69003 France. INSA-Lyon, IMP, Villeurbanne, F-69621 France.

CNRS, UMR 5523, Ingénierie des Matériaux Polymères, Villeurbanne, F-69621, France.

The growing interest in utilizing biohybrid materials arises from the possibility to benefit the best features of each component to reach new tunable and adaptable materials. Indeed these material are constituted of molecular or polymeric species, of biologic origin with other components (e.g. synthetic polymers, ceramics, metal and metal oxides…) and their combinations are in theory infinite.1 Herein we aim at generating non-conventional biobased solid electrolytes in exploiting the synergistic interactions between galactomannan chains and hydrophilic imidazolium ionic liquids (IL). Indeed the latter have unique attributes: thermal and chemical stability, non-inflammability, non-volatility and high conductivity.2,3 Galactomannans, especially guar, are abundant non-toxic polysaccharides with high thermal stability and commercial availability of very high molecular weights (up to 3 million g.mol-1). We demonstrated that such guar/IL association leads to solid-like gels. 4,5 Then we focused on the development of ternary blends presenting a higher degree of sophistication by incorporating additional reinforcing building-blocks, such as imidazolium-based poly(ionic liquid) (PIL). PIL are promising synthetic polymers which combine the properties of ILs and the ones of polymers in terms of mechanical reinforcement, and dimensional stability. We synthesized a series of PIL by RAFT polymerization and we particularly show the excellent control of the polymerization.6 finally, we generated biohybrid guar-based grafted copolymer, which can keep the intrinsic properties and also bring new properties of PILs that raw guar gum does not have. Structure/properties relationships of the resulting multicomponent systems were in-depth investigated. The rheological, thermal and conductive properties were methodically studied and correlated with the morphology of the biohybrids by means of synchrotron scattering measurements. The concept presented herein, based on biosourced polymer-containing multi-component systems represents a promising route for the design of advanced conductive materials.

References: 1 Gao J., Maruyama A., Encyclopedia of Polymeric Nanomaterials, DOI: DOI 10.1007/978-3-642-

36199-9_231-1 2 Pinkert A., Marsh K. N., Pang S., Staiger M. P., Chem. Rev., 2009, 109, 6712–6728. 3 Hayes R., Warr G. G., Atkin R., Chem. Rew., 2015, 115, 6357-6426. 4 Lacroix C., Sultan E., E. Fleury, Charlot A. Polymer Chemistry, 2012, 3, 538 5 Verger L., Corre S., Poirot R., Quintard G., Fleury E., Charlot A., Carbohydr. Polym., 2014,10,932. 6 Zhang B., Yan X., Alcouffe P., Charlot A., Fleury, E., Bernard. J. ACS Macro Lett. 2015, 4, 1008-1011

15

CI-17 — Prix GFG 2016 —

CI-18

Enzymatic synthesis of thioglycoconjugates: our recent progresses

Richard Daniellou 1,2

1 ICOA, UMR CNRS 7311, Université d’Orléans, rue de Chartres, BP6759, 45067 Orléans cedex 2

2 Cosm’actifs, GDR CNRS 3711

Carbohydrates play an important part in a vast array of biological processes and therefore glycomimetics are currently becoming a powerful class of novel therapeutics.1 Amongst them, thioglycosides, in which a sulfur atom has replaced the glycosidic oxygen atom, are tolerated by most biological systems. Their major advantages rely in the fact that they adopt similar conformations than the corresponding O-glycosides and especially that they prove to be less sensitive to acid/base or enzyme-mediated hydrolysis. Such compounds have already demonstrated to be valuable tools as good chemical donors for synthetic purposes,2,3 as stable intermediates in X-ray crystallographic analysis of proteins4 and, of particular interest, as competitive inhibitors of a wide range of glycosidases involved in numerous diseases.5 Besides the synthetic methodologies developed throughout the years by organic chemists, the presence of natural S-glycoconjugates was recently assessed and lead to the discovery of some glycosyltransferases involved in such rare biocatalytic processes. In parallel, the increases of knowledge on the mechanism and the structure of glycoside hydrolases have conducted to the development of original catalysts with greatly improved synthetic properties for thioglycosidic linkages. However biocatalyzed procedures of thioglycosylation still represent an emerging area.6

Herein, we will discuss our recent findings in this tremendous field. Firstly we will show our results dealing with the glycosyltransferase S-UGT74B1 from Arabidopsis thaliana and demonstrate its ability to promote the synthesis of various desulfoglucosinolates. Then, a small library of mutants of glycoside hydrolases from Dictyoglomus thermophilum will be used to demonstrate their ability to prepare the S-glucosylated thiotyrosine. In addition, comparisons of the tridimensional structures and the mechanisms of these original enzymes will open us powerful synthetic perspectives. References : [1] B. Ernst and J. L. Magnani, Nat. Rev. Drug Discovery, 2009, 8, 661-677. [2] X. Zhu and R. R. Schmidt, Angew. Chem., Int. Ed., 2009, 48, 1900-1934. [3] J. D. C. Codee, R. Litjens, L. J. van den Bos, H. S. Overkleeft and G. A. van der Marel, Chem.

Soc. Rev., 2005, 34, 769-782. [4] H. Driguez, Chembiochem, 2001, 2, 311-318. [5] D. J. Wardrop and S. L. Waidyarachchi, Nat. Prod. Rep., 2010, 27, 1431-1468. [6] L. Guillotin, P. Lafite and Daniellou, R., Chapter 10 Enzymatic thioglycosylation: current

knowledge and challenges. In Carbohydrate Chemistry: Volume 40, The Royal Society of Chemistry: 2014; Vol. 40, pp 178-194.

Glycoengineering therapeutic biologics : optimization of next generation antibodies

Catherine Ronin

Siamed’Xpress

The N-linked glycan profiles of recombinant therapeutics significantly affect the biological functions of the protein of interest, most often its duration in the circulation. The glycome of a human protein drug engineered in host cells is largely determined by both the cellular genotypes and culture settings. More particularly, antigenic sugar determinants are by now formally prohibited. With pressure from pricing by the regulatory agencies and biosimilars looming, more efficient and effective approaches are actively sought, among which the field of glycoengineering is especially attractive because it also allows improvement of the 1st generation molecules. Over the past decade, the segment of therapeutic antibodies has become the highest selling class of recombinant biological products: 7 out of 10 worldwide prescription drugs have been antibodies in 2015. Most of them are of the IgG1 isotype. Biological studies have shown that the distribution of the 27 glycans in the Fc fragment can significantly impact antibody efficacy, stability and effector function. Indeed, the IgG glycome alternatively encodes pro-inflammatory or anti-inflammatory activities. This sugar switch is largely based on truncated unusual biantennary glycans and core fucose. Adverse immunogenicity has been noticed along with the earliest generation of engineered IgG scaffolds as well as from the use of various expression systems. Today, there is a clear need to solve several of these different issues. A key step in development for monoclonal antibodies undoubtedly involves optimization and control of N-glycan profiles to produce next generation antibodies.

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CI-20

1

Popular Glycoscience: Building, Seeing and Playing with Complex Carbohydrates

Serge Pérez

Department of Molecular Pharmacochemistry, CNRS-Univeristé de Grenoble Alpes, Grenoble, France.

In two recently published monographs, “A road-map for Glycoscience in Europe” and “Transforming Glycoscience: A Roadmap for the future” published respectively under the auspices of the European Science Foundation and the National Academies USA, a selected number of goals were identified. One of particular importance was the need for “establishment of long term databases and bio-informatics and computational tools to enable accurate carbohydrate and glycoconjugate structural predictions”. One of the challenges facing Glycoscience is the development and implementation of robust and validated informatics toolbox enabling accurate and fast determination of complex carbohydrate sequences extendable to 3D prediction,

computational modeling, data mining and profiling. The concomitant expansion of stable and integrated databases,

cross-referenced with popular bioinformatics resources should contribute to connecting glycomics with other –

omics.

Glyco3D (2) features a family of databases covering the 3D features of mono-, di-, oligo-, poly-

saccharides, glycosyltransferases, lectins, monoclonal antibodies and glycosaminoglycan-binding proteins. This ensemble offers a unique opportunity to characterize the 3D features that a given oligosaccharide can assume in different

environments. A common nomenclature has been adopted that conforms to the recommendations for

carbohydrates and including the constraints required by the developing field of glycobiology in terms of visualization and encoding. A search engine has

been developed that scans the full content of all the data bases for queries related to sequential information of the carbohydrates or other related descriptors.

Whereas macromolecular builders are also made available for generating three-dimensional structures of polysaccharides and complex carbohydrates, there was a clear need to develop a molecular visualization program that would cope with the uniqueness of the range of carbohydrate structural features, either alone or in complex environments in particular with proteins and lipids. To this aim, video game-based computer graphic software (SweetUnityMol (3)) was developed. All the specific structural features displayed by the simplest to the most complex carbohydrate-containing molecules have been taken into account and can be conveniently depicted. This concerns the identification of monosaccharides types, conformations, location in single chain or multiple branched chains, depiction of secondary structural elements and the essential constituting elements in very complex structures. In all these instances, particular attention was given to cope with the accepted nomenclature and pictorial representation used in carbohydrate chemistry, biochemistry and glycobiology. This program closely follows the most accepted symbolic representations for monosaccharides and existing formats for atomic coordinates and opens the route to pictorial representation of carbohydrates when studied at the “coarse-grain” level. References : [1] http://glycopedia.eu/IMG/pdf/white_paper_feb2015.pdf [2] Glyco3D. http://www.glyco3D.cermav.cnrs.fr [3] S. Perez, T. Tubiana, A. Imberty, M. Baaden, Glycobiology, 2015, 25, 483-491.

Interaction of glycosaminoglycans with cytokine biochips probed by Surface Plasmon Resonance Imaging coupled

with Mass Spectrometry (SPRi-MS)

Cédric Przybylski1* 1 Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement (LAMBE), Université Evry-Val-d’Essonne, CNRS UMR 8587, Bld François Mitterrand, 91025 EVRY Cedex, France

* Present adress : Institut Parisien de Chimie Moléculaire (IPCM), Université Pierre et Marie Curie, CNRS UMR 8232, 4 place Jussieu, 75252 PARIS Cedex 05, France

The non-covalent interactions between proteins and anionic polysaccharides such as glycosaminoglycans (GAGs) are involved in several physio-patholological processes including cell signalling and recognition, bacterial and viral infections, and cancer progression. One of the main barriers in understanding the molecular mechanisms involved in these interactions hold in decoding the structural information contained in GAGs sequences. This task remains trick especially because of variable level of acetylation, sulfation, and epimerization hindering further advances of the glycobiology field.[1-4]

To determine featuring parameters of complexes such as stoichiometry, kinetic constants and structural determinants involved in interaction, several analytical methods such as NMR and isothermal calorimetry titration can be used.[5] Nonetheless, most of them meet only one or two of the aforementioned parameters, while requiring extensive analysis times and/or large volumes/amounts.

During this last decade, biochips technology has experienced an unprecedented development coinciding with the spreading of the “omics” sciences. These developments were motivated by the ability to miniaturize and achieve the high-throughput and parallel screening of thousands of interactions. In glycobiology, biochips are increasingly regarded as a reliable tool for glycome exploration, study of protein/glycan interaction, and the discovery of new enzyme activities. Most of the reported glycobiochip approaches required glycan of pure and defined structures.[6-11] Moreover, such biochips required labelling of one partner for detection, provided relative data on interaction strength, and failed to allows kinetics constant determination and ligand identification.

To tackle these limitations, we have developed an original platform where interactions of oligo/polysaccharides with proteins immobilized on biochips are probed by Surface Plasmon Resonance experiments and further analysed by on-chip mass spectrometry. This strategy has been successfully applied to the study of cytokine/GAGs interactions. References: [1] Gandhi, N.S., Mancera, R.L. Biol. Drug Des., 2008, 72(6): 455-482. [2] Handel, T.M., Johnson Z., Crown S.E., Lau E.K., Proudfoot, A.E. Annu. Rev. Biochem., 2005,

74385- 74410. [3] Sasisekharan, R., Raman, R., Prabhakar,V. Annu. Rev. Biomed. Eng., 2006, 8181-8231. [4] Capila, I., Linhardt, R.J. Angew. Chem., Int. Ed., 2002, 41(3): 390-412. [5] Harding, S.E., Chowdhry, B.Z., Protein-Ligand interactions Vol. 1 and 2, Oxford University press,

New York, 2001, 354 and 446 pp. [6] Krishnamoorthy, L., Mahal L. K., ACS Chem. Biol., 2009, 4(9): 715-732. [7] Paulson, J.C., Blixt, O., Collins B.E. Nat. Chem. Biol., 2006, 2(5): 238-248. [8] Beloqui, A., Sanchez-Ruiz, A., Martin-Lomas, M., Reichardt, N.C., Chem. Commun., 2012,

48(11): 1701-1703. [9] Feizi, T., Chai, W. Nat. Rev. Mol. Cell Biol., 2004, 5(7): 582-588. [10] Fukui, S., Feizi, T., Galustian, C., Lawson, A.M., Chai, W. Nat. Biotechnol., 2002, 20(10): 1011-1017.

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1,2-cis-glycosylation: the 2-azido-2-deoxy-D-gluco case in Heparan Sulfate fragment synthesis

David Bonnaffé 1

1 Institut de Chimie Moléculaire et des Matériaux d'Orsay, UMR 8182 Équipe Méthodologie, Synthèse et Molécules Thérapeutiques, LabEx LERMIT, Univ Paris Sud,

CNRS, Université Paris-Saclay, Orsay, France

Heparan sulfate (HS), the glycan part of proteoglycans found at the cell surface and in the extracellular matrix, is a negatively charged linear polysaccharide that displays one of the highest information potential amongst biomolecules. The regulated HS biosynthesis machinery allows generating up to 48 dp2 units (figure 1) and combining them into selective docking sites for more than 500 proteins, with the presumed aim to finely regulate and tune their bioactivities depending on the needs of the cell where they are produced [1]. Within the exception of the fully characterized heparin/AT-III interaction, involving a specific pentasaccharide sequence, there is much debate on the mechanisms allowing specific HS/protein interactions [2]. Chemists have thus a large playground to conceive tools to challenge hypotheses on HS-protein interactions [3]. However, HS fragment syntheses is not trivial and one key points in the total synthesis of HS fragments is to control the 1,2-cis stereoselectivity of the glycosylation reaction involving 2-azido-2-deoxy-D-gluco donors. In this regard, we will discuss how systematic studies of the influence of donors and acceptors on the stereochemical outcome of the glycosylation as well as low temperature NMR experiments can shed light on this important reaction.

Figure 1: HS theoretical molecular diversity

and D-glucosaminyl-1,2-cis linkage References : [1] Xu, J-D. Esko. Annu. Rev. Biochem. 2014, 83, 129–57. U. Lindahl, L. Kjellén. J. Internal Medicine

2013, 273, 555-571. [2] A. Sarkar A, U-R. Desai. PLoS One 2015, 10, e0141127D. H. Lortat-Jacob, A. Grosdidier, A.

Imberty. Proc. Natl. Acad. Sci. USA. 2002, 99, 1229–1234. [2] S-B. Dulaney, Y. Xu, P. Wang, G. Tiruchinapally, Z. Wang, J. Kathawa, M-H. El-Dakdouki, B.

Yang, J. Liu, X. Huang . J. Org. Chem. 2015, 80, 12265−12279. Y-P. Hu, Y-Q. Zhong, Z-G. Chen, C-Y. Chen, Z. Shi, M-M-L. Zulueta, C-C. Ku, P-Y. Lee, C-C. Wang, S-H. Hung. J. Am. Chem. Soc. 2012, 134, 20722–20727. D. Bonnaffé. C. R. Chimie 2011, 14, 29-73. F. Baleux, L. Loureiro-Morais, Y. Hersant, P. Clayette, F. Arenzana-Seisdedos, D. Bonnaffé, H. Lortat-Jacob. Nature Chemical Biology 2009, 5 (10), 743-748. A. Dilhas, R. Lucas, L. Loureiro-Morais, Y. Hersant, D. Bonnaffé. J. Comb. Chem. 2008, 10, 166–169. A. Lubineau, H. Lortat-Jacob, O. Gavard, S. Sarrazin, D. Bonnaffé, Chem. Eur.J. 2004, 10, 4265-4282.

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COMMUNICATIONS ORALES (CO + Duo)

21

Duo-01

CO-01 — Prix Bernard Fournet-André Verbert —

DUO-O1

A key pyranose-2-phosphate motif is responsible for both antibiotic import and quorum-sensing regulation in

Agrobacterium tumefaciens

Abbas El Sahili1, Si-Zhe Li2, Julien Lang1, Cornelia Virus3, Sara Planamente1, Mohammed Ahmar2, Beatriz G. Guimaraes4, Magali Aumont-Nicaise1, Armelle Vigouroux1, Laurent

Soulère2, John Reader3, Yves Queneau2, Denis Faure1 & Solange Moréra1

1Institute for Integrative Biology of the Cell (I2BC), CNRS CEA Univ. Paris-Sud, Université Paris-Saclay, Avenue de la Terrasse, Gif-sur-Yvette 91198, France

2Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, ICBMS, Université de Lyon, INSA Lyon, UMR 5246, CNRS, Université Lyon 1, INSA Lyon, CPE-Lyon;

Bât J. Verne, 20 av A. Einstein, 69621 Villeurbanne, France 3Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill,

Chapel Hill, NC 27599, USA 4Synchrotron SOLEIL, 91192 Gif sur Yvette, France

We succeeded in understanding how the periplasmic protein AccA from the pathogen A. tumefaciens could bind both the plant compound agrocinopine and the antibiotic agrocin 84. Whereas agrocinopine acts as a nutrient and regulatory signal in A. tumefaciens, agrocin 84 is lethal once degraded by the enzyme AccF into a toxic moiety. We identified the pyranose-2-phosphate-like moiety (arabino for agrocinopine and gluco for agrocin 84) shared by these two ligands as the key recognition template for AccA. We hypothesized that agrocin 84 will kill all agrobacteria possessing AccA and AccF and that AccA is a gateway allowing the importation of any compound possessing such a pyranose-2-phosphate motif, and this was confirmed using new synthetic analogs of agrocinopine specifically prepared. Furthermore, among these analogs, arabinose-2-phosphate, resulting from the cleavage of agrocinopine by AccF, was proved, using affinity and in vivo assays, to be the effector of the transcriptional repressor AccR, which controls quorum-sensing and virulence plasmid propagation. Overall, through an interdisciplinary approach, we could identify an original and specific key pyranose-2-phosphate motif that not only allows selective passage of active compounds into the pathogen cells, but also, once these compounds are cleaved, keeps to the matured products their ability to act as signals. Our work opens up new opportunities to rationally design novel antibiotics.

Figure 1: recognition of a key pyranose-2-phosphate motif in AccA ligand binding site

Reference : El Sahili A, Li SZ, Lang J, Virus C, Planamente S, Ahmar M, Guimaraes BG, Aumont-Nicaise M, Vigouroux A, Soulère L, Reader J, Queneau Y, Faure D, Moréra S. (2015). PLoS Pathogens 11(8):e1005071.

Regulation of hepatic Fatty Acid Synthase properties by O-GlcNAcylation in vivo and ex vivo

Baldini Steffi1, Wavelet Cindy1, Anne-Marie Mir1, Marlène Mortuaire1, Hainault Isabelle2,

Postic Catherine2, Guinez Céline3 & Lefebvre Tony1.

1 CNRS-UMR 8576, Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), FRABio FR3688 , Villeneuve d'Ascq, France

2 INSERM, U1016, Institut Cochin, Paris, France 3 Unité Environnement Périnatal et santé UPRES EA 4489, IFR 114, Villeneuve d’Ascq, France

During meal intake, two metabolic pathways are activated in the liver, the glycolysis and the lipogenesis, to drive the production of fatty acids. The Hexosamine Biosynthesis Pathway (HBP), which end product is UDP-GlcNAc the substrate of OGT (O-GlcNAc Transferase) to O-GlcNAcylate proteins, is also activated. O-GlcNAcylation is a dynamic post translational modification (PTM) that controlled a plethora of protein properties. Disturbance in the O-GlcNAcylation dynamism is implicated in several pathologies. Numerous studies link metabolic disorders emergence to O-GlcNAcylation mechanisms deregulation. Knowing that there is a close relationship between glucose, O-GlcNAcylation levels and activation of the glucido-lipid metabolism, a link between the activation enzymes and O-GlcNAcylation should exist. More precisely we focused on Fatty Acid Synthase, FAS which produces fatty acids. In this study, O-GlcNAcylation levels and FAS expression were analyzed in liver of C57BL6 mice fed a Chow Diet (CD) or High Carbohydrate Diet (HCD), in liver of mice harboring an inhibition of OGA and in primary hepatocytes of mice cultured in different O-GlcNAcylation levels. Co-immunoprecipitation experiments showed that OGT and FAS interacted physically and O-GlcNAcylation plays an essential role on FAS expression and activity. Indeed, a correlation between FAS expression and O-GlcNAcylation level was shown and an increase of O-GlcNAcylation levels paralleled the protection of FAS against this degradation, increasing the interaction between FAS and its deubiquitinylase USP2a (Fig. 1). Moreover FAS activity was increased in fasted HCD mice compared to fasted CD mice. Taken together, our results suggest that O-GlcNAcylation may represent indirectly a new regulation of FAS protein content and activity in liver under both physiological and physiopathological conditions.

Figure 1 : Regulation of Fatty Acid Synthase properties by O-GlcNAcylation

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CO-02

CO-03

The MG system as a ligation tool in biological chemistry

Giuliano Cutolo,1 Franziska Reise,2 Jasna Brekalo,1, 2 Pierre-Yves Renard,3

Marie Schuler,1 Thisbe K. Lindhorst2 & Arnaud Tatibouet1 1ICOA-UMR7311, Université d’Orléans, Rue de Chartres, BP6759, 45067 Orléans Cedex 2, France. 2Otto Diels Institute of Organic Chemistry, Christiana Albertina Univ; of Kiel, D-24098 Kiel, Germany.

3COBRA UMR 6014 & FR 3038, UNIV Rouen, INSA Rouen, CNRS, IRCOF, 1 Rue Tesnieres, 7682, Mont-Saint-Aignan Cedex, France ;

The myrosinase-glucosinolate (MG) tandem is a well-known mechanism of defense in plants, restricted to species of the order Brassicales.[1,2] This biochemical system is unique in that myrosinase acts as a thioglucoside glucohydrolase cleaving the anomeric C-S bond of glucosinolates (GLs) to liberate transient species that spontaneously form isothiocyanates (ITCs). This reaction sequence generates a toxic, strong electrophile from a stable non-toxic precursor. Thus, myrosinase-catalysed cleavage of glucosinolates generating isothiocyanates is orthogonal to other glycosidases as well as classical chemical ligation methods. We want to explore the myrosinase-glucosinolate (MG) tandem as an enzymatically driven bioorthogonal ligation system. As a therapeutically relevant target, the bacterial lectin FimH will be employed to develop the MG reaction as a new methodology for site-selective bioconjugation of proteins.[3,4]

Figure 1: Myrosinase-glucosinolate system as a ligation tool

Herein we will disclose the different approaches developed towards the synthesis of these complex glucosinolates analogues and our first set of ligations. References : [1] Fahey, J. W.; Zalcmann, A. T.; Talalay, P. Phytochemistry 2001, 56, 5-51. [2] Rollin, P.; Tatibouët, A. C. R. Chimie, 2011, 14, 194-210. [3] Hartmann, M.; Lindhorst, Th. K. Eur. J. Org. Chem., 2011, 3583-3609. [4] Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. ACS Chem. Biol. 2014, 9, 592−605.

Tectonin2 from Laccaria bicolor is designed for methylated glycans recognition

Roman Sommer1, Silvia Bleuer2, Olga N. Makshakova3,4, Alexander Titz1,

Markus Künzler2, & Annabelle Varrot3 1 Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical

Research Saarland (HIPS), D-66123 Saarbrücken, Germany 2 Institute of Microbiology, Swiss Federal Institute of Technology (ETH), 8093 Zürich, Switzerland

3 University Grenoble Alpes, CERMAV-CNRS-UPR5301, F-38000 Grenoble, France 4 present position: Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center,

Russian Academy of Sciences, 420111 Kazan, Russia The tectonin family of lectins has been associated with innate immunity where members would act as defense effector molecules or recognition factors. Members present multiple copies of the so-called tectonin domain predicted to form β-propeller structures but experimental data are scarce. Tectonin2 from the mushroom Laccaria bicolor (Lb-Tect2) is a nematotoxic lectin that is also able to aglutinate gram-negative bacteria suggesting a role in fungal defense [2]. These properties depend on the recognition of O-methylated glycans present on bacterial LPS or nematode cell surface by Lb-Tect2. Thanks to the synthesis of 2-O-methyl-methyl-seleno-L-fucopyranoside, its structure could be solved by MAD using the selenium signal at 1.65 Å. A Lb-Tect2 structure in complex with 4-O-methyl-α-D-mannopyranoside (4MeMan) was also solved at 1.95 Å. Lb-Tect2 forms a highly symmetrical six-bladed β-propeller. One binding side is found per blade and not at the blade interface like in many other lectins. The six binding sites present a hydrophobic pocket designed to accommodate the methyl group. Modelling studies have shown that it would be difficult to kill the methyl recognition. Lb-tect2 has a unique quaternary arrangement with three molecules in the bottom and one on the top forming a pseudo three fold axis in the tetramer. This allows Lb-Tect2 to have a uniform presentation of its 24 binding sites and to be compared to a sea mine. This explains the better affinity observed for 4MeMan on a surface rather than in solution and reflects again the importance of lectin multivalency.

Figure 1 : Left: Representation of Lb-Tect2 β-propeller fold. Right: zoom on one Lb-Tect2 binding site

References : [1] Varrot A, Basheer SM, Imberty SM. Fungal lectins: Structure, function and potential applications,

Curr Opin Struct Biol, 2013, 23(5): 678-85. [2] Wohlschlager, T. et al. Methylated glycans as conserved targets of animal and fungal innate

defense. Proc Natl Acad Sci U S A,2014, 111, E2787-96.

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CO-05

Multivalent iminosugars to modulate glycosidase activity

Yoan Brissonnet, David Deniaud & Sébastien G. Gouin

Université de Nantes, Laboratoire CEISAM, UMR CNRS 62302 2, rue de la Houssinière, BP 92208, 44322Nantes Cedex 3, France

During recent decades, tremendous efforts have been dedicated to designing potent and selective glycosidase inhibitors. Potential candidates often lack glycosidase selectivity and the resulting non- specific inhibition generally leads to severe side-effects. Limiting selectivity issues due to unwanted inhibition of related glycosidases is a challenge not fully achieved with the first generation of inhibitors.

We explored an alternative strategy to the traditional “lock and key” concept for the design of glycosidase inhibitors. Iminosugars were grafted in a multiple fashion onto a common scaffold, potentially to provide cooperative effects, leading to a greater affinity enhancement with glycosidase targets than predicted from the sum of the constitutive interactions. This phenomenon, called the “multivalent” or “clustering” effect has been successfully exploited to design potent inhibitors of carbohydrate-binding proteins (lectins) but has rarely been investigated for carbohydrate-processing enzymes. In a first systematic study, we observed a small, but significant clustering effect on jack bean α-mannosidase with a gain in selectivity.1 Since, several research groups have reported synthetic multivalent inhibitors for glycosidases and glycosyltransferases with strong inhibitory activities.2 The presentation will be focused on our efforts to rationalize the multivalent inhibition observed.3 Importantly, this led to the serendipitous discovery that polymeric iminosugars could also activate specific glycosidases.4 The concept of glycosidase activation is largely unexplored, with a unique recent example of small-molecules activators of a bacterial O-GlcNAc hydrolase.5 The possibility of using these polymers as “artificial enzyme effectors” may therefore open up new perspectives in therapeutics and biocatalysis.

Figure 1 : Polymeric iminosugars can improve glycosidase activity.

References : [1] Diot, J.; Carcía-Moreno, I.; Gouin, S.G.; Ortiz Mellet, C.; Haupt, K.; Kovensky, J. Org. Biomol.

Chem. 2009, 7, 357-363. [2] Gouin S.G. Chem. Eur. J. 2014, 20, 11616-11628. [3] Brissonnet Y., Ortiz-Mellet C., Morandat S., Garcia Moreno I., Deniaud D., Matthews S. E., Vidal

S., El Kirat K., Gouin. S. G.J. Am. Chem. Soc. 2013, 135, 18427-18435. [4] Brissonnet,Y.; Tezé, D.; Fabre, E.; Deniaud, D.; Daligault, F.; Tellier, C.; Šesták, S.; Ladévèze, S.;

Remaud-Simeon, M.; Potocki- Véronèse, G.; Gouin, S. G. Bioconjugate chem. 2015, 26, 766-772 [5] Darby, J. F.; Landström, J.; Roth, C.; He, Y.; Davies, G. J.; Hubbard, R. E., Angew. Chem. Int.

Ed. 2014, 53, 13419-13423.

Exploration of the lignocellulolytic potential of invertebrate microbiome

Gregory Arnal1, Pablo Alvira1, Sophie Bozonnet1, Silvia Melgosa-Vidal2, William G. T. Willats2, Regis Faure1, Bernard Henrissat3, Claire Dumon1

& Michael O’Donohue1 1. LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France

2.Department of Plant Biology and Biotechnology, University of Copenhagen, Denmark 3.Université Aix Marseille, CNRS, UMR6098, F-13288 Marseille, France.

Biocatalysts are essential for the development of bioeconomy. Recently, a function-based metagenomic approach from diverse termite gut microbiota revealed hundreds of new glycoside hydrolases including 63 non-redundant hypothetical glycoside hydrolases (GH) in the gut of P. militaris, a fungus-growing termite [1]. Most of these enzymes were found to be encoded by gene clusters or Polysaccharide Utilization Loci (PUL) [2] and many were found to be multimeric, displaying a variety of modular configurations often combining more than one catalytic module, with or without CBMs, or modules of unknown function (UNK). Therefore, faced with this wealth of new sequences, we have deployed a series of so-called high-throughput methods that have somewhat accelerated steps such as cloning, expression and characterization of both full-size enzymes and truncated forms thereof. Starting with a hundred of target sequences, 48 proteins were expressed as soluble protein. A set of miniaturized assays including sugar-coated microarrays, binding assay and hydrolysis of chromogenic substrates revealed activity for all the soluble proteins in their full-size or truncated form (CBM or UNK modules). Enzymes were also shown to complement cellulase cocktail on complex biomass such as wheat straw and wheat bran. This study revealed promising biocatalysts such as multimeric xylanases and esterases, and the activity of two UNK domains were clearly demonstrated. References : [1] Bastien, G., et al. (2013) Mining for hemicellulases in the fungus-growing termite

Pseudacanthotermes militaris using functional metagenomics. Biotechnol. Biofuels 6(1):78. [2] Arnal, G., et al. (2015). Investigating the Function of an Arabinan Utilization Locus Isolated from a

Termite Gut Community. Appl. Environ. Microbiol. 81, 31–39.

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CO-07

Selective oxidation of free carbohydrates to corresponding aldonates using gold supported catalysts under

microwave-irradiation Mehdi Omri 1, Gwladys Pourceau 1, Matthieu Becuwe 2 & Anne Wadouachi 1

1 Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources, UMR 7378 CNRS 2 Laboratoire de Réactivité et Chimie des Solides, UMR CNRS 7314

The useful chemicals prepared from biomass feedstock such as carbohydrates is becoming more and more attractive to prepare environmentally friendly products used in various fields (cosmetics, pharmaceutical, medical...). In fact, a variety of high-value products such as detergents, pharmaceuticals, cosmetics can be easily obtained from carbohydrates by different chemical modifications (oxidation, hydrogenation, isomerization, glycosylation…). Among these transformations, selective oxidation of anomeric position of free carbohydrates is of particular interest since it led to aldonic acids, which are biocompatible and biodegradable compounds widely used in several areas such as food, paper, cosmetics and pharmaceutical industries, and important platform chemicals1. Conventional oxidation methods require the use of homogenous catalysis2 which exhibits several drawbacks such as separation of the products, environmental toxicity of catalysts or oxidizing reagent, limited selectivity leading to multiple-step protocols with protection/deprotection steps. These last are not in accordance with the green chemistry principles. Therefore, the development of novel oxidation methodologies using reusable heterogeneous catalysts is a very interesting alternative allowing reducing waste. Herein, we report an efficient methodology for selective oxidation of free carbohydrates to corresponding aldonates using supported gold catalysts combined with hydrogen peroxide under microwave-irradiation3 . The proposed methodology was applied to several sugars (monosaccharides or oligosaccharides, neutral or acidic sugars) leading to good to excellent conversion yields and selectivity to corresponding aldonates. The influence of several experimental conditions and recyclability of catalyst were investigated.

Figure 1: D-glucose oxidation using gold supported catalysts under microwave.

References : [1] H. Hustede, H. J. Haberstroh, E. Schinzig, Ullmann's Encyclopedia of Industrial Chemistry,

6th ed, Wiley-VCH: Weinheim, 2000, vol A 12, 449 [2] S.J. Mantell, P.S. Ford, D.J. Watkin, G.W.J.Fleet, D.Brown. Tetrahedron, 1993, 49, 3343. [3] M. Omri, G. Pourceau, M. Becuwe, A. Wadouachi ; ACS Sus. Chem. Eng. 2016. In Press

O2 orH2O2

AuNps

MOx

MOx

MOx Glucose Gluconate

MW

MOx=CeO2,Al2O3,TiO2

Assembly of a marine exopolysaccharide into microgels for protein delivery applications

Agata Zykwinska,1 Mélanie Marquis,2 Corinne Sinquin,1

Stéphane Cuenot,3 & Sylvia Colliec-Jouault1

1 Ifremer, Laboratoire Ecosystèmes Microbiens et Molécules Marines pour les Biotechnologies, 44311 Nantes, France

2 INRA, UR1268 Biopolymères Interactions Assemblages, F-44300 Nantes, France 3 Institut des Matériaux Jean Rouxel (IMN), Université de Nantes-CNRS, 44322 Nantes, France

Assembly of biopolymers into microgels is an elegant strategy for bioencapsulation with various potential biomedical applications. Such biocompatible and biodegradable microassemblies are developed not only to protect the encapsulated molecule but also to ensure its sustained local delivery. In the present study, an unusual polysaccharide from marine origin, namely HE800 EPS was structured for the first time using microfluidics in functional microcarriers that can be used as protein delivery systems.1,2 The significant advantage of the present delivery system is based on peculiar polysaccharide glycosaminoglycan (GAG)-like structure and its biological properties, which can both be explored to create an innovative biomaterial for tissue engineering applications. This high-added value polysaccharide was shown to be able to form microparticles and microfibers, through physical cross-linking with copper ions, using microfluidics.3 It was shown that the microparticle morphology could be modulated by the polysaccharide concentration and its chain length, and that either homogeneous or heterogeneous structures could be obtained. A model protein, namely Bovine Serum Albumin (BSA) was subsequently encapsulated within HE800 microparticles in one-step process using microfluidics. The protein release was tuned by the microparticle morphology with a lower protein amount released from the most homogeneous structures. Our findings demonstrate the high potential of HE800 EPS based microassemblies as innovative protein microcarriers for further biomedical applications.

References :

1. Senni et al. (2013). Unusual glycosaminoglycans from a deep sea hydrothermal bacterium improve fibrillar collagen structuring and fibroblast activities in engineered connective tissues. Marine Drugs, 11, 1351-1369.

2. Zykwinska et al., (2016). Assembly of HE800 exopolysaccharide produced by a deep-sea hydrothermal bacterium into microgels for protein delivery applications. Carbohydrate Polymers, 142, 213-221.

3. Marquis et al. (2015). Microfluidics assisted generation of innovative polysaccharide hydrogel microparticles. Carbohydrate Polymers, 116, 189-199.

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CO-08

CO-09

Preparation of New Nanovectors by Synthesis of Glycerolipidyl and Phosphoramidyl-cyclodextrins

Véronique Bonnet1, Florian Nolay1, Florence Djedaïni-Pilard1,

Catherine Sarazin,2 Karim El Kirat3 & Sandrine Morandat2

1 Laboratoire de Glycochimie des Antimicrobiens et des Agroressources UMR CNRS 7378 Université de Picardie Jules Verne, 33 R. Saint-Leu, 80039 Amiens Cedex

2 Unité Génie Enzymatique et Cellulaire FRE CNRS 3580 UPJV, 33 R. Saint-Leu 80039 Amiens Cedex 3 BMBI UMR 7338 CNRS UTC, Compiègne, France

Numerous chemical modifications of CD were reported to form safer compounds or new structures able to self-organize in water. Among CD derivatives, amphiphilic cyclodextrins which have capacity to form nanoparticles without cosolvent, could be excellent drug delivery systems [1]. To create a new family of amphiphilic cyclodextrin easily available, the grafting of esters by lipases catalyzed reactions in non-conventional media was promising. In previous work, we reported that the reaction of permethylated 6-amino-6-deoxy-β-cyclodextrin and vinyl esters catalysed by various lipases without solvent led to acyl permethylated β-CD [2]. Using vinyl esters, polyenyl-derivatives of cyclodextrins were obtained and fully characterized [3].

After optimization of enzymatic reaction with few simple substrates, we have worked with 6-(glyceryl)-amidosuccinamide-6-deoxy-permethylated β-CD, 6-(1,3-dihydroxy-propionamidyl)-6-deoxy-permethylated β-CD and, 6-(2,3-dihydroxy-propionamidyl)-6-deoxy-permethylated β−CD. The chemo-enzymatic synthesis and the influence of the nature of the cyclodextrins will be discussed. A second family of amphiphilic cyclodextrin will be presented, the lipophosphoramidyl cyclodextrins which are synthesized in one step Atherton Todd reaction from 6-β-alanyl-6-deoxy permethylated β-CD [4]. Potential capacity of these compounds to be used as nanovectors in therapeutics will be discussed. Studies of preparation and stability of nanoparticles in water or physiological media will be presented.

O

O OMeO

OMe OMe

OMeMeO

O 6

HN

O

NHPOO

O

O

O O

HN

MeOOMe OMe

OMeMeO

O 6

O

O

HN

OR'OR

O

O O

HN

MeOOMe OMe

OMeMeO

O 6

O

O

HN

OR

OR'

R,R' = H, COC7H15, COC17H35

This work was supported by Conseil Régional de Picardie under Agroressources Program.

[1] Bonnet V., Gervaise C., Djedaïni-Pilard F., Furlan A., Sarazin C., 2015, Drug Discovery Today, doi 10.1016/j.drudis.2015.05.008.

[2] Favrelle, A.; Bonnet, V.; Sarazin, C.; Djedaıni-Pilard, F. 2007. J Incl Phenom Macrocycl Chem, 57, 15-20., Favrelle, A.; Bonnet, V.; Avondo, C.; Aubry, F.; Djedaïni-Pilard, F.; Sarazin, C. 2010. Journal of Molecular Catalysis B: Enzymatic, 66, 224-227.

[3] Favrelle, A.; Bonnet, V.; Sarazin, C.; Djedaïni-Pilard, F. 2008. Tetrahedron: Asymmetry, 19, 2240-2245. [4] Gervaise, C. ; Bonnet, V. ; Wattraint, O.; Aubry, F.; Sarazin, C. ; Jaffrès, P.A. ; Djedaïni-Pilard, F.,

2012. Biochimie, 94,66-74.

Bacterial synthesis of polysialic acid lactosides in recombinant Escherichia coli K-12

Emeline Richard, Laurine Buon, Sophie Drouillard, Sébastien Fort, & Bernard Priem

Univ. Grenoble Alpes, CERMAV, F-38000 Grenoble, France Polysialic acids are sialic acid based polysaccharides, mostly found in the nervous system and largely overexpressed on tumour cells1, that confer interesting properties for medical applications. Polysialic acids are also bacterial capsular polysaccharide, synthesized by bacterial polysialyltransferases2. These processive enzymes are also known to synthesize in vitro polysialic acid from disialylated and trisialylated lactosides acceptors3. Here, we present the engineering of a non-pathogenic Escherichia coli strain, overexpressing recombinant sialyltransferases and sialic acid synthesis genes, able to perform the bacterial conversion of an exogenous lactoside into polysialyl lactosides4. Several bacterial polysialyltransferases encoding genes were assayed for their ability to perform the synthesis of polysialyl lactosides in the recombinant strains. Fed-batch-cultures allowed us to produce several grams per liter of α-2,8 polysialic acid and alternate α-2,8-2,9 polysialic acid. Bacterial culture in presence of propargyl-β-lactoside as exogenous acceptor led to the production of conjugatable polysaccharides by mean of copper assisted click-chemistry.

Figure 1: Polysialic acid strucure

References : [1] C. Sato et al: Disialic, oligosialic and polysialic acids: distribution, functions and related disease.

J. Biochem. (2013) 154(2): 115-136. [2] Steenbergen et al: Functional relationships of the sialyltransferases involved in expression of the

polysialic acid capsules of Escherichia coli K1 and K92 and Neisseria meningitidis groups B or C. J Biol Chem. (2003) 278:15349-59.

[3] L. Willis et al: Characterization of the α2-8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme. Glycobiology (2008) 18:177-186.

[4] E. Richard et al: Bacterial synthesis of polysialic acid lactosides in recombinant Escherichia coli K-12. Glycobiology (2016).

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Synthesis and property of N-oxyamide-linked glycoconjugates

Na Chen, Joanne Xie PPSM, ENS Cachan, CNRS, Université Paris-Saclay, Cachan, 94235 France

Glycoconjugates like glycolipids and glycoproteins are involved in a variety of important biological, physiological and pathological processes.[1] Synthesis of glycoconjugate mimics has attracted increasing research interest for biological and pharmaceutical applications, especially in diagnostics, vaccines and therapeutics.[2] Diversely functionalized carbohydrate building blocks could provide a versatile platform for the generation of carbohydrate mimics and various conjugates. Aminooxy acid derived peptides can easily organize into turns and helices structures through intramolecular H bond formation, and the N-oxyamide linkage is resistant to chemical and enzymatic hydrolysis.[3,4] This unique property makes N-oxyamide linkage attractive for the design of new glycoconjugates. Moreover, the oxyamine function could be readily used for generating libraries of conjugates through chemoselective neoglycosylation with reducing sugars,[5] oxime ligation with carbonyl compounds,[6] in addition to coupling with carboxylic acids. Starting from O-glycosyl glycerol, we have developed a methodology for the synthesis of N-oxyamide-linked glycoglycerolipids, the (2R)- and (2S)-aminooxy analogues of β-O-glucosylserine and N-oxyamide-linked glycopeptides.[7-9] Synthesis, glycolipid assembly with gold nanoparticles for receptor targeting imaging and drug delivery will be presented.

Figure 1: Structure of glycosyl aminooxy esters and N-oxyamide-linked glycoconjugates

References : [1] D. Kolarich, B. Lepenies, P. H. Seeberger, Curr. Opin. Chem. Biol. 2012, 16, 214-220 [2] J.E. Hudak, C.R. Bertozzi, Chem. Biol. 2014, 21, 16-37 [3] X. Li, Y.-D. Wu, D. Yang, Acc. Chem. Res. 2008, 41, 1428-1438 [4] F. Chen, B. Ma, Z.-C. Yang, G. Lin, D. Yang, Amino Acids 2012, 43, 499-503. [5] R.D. Goff, J. S. Thorson, Med. Chem. Commun. 2014, 5, 1036-1047 [6] E.L. Smith, J. P. Giddens, A.T. Iavarone, K. Godula, L.X. Wang, C.R. Bertozzi, Bioconjugate

Chem. 2014, 25, 788-795 [7] N. Chen, J. Xie, J. Org. Chem. 2014, 79, 10716-10721 [8] N. Chen, J. Xie, Org. Biomol. Chem., 2016, 14, 1102-1110 [9] N. Chen, Z.H. Yu, D. Zhou, X.L. Hu, Y. Zang, X.P. He, J. Li, J. Xie, Chem. Commun. 2016, 52, 2284-2287

Activity and structural characterization of Candida albicans β-1,2 mannosyltransferase CaBmt3 involved in the elongation of the cell-wall phosphopeptidomannan

Thomas Hurtaux1,2, Ghenima Sfihi-Loualia1, Emeline Fabre1, Florence Delplace1, Coralie Bompard1,

Anaïs Mée3, Sébastien Gouin4, Jean-Maurice Mallet3, Boualem Sendid2 & Yann Guérardel1 1 Univ. Lille, CNRS, UMR 8576 UGSF Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France

2 Univ. Lille, Inserm, CHU Lille, U995 LIRIC Lille Inflammation Research International Center, 59000 Lille, France 3 UMR 7203, Laboratoire des BioMolécules, Ecole Normale Supérieure, 75231 Paris

4 CEISAM, LUNAM Université, UMR CNRS 6230, 44322 Nantes

Candida albicans is a saprophytic yeast found in the flora of the human gastro-intestinal tract. It can however become pathogenic in immunocompromised individuals and cause severe infections, especially in a nosocomial environment, associated with high morbidity and mortality rates. The cell wall of C. albicans, in contact with the host, contains β-1,2 oligomannosides (β-Man) that are linked to several parietal structures such as phospholipomannans (PLM) and phosphopeptidomannans (PPM). These β-Man are found in every pathogenic species of Candida and are considered as virulence factors. The identification of a family of 9 genes coding for β-mannosyltransferases (CaBmt) led to a better understanding of the role of the enzymes [1]. Out of these, CaBmt1 adds the first β-Mannosyl residue [2] whereas CaBmt3 adds a second one [3] onto the acid-stable fraction of the PPM. Characterization of activity and structure of CaBmt3 is underway using recombinant forms of this enzyme. Substrate specificity was determined on pyridylamino or mantyl-tagged oligomannosides and observed with HPLC-fluorometric detection. We established that CaBmt3 requires an acceptor substrate capped with βMan(1-2)αMan motif to add a single βMan(1-2) residue. Crystallogenesis screenings and SAXS studies have allowed us first insight in CaBmt general surface organization. Furthermore, mutagenesis targets identified in silico allowed the expression in Escherichia coli of enzymes mutated in the active site. This led to a better understanding of the role of these specific amino acids in the catalytic mechanism of CaBmt3.In parallel, we have assessed the modulating activities of monovalent and multivalent iminosugar analogs on CaBmt1 and CaBmt3 in order to control the enzymatic biosynthesis of β-Man [4].Ultimately, the goal is to develop CaBmt inhibitors in order to facilitate the struggle against invasive candidiasis.

Figure 1: Schematic representation of CaBmt3 enzymatic reaction References: [1] Mille C, et al. (2008), J Biol Chem., 283, 9724-36. [2] Fabre E, Sfihi-Loualia G, et al. (2014), Biochem. J, 457, 347-360 [3] Sfihi-Loualia G, Hurtaux T, et al. (2016), Glycobiology, 26(2), 203-14 [4] Hurtaux T, Sfihi-Loualia G, et al. (2016), Carbohydr Res. (in press)

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How to tip the balance from hydrolysis toward transglycosylation: molecular basis in retaining GHs

Bastien Bissaro1, Julien Durand1, Xevi Biarnés2, Tobias Tandrup3, Claire Dumon1, Pierre Monsan1,4,

Leila Lo Leggio3, Antoni Planas2, Sophie Bozonnet1, Michael J. O’Donohue1 & Régis Fauré1 1 LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France

2 Lab. of Biochemistry, Inst.Químic de Sarrià, Univ. Ramon Llull, Via Augusta, 08017 Barcelona, Spain 3 Dept. of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Kbh Ø, Denmark

4 Toulouse White Biotechnology, UMS INRA/INSA 1337, UMS CNRS/INSA 3582, 3 Rue des Satellites, 31400 Toulouse, France

Transglycosylases (TGs) are scattered among several glycoside hydrolase (GH) families within the CAZy classification and are almost indistinguishable from their hydrolytic counterparts, meaning that any given TG is structurally more related to other members of its CAZy family than to other TGs from other families. For this reason it is a challenge to understand how these enzymes can perform transglycosylation reactions in aqueous medium, where the molarity of water is overwhelming, and thus the propensity to perform hydrolysis is theoretically enormous.[1] Accordingly, considering this lack of rationale, the design of new TGs using the vast array of GHs as protein templates is extremely difficult, and thus the development of new tools for chemoenzymatic glycosynthesis is arduous. In our work, we have used random and semi-rational techniques to engineer two hydrolytic retaining GHs. This has provided us with two successes that have progressed our understanding of the TG-GH conundrum and allowed us to make new hypotheses about how TGs overcome the omnipresence of water. In a first example we created finely-tuned evolved GH51-based TGs that can be qualified as the first non-Leloir transarabinofuranosylases. When acting on nitrophenyl-activated donor sugars these enzymes display an almost exclusive transglycosylating phenotype, transferring the sugar moiety bound in subsite -1 to carbohydrate acceptors at high yield (up to 80%).[2] Additionally, we engineered a pH-control feature that provides the means to obtain a perfectly stable product.[3] In a second example, site-saturation mutagenesis was used to target active site residues in a GH5 endo-glycoceramidase. This procured a mutant that is able to transfer cellobiosyl onto aliphatic diols and alcohols bearing a δ-hydroxyl ketone function, producing functionalized alkyl cellobiosides in up to 93% yields (unpublished data). Our achievements and recent analysis of accumulated bibliographic data,[1] now provide us with a much clearer understanding of how the T/H partition is established in retaining GHs. In turn, this knowledge allows us to propose rules for the rational design of TGs. Acknowledgement: The PhD fellowship of B.B. was supported by INRA (CJS). A part of this research was supported by the project ‘BioSurf - Novel production strategies for biosurfactants’ (ERA-NET grant no. 0315928A, ERA-IB10.039). Contribution from R.F. was partially supported by the Région Midi-Pyrénées grant DESR-Recherche 14052246 (CTP-B) and the research mobility grants from INSA Toulouse (2015). C.D. and R.F. thank the French-Danish Research Collaboration Program (IFD) for a travel grant. References : [1] B. Bissaro, P. Monsan, R. Fauré, M.J. O’Donohue, Biochem. J., 2015, 467(1):17-35 [2] B. Bissaro, J. Durand, X. Biarnés, A. Planas, P. Monsan, M.J. O'Donohue, R. Fauré, ACS Catal.,

2015, 5(8):4598-4611 [3] B. Bissaro, O. Saurel, F. Arab-Jaziri, L. Saulnier, A. Milon, M. Tenkanen, P. Monsan, M.J.

O'Donohue, R. Fauré, BBA-Gen. Subjects, 2014, 1840(1):626-636

Unraveling the multivalent binding of a marine family 6 carbohydrate-binding module

with its native laminarin ligand

Elizabeth Ficko-Blean1, Murielle Jam1, Aurore Labourel1, Robert Larocque1, Mirjam Czjzek1 & Gurvan Michel1

1 Sorbonne Université, UPMC Univ Paris 06, CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, F-29688, Roscoff cedex, Bretagne, France

Brown macroalgae are important primary producers from the marine ecosystem and a large proportion of their organic biomass is recycled through the food chain. Laminarin is an abundant brown algal storage polysaccharide and marine microorganisms, such as Zobellia galactanivorans, produce laminarinases for its degradation. These laminarinases are often modular, as is the case with ZgLamC which has an N-terminal GH16 module, a central family 6 carbohydrate-binding module (CBM) and a C-terminal PorSS module. This is the first study characterizing the interactions between a true marine CBM6 and its natural laminarin ligand. The crystal structure of ZgLamC_CBM6 indicates that this CBM has two clefts for binding sugar (Variable loop site, VLS, and concave face site, CFS). The VLS binds in an exo-manner and the CFS interacts in an endo manner with laminarin. Isothermal titration calorimetry experiments confirm that these binding sites have different modes of recognition for laminarin. Based on the isothermal titration calorimetry data and structural data we propose a model of ZgLamC_CBM6 interacting with different chains of laminarin in a multivalent manner, forming a complex cross-linked protein-polysaccharide network1 (Figure 1).

Figure 1 : Complex multivalent CBM6 interactions with laminarin polysaccharide References : [1] Elizabeth Ficko-Blean, Murielle Jam, Aurore Labourel, Robert Larocque, Mirjam Czjzek, Gurvan

Michel. Unraveling the multivalent binding of a marine family 6 carbohydrate-binding module with its native laminarin ligand. FEBS Journal, 2016. In press.

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Polygalacturonase from Arabidopsis thaliana: to new enzymes for industrial applications

Corinne Pau-Roblot1, Olivier Habrylo1, Ludivine Hocq1, Françoise Fournet1,

Michelle Pillon-Lequart1, Jean-Marc Domon1, Catherine Rayon1, Grégory Mouille2, Aline Voxeur2, Valérie Lefebvre1 & Jérôme Pelloux1

1Unité de biologie des plantes et innovation, EA 3900, Université de Picardie Jules Verne, UFR des sciences, 33 rue saint Leu, 80039 Amiens, France

2 Institut Jean-Pierre Bourgin, UMR1318, INRA-AgroParisTech, ERL3559 CNRS, INRA Centre de Versailles-Grignon, Route de St-Cyr (RD10), 78026 Versailles Cedex, France

The plant cell wall not only has a structural role in determining the texture and mechanical properties of plants and their organs. In fact, it also plays a critically role in growth and differentiation. Indeed, pectins are major components of plant primary cell walls and constitute a valuable biomass for food and non-food applications but was also shown to be involved in major events on plant development [1]. Homogalacturonan (HG) is the most abundant pectic polysaccharide in the primary cell wall of dicotyledons. It can be acetylated and/or methyl-esterified on specific carbons [1]. The degree of methyl-esterification (DM) and acetylation (DA) is controlled within the plants by specific enzymes such as pectin methylesterases (PME) or pectin acetylesterase (PAE) respectively. Changes in DM can have dramatic consequences on the rheological and chemical properties of the cell wall, modulating, for instance, the sensitivity of HG to phytopathogens degrading enzymes such as polygalacturonases (PG). During pathogen infection, action of PG induces cell wall degradation and promotes colonization of host tissues by pathogens (bacteria and fungi) [2]. So far, substrates specificity and enzymes (PME and PG) mode of action are so far largely unknown in plants. In order to bring out new potential applications of substrates (medicine, preventive treatment of crops against pathogens), a better understanding of the relationships between their structure, modulated via the action of specific enzymes, and their properties is required.

Using a multidisciplinary approach, we characterised a PG of A. thaliana to explore its biochemical activity and functions in planta. The enzyme was expressed in P. pastoris as secreted a protein. After purification by chromatography, the biochemical characterization was performed, giving new insights into the enzymatic properties of this enzyme. References : [1] Wolf S. et al. (2009). Mol. Plant, 2: 851-860. [2] Shah P. et al. (2009). Proteomics, 9: 3126-3135

Chemo-enzymatic synthesis of innovant glycolipids for cosmetic formulation

Cédric Peyrot 1,2, Perrine Cancellieri 3, Laure Guillotin1, Pierre Lafite 1,

Ludovic Landemarre 3, Loïc Lemiégre 2 & Richard Daniellou 1 1 Univ, Orleans, ICOA, UMR 7311, rue de Chartres F-45067 Orléans, France

2 ENSC-Rennes, Equipe COS, UMR 6226, 11, allée de Beaulieu 35708 Rennes cedex 7, France 3 GLYcoDiag, 520 rue de Chanteloup, 45520 Chevilly

For twenty years, some low molecular weight carbohydrates have emmerged as a new class of hydrogels. These compounds, such as glycolipids, can create weak intermolecular interactions to trap a large amount of water. Synthetic methods are expensive, tedious and generally leads to low yields.This represent an obstacle to their use in cosmetic industry. Our challenge is to produce glycolipids using environmentally benign methods like enzymatic reaction. Thanks to protein engineering we are able to produce new biocatalysts to obtain these glycolipids. The structure of targeted glycolipids will be made up of three distinct parts : - a saccharidic part attached thanks to a thioglycoligase, the specificity owing of forming a

thioglycosidic bond, more stable to chemical and enzymatic hydrolysis. The great structural diversity concerning the sugar part allows us to imagine many different compounds,

- a linker, mainly in the form of thioarylic derivate, which can be further functionalized in meta or para position by different functions,

- a fatty chain attached to the linker by esterification or amidation . The length of the chain, the presence of branching, unsaturation may vary, so as to equilibrate the hydrophobic/hydrophilic balance essential to obtain a hydrogelator.

Our goal is to develop a new chemo-enzymatic method of glycolipid synthesis, access to structural diversity and test all the products as new hydrogelator (figure 1).

Figure 1 : Structural diversity glycolipids targeted

References : Estroff, L . Hamilton A. D., Chem. Rev, 2004, 104 (3), pp 1201-1218 Guillotin, L., Lafite, P., Daniellou, R., Carbohydr.Chem, 2014, 10 (40) pp 178-194

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Infrared Multiple Photon Dissociation Spectroscopy : a new powerful technique for structural characterization

of carbohydrates

Baptiste Schindler 1, Loic Barnes 1, Abdul-Rahman Allouche 1, Stéphane Chambert 2 & Isabelle Compagnon 1,3

1 Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne, France; Institut Lumière Matière, UMR5306 Université Lyon 1-CNRS; Université de Lyon 69622 Villeurbanne Cedex, France.

2 Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne, France. Laboratoire de Chimie Organique et Bioorganique, INSA Lyon, CNRS, UMR5246, ICBMS,

Bât. J. Verne, 20 Avenue A. Einstein, 69621 Villeurbanne Cedex, France 3 Institut Universitaire de France IUF, 103 Blvd St Michel, 75005 Paris, France

We have built an instrument coupling Mass Spectrometry and vibrational spectroscopy (IRMPD), dedicated to the structural characterization of carbohydrates. We present the molecular fingerprint obtained by IR spectroscopy as an universal metric to resolve carbohydrate isomerisms, whereas previously reported hyphenated methods yielded partial structural information. Using this metric, we can resolve all structural information of underivatized carbohydrates, including the monosaccharide content, regiochemistry and stereochemistry of the glycosidic linkages. With the combination of mass spectrometry sensitivity and spectroscopic structural resolution, our method requires typical MS conditions, that is small amount of sample, minimal chemical purification and applies to underivatized analytes, which represents a major breakthrough in high-throughput analysis of natural carbohydrates. Example of recent applications include glycosaminoglycanes and chitines. This instrument is open to external users via the glycophysics network. We expect that making this new structural tool available to the glycochemistry and glycobiology communities will foster the full development of glycosciences applications.

Figure 1 : example of application: spectroscopic elucidation of isobaric functional modifications of glucosamine

References : B. Schindler, J.Joshi, A.-R. Allouche, D.Simon, S. Chambert, V.Brites, M.-P. Gaigeot & I. Compagnon. Distinguishing isobaric phosphated and sulfated carbohydrates by coupling of mass spectrometry with gas phase vibrational spectroscopy, Phys Chem Chem Phys, 2014, 16, 22131-22138.

Chemical synthesis and development of modified xylosides as potential inhibitors targeting β4GalT7, a key enzyme

in glycosaminoglycan biosynthesis initiation

Samir Dahbi1, Isabelle Bertin-Jung2, Anne Robert2, Jean-Claude Jacquinet1, Sandrine Gulberti2, Nick Ramalanjaona2, Sylvie Fournel-Gigleux2 & Chrystel Lopin-Bon1

1 ICOA, UMR 7311 CNRS Université d’Orléans, Pôle de Chimie, Rue de Chartres, 45100 Orléans 2 IMoPA, UMR 7365 CNRS Université de Lorraine, Biopôle-Campus Biologie Santé, Faculté de

Médecine, 54505 Vandoeuvre-lès-Nancy

Proteoglycans (PGs) consist of linear anionic polysaccharide chains called glycosaminoglycans (GAGs), covalently attached to serine residues of a core protein (Figure 1). PGs are localized in extracellular matrix, but also on cell surfaces. Because of their anionic characteristics and their structural diversity, GAG chains have the ability to interact with lots of soluble effectors (as growth factors). This property explains their important roles in cellular processes like migration, proliferation and cell signaling. However, deregulations of PG metabolism are involved in pathological contexts such as cancer, osteoarticular and cardiovascular disorders, as well as severe genetic diseases (Ehlers-Danlos syndrome).

Figure 1: Structure and biosynthesis of PGs Figure 2: Structure of modified xylosides

PG biosynthesis is initiated by the formation of a tetrasaccharide linker GlcA(β1→3)Gal(β1→3)Gal(β1→4)Xylβ−, which acts as a primer for the elongation of GAG chains. Among the glycosyltransferases involved, the β1,4-galactosyltransferase 7 (β4GalT7) catalyses the transfer of the first Gal residue of the linkage region onto the xylose residue. Because all GAGs share the same initiating tetrasaccharide, β4GalT7 is a key enzyme of GAG initiation and a prime target for the study of PG biosynthesis to be potentially investigated for the development of therapeutic agents. We have previously reported the structure-guided design of inhibitors which target β4GalT7 [1]. In order to study the influence of each of the three positions of the xylose on the inhibitory potency, we have synthesized a set of 4-methylumbelliferyl β-D-xylopyranosides (4MU-Xyl) modified at C-2, C-3 or C-4 (Figure 2). These compounds have been tested as substrates and/or inhibitors of β4GalT7. We have shown that the 2-modified xylosides are not inhibitors, but poor β4GalT7 substrates. The best inhibitors are 4-modified xylosides, more particularly the 4-Fluoro-Xyl-MU, which is able to inhibit β4GalT7 in vitro activity and in cellulo GAG biosynthesis [1]. To summarize, modified xylosides have the ability to impact in vitro galactosyltransferase activity and to affect GAG synthesis in cells. These compounds that specifically target β4GalT7, represent valuable chemical-biological tools to explore β4GalT7 active site and to evaluate the biological consequences of GAG modulation in cellulo. These molecules have also significant potential towards pharmaceutical and therapeutic applications. References : [1] M. Saliba, N. Ramalanjaona, S. Gulberti, I. Bertin-Jung, A. Thomas, S. Dahbi, C. Lopin-Bon, J.-C.

Jacquinet, C. Breton, M. Ouzzine, S. Fournel-Gigleux, J. Biol. Chem., 2015, 290, 7658.

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P-02

Degradation of wood by the Carbohydrate-Active Enzyme set of the fungus Pycnoporus coccineus

Marie Couturier 1,2,3,4 David Navarro 1,2,3, Didier Chevret 5, Bernard Henrissat 6,7,8, François

Piumi 1,2,3, Francisco J Ruiz-Dueñas 9, Angel T Martinez 9, Igor V Grigoriev 10, Robert Riley 10, Anna Lipzen 10, Jean-Guy Berrin 1,2,3, Emma R Master 4 & Marie-Noëlle Rosso 1,2,3

1 Aix Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques,

163 avenue de Luminy, F-13288 Marseille, France 2 INRA, UMR1163 Biodiversité et Biotechnologie Fongiques,

163 avenue de Luminy, F-13288 Marseille, France 3 Polytech'Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques,

163 avenue de Luminy, F-13288 Marseille, France 4 Department of Chemical Engineering and Applied Chemistry,

University of Toronto, Toronto, Ontario, Canada 5 INRA, UMR1319 Micalis, Plateforme d’Analyse Protéomique de Paris Sud-Ouest,

78352, Jouy-en-Josas, France 6 Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257 CNRS, Université Aix-Marseille, 13288 Marseille, France

7 Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia 8 INRA, USC 1408 AFMB, 13288 Marseille, France

9 CIB, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain 10 US Department of Energy Joint Genome Institute (JGI), Walnut Creek, California, USA

White-rot basidiomycete fungi are potent degraders of plant biomass (i.e. lignocellulose) which is mineralized through the combined action of a wide range of carbohydrate active and lignin active enzymes[1]. Genomic studies and functional analyses have started to unveil the enzymatic mechanisms leading to lignocellulose breakdown by fungi, but their ability to preferentially degrade some substrates is not well understood[2]. The Polyporale fungus Pycnoporus coccineus BRFM310 displays the interesting capability to grow well on both coniferous and deciduous wood. In the present study we analyzed the early response of the fungus to softwood (pine) and hardwood (aspen) feedstocks and tested the effect of the secreted enzymes on lignocellulose deconstruction. To do so, we performed transcriptomic and proteomic analyses of P. coccineus grown on pine or aspen to identify the sets of enzymes potentially involved in lignin and polysaccharide degradation. In parallel, the enzymes were used in wood hydrolysis experiments. The combined analyses of soluble sugars and solid residues showed the suitability of P. coccineus secreted enzymes for softwood degradation[3]. Beyond the variety of CAZymes identified in its genome, transcriptome and secretome, other parameters such as the abundance of many proteins of unknown function could be involved in the efficiency of P. coccineus for softwood conversion. [1] Kubicek CP, (2012) The Actors: Plant Biomass Degradation by Fungi, in Fungi and

Lignocellulosic Biomass, Wiley-Blackwell, Oxford, UK. [2] Blanchette R (1991) Delignification by wood-decay fungi. Ann Rev Phytopath 29: 381-398. [3] Couturier M et al (2015) Enhanced degradation of softwood versus hardwood by the white-rot

fungus Pycnoporus coccineus. Biotechnol Biofuels 8:216.

From Carbohydrate-Based Thioimidate N-Oxides to

Iminosugars Derivatives

Marie Schuler, Stéphanie Marquès, Domenico Romano, Maria Domingues & Arnaud Tatibouët 1

1 ICOA-UMR7311, Université d’Orléans, Rue de Chartres, 45067 Orléans, France. Our group has recently revealed an unusual thiofunction: the ThioImidate N-Oxide function (TINO).[1] Encouraged by the synthetic potential of this “thionitrone” analogue, we designed a general method for the preparation of thioimidate N-oxides (II) with a view to further exploring the potential of this rarely known functional group. Herein, we would like to report the synthesis of a small library of various furanose- and pyranose-based thioimidate N-oxides, representatives of both pentoses and hexoses.[2] Our approach relies on the cyclisation of a suitably functionalised thiohydroximate (I) either through a halocyclisation reaction (route A) or a nucleophilic substitution (route B). The reactivity of these novel structures has then been studied, more specifically in pallado-catalysed Liebeskind-Srogl cross-couplings, thus giving access to original ketonitrones (III).

Scheme 1: Synthesis of carbohydrate-based thioimidates N-oxides

These enantiomerically pure backbones constitute valuable intermediates in the synthesis of polyhydroxylated biologically active compounds, such as novel iminosugars and imino-C-nucleosides. References: [1] a) J. Schleiss, D. Cerniauskaite, D. Gueyrard, R. Iori, P. Rollin, A. Tatibouët Synlett 2010, 725-778;

b) J. Schleiss, P. Rollin & A. Tatibouët Angew. Chem. Int. Ed. 2010, 49, 577-580. [2] S. Marquès, M. Schuler, A. Tatibouët Eur. J. Org. Chem. 2015, 11, 2411-2427. [3] a) Iminosugars: From Synthesis to Therapeutic Applications (Eds: P. Compain and O.R. Martin),

Wiley-VCH: Weinheim, 2007; b) Stambasky, J.; Hocek, M.; Kocovsky, P. Chem. Rev. 2009, 109, 6729–6764.

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Enzym’n click synthesis of chitinoligosaccharide probes for plant biology

Arnaud Masselin, Stéphanie Pradeau, Sylvain Cottaz, Sébastien Fort

CERMAV CNRS, Univ. Grenoble Alpes, 38000 Grenoble, France

Plants have evolved sensitive and intricate mechanisms to discriminate beneficial and harmful microorganisms via the signals that these microorganisms produce. Such signals include chitin-related molecules with huge potential for sustainable agriculture, because of their abilities to enhance plant nutrition and growth, and to incite plants to defend themselves against pests.

Lipochitinoligosaccharides (LCOs) are symbiotic signals essential for nodulation in legumes.1 They also activate plant root development and stimulate the establishment of the arbuscular mycorrhizal (AM) symbiosis in leguminous and non leguminous plants.2 Short chain chitinoligosaccharides CO 3-5, have also recently been described as signal molecules involved in the AM symbiosis.3 In contrast, long chain chitinoligosaccharides CO 6-8 commonly produced by pathogenic fungi, have long been described as potent elicitors of plant defence.4, 5

Pure and well-defined chitinoligosaccharides probes are thus required to address the fundamental biological question of how these closely related molecules can trigger such different and sometimes contradictory plant responses. Biocatalysis in combination with click chemistry offers an efficient way to synthesize complex oligosaccharide probes. In this context we will describe the enzymatic synthesis of short and long chain chitinoligosaccharides and their chemical modification in aqueous media with conjugatable groups. The newly synthetic glycoconjugates will provide molecular tools to decipher plant-microbes communication.

Figure 1 : Enzym’n click synthesis of chitinoligosaccharide probes References : [1] Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Promé JC, Dénarié J (1990) Symbiotic host-

specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature 344: 781-784

[2] Maillet F, Poinsot V, André O, Puech-Pages V, Haouy A, Gueunier M, Cromer L, Giraudet D, Formey D, Niebel A, Martinez EA, Driguez H, Bécard G, Dénarié J (2011) Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469: 58-63

[3] Genre A, Chabaud M, Balzergue C, Puech-Pagès V, Novero M, Rey T, Fournier J, Rochange S, Bécard G, Bonfante P, Barker DG (2013) Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone. New Phytologist 198: 190-202

[4] Boller T (1995) Chemoperception of Microbial Signals in Plant Cells. Annu Rev Plant Physiol Plant Mol Biol 46: 189-214

[5] Shibuya N, Minami E (2001) Oligosaccharide signalling for defence responses in plant. Physiological and Molecular Plant Pathology 59: 223-233

Subcellular localization of heparan 3-OST2, 3A and 3B

Maxime Delos, François Foulquier, Charles Hellec, Fabrice Allain and Agnès Denys Structural and Functional Glycobiology Unit, UMR8576 CNRS/USTL, 59655 Villeneuve d’Ascq

Through their ability to interact with many proteins, heparan sulfates (HS) play an important role in many physiopathological processes. They are composed of repeat dissacharide units, which can be sulfated in various positions. The reaction of 3-O-sulfation of glucosamine residues is the last modification in HS moieties, which can be catalyzed by seven 3-O sulfotransferases (3-OSTs). Each of them is distinct by virtue of its fine substrate specificity and tissue distribution. Indeed, 3-OST1 is known to generate the HS-binding motif for antithrombin-III, while 3OST2, 3A, 3B, 4 and 6 generate an HS motif that may serve as an entry receptor for the gD protein of HSV-1. While 3-OST4 and 3-OST6 are mainly restricted to embryonic tissues, 3-OST2 is highly expressed in neurons and anti-inflammatory macrophages, while 3-OST3B is expressed in pro-inflammatory cells [1]. In contrast, 3-OST3A has a more ubiquitous distribution. In addition, recent studies have reported a role for 3-OST2, 3A and 3B in tumor progression, but the biological role of 3-O-sulfated HS is not yet understood. In the general scheme of HS biosynthesis, HS sulfotransferases were considered as resident enzymes of the Golgi apparatus [2]. However, such a subcellular localization of 3-OST2, 3A and 3B has not been yet confirmed by experimental approaches. In this context, we decided to explore the localization of these isoenzymes by using confocal microscopy. For each enzyme, we constructed fluorescent probes, which correspond to the red fluorescent protein (RFP) fused to either the full-length enzymes or to the enzymes deprived of their catalytic domains. As expected, our results showed a restricted localization of both 3-OST3B constructs in the Golgi apparatus. In contrast, 3-OST3A was found in the proximity of the plasma membrane, in addition to a normal localization in the Golgi apparatus. Moreover, 3-OST2 was found in some specific areas in the plasma membrane. These results suggesting a specific role for these isoforms, we are now investigating a possible co-localization and/or trafficking of 3-OST3A and 3-OST2 with cell surface HS proteoglycans. Our first results show 3-OST3A in a close proximity to syndecan-4, while 3-OST2 co-localized with syndecan-2. This study will allow to better understand the specific function of 3-OST isoenzymes in making 3-O-sulfated HS with distinct activities. References : [1] Martinez P., Denys A., Delos M., Sikora AS., Carpentier M., Julien S., Pestel J. and Allain F.

(2015) Macrophage polarization alters the expression and sulfation pattern of glycosaminoglycans. Glycobiology 25, 502-13

[2] Pinhal M. A., Smith B., Olson S., Aikawa J., Kimata K. and Esko J. D. (2001). Enzyme interactions in heparan sulfate biosynthesis: uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo, Proc. Natl. Acad. Sci. USA 98, 12984-12989.

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Characterization of Fungal Lytic Polysaccharide MonoOxygenases

Simon Ladevèze 1,2, Bernard Henrissat 3, & Jean-Guy Berrin 1,2

1 INRA, UMR1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France 2 Aix-Marseille Université, Polytech Marseille, UMR1163 Biotechnologie des Champignons

Filamenteux, 13288 Marseille, France 3 Aix-Marseille Université, UMR7257 Architecture et Fonction des Macromolécules Biologiques,

13288 Marseille, France Lignocellulosic biomass is a central resource for biofuel and chemistry industries. In the last years, biomass recalcitrance, i.e the natural resistance of plant cell wall degradation by enzymatic processes has undergone striking evolutions. Lytic Polysaccharide MonoOxygenases (LPMOs), a new class of secreted enzymes were identified as boosters of biomass deconstruction through the oxidative cleavage of polysaccharides. AA9 LPMOs are cellulose-active enzymes of fungal origin, of which several members have been characterized [1]. The yeast Geotrichum candidum, which is readily used in the cheese industry, is able to grow on wooden boxes of cheese. Some strains have been demonstrated to be able to degrade filter paper and cotton more efficiently than some industrial enzyme preparations, primarily due to an efficient GH7 cellobiohydrolase [2]. Recently, comparative genomics revealed 4 AA9 LPMOs in its genome [3]. Our work presents the first yeast AA9 enzymes characterization, demonstrating the involvement of LPMOs in the ability of G. candidum to degrade cellulose and xyloglucan. Moreover, the use of Pichia Pastoris as expression host for these yeast AA9 LPMOs also grant access to higher protein production yields that can greatly reduce costs and increase the efficiency of the industrial cocktails used for lignocellulose degradation. References : [1] Bennati-Granier C, Garajova S, Champion C, Grisel S, Haon M, Zhou S, Fanuel M, Ropartz D,

Rogniaux H, Gimbert I, Record E, Berrin J-G (2015) Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina. Biotechnol Biofuels. doi: 10.1186/s13068-015-0274-3

[2] Borisova AS, Eneyskaya EV, Bobrov KS, Jana S, Logachev A, Polev DE, Lapidus AL, Ibatullin FM, Saleem U, Sandgren M, Payne CM, Kulminskaya AA, Ståhlberg J (2015) Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C. FEBS J 282:4515–4537. doi: 10.1111/febs.13509

[3] Morel G, Sterck L, Swennen D, Marcet-Houben M, Onesime D, Levasseur A, Jacques N, Mallet S, Couloux A, Labadie K, Amselem J, Beckerich J-M, Henrissat B, Van de Peer Y, Wincker P, Souciet J-L, Gabaldón T, Tinsley CR, Casaregola S (2015) Differential gene retention as an evolutionary mechanism to generate biodiversity and adaptation in yeasts. Sci Rep 5:11571. doi: 10.1038/srep11571

Total synthesis of modified oligosaccharides from the linkage region of proteoglycans as potential

inhibitors or effectors of the enzyme CSGalNAcT-1 B. Ayela1, T. Poisson2, X. Pannecoucke2, C. Lopin-Bon1

1 ICOA-UMR 7311, Université d’Orléans, Orléans, France

2 Université de Normandie, COBRA,UMR 6014 ,FR 3038; Université de Rouen, France Proteoglycans are complex macromolecules which consist of a protein backbone (or core protein) covalently linked to characteristic linear polysaccharidic chains called glycosaminoglycans (GAGs). GAGs are natural polysaccharides constituted of a repetitive disaccharidic unit, and they are implicated in many biological processes such as cell growth and proliferation. Proteoglycans also appear to be involved in various diseases such as arthritis, some forms of cancer and even Alzheimer’s disease. Proteoglycan synthetic pathways are still not well known and generate a growing interest in fundamental research [1].

Figure 1 : Biosynthesis of the Proteoglycans

GAG biosynthesis is initiated by the formation of a tetrasaccharide linkage region covalently linked to serine residues of the PG core protein. CSGalNacT-1, one of the enzymes involved in the biosynthesis of proteoglycan, initiates the elongation of the GAG as chrondroitin sulfate chains.

In order to study the influence of the last disaccharide unit (GlcA-Gal) of the tetrasaccharide linkage on the activity of CSGalNacT-1, we synthetized both natural and chemically modified disaccharides and trisaccharides. Starting from monosaccharides such as D-glucose and D-galactose, we were able to quickly have access to a large library of oligosaccharides. These compounds will be further tested in collaboration with our biologist partners, as potential acceptors or inhibitors of CSGalNacT-1.

Figure 2 : Synthetic pathway toward the modified oligosaccharides References : [1] Aït-Mohand K; Lopin-Bon C; Jacquinet JC; Carbohydrate Res. 2012 353 ; 33-48

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Catalytic aerobic oxidation of reducing sugars issued from softwood hemicellulose acid hydrolysis

Yves Queneau 1,3, Elie Derrien,1,2,5, Catherine Pinel,1,2 Michèle Besson,1,2

Mohammed Ahmar,1,3 Emilie Martin-Sisteron,1,4 Guy Raffin,1,4 Philippe Marion5

1 Université de Lyon, France 2 IRCELYON, Institut de recherches sur la catalyse et l’environnement de Lyon,

UMR5256 CNRS-Université Lyon1, Villeurbanne 69626 3 INSA Lyon, ICBMS, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires,

UMR5246 CNRS-Université Lyon1,INSA Lyon, CPE Lyon, Villeurbanne 69621 4 ISA, Institut des Sciences Analytiques, UMR 5280 CNRS-Université Lyon1, Villeurbanne 69626

5 SOLVAY Research and Innovation Centre of Lyon, Saint-Fons 69192

Sugars, the primary constituents of cellulose and hemicelluloses polysaccharides in wood can be recovered from easily depolymerized hemicelluloses by hydrolysis techniques, and then used for the production of high added-value compounds. In this work, we will report the catalytic oxidation with air of aldoses released in a softwood hemicellulosic hydrolysate to the corresponding aldaric acids. Among these sugar-derived diacids, glucaric acid has been targeted as a “top value-added chemical from biomass” by the US Department of Energy [1]. Aerobic oxidation over supported metallic catalysts has been reported for the aqueous phase conversion of D-glucose to D-glucaric acid [2,3].

After 2-step hydrolysis of pine wood chips containing mainly galactoglucomannans and some arabinoglucuronoxylans, the resulting filtrated aqueous stream consisted of ca. 45 g L-1 monosaccharides with a high proportion of mannose (44%), together with glucose (13%), galactose (15%), xylose (21%), and arabinose (7%). Synthetic solutions of the different aldoses, separately or in mixture, and the hydrolysate were oxidized with alkaline pH control over Pt/C ([aldose]0 = 0.25M, aldose/Pt (n/n) = 157, pH 9 (NaOH 10wt.%, T = 60°C, air flow 0.5 L min-1) or in non-neutralized conditions over AuPt bimetallic catalyst ([aldose]0 = 0.25M, aldose/metal = 40, T= 100°C, air pressure = 40 bar). Precise quantification of the outcome of the oxidation reactions was made possible by analysis by ionic chromatography with an amperometric detector and by comparison with authentic aldaric samples; these were either commercially available, or were prepared from the native monosaccharides by unambiguous multistep protocols, notably via dimethylamide intermediates [4].

The poster will describe the organic synthesis of the sugar-diacids and will compare the yields in the different aldaric acids for the different sugar-rich solutions in basic and neutral conditions. References: [1] Werpy, J. T. and Petersen, G., “Top Value Added Chemicals from Biomass”, US Department of

Energy, Vol. 1, August 2004, pp. 36-38. [2] Besson, M.; Flèche, G.; Fuertes, P.; Gallezot, P. ; Lahmer, F. Recl. Trav. Chim. Pays-Bas 1996,

115, 217-221. [3] Murphy, V. J. et al., US 2011/0306790 (2011) [4] Carpenter, C. A.; Hardcastle, K. I.; Kiely, D. E. Carbohydrate Res. 2013, 376, 29-36.

Deciphering the glycosylation changes occurring during the differentiation and the activation of monocytic THP-1

cell line into macrophages Clément P. Delannoy1, Yoann Rombouts1, Sophie Groux-Degroote1, Stephanie Holst2,

Bernadette Coddeville1, Anne Harduin-Lepers1, Manfred Wuhrer2, Elisabeth Elass1 and Yann Guérardel1

1 Structural and Functional Glycobiology Unit, UMR CNRS 8576, Univ. of Lille, 59655 Villeneuve d’Ascq, France 2 Center for Proteomics and Metabolomics, Leiden Univ. Medical Center, 2300 RC Leiden, The Netherlands

Macrophages mediate innate immune system through the initiation and regulation of inflammation and contribute to adaptive immunity via antigen processing [1]. Cell surface glycosylation has been widely described to be involved in different physiological or pathological processes, such as host defense, immunological and inflammatory responses [2] but much less is known about the variations of glycosylation related to the differentiation of monocytes into macrophages. Human monocytic cell line THP-1 is frequently used as macrophage-like models [3], after treatment with phorbol myristate acetate (PMA), allowing the prediction of both function and behavior of these phagocytes. Based on these observations, the aim of this study was to highlight the glycome variation during the differentiation of the human monocytic THP-1 cell line into macrophages. In this study, MALDI-TOF data analysis showed that the differentiation of monocytic THP-1 cells into macrophages induced gangliosides biosynthesis, but also an increase of complex N-glycan and the degree of branching. This data were correlated with the expression pattern of glycosyltransferases and glycosidases involved in glycan elongation and trimming. In the other hand, the differentiated THP-1 cells were exposed to inflammatory agents. The 19-kDa lipoprotein, a component of cell-wall of Mycobacterium tuberculosis, has an important role in the induction of immune response in macrophages. This lipoprotein induces a pro-inflammatory response through Toll-like receptor 2 [4]. However, nothing is known about the influence of the 19-kDa lipoprotein on macrophage glycosylation. To investigate the impact of this lipoprotein, a synthetic lipopeptide has been used to mimic the lipid moiety of the cell-wall associated 19-kDa lipoprotein [5]. By treating macrophages with the synthetic lipopeptide, the N-glycosylation pattern has been impacted. MALDI-TOF data showed that this cell-wall component of mycobacterium induced a decrease of complex-type N-glycans.

Figure 1 : Alteration of glycosphingolipids composition of THP-1 cells after PMA-treatment.

References : [1] Gordon, S., and Taylor, P. R. (2005). Nat. Rev. Immunol. 5, 953–964 [2] Ryan, S. O., and Cobb, B. A. (2012). Microbes Infect. 14, 894–903 [3] Auwerx, J. (1991). Experientia 47, 22–31 [4] Sánchez, A et al. (2012). Clin. Dev. Immunol. 2012, 950503 [5] Schromm, A. B. et al. (2010). Innate Immun. 16, 213–225

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Structural and functional characterization of a hypothetical new glycoside hydrolase

Barbara Guyez1,2, Franck Moncassin1,2, Claire Raingeval1,2, Sophie Bozonnet 2,

Bernard Henrissat3, Lionel Mourey1, Michael O’Donohue2, Samuel Tranier1 & Claire Dumon2

1Institut de Pharmacologie et de Biologie Structurale, UMR5089 Toulouse 2 LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France

3 Laboratoire d‘Architecture et Fonction des Macromolécules Biologiques, UMR 7257 Case 932

Plant biomass is a renewable and inexhaustible carbon source, and to maximize its valorization it is necessary to develop new biocatalysts able to hydrolyze efficiently the cellulose and hemicellulose content [1] [2]. To do so, glycosides hydrolases (GH) are the biocatalysts of choice. They catalyze hydrolysis of the very stable O-glycosidic bound. To degrade hemicellulose, microorganisms produce a panel of GH such as xylanases, xylosidases, or arabinofuranosidases [3].

New potential GHs were discovered from the functional screening of an earthworm gut metagenomic library. One metagenomic clone active on cellobiose revealed three putative GH: a GH1, a GH4 and the third one annotated as a hypothetical GH.

This latter putative GH is particularly interesting, because it could not be assigned to any of the 135 existing GH families of the CAZy database. Here, we describe the structural and functional characterization of this GH named after GH-star. A large range of substrates was tested and activity was observed with arabinoxylan and xylooligosaccharides as substrates. Additionally, X-ray structure of this protein was solved at 1.6Å resolution and even if the overall folding is very similar to GH5 enzymes, a major difference is the lack of one of the catalytic residues. Taken together, results suggest that this enzyme could be the first characterized member of a new Glycoside hydrolase family. References : [1] Himmel, Ding, Johnson, Adney, Nimlos, Brady and Foust (2007). Science, Vol. 315, Issue 5813,

pp. 804-807 DOI: 10.1126 [2] Sticklen (2008). Nature Reviews Genetics 9, 433-443 doi:10.1038/nrg2336 [3] Dumon, Song, Bozonnet, Fauré and O’Donohue (2012). Process Biochemistry. 47 346–357

DOI:10.1016/j.procbio

Synthesis of putative inhibitors for the human

endosulfatase, H-sulf 2, a new therapeutic target in cancer and inflammatory diseases

Mock-Joubert Maxime 1, Christine Le Narvor 1, David Bonnaffé 1 & Romain Vivès 2 .

1 Equipe Méthodologie, Synthèse, et Molécules Thérapeutiques, Institut de Chimie Moléculaire et des Materiaux d’Orsay, Université Paris Sud, CNRS, Université Paris-Saclay

2 Groupe Structures et Activités des Glycosaminoglycanes, Institut de Biologie Structurale, CEA Grenoble Heparan sulphate proteoglycans (HSPGs) interact with many proteins, especially growth factors or cytokines via specific sulfation patterns, which are due to its highly regulated biosynthetic machinery. HS can also be remodeled at the cell surface and in the extracellular matrix by a novel class of extracellular enzymes, the endosulfatases (HSulf 1 and 2), which selectively remove 6-O-sulfo groups from glucosamine residues within HS. [1-3] HSulfs expression/production are deregulated in many human cancers including breast, lung, ovarian and hepatocarcinoma.[4-5] HSulf-2 is strongly induced in lung squamous cell carcinoma and lung carcinoma, which are cancers with poor prognosis. Thus, this enzyme represents an interesting new therapeutic target. Sulfatases belongs to a rather large family of enzymes and, although many aspects remain to be clarified concerning their mechanism of action, the amino acid sequences are relatively conserved, especially the residues involved in the catalysis.[6] Indeed, all these enzymes seem to share a common catalytic mechanism, which involved a Cα-formylglycine residue and efficient inhibitors have been designed by replacing the sulfate’s moiety by a sulfamate function. [7-8] Here, we report the synthesis of potential HSulf inhibitors based on the incorporation of the sulfamate residue in heparin sulfate fragments. Starting from the trisaccharide A, we describe the synthesis of compounds B,C.

References: [1] A. Seffouh, et al, FASEB J. 2013, 23, 2431-2439. [2] R. R. Vivès, et al, Front. Oncol. 2014, 3, 331, 1-11. [3] M. Buono et al, Cell. Mol. Life Sci. 2010, 67, 769-780. [4] S. Rosen et al, Expert. Opin. Ther. Targets. 2010, 14, 935-949. [5] X. Zheng et al, Genes Chromosom. Cancer 2013, 52, 225–236. [6] H. Lemjabbar-Alaoui, Oncogene 2010, 29, 635–646 [7] S. R. Hanson, et al, Angew. Chem. Int. Ed. 2014, 43, 5736–5763. [8] M. Schelwies, et al, ChemBioChem 2010, 11, 2393–2397

Acknowledgements: This work was funded by the grant ANR-10-LABX-33 as members of the Laboratory of Excellence LERMIT

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Preparation of various sulfoforms of oligosaccharides for the study of proteoglycans biosynthesis

Hélène Ledru1, Chrystel Lopin-Bon1

1 Institut de Chimie Organique et Analytique (ICOA) – UMR 7311, Université d’Orléans, France

Proteoglycans (PGs) are macromolecular glycoproteins composed of glycosaminoglycan chains (GAGs) covalently linked to L-serine residues. GAGs play important roles in many biological processes, such as cell growth and proliferation. However they are also involved in several diseases including arthropathies, Alzeimer’s disease and cancer. Their biosynthesis involves the action of glycosyltransferases (GTs) and starts with the formation of a tetrasaccharidic sequence GlcA-β-1,3-Gal-β-1,3-Gal-β-1,4-Xyl-β-O attached to a core protein (Figure 1). This GAG-linkage region initiates the formation of two types of GAG chains, heparin/heparan sulfates (Hep/HS) with the addition of α-D-GlcNAc and chondroitin sulfates/dermatan sulfates (CS/DS) with addition of β-D-GalNAc. During the biosynthesis, the linkage region may be modified by sulfation on D-Gal units but the role of these substitutions is not yet fully understood.

Figure 1 : Linkage region of proteoglycans

Our project aims at preparing potential substrates of GTs and particularly of human EXTL3 and CSGalNAcT-1, which orientate the biosynthesis toward Hep/HS or CS/DS chains respectively. We are currently developing stereo- and regio-controlled syntheses of sulfated and unsulfated disaccharides (D-GlcA-β-1,3-D-Gal-β) of the linkage region and the corresponding trisaccharides (transfer products), with the first aminosugar of each GAG chains (D-GlcNAc or D-GalNAc) (Figure 2). Moreover we are currently studying methodologies of sulfation with different techniques like microwaves, flow reactions and chemoenzymatic reactions.

Figure 2 : Various molecules under investigation

C-type lectins receptors (CLRs) arrays to screen immunocompatibility and reactivity of biological sample

Silvia Achilli1,2,3, Blanka Didak1,2,3,4, Corinne Vivès1,2,3, Michel Thépaut1,2,3,

Ludovic Landemarre4, Franck Fieschi1,2,3

1Univ. Grenoble Alpes, Inst. de Biologie Structurale, Grenoble, France

2 CNRS, IBS, F-38044, Grenoble, France 3 CEA, IBS, F-38044 Grenoble, France 4 GLYcoDiag, 45067 Orléans, France

Lectins are unique among proteins in that they bind specifically carbohydrates. Among all of the animal lectins that have been defined, one family include a large group of calcium dependent carbohydrate-binding molecules, known as C-type lectins receptors (CLRs) [1].

CLRs are largely present at the surface of antigen presenting cells were they play crucial in the specific recognition of carbohydrate-based PAMPs (pathogens associated molecular pattern) or DAMPs (danger associated molecular patters). Thus they are directly involved in the immune activation and adapted response as a function of the situation (immune activation or tolerance). Indeed, they offer tremendous potential to enhance the efficacy of vaccines and as therapeutic targets in infectious and non-infectious diseases. However CLRs functions are still not perfectly understood and critical questions remain, such as how CLR responses are regulated, how responses from multiple CLRs are integrated [2]. A major objective would be to use these CLRs as modulators in order to tailor the immune system response. To do so, molecules selective to each individual CLRs have to be developed.

Here, we produce several recombinant human CLRs in bacteria, in order to test their interaction with selective carbohydrate immunomodulators and to develop new lead structures for highly selective glycan based multivalent immunotherapeutics relevant for the development of cancer, autoimmune diseases and allergy treatment. In order to foster the identification of CLRS specific ligand we aim to develop a human C-type lectin arrays. In the present study, performed in collaboration with the company GLYcoDiag (France), preliminary data of interaction between CLRs and a panel of natural carbohydrates are presented.

References: [1] Elizabeth J. Soilleux ; « DC-SIGN and DC-SIGN R : friend or foe ? » Clinical Science (2003) ; 104 :

437-446 [2] Ivy M. Dambuza and Gordon D. Brown « C-type lectins in immunity : recent developments »,

Current Opinion in Immunology 2015 (32) : 21-27

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Le catabolisme de la paroi mycobactérienne

Alexandre Méry1, Lin Shen1, Albertus Viljoen2, Sydney Villaume3, Christophe Mariller1, Stéphane Vincent3, Laurent Kremer2 & Yann Guérardel1

1 Univ. Lille, CNRS, UMR8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle,

59000 Lille, France 2 Mycobacterial Pathogenesis and Novel Therapeutic Targets, CNRS-FRE3689, 34000 Montpellier, France

3 Université de Namur, Laboratoire de Chimie Bio-organique, Namur, Belgium

L’Arabinogalactane (AG) est un élément clé de la paroi myctobactérienne, représentant approximativement 35 % de ses composants totaux. L’AG se distingue essentiellement par sa structure glycannique unique composée de D-Ara et de L-Gal, tous deux sous forme furanose. Contrairement à la plupart des polysaccharides bactériens, l’AG ne possède pas d’unités de répétition mais comprend plutôt des motifs structuraux bien distincts. Ce polysaccharide a une fonction essentielle car il permet la connexion entre la couche des acides mycoliques et la couche interne du peptidoglycane pour former le complexe mycolyl-arabinogalactane-peptidoglycane (mAGP). De par son importance cruciale dans le mode de vie des mycobactéries, la compréhension de la biosynthèse du complexe mAGP a toujours été essentielle pour le développement de nouvelles cibles médicamenteuses. De plus, de récentes études ont mises en avant l’existence d’une D-arabinase endogène chez Mycobacterium smegmatis montrant ainsi que le catabolisme de la paroi mycobactérienne, et plus particulièrement du mAGP est une voie innovante vers la découverte de nouveaux traitements anti-tuberculeux. Dans ce contexte, ce projet a pour but de prouver que les mycobactéries peuvent dégrader leur propre mAGP en se focalisant principalement sur les enzymes capables de cliver les parties arabinane et galactane de l’AG. En utilisant la chromatographie ionique couplée à l’analyse par spectrométrie de masse des produits enzymatiques, nous avons développé un outil pour la détection des activités glycosidasiques chez les mycobactéries. Pour le moment, cette méthode est principalement utilisée pour contrôler l’activité endo-D-arabinase afin de la purifier et de l’identifier. Nous avons également cherché des gènes codant des glycoside hydrolases en criblant le génome mycobactérien et en concentrant nos recherches sur l’identification de glycoside hydrolases utilisant le D-Araf et le L-Galf comme substrats grâce à la base de données CAZY (http:www.cazy.org). Cette seconde approche nous a permis d’identifier et d’exprimer chez E.coli la protéine Rv3096 de M. tuberculosis. L’analyse fonctionnelle a montré que cette protéine est une galactofuranohydrolase dégradant de façon récurrente la chaîne galactane lorsqu’elle est incubée avec de l’AG. Nous avons donc développé avec succès une méthodologie simple pour le screening d’activités glycosidasiques et d’identification d’enzymes. Cela nous a ainsi déjà permis d’identifier l’activité de la galactofuranohydrolase Rv3096.

OZO derived iminosugars The one-pot Retro-Michael/Michael addition solution

Maria Dominguès,1,2 Marie Schuler, 1 Justyna Jaszczyk1, Pierre Lafite1, Richard Daniellou1,

Isabel Ismael,2 Arnaud Tatibouët1 1 Rue de Chartres, BP 6759, Université d'Orléans et CNRS, ICOA, UMR 7311, 45067 Orléans, France

2 Chemistry Department, Textile and Paper Materials Unity, University of Beira Interior, 6200-001 Covilhã, Portugal

1,3-oxazolidine-2-thiones (OZT) are simple heterocycles which have shown various interests and applications. In stereoselective synthesis, it has been compared to the chiral auxiliary 1,3-oxazolidine-2-one (OZO) of Evans with the main work of Crimmins, but also has shown various applications in Michael type addition or sulfur transfer reaction.[1-3] This simple heterocycle could also be found in Nature as the degradation product of glucosinolates and acts as a biological marker.[4] Over the years, our group developed methods to anchor this structure on various carbohydrate backbones to study their chemical reactivities and develop new bioactive molecules.[5] More recently we have explored the chemistry toward iminosugars analogues of the indolizidine-type structures related to castanospermine. [6] Over the years two main approaches have been used to introduce an OZT, from a β-aminoalcohol with a thionocarbonyl source or directly on reducing sugars by reacting with thiocyanic acid.

Figure 1 : Access to aminal type iminosugars

This last approach has been one of the main stream in our laboratory and we have shown the possible balance of reactivity of an α-hydroxycarbonyl with thiocyanic acid to the formation of either an OZT or a 1,3-oxazine-2-thione (OXT). This reactivity led to various structures depending on the carbohydrate series and the nature of the protecting groups. We have further explored the potential of these heterocyclic moieties in developing the transformation to oxazolidine-2-one derivatives and its application using a one-pot retro-Michael/Michael type addition to the synthesis of iminosugars, analogues of deoxynojirimycin. References: [1] Han, Y.-Y.; Chen, W.-B.; Han, W.-Y.; Wu, Z.-J.; Zhang, X.-M.; Yuan, W.-C. Org. Lett., 2012, 14

490–3. [2] Cano, I.; Gomez-Bengoa, E.; Landa, A.; Maestro, M.; Mielgo, A.; Olaizola, I.; Oiarbide, M.; Palomo,

C. Angew. Chem., Int. Ed., 2012, 51, 10856–60. [3] Munive, L.; Rivas, V.M.; Ortiz, A.; Olivo, H.F. Org. Lett., 2012, 14, 3514–7. [4] Agerbirk, N.; Olsen, C.E. Phytochemistry, 2015, 115, 143–51. [5] Simao, A.C.; Rousseau, J.; Silva, S.; Rauter, A.P.; Tatibouët, A.; Rollin, P. Carbohydr. Chem.,

2009, 35, 127–72. [6] Silva, S.; Sanchez-Fernandez, E.M.; Ortiz Mellet, C.; Tatibouët, A.; Pilar Rauter, A.; Rollin, P. Eur.

J. Org. Chem., 2013, 7941–51.

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Synthesis of glycosides restrained in a 1,4B boat conformation. Impact on the in vitro activity of the β-N-acetylhexosaminidase C

(O-GlcNAcase) and the intracellular O-GlcNAcylation levels in human cell lines

Anne-Sophie Vercoutter-Edouart1, Olivier Massinon2,

Marlène Mortuaire1, Maïté Leturcq1, Tony Lefebvre1 & Stéphane Vincent2

1 Unité de Glycobiologie Structurale et Fonctionnelle, UGSF, Univ. Lille, CNRS, UMR 8576, Lille, France 2 Unité de Chimie Organique, Université de Namur, Namur, Belgique

O-GlcNAcylation is a dynamic and reversible glycosylation on serine and threonine residues of nuclear, cytoplasmic and mitochondrial proteins. O-GlcNAc cycling is regulated by two single enzymes: O-GlcNAc Transferase (OGT or UDP-N-acetylglucosamine-peptide N-acetyl-glucosaminyltransferase) and O-GlcNAcase (OGA or β-N-acetylhexosaminidase, GH84). O-GlcNAcylation is involved in the regulation of fundamental cellular processes, including signal transduction, the cell cycle and proteasomal degradation. Dysregulation of O-GlcNAc levels is associated with various human diseases, such as diabetes, cancer, neurodegenerative and cardiovascular diseases [1]. To better understand the molecular and cellular mechanisms regulated by O-GlcNAc post-translational modification, numerous efforts have been made in the last few years to develop small-molecules inhibitors targeting OGT or OGA activity. In this way, and thanks to the elucidation of the mechanism of action of these glycosyl-processing enzymes at the atomic level and particularly at the transition state [2], we developed a new class of synthetic GlcNAc analogues restrained in a 1,4B conformation [3].The selectivity of these bicyclic compounds towards the enzymatic activity of β-N-acetylhexosaminidases was measured in vitro. We also evaluated the impact of these synthetic glycomimetics on the intracellular O-GlcNAc levels, the cell cycle progression and the proliferation rate of normal and cancerous human cell lines. Although most of them show a moderate inhibition of O-GlcNAcase, these glycoside analogues may be the leaders of a new class of pharmacological inhibitors for therapeutical purposes.

Figure: Rational design of glycomimetics locked in a 1,4B boat conformation.

References: [1] Lefebvre T, Issad T. (2015) 30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of

Cell Signaling and Metabolism by O-GlcNAcylation. Front Endocrinol (Lausanne), 9;6:17 [2] Macauley MS, Vocadlo DJ. (2010) Increasing O-GlcNAc levels: An overview of small-molecule

inhibitors of O-GlcNAcase. Bioch. Biophys Acta 1800; 107–121. [3] Thiery E, Reniers J, Wouters J & Vincent SP. (2015) Stereoselective synthesis of boat-locked

glycosides designed as glycosyl hydrolase conformational probes. Eur. J Org. Chem. 7 1472-1484.

Role of heparan sulfate 3-O-sulfotransferases in cancer cell proliferation and survival

Charles Hellec, Agnès Denys, Maxime Delos, Mathieu Carpentier & Fabrice Allain

Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq, France

Heparan sulfates (HS) are linear and sulfated polysaccharides, for which the sulfation pattern determines the biological properties. During the polymerization of the chains, several sulfotransferases transfer sulfate groups at different positions. The last step of HS biosynthesis is catalyzed by 3-O-sulfotransferases (3-OSTs), which transfer sulfate group to the OH at the position C3 of glucosamine residues. The 3-OST family is represented by seven isoenzymes, which trigger different substrate specificities and cell-type specific expression [1]. The role of 3-OSTs in cancer is still misunderstood. Previous studies reported that cancer cells do not express 3-OSTs and their ectopic expression reduced cell growth and survival, suggesting an anti-tumoral activity. However, these findings are contradictory with recent studies arguing that 3-O-sulfated HS may act as pro-tumoral factors. Indeed, it was recently demonstrated that overexpression of 3-OST3B and 3-OST2 respectively in leukemia cells and breast cancer cells promoted cell proliferation and migration, two typical features of invasive cancer cells [2, 3]. Moreover, 3-OST4, which is normally highly expressed in embryonic neuronal tissues, is re-expressed in some cancer cells, and this over-expression correlates with immune escape in vivo [4]. Nevertheless, the underlying mechanism involving 3-OSTs in tumor growth and expansion remains unknown. Here, we analyzed the effect of an overexpression of 3-OST2, 3-OST3B and 3-OST4 in MDA-MB-231 and BT-20 cancer cells. We find that the three enzymes efficiently enhanced cell proliferation and survival, which was related to an increase in the activation of c-Src and Akt. Furthermore, 3-OST overexpression leads to cell protection against apoptosis induced by either staurosporin or the combination anti-Fas/TNF-α. These effects are similar for the three isoenzymes, indicating a general pro-tumoral activity of 3-O-sulfated HS. Taken together, our findings are supporting a model in which 3-OSTs display pro-tumoral activity, thus suggesting that an increase in the reaction of HS 3-O-sulfation in cancer cells could be associated with a bad prognosis. References : [1] Esko JD, Selleck SB. Order out of chaos: assembly of ligand binding sites in heparan sulfate.

Annu. Rev. Biochem. 2002;71:435-71 [2] Zhang L, Song K, Zhou L, et al. Heparan Sulfate D-glucosaminyl 3-O-sulfotransferase-3B1

(HS3ST3B1) promotes angiogenesis and proliferation by induction of VEGF in acute myeloid leukemia cells. J. Cell. Biochem. 2015 Jun;116(6):1101-12

[3] Vijaya Kumar A, Salem Gassar E, Spillmann D, et al. HS3ST2 modulates breast cancer cell invasiveness via MAP kinase – and Tcf4 (Tcf7I2)-dependent regulation of protease and cadherin expression. Int. J. Cancer. 2014 Dec 1;135(11):2579-

[4] Birrocio A, Cherfils-Vicini J, Augereau A, et al. TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells. Nat. Cell. Biol. 2013 Jul;15(7):818-28

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GLYcoPROFILE® : deciphering the sweet side of cells

Blanka Didak1, Alexiane Decout1, Eric Duverger1 and Ludovic Landemarre1

1 GLYcoDiag, 45520 Chevilly The cell-cell or cell-matrix interactions/communications processes are initiated in the first time of recognition mainly via specific affinities between a glycan moiety and a carbohydrate recognition region of a protein or glycoprotein (lectin, glycan receptor). These glycobiological interactions are involved in a number of key biological phenomena occuring in living organisms. Indeed, glycobiological interactions drive the key steps of life from the beginning with the first communication between the two parent cells, to the pathological states with the expression of specific glycans (“glycobiomarkers”), or during the aging with the change of some glycan structures (glycosaminoglycans) which allow modifications of their recognition by glycans receptors. Hence, further studying of those glycans-proteins recognitions could be of great interest for the discovery of new glycan biomarkers on cells.

Since the beginning of the 21st century, lectin array technology in increasingly used to generate relevant information related to glycan motifs, accessibility and a number of other valuable insights of molecules (purified and non-purified) or cells. GLYcoDiag has developed a technology platform called GLYcoPROFILE ® intended for the determination of interaction profiles with lectins or glycans allowing to identify "glycan signatures" on the surface of molecules or cells .

The nature of cell surface glycans can help to distinguish between cell-types, and for a single cell type, its glycan signature can vary during growth, differentiation and pathological transformation. Thus, GLYcoPROFILE® could represent a powerful technology for the monitoring and characterisation of specific glyco-biomarkers related to cells-type, cells differentiation state, cells behaviour, cells environment or cells interactions. Thus, primary cells glyco-signatures under the presence of products (glyco or not) can be connected with specific glycans signatures. Expression of therapeutic recombinant molecules by cells such as CHO also induces glycan signature modification that can be used to select a specific clone (lectin-aided capture) and characterize a productive clone during recombinant protein production process. Finally, GLYcoPROFILE® technology can be applied to germinal cells and obtention of specific glycan signature (cell surface glycan accessibility) could open the way to medical applications such as diagnostics of infertility.

Iminosugars-based macrocycles to deliver new sweet azacrowns

A. Bordes, N. Fontelle, J. Désiré, F. Lecornué, J. Guillard and Y. Blériot

Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP),

UMR CNRS 7285, Université de Poitiers, Equipe E5 “Synthèse Organique”, 4 rue Michel Brunet, 86073 Poitiers Cedex

Iminosugars, sugar analogs in which the endocyclic oxygen has been replaced by nitrogen, constitute a major class of sugar mimetics. Their use has been limited to the biological field so far as these compounds have shown promising therapeutic properties[1]. Interestingly, their incorporation into macrocycles could deliver innovative scaffolds that could display chelation properties as well as catalytic potential when bound to metals. For this purpose, an efficient synthesis of iminosugar C-glycosides[2] displaying two arms at C-5 and C-1 positions is necessary. Our last results toward the development of a robust synthesis of six membered iminosugars C-glycosides using a highly diastereoselective tandem Staudinger-Aza-Wittig reaction will be presented. The conversion of these structures into unprecedented iminosugar duplexes displaying various linkages between the two iminosugar units and the preliminary chelation properties of these iminosugar aza-crowns (ISAC) will be disclosed.

Figure 1: iminosugar-aza-crowns developed in this work

References: [1] Li, H.; Blériot, Y et al.; Bioorg. Med. Chem. 2009, 17, 5598. [2] Mondon, M.; Fontelle, N.; Désiré, J.; Lecornué, F.; Guillard, J.; Marrot, J.; Blériot, Y. ; Org. Lett.

2012, 14, 870.

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Structural investigation of cell surface assossiated polysaccharides of Lactobacillus delbrueckii subsp.

bulgaricus 17 as potential substrates for bacteriophage Ld17 glycerophosphodiesterase

Irina Sadovskaya1, Evgeny Vinogradov2, Anneleen Cornelissen3, Thierry Grard1,

Stéphanie Blangy4.5, Silvia Spinelli4.5, Eoghan Casey3, Jennifer Mahony3, Jean-Paul Noben6, Fabio Dal Bello7, Christian Cambillau4.5 & Douwe van Sinderen3,8*

1 Equipe BPA, Université du Littoral-Côte d’Opale, Institut Régional Charles Violette EA 7394, USC Anses-ULCO, Bd Bassin Napoléon, BP 120, 62327 Boulogne-sur-mer, France

2 National Research Council, 100 Sussex Dr, K1A 0R6, Ottawa, Canada 3 School of Microbiology & 8 APC Microbiome Institute, University College Cork, Cork, Ireland

4 Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, Campus deLuminy, Marseille, France

5 Centre National de la Recherche Scientifique, Architecture et Fonction des Macromolécules Biologiques, UMR 6098, Campus de Luminy,Marseille, France

6 Biomedical Research Institute (Biomed) and School of Life Sciences, Transnationale Universiteit Limburg, Hasselt University, Agoralaan-Building C, BE-3590 Diepenbeek, Belgium

7 Sacco srl, Cadorago, Italy Bacteriophages are the largest cause of fermentation failure in the dairy industry. The study of interactions between lactic acid bacteria (LAB), in particular lactobacilli, and their infecting bacteriophages are the focus of intensive research. The cell surface-associated polysaccharides (sPSs) of LAB were shown to act as receptors for bacteriophages. In the current work, we identified three different sPSs of an industrial strain Lb. delbrueckii subsp. bulgaricus 17 (Ldb17), and established their chemical structure by 2D NMR spectroscopy and methylation analysis : (1) a neutral branched sPS1, composed of hexasaccharide repeating units (-[α-D-Glcp-(1-3)-]-4-β-L-Rhap2OAc-4-β-D-Glcp-[α-D-Galp-(1-3)]-4-β-Rhap-3-β-D-Galp-), (2) an acidic sPS2, a linear D-galactan with the repeating unit having a structure (-[Gro-3P-(1-6)-]-3-β-Galf-3-α-Galp-2-β-Galf-6-β-Galf-3-β-Galp-), and (3) short chain poly(glycerophosphate) teichoic acids. We have shown that the sPS2 is the major substrate for a glycerophosphodiesterase (GDPD) enzyme, derived from the Lb. delbrueckii ssp bulgaricus group b bacteriophage 17. Further research will help developing new strategies to prevent lysis of starter cultures during dairy fermentation. * Published in part: Vinogradov, E., Sadovskaya, I., Cornelissen, A., van Sinderen, D. (2015) Carbohydr. Res. 413, 93-99

Structural aspects and membrane binding properties of MGD1, the major galactolipid synthase in plants

Joana Rocha1, Milène Nitenberg,1 Eric Maréchal2, Agnès Girard-Egrot3,

Maryse Block2 & Christelle Breton1 1 CERMAV-CNRS, Univ. Grenoble Alpes, Grenoble, France

2 LPCV, UGA-CEA-CNRS-INRA UMR 5168, Grenoble, France 3GEMBAS Team, ICBMS, UMR CNRS 5246, University of Lyon, 69622 Villeurbanne, France

Galactolipids, such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are a unique lipid class ubiquitously found in photosynthetic organisms, from cyanobacteria to land plants. They constitute the most profuse lipid class on earth and they are essential for the assembly and function of the photosynthetic apparatus. MGDG synthesis is catalyzed in a single step by a MGDG synthase (called MGD), which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG). MGD1 is the major galactolipid synthase in Arabidopsis and is essential for the massive expansion of membrane thylakoids. It is a monotopic protein localized into the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids.

The catalytic domain of MGD1 has been successfully expressed as an active and soluble form into E. coli and conditions for activity tests and effects of known positive effectors such as phosphatidic acid (PA) and phosphatidylglycerol (PG) were reassessed [1]. The crystal structures of the catalytic domain of MGD1, free and in complex with UDP, have been recently solved [2]. MGD1 displays the canonical GT-B fold with two distinct Rossmann-type domains. These structures give insight into residues critical for binding UDP-Gal and clues for DAG recognition. In addition, we identified a few amino acid residues that are expected to bind PG. Using a Langmuir membrane model which allows tuning of both lipid composition and packing, we investigated the membrane binding properties of MGD1 [3]. Interestingly, MGD1 has a large disordered loop in its N-terminal domain (~50 amino acids) that was shown to be important for DAG binding.

References: [1] Rocha et al., (2013) Biochimie, 95, 700-708 [2] Rocha et al., (2016) Plant J., 85, 622-633 [3] Sarkis et al., (2014) FASEB J, 28, 3114-3123

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Mutations within a water channel change the balance between transglycosylation and hydrolysis in Agarase

Franck Daligault1, Romain Irague1, Benoît David1, Diane Jouanneau2,

Mirjam Czjzek2, Yves-Henri Sanejouand1 & Charles Tellier1 1UFIP, UMR-CNRS 6286, Université de Nantes, 2 rue de la Houssinière, 44322 Nantes, France

2LBI2M CNRS-UPMC 8287, Station biologique de Roscoff, France

Up to now, most mechanistic enzyme studies have focused on the protein sequence and the impact of the spatial arrangement of aminoacid sidechains on the mechanism. On the other hand, although the role of hydration in proteins is largely recognized for protein folding, stability and dynamics, few studies have been dedicated to the hypothesis that water may have a more direct role in enzymatic catalysis. This later point could indeed prove particulary important in the case of hydrolysis, since water is also a substrate of the reaction.

Known for their ability to hydrolyse glycosidic linkages, numerous retaining glycoside hydrolases are also able to catalyse transglycosylation reaction which can be harnessed for the synthesis of complex oligosaccharides [1]. Although in the vast majority of cases hydrolysis prevails over transglycosylation reaction, propensity has already been increased through mutagenesis and directed evolution experiments [2,3,4]. However, little is known about the regulation of the balance between both activities.

We discover, via molecular dynamics (MD) simulations, a potential intermittent water channel connecting the bulk to the active site in a β-agarase belonging to glycoside hydrolases families GH16 which supports the hypothesis of a possible role of internal water dynamics in the hydrolytic activity of glycosidases [5,6]. Mutagenesis of specific buried amino acid residues in the vicinity of these water channels coupled with the biochemical characterization of the corresponding mutants allowed us to identify four specific residues in the enzyme involved in the regulation of the activity balance between hydrolysis and transglycosylation. In this enzyme, two of those functional residues tend to form a bottleneck at the end of the channel at the interface with the catalytic pocket. Within this context, it is tempting to speculate that those residues may be involved in controlling water release from the core channel to the active site, thus regulating the balance between hydrolysis and transglycosylation in those β-glycosidases. References: [1] Bissaro B., Monsan P., Fauré R., and O’Donohue M.J. (2015). Glycosynthesis in a waterworld: new

insight into the molecular basis of transglycosylation in retaining glycoside hydrolases. Biochem. J. 467, 17-35.

[2] Feng H.-Y., Drone J., Hoffmann L., Tran V., Tellier C., Rabiller C., and Dion M. (2005). Converting a β-glycosidase into a β-transglycosidase by directed evolution. J. Biol. Chem. 280, 37088–37097.

[3] Teze D., Hendrickx J., Czjzek M., Ropartz D., Sanejouand Y.-H., Tran V., Tellier C., and Dion M. (2014). Semi-rational approach for converting a GH1 β-glycosidase into a β-transglycosidase. Protein Eng. Des. Sel. 27,13–19.

[4] Teze D., Daligault F., Ferrières V., Sanejouand Y.-H., and Tellier C. (2015). Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase. Glycobiology 25(4), 420–427.

[5] Hehemann, J.-H., Correc, G., Thomas, F., Bernard, T., Barbeyron, T., Jam, M., Helbert, W., Michel, G., and Czjzek, M. (2012). Biochemical and structural characterization of the complex agarolytic enzyme system from the marine bacterium Zobellia galactanivorans. J. Biol. Chem. 287, 30571–30584.

[6] Teze, D., Hendrickx, J., Dion, M., Tellier, C., Woods, V.L., Tran, V., and Sanejouand, Y.-H. (2013). Conserved water molecules in family 1 glycosidases: a DXMS and molecular dynamics study. Biochemistry 52, 5900–5910

Synthetic access to MecPP and analogues thereof using D-galactose as a chiral scaffold

Marie Buchotte 1, Petra Hellwig 2, Franck Borel 3, Jean-Luc Ferrer 3,

Myriam Seemann 4 & Jean-Bernard Behr 1 1 Institut de Chimie Moléculaire de Reims, UMR-CNRS 7312, 51687 Reims

2 Laboratoire de bioelectrochimie et spectroscopie, UMR 7140 CMC, Université de Strasbourg-CNRS, 1 rue Blaise Pascal, 67000 Strasbourg

3 Univ. Grenoble Alpes, CNRS, CEA, IBS, F-38044 Grenoble, France 4 Laboratoire Chimie Biologique et Applications Thérapeutiques, Institut Le Bel, 67070 Strasbourg

The discovery of the methylerythritol phosphate (MEP) pathway in bacteria in the early 90’s offered new opportunities to overcome the issue of resistance to standard antibiotic treatment.[1] The MEP pathway is built on seven consecutive enzymatic activities, all of them being fundamental for bacterial survival. It has been clearly demonstrated that deletion or inhibition of any of these enzymes in E. coli is lethal for the microorganism. The ANR-project in which we are involved (Antiobio-T), aims at developing new antibacterial agents with unprecedented mode of action to enlarge the therapeutic repertoire. To this goal, we wish to design, synthesize and assay potent inhibitors of GcpE and LytB, the two last enzymes involved in the MEP pathway (Figure 1). This goes through an accurate knowledge of the structures and the mode of action of both enzymes, notably through crystallographic analysis, which requires molecular tools like substrate and substrate analogues in preparative amount. We present here the synthetic route towards a series of analogues of MEcPP (the natural substrate for GcpE), which will serve as mechanistic probes in our investigations. Galactose was used as a building block for the enantioselective synthesis of the target compounds.

Figure 1 References : N. Campos, M. Rodríguez-Concepción, M. Seemann, M. Rohmer & A. Boronat (2001) FEBS Letters, 488, 170; M. Seemann, B. Tse Sum Bui, M. Wolff, D. Tritsch, N. Campos, A. Boronat, A. Marquet & M. Rohmer (2002) Angew. Chem. Int. Ed. 41, 4337; M. Seemann, K. Janthawornpong, J. Schweizer, L. H. Böttger, A. Janoschka, A. Ahrens-Botzong, E. Ngouamegne Tambou, O. Rotthaus, A. X. Trautwein, M. Rohmer & V. Schünemann (2009), J. Am. Chem. Soc., 131, 13184; A. Ahrens-Botzong, K. Janthawornpong, J. A. Wolny, E. Ngouamegne Tambou, M. Rohmer, S. Krasutsky, C.D. Poulter, V. Schünemann, M. Seemann (2011), Angew. Chem. Int. Ed. 50, 11976. K. Janthawornpong, S. Krasutsky, P. Chaignon, M. Rohmer, C.D. Poulter, M. Seemann (2013), J. Am. Chem. Soc. 135, 1816. Faus, I.; Reinhard, A.; Rackwitz, S.; Wolny, J. A.; Schlage, K.; Wille, H.-C.; Chumakov, A.; Krasutsky, S.; Chaignon, P.; Poulter, C. D.; et al (2015) Angew. Chem. Int. Ed., 54, 12584.

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PAGés platform : a tool for glycan analysis

Bernadette Coddeville, Yann Guérardel, Frédéric Krzewinski, Emmanuel Maes, Dounia Mouajjah, Olga Plechakova, Martine Ratajczak, Xavier Trivelli & Nao Yamakawa

Plateforme PAGés, UGSF, Av. Mendeleiev, Bat C9, Université de Lille 1, 59655 Villeneuve d’Ascq

Established in december of 2012 and supported by a French national scheme for platform coordination (IBiSA) that assesses national and international standards of analytical quality. The PAGés platform is expert in glycans analysis whatever their origin. PAGés is located in the “Unité de Glycobiology Structurale et Fonctionnelle” UMR 8576 at the University of Lille1. It is composed of nine persons who ensure the continuity of service. The platform operates on the principle of customers-service and the customer can be categorized into three groups, from the private sector, from the academic sector or sector from FraBio federation (“federation of research”) which financially supports the platform. The collaboration needs to be in form of contract or service delivery. Nevertheless the establishment of a project file (downloaded on the website) is required before quote the work. The main objective of the platform is to provide an engineering service for private and academic research in the field of structural analysis of carbohydrate. PAGés is able to afford glycan mapping of a purified protein and identify the both position and nature of glycan sequences carried by glycopeptides. It is also able to describe i) the primary structure of polysaccharides under specific conditions, ii) to analyze complex mixtures and extract sweetened information (e.g. : milk, serum, freeze-dried products of diverse and various origins etc ...) iii) offer, under certain conditions, guidance in the development and analysis strategy. Meanwhile, PAGés is engaged in technological development for example in the miniaturization and optimization of specific methods to sugars, analyzes strategies, development of analytical methods in all areas of expertise which are affordable through the platform. To carry out its work, the platform has an infrastructure perfectly suited to the analysis of glycans. Also the location on two laboratories or chemical analyzes and derivations can be made, it has at its disposal the perfect tools to overcome glycan sequences including mass spectrometry in different modes and nuclear magnetic resonance with different fields both available on site or on the technology platforms of the University of Lille. Finally, all information regarding the platform or work’s requests are available on its website (plateforme-pages.univ-lille1.fr).

Figure 1: That we can do for you!

New nanomolar biphenyl C-mannopyranoside ligands reveal unprecedented binding modes in the FimH adhesin

of Escherichia coli

Eva-Maria Krammer 1, Emmanuel Maes 1, Nao Yamakawa 1, Jérôme De Ruyck 1, Gérard Vergoten 1, Stefan Oscarson 2, Mohamed Touaibia 3, René Roy 3, Julie Bouckaert 1

1 Unité Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS Univ. Lille, 59000 Lille, France 2 Center for Synthesis and Chemical Biology, Univ. College Dublin, Belfield, Dublin 4, Ireland

3 Pharmaqam, Dept of Chemistry, Univ. du Québec, Montréal, Québec, H3C 3P8, Canada

Selective inhibitors for the type-1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against acute or recurrent uropathogenic Escherichia coli infections.

The specificity and affinity of a small comprehensive library of FimH inhibitors including five different families of mannopyranoside derivatives harboring hydrophobic aglycons was systematically investigated to derive a set of structure-activity relationships.

Functionalities were suitably positioned to fit within the “tyrosine gate” of the FimH carbohydrate binding site, by amide, sulfamide, thioalkyl or alkyl spacers connected through a O- or C-glycosidic bond to alpha-D-mannopyranoside.

Alkylated alpha-D-mannosides, that do not contain a sulfur, and those alpha-anomeric D-mannosides coupled via an O-glycosidic linkage with para- and ortho- substituted biphenyl alkenes were among the most potent ligands described (Kd’s of near 3 nM). Importantly, alpha-D-mannosides with C-glycosidic linkage showed similar affinity as their O-linked analogs, with a Kd near 7 nM. This finding is of interest for therapeutical purposes because C-glycosidic bonds are better resistant to enzymatic degradation.

The three-dimensional solution structure of such a compound has been determined using NMR: NOESY, TOCSY and heteronuclear multiple-bond correlation spectroscopy (HMBC). This conformation was then docked into the FimH binding site of crystal structures with different Tyr48 side chain conformations. The mannose saccharide makes the usual interactions and the first phenyl ring stacks with Tyr48. Remarkably, the second ortho-placed phenyl ring has pushed out of the way Tyr48 to take over its interaction with Ile13 at the lower lip of the mannose-binding pocket.

Binding of anti-adhesive compounds at this site where large shear-force induced changes occur, and by the exchange with the tyrosine 48 side chain, is a mechanism as yet unexplored in drug discovery of FimH antagonists of Escherichia coli adhesion.

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Carbohydrate ofinterest

Fluorescent function coupling

Fluorescent dye Interacting

properties validation

+ +

Bioactive molecule

Protein ofinterest

Fluorescent screening of glyco-active compounds with GlycoFluo technology

Isabelle Bertin-Jung1, Anne Robert1, Sandrine Gulberti1, Chrystel Lopin-Bon2, Jean-Claude Jacquinet2, Sylvie Fournel-Gigleux1

1 UMR 7365 CNRS-Université de Lorraine - Ingénierie Moléculaire et Physiopathologie Articulaire

(IMoPA) - Groupe MolCelTEG (Molecular, Cellular, Therapeutic Engineering & Glycosyltransferases) - Biopôle de l'Université de Lorraine - 54505 Vandoeuvre-lès-Nancy, France

2 UMR CNRS 7311 CNRS - Université d’Orléans - Institut de Chimie Organique et Analytique (ICOA) – Equipe Glycochimie et protéoglycanes - Rue de Chartres - 45067 Orléans

GlycoFluo is a new generation of fluorescence-based screening technology for the identification of bioactive compounds targeting interactions between proteins and carbohydrates. The involvement of protein-carbohydrate interactions has been indeed described in many pathophysiological situations, like tumor cell adhesion, cell migration or host-pathogen recognition. The development of therapeutics targeting these interactions has lagged behind due, at least in part, to the lack of convenient screening technologies. The GlycoFluo technology uses a fluorescent probe (a carbohydrate labeled with N-methylanthranilate, N-MANT) which specifically interacts with the target protein (Figure 1). This interaction leads to a modification in the probe fluorescence spectrum properties, which can be measured by direct fluorescence, anisotropy or FRET (Förster / fluorescence Resonance Energy Transfer).

Figure 1 : Principle of GlycoFluo technology

GlycoFluo represents a low cost, rapid and effective technology, proposing an original alternative for identifying bioactive molecules targeting carbohydrate-protein interactions. Current available services: (i) Available on request: feasibility study of functional analysis of protein-carbohydrate interactions, including the development of specific fluorescent ligands targeting the proteins of interest (in collaboration with our partners which are specialists of chemical synthesis of oligosaccharides-ICOA, Orléans, France); (ii) Fluorescence-based screening of glycoactive compounds and hit selection; (iii) In vitro functional characterization of ligands targeting the protein of interest (Kd and Ki determination) Latest development of the “Nancy” glycobiology platform: The valorization of GlycoFluo technology is being realized within an integrated platform dedicated to glycobiology. This platform will propose innovative services in glycobiology, including glycoactive compounds screening and/or glycan analysis and glycosyltransferase assays in vitro and in cellulo. The new patented and original GlycoFluo technology represents an added-value within this platform in terms of carbohydrate-protein interaction studies and in cellulo analysis of biological effects of bioactive compounds. Latest services of the “Nancy” glycobiology platform: (i) In cellulo GAG anabolism evaluation, (ii) Disaccharidic analysis of neo-synthesized GAG chains, (iii) Glycosyltransferase assays and kinetics.

This work has been supported by Région Lorraine, Fédération de Recherche FR3209 BMCT-CNRS Université de Lorraine and SATT Grand Est. Patent: Fournel-Gigleux S, Gulberti S, Bertin-Jung I, Ramalanjaona N, Lopin-Bon C, Jacquinet JC, Ouzzine M. Conjugués glucidiques fluorescents, leur procédé de préparation et leurs utilisations. Pat2503562fr00. 2014

Chemical synthesis of multivalent chemical probes and their study as modulators of multivalent

glycan-protein interactions U. Alali,1 M. Taouai,1 S. Kravchenko,1 C. Epoune,1 D. Arosio,2 A. Bernardi,3 A. Siriwardena1

1 Le Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources (LG2A) UMR 7378 CNRS,33 Rue St Leu, 30083 Amiens, France

2 CNR-ISTM MI, c/o Dipartimento di Chimica, Università di Milano, Via C. Golgi,19, 20133Milano, Italy 3 Universita' degli Studi di Milano, Dipartimento di Chimica, via Golgi 19, 20133 Milano, Italy

We have recently demonstrated that simple monosaccharides conjugated to nanodiamond particles are not only stable to the action of glycosyl hydrolases but inhibit these enzymes [1]. The present work seeks to investigate the behaviour of glyco-gold nanoparticles (GNPs, Fig1) towards the hydrolytic action of these same enzymes in the hope of shedding further light on the unexpected activity of multivalently glycostructures as glycosidase inhibitors.

. Figure1. The monosaccharide-grafted gold nanoparticles targeted in the present work

The required glyco-GNPs have been obtained using the biofunctionalised linker, SAc-TEG-Undecene-N3, synthesised adapting a previously developed method [2]. A “click” coupling has been exploited for the conjugation of propargyl-functionalised monosaccharides to the linker. The target glyco-GNPs were obtained using the classical route [3]. References: [1] A. Siriwardena, et al, RSC Adv., 2015, 5, 100568 [2] A. Barrientos, et al., Chem. Eur. J., 2003, 9, 1909 [3] B. V. Enustun and J. Turkevich, J. Am. Chem. Soc., 1963, 85, 3317. �

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Infrared Multiple Photon Dissociation Spectroscopy: a new powerful technique

for structural characterization of carbohydrates

Baptiste Schindler 1, Loic Barnes 1, Abdul-Rahman Allouche 1, Stéphane Chambert 2 & Isabelle Compagnon 1,3

1 Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne, France; Institut Lumière Matière, UMR5306 Université Lyon 1-CNRS; Université de Lyon 69622 Villeurbanne Cedex, France.

2 Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne, France. Laboratoire de Chimie Organique et Bioorganique, INSA Lyon, CNRS, UMR5246, ICBMS,

Bât. J. Verne, 20 Avenue A. Einstein, 69621 Villeurbanne Cedex, France 3 Institut Universitaire de France IUF, 103 Blvd St Michel, 75005 Paris, France

We have built an instrument coupling mass spectrometry and vibrational spectroscopy (IRMPD), dedicated to the structural characterization of carbohydrates. We present the molecular fingerprint obtained by IR spectroscopy as an universal metric to resolve carbohydrate isomerisms, whereas previously reported hyphenated methods yielded partial structural information.

With the combination of mass spectrometry sensitivity and spectroscopic structural resolution, our method requires typical MS conditions that is small amount of sample, minimal chemical purification and applies to underivatized analytes, which represents a major breakthrough in high-throughput analysis of natural carbohydrates.

Using this metric, we can resolve all structural information of underivatized carbohydrates, including position of functional modifications (sulfate in HS/CS disaccharides), monosaccharide content (in particular the nature of uronic acid in glycosaminoglycans), regiochemistry and stereochemistry of the glycosidic linkages.

Angiogenesis in hypoxic conditions: implication of the syndecan-4 ectodomain shedding

Amena Butt1, Hanna Hlawaty1, Oualid Haddad1, Erwan Guyot1,2, Christelle Laguillier-Morizot1,2, Carole Planès3,4, Olivier Oudar1, Nathalie Charnaux1,2, Angela Sutton1,2.

1 INSERM U1148, UFR SMBH, Université Paris 13, PRES Paris Sorbonne Cité, Bobigny, France 2 Service de Biochimie, Hôpital Jean Verdier, APHP, Bondy, France

3 EA2363, UFR SMBH, Université Paris 13, PRES Paris Sorbonne Cité, Bobigny, France 4Explorations fonctionnelles, Hôpital Avicenne, APHP, Bobigny, France

The atheroma plaques induce vessel obstruction, leading to a reduced blood flow and a local hypoxia. To counteract this hypoxia, the formation of new blood vessels from pre-existing ones takes place, this phenomenon being called angiogenesis. Our laboratory demonstrates that syndecan-4, a heparan sulfate chains proteoglycan, exerts a pro-angiogenic effect [1]. High levels of syndecan-4 in the serum of patients with cardiovascular diseases has been observed, suggesting a syndecan-4 ectodomain shedding whose role is still undetermined [2]. The purpose of our work is to assess whether hypoxia induces the shedding of syndecan-4 and to evaluate the role of the shedded ectodomain in the formation of vascular networks in vitro. The human umbilical vein endothelial cells (HUVECs) are placed under hypoxia for 24 hours in 1% O2 or under normoxia in 21% O2. The expression of syndecan-4 is evaluated by qRT-PCR, flow cytometry and western blot. Then, the formation of vascular networks is studied on a 2D matrigel angiogenesis assay and the syndecan-4 ectodomain shedding is measured by dot blot using the cell conditioned media. The expression of metalloproteinases is studied by qRT-PCR and their activity by zymography. The conditionned media of endothelial cells placed in hypoxia promotes the formation of vascular networks. Our results demonstrate that hypoxia induces an increase in gene and protein expression of syndecan-4, as well as its ectodomain shedding. Furthermore, hypoxia induced the over-expression of MMP2, which could be responsible for syndecan-4 ectodomain shedding. Our results suggest the involvement of syndecan-4 ectodomain shedding in the vascular networks formation under hypoxia that could be induced by the MMP2. Ultimately, this issue will increase our knowledge of the mechanisms involved in angiogenesis and favor to consider new therapeutic strategies for cardiovascular disease.

Figure 1: The implication of syndecan-4 shedding in the formation of vascular networks References : [1] L. Maillard, N. Saito, H. Hlawaty, V. Friand, N. Suffee, F. Chmilewsky, O. Haddad, C. Laguillier, E.

Guyot, T. Ueyama, O. Oudar, A. Sutton, and N. Charnaux, “RANTES/CCL5 mediated-biological effects depend on the syndecan-4/PKCα signaling pathway.,” Biol. Open, 3(10), 995-1004, 2014.

[2] T. Kojima, A. Takagi, M. Maeda, T. Segawa, A. Shimizu, K. Yamamoto, T. Matsushita, and H. Saito, “Plasma Levels of Syndecan-4 (Ryudocan) Are Elevated in Patients with Acute Myocardial Infarction,” Thromb. Haemost., 85(5), 793-799, 2001.

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Expression of OGT correlates with migration and proliferation of colon cell lines

Agata Steenackers1, Vanessa Dehennaut1, Stéphanie Olivier-Van Stichelen1, Tony Lefebvre1 & Ikram El Yazidi-Belkoura1

1CNRS/UMR 8576, Unit of Structural and Functional Glycobiology (UGSF), Lille 1 Université, Villeneuve d’Ascq, France

The O-GlcNAc transferase (OGT) is a key regulator of the post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) onto Ser/Thr residues. OGT uses the end product of the hexosamine biosynthetic pathway (HBP), UDP-GlcNAc, as a donor for O-GlcNAcylation processes. It is reported that OGT and O-GlcNAcylation levels are increased in cancers. We showed that in the colorectal cancers (CRC) cell lines (HT29, HCT116) the expression of OGT and O-GlcNAcylation level were elevated, and that O-GlcNAcylation directly interfered with β-catenin stability and proliferation of cells. Previous studies showed that oncogenic factors such as p53, MYC or β-catenin are O-GlcNAcylated. The Wnt/β-catenin pathway is modified in most CRC by genetic alteration of β-catenin or one member of the destruction complex. Consequently, β-catenin is protected from proteasomal degradation and therefore induces cell proliferation. A similar observation was made when HBP flux was increased by culturing cells in high glucose medium. In these conditions, β-catenin was protected against the degradation thus accelerating cell proliferation. In a recent study, we identified four O-GlcNAcylation sites at the N-terminus of β-catenin, one of those (T41) localized in the destruction box is crucial for the control of β-catenin degradation. In that context we studied the effect of OGT silencing in CRC cell lines and non-cancerous cells CCD841CoN. We reported that silencing of OGT halved proliferative and migratory capacities of cancer cells. OGT knock-down also diminished cell adhesion corroborating previous observations that inhibiting O-GlcNAcylation decreases β-catenin/α-catenin interactions necessary for mucosa integrity, which suggests that O-GlcNAcylation also affects localization of β-catenin at adherens junction level.

Impact of sialic acids on the molecular dynamic of bi-antennary and tri-antennary glycans

Alexandre Guillot1, Manuel Dauchez1,2, Nicolas Belloy1,2, Jessica Jonquet1,2,

Laurent Duca1, Béatrice Romier1, Pascal Maurice1, Laurent Debelle1, Laurent Martiny1, Vincent Durlach1,3, Sébastien Blaise1, Stéphanie Baud1,2

1 Université de Reims Champagne-Ardenne, UMR CNRS 7369,

Matrice extracellulaire et Dynamique Cellulaire, Reims 2 Plateau de Modélisation Moléculaire Multi-échelle, Reims

3 Pôle Thoracique-Cardio-Neuro-Vasculaire, CHU de Reims Sialic acids (SA) are monosaccharides that can be located at the terminal position of glycan chains on a wide range of proteins [1]. The post-translational modifications, such as N-glycan chains, are fundamental to protein functions. Indeed, the hydrolysis of SA by specific enzymes such as neuraminidases can lead to drastic modifications of protein behaviour [2] [3]. However, the relationship between desialylation of N-glycan chains and possible alterations of receptor function remains unexplored. Thus, we aimed to establish the impact of SA removal from N-glycan chains on their conformational behaviour. We therefore undertook an in silico investigation using molecular dynamics to predict the structure of an isolated glycan chain. We performed, for the first time, 500 ns simulations on bi-antennary and tri-antennary glycan chains displaying or lacking SA. We showed that desialylation alters both the preferential conformation and the flexibility of the glycan chain. We also developed an original visualization method allowing to estimate the covered area provided by the glycan on proteins. With this tool, we showed that the removal of SA causes modifications of the protein surface protected by the glycan. These results suggest that the dynamic of glycan chains induced by presence or absence of SA may explain the changes in the protein function.

Figure: monofucosylated disialylated bi-antennary glycan chain References: [1] Schauer, R. Sialic acids as regulators of molecular and cellular interactions. Current Opinion in

Structural Biology 19, 507–514 (2009). [2] Hinek, A., Bodnaruk, T. D., Bunda, S., Wang, Y. & Liu, K. Neuraminidase-1, a subunit of the cell

surface elastin receptor, desialylates and functionally inactivates adjacent receptors interacting with the mitogenic growth factors PDGF-BB and IGF-2. Am. J. Pathol. 173, 1042–1056 (2008)

[3] Blaise, S. et al. Elastin-Derived Peptides Are New Regulators of Insulin Resistance Development in Mice. Diabetes 62, 3807–3816 (2013)

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The MCM2-7 helicase complex is glycosylated by O-GlcNAc Transferase. Towards a new role of OGT in the

regulation of DNA replication

Maïté Leturcq, Marlène Mortuaire, Tony Lefebvre, Anne-Sophie Vercoutter-Edouart

Unité de Glycobiologie Structurale et Fonctionnelle, UGSF, Univ. Lille, CNRS, UMR 8576, Lille, France

O-GlcNAcylation (O-linked N-acetylglucosaminylation) is a dynamic and reversible post-translational modification regulated by OGT (O-GlcNAc Transferase) and OGA (O-GlcNAcase). This glycosylation consists in the addition of a single residue of β-D-N-acetylglucosamine (GlcNAc) to the hydroxyl group of serine and threonine residues of cytosolic, nuclear and mitochondrial proteins and can compete with phosphorylation to regulate the activity of target-proteins [1]. Several works, including those of our lab, showed that a disruption of the dynamic of O-GlcNAcylation affects mitotic events and cellular division. In addition, overexpression of OGT and increase of its activity contribute to tumorigenesis by promoting growth and invasion of cancer cells, both in vitro and in vivo [2]. We previously described for the first time the cell cycle-dependent O-GlcNAcylation of the Mini-Chromosome Maintenance Proteins MCM2, MCM3, MCM6 and MCM7 which are key proteins involved in the formation of the pre-replicative complex [3]. The aim of our work is now to understand the role of O-GlcNAcylation and OGT on the formation of the MCM2-7 complex and its recruitment to the chromatin. By WGA affinity chromatography and click-chemistry approaches, we showed that the O-GlcNAcylated forms of MCM are mainly detected in the chromatin-bound protein fraction. Co-immunoprecipitation and GST pull-down experiments further showed that OGT preferentially interacts with some of the MCM proteins. Finally, we are currently investigating the crosstalk between phosphorylation and O-GlcNAcylation of the MCM proteins by using two-dimensional electrophoresis and western-blotting combined with Click-chemistry strategy. This study will bring new elements to understand the role of OGT and O-GlcNAc modification in the molecular mechanisms involved in the initiation of DNA synthesis. The question remains whether a pathological dysregulation of O-GlcNAc status of the MCM2-7 complex could disrupt the control of the initiation of the genome replication and thus contribute to the uncontrolled proliferation of cancerous cells. References: [1] Lefebvre T, Issad T. (2015) 30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of

Cell Signaling and Metabolism by O-GlcNAcylation. Front Endocrinol (Lausanne), 9;6:17 [2] Ma Z, Vosseller K. (2014) Cancer metabolism and elevated O-GlcNAc in oncogenic signaling. J

Biol Chem. 289(50):34457-65. [3] Drougat L, Olivier-Van Stichelen S, Mortuaire M, Foulquier F, Lacoste AS, Michalski JC, Lefebvre

T, Vercoutter-Edouart AS. (2012) Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition. Biochim Biophys Acta, 1820(12):1839-48.

Iminosugar-Based Galactoside Mimics as Pharmacological Chaperones for Lysosomal β-Galactosidases

Estelle Gallienne 1, Anna Biela-Banas 1, Sophie Front 1 & Olivier R. Martin 1

1 ICOA, Université d’Orléans & CNRS, Orléans, France

As part of our research program dedicated to the design of new iminosugars as therapeutic agents for lysosomal storage disorders (LSD), we synthesized iminosugar-based galactoside mimics as pharmacological chaperones (PCs) for lysosomal β-galactosidases, responsible for GM1-gangliosidosis, Morquio B and Krabbe diseases. Pharmacological chaperone therapy is a new and innovative strategy, which consists in the administration at very low concentrations of small molecules having strong interactions with the enzyme. These compounds, which are paradoxically potent inhibitors of the glycosidase involved, help stabilize the mutant protein and save it from degradation.[1] This results in an increased enzymatic activity in the lysosome and a concomitant decrease of the symptoms.

Given our very promising results in the design of 1-C-alkyl imino-D-xylitols as PCs for β-glucocerebrosidase,[2] the enzyme involved in Gaucher disease, we turned our efforts to the synthesis of 1-C-alkyl imino-L-arabinitols such as 1,[3] which were found to be deprived of inhibitory activity against the three tested lysosomal galactosidases.[4] The challenging synthesis of 1-C-alkyl imino-D-galactitols of type 2 [5] was then investigated leading to potent inhibitors of lysosomal α-galactosidase A, responsible for Fabry disease.[4] As 1-N-iminosugars are known to preferably inhibit β-glycosidases, 4-epi-isofagomine 3 was synthesized and evaluated towards lysosomal galactosidases. It was found to be a potent inhibitor of lysosomal β-galactosidase and the first iminosugar reported to inhibit the β-galactocerebrosidase, the enzyme involved in Krabbe disease, a devastating neurological LSD.[4] In order to test the influence of an alkyl chain on the inhibitory properties, 1-C- and 5-C-alkyl-iminoribitols 4a and 4b, as simplified alkylated analogs of 3, were synthesized and tested against various galactosidases.[6] Results of this evaluation and further perspectives will be described in this communication.

4bGalactoside 4a31 2

( )4 ( )4

Figure 1: Iminosugar-based galactoside mimics

References: [1] J.-Q. Fan, Biol. Chem. 2008, 389, 1-11. [2] W. Schönemann, E. Gallienne, K. Ikeda-Obatake, N. Asano, S. Nakagawa, A. Kato, I. Adachi,

M. Górecki, J. Frelek, O.R. Martin, ChemMedChem 2013, 8, 1805-1817. [3] A. Biela, F. Oulaïdi, E. Gallienne, M. Górecki, J. Frelek, O. R. Martin, Tetrahedron 2013, 69,

3348-3354. [4] A. Biela-Banaś, F. Oulaïdi, S. Front, E. Gallienne, K. Ikeda-Obatake, N. Asano, D. A. Wenger,

O.R. Martin, ChemMedChem 2014, 9, 2647-2652. [5] A. Biela-Banaś, E. Gallienne, S. Front, O. R. Martin, Tetrahedron Lett. 2014, 55, 838-841. [6] S. Front, E. Gallienne, J. Charollais-Thoenig, S. Demotz, O.R. Martin, ChemMedChem 2016, 11,

133-141.

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Anti-Metastatic Properties of a Marine Bacterial Exopolysaccharide-Based Derivative Designed

to Mimic Glycosaminoglycans Dominique Heymann 1, Carmen Ruiz-Velasco 1, Julie Chesneau 1, Jacqueline Ratiskol 2, Corinne Sinquin 2 & Sylvia Colliec-Jouault 2

1 INSERM, UMR 957, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Equipe Ligue Contre le Cancer 2012, Nantes 44035, France

2 IFREMER, Laboratoire EM3B Ecosystèmes Microbiens et Molécules Marines pour les Biotechnologies, Centre Atlantique, BP21105, Nantes 44311, France

Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases.

In this study, the effect of three oversulfated low molecular weight marine bacterial exopolysaccharides (OS-EPS) with different molecular weights (4, 8 and 15 kDa) were first evaluated in vitro on human and murine osteosarcoma cell lines. Different biological activities were studied: cell proliferation, cell adhesion and migration, matrix metalloproteinase expression. This in vitro study showed that only the OS-EPS 15 kDa derivative could inhibit the invasiveness of osteosarcoma cells with an inhibition rate close to 90%. Moreover, this derivative was potent to inhibit both migration and invasiveness of osteosarcoma cell lines; had no significant effect on their cell cycle; and increased slightly the expression of MMP-9, and more highly the expression of its physiological specific tissue inhibitor TIMP-1. Then, the in vivo experiments showed that the OS-EPS 15 kDa derivative had no effect on the primary osteosarcoma tumor induced by osteosarcoma cell lines but was very efficient to inhibit the establishment of lung metastases in vivo.

These results can help to better understand the mechanisms of GAGs and GAG-like derivatives in the biology of the tumor cells and their interactions with the bone environment to develop new therapeutic strategies.

Figure 1 : Effect of OS-EPS 15 kDa derivative on the lung

metastatic incidence: (A) metastatic incidence in treated animals (OS-EPS derivative or heparin; s.c. 6 mg/kg daily) vs.

control; (B) histological analyses of the lung tissue of treated

animals or not (* metastatic foci) and (C) survival rate (%) of

treated animals (OS-EPS derivative or heparin) vs.

control. ** p < 0.01.

Deciphering the complex alginolytic system of the marine bacterium Zobellia galactanivorans

François Thomas 1, Robert Larocque 2, Yongtao Zhu 3, Mark J. McBride 3, Tristan Barbeyron 1, Mirjam Czjzek 1 & Gurvan Michel 1

1 Sorbonne Université, UPMC, CNRS, UMR 8227, Integrative Biology of Marine Models,

Station Biologique de Roscoff, Roscoff, France 2 FR2424 CNRS, Station Biologique de Roscoff, Roscoff, France

3 Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, USA The marine flavobacterium Zobellia galactanivorans is able to degrade a great variety of polysaccharides from marine macroalgae, and to use them as carbon and energy sources. In this study, we have focused on the degradation of alginate. This main cell wall polysaccharide from brown algae consists of β-D-mannuronate (M) and α-L-guluronate (G) monomers and is widely used as gelling agent in industrial applications. Z. galactanivorans possesses a complex alginolytic system, comprising 12 degradation enzymes, three import proteins and a regulation factor. The majority of these proteins are encoded within two operons conserved in other heterotrophic bacteria and their expression is induced by the presence of alginate [1]. Z. galactanivorans possesses notably seven alginate lyases (2 PL6, 3 PL7, 1 PL14 and 1 PL17), questioning their potential redundancy or synergistic action. To elucidate their functions, we therefore cloned the seven genes and obtained recombinant proteins for biochemical and structural characterization. In-depth study of the alginate lyases AlyA1 and AlyA5 showed that although belonging to the same PL7 family, they displayed drastically different modes of action. Namely, AlyA1 is an endolytic guluronate lyase, whereas AlyA5 cleaves monomers from the non-reducing end of oligo-alginates in an exolytic fashion. Crystal structures revealed a common jelly-roll fold for the two enzymes. However, additional loops in AlyA5 obstruct the cleft and create a pocket topology contrasting with that of AlyA1, thus explaining the different modes of action [2]. Genetic tools have been recently developed for Z. galactanivorans in collaboration with Dr McBride’s group and, to further decipher the biological role of AlyA1, a first deletion mutant (ΔalyA1) has been obtained. Compared to the wild type strain, ΔalyA1 showed a strong growth delay on gelified alginate and an impaired liquefaction efficiency. Interestingly, among the seven alginate lyases in the system, AlyA1 was the only one containing a carbohydrate binding module (CBM) and found secreted in the medium. Therefore, it might be a crucial enzyme to initiate the degradation pathway of alginate when present in the context of an algal cell wall. Characterization of the other alginate lyases is underway and will help decipher this complex catabolic system. References : [1] Thomas, F. et al. Characterization of the first alginolytic operons in a marine bacterium: from their

emergence in marine Flavobacteriia to their independent transfers to marine Proteobacteria and human gut Bacteroides. Environ. Microbiol. 14, 2379-94 (2012).

[2] Thomas, F. et al. Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct modes of action and exquisite adaptation to their natural substrate. J. Biol. Chem. 288, 23021-37 (2013).

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B3GALT6 mutations causes a pleiotropic form of Ehlers-Danlos syndrome (EDS) due defects in glycosaminoglycan biosynthesis

Xiaomeng Pang1, Anne Robert1, Isabelle Bertin-Jung1, Tim Van Damme2, Fransiska Malfait2, Sandrine Gulberti1, Sylvie Fournel-Gigleux1

1 UMR 7365 CNRS-Univ. Lorraine, MolCelTEG group, Biopôle de l'Université de Lorraine, 54505 Vandoeuvre-lès-Nancy, France ; 2 Center for Medical Genetics, Ghent Univ. Hospital, De Pintelaan 185, 9000 Gent, Belgium

Introduction: Proteoglycans (PGs) are major components of cell plasma membranes and extracellular matrices (ECM). These complex macromolecules play important pathophysiological roles in the organization of ECM of connective tissues as well as in cell signaling and embryonic and post-natal development. PGs are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein through a tetrasaccharide linkage [Glucuronic acid-β1,3-Galactose-β1,3-Galactose-β1,4-Xylose-β1-O-]. The addition of the third residue (galactose) is catalyzed by β1,3-galactosyltransferase 6 (β3GalT6), a key glycosyltransferase in GAG initiation [1]. Recently, mutations of β3GalT6 have been associated to a pleiotropic form of Ehlers-Danlos Syndrome (EDS), a severe connective tissue disorder characterized by skin and bone fragility, musculoskeletal malformations, delayed wound healing, joint hyperlaxity and intellectual disabilities [2; 3]. Objective and strategy: The objective of this work is to understand the consequences of β3GalT6 defects in the development of EDS clinical symptoms, starting with evaluation of GAG anabolism and cell migration in patient dermal fibroblasts. β3GalT6 gain- and loss-of-function studies have been conducted to better decipher the implication of this galactosyltransferase in EDS pathophysiology. Experimental methods: GAG anabolism has been determined by quantifying incorporation of a radiolabelled precursor (35S) in neosynthesized GAG chains in patient fibroblasts or control cells. Fibroblast migration has been evaluated by wound healing tests. Over expression of β3GalT6 in defective cells has been carried out by electroporation of B3GALT6 cDNA in patient fibroblasts. The evaluation of GAG anabolism and cell migration in the genetically modified cells has been performed as described above. Main results: β3GalT6 defective fibroblasts exhibited a marked reduction in GAG anabolism. An impaired glycanation of decorin core protein was also observed in patient cells, confirming that the GAG defect is due to β3GalT6 loss of function. In vitro wound healing tests revealed a significant delay in defective fibroblast migration compared to control cells, which possibly explains some phenotypic aspects of the disease, such as defective wound closure in relation to GAG defect. The impact of mutations (point mutations, deletions) will be discussed in terms of GAG anabolism and cell migration, in attempt to establish a possible correlation between β3GalT6 loss of function in patient cells and the severity of clinical symptoms. Overexpression of wild-type β3GalT6 has been conducted in patient defective cells. Enzyme expression is stable up to 72h after electroporation in genetically modified cells. Interestingly, GAG anabolism and cell migration were partially restored (around 30%) when β3GalT6 is overexpressed in patient fibroblasts, which could be the starting point to the development of therapeutic strategies including gene therapy and enzyme replacement therapy. Conclusions: This work has shown that β3GalT6 is a key glycosyltransferase in GAG initiation. Mutations of this galactosyltransferase are responsible for a unique combination of severe generalized symptoms in EDS, characterized by important connective tissue disorders. This work provides a better understanding of the crucial role of β3GalT6 in EDS pathophysiological process, more precisely, in terms of GAG anabolism and cell migration. AMSEDgenetique Association is gratefully acknowledged for its financial support to our research and Valérie Gisclard for her constant support. References: [1] X. Bai et al., 2001, J. Biol. Chem., 276, 48189-48195 [2] F. Malfait, et al., 2013, Am. J. Hum. Genet., 92, 935-945 [3] M. Nakajima et al., 2013, Am. J. Hum. Genet., 92, 927–934

Structural and Functional Studies of a Trehalulose Hydrolase MutA from Rhizobium sp.

Alexandra Lipski1, Sébastien Violot1, Hildegard Watzlawick2, Richard Haser1,

Ralf Mattes2 & Nushin Aghajari1

1 Laboratory for Biocrystallography and Structural Biology of Therapeutic Targets, Molecular Microbiology and Structural Biochemistry, CNRS and University of Lyon 1, UMR 5086,

7 passage du Vercors, F-69367 Lyon Cedex 07, France 2 Universität Stuttgart, Institut für Industrielle Genetik, Allmandring 31, D-70569 Stuttgart, Germany

Trehalulose (α-D-glucopyranosyl-1,1-D-fructose) is one of the natural occurring isomers of sucrose (α-D-glucopyranosyl-1,2-β-D-fructofuranoside) and is found in honey and sugar cane in small amount. This disaccharide possesses physical and organoleptic properties similar to sucrose. The production of trehalulose by Rhizobium sp. from sucrose is catalyzed by sucrose isomerases (SI), namely by the trehalulose synthase (TS), MutB. Recently, an adjacent TS homologous gene, mutA, encoding a hydrolytic enzyme was identified (Watzlawick & Mattes, 2009). This enzyme catalyzes the hydrolysis of trehalulose, isomaltulose (α-D-glucopyranosyl-1,6-D-fructose), and sucrose into glucose and fructose, with a highest activity on trehalulose. The enzyme MutA belongs to the GH13 family and possesses the common characteristics found in this family. The genes mutA and mutB are putatively responsible for the uptake and utilization of trehalulose and isomaltulose in the bacterium and could possibly be involved in transcriptional regulation. Gene regulation is essential for prokaryotes to increase versatility and adaptability regulation of transcription, an issue necessary for the cell to adapt rapidly to the changes in the outer environment as eg. stress, and the availability of nutrients. We have crystallized and solved the three-dimensional structure of MutA to 2.5 Å resolution, and functional studies have been carried out as well for this enzyme. Comparative studies with SIs SmuA (isomaltulose synthase) (Ravaud et al., 2009) and MutB (trehalulose synthase) (Ravaud et al., 2007), with Saccharomyces cerevisiae isomaltase (Yamamoto et al., 2010; 2012) and with Bacillus cereus oligo-1,6-glucosidase OGL (Watanabe et al., 1997) have been performed and show overall structural similarity to these enzymes. The atypical form of the active site of MutA seems to be important for substrate hydrolysis and minor transferase activity. Based on primary- and tertiary structure analysis, four active (P232F, F266A, F290A and F266A-F290A) and two inactive mutants (D210N and E264Q) were generated in order to gain a better understanding of the structure/function/activity relationships of these enzymes. MutA is the first enzyme described as being able to hydrolyse trehalulose, and could potentially find its use in industry, e.g. in the treatment of sticky cotton fibers in textile factories. This work was supported by the CNRS (PhD scholarship of AL) and by the University of Lyon1. We acknowledge access to beamlines at the European Synchrotron Radiation facility (ESRF, Grenoble, France) and at Swiss Light Source (SLS, Paul Sherrer Institute, Switzerland) and the excellent support by the beamline scientists.

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Liste des posters

P-01 Marie Couturier, David Navarro, Didier Chevret, Bernard Henrissat, François Piumi, Francisco Ruiz-Dueñas, Angel Martinez, Igor Grigoriev, Robert Riley, Anna Lipzen,

Jean-Guy Berrin, Emma R Master & Marie-Noëlle Rosso : Degradation of wood by the Carbohydrate-Active Enzyme set of the fungus Pycnoporus coccineus ......................... 33

P-02 Marie Schuler, Stéphanie Marquès, Domenico Romano, Maria Domingues & Arnaud Tatibouët : From Carbohydrate-Based Thioimidate N-Oxides to Iminosugars Derivatives .............................................................................................................................................................................................................................................................................................. 33

P-03 Arnaud Masselin, Stéphanie Pradeau, Sylvain Cottaz & Sébastien Fort : Enzym’n click synthesis of chitinoligosaccharide probes for plant biology .............................................. 34

P-04 Maxime Delos, François Foulquier, Charles Hellec, Fabrice Allain & Agnès Denys : Subcellular localization of heparan 3-OST2, 3A and 3B ........................................................... 34

P-05 Simon Ladevèze, Bernard Henrissat & Jean-Guy Berrin : Characterization of Fungal Lytic Polysaccharide MonoOxygenases ................................................................................... 35

P-06 B. Ayela, T. Poisson, X. Pannecoucke & C. Lopin-Bon : Total synthesis of modified oligosaccharides from the linkage region of proteoglycans as potential inhibitors or effectors of the enzyme CSGalNAcT-1 ............................................................................................................................................................................................................................................. 35

P-07 Yves Queneau, Elie Derrien, Catherine Pinel, Michèle Besson, Mohammed Ahmar, Emilie Martin-Sisteron, Guy Raffin & Philippe Marion : Catalytic aerobic oxidation of reducing sugars issued from softwood hemicellulose acid hydrolysis ......................................................................................................................................................................................... 36

P-08 Clément Delannoy, Yoann Rombouts, Sophie Groux-Degroote, Stephanie Holst, Bernadette Coddeville, Anne Harduin-Lepers, Manfred Wuhrer, Elisabeth Elass & Yann Guérardel : Deciphering the glycosylation changes occurring during the differentiation and the activation of monocytic THP-1 cell line into macrophages .................................... 36

P-09 Barbara Guyez, Franck Moncassin, Claire Raingeval, Sophie Bozonnet, Bernard Henrissat, Lionel Mourey, Michael O’Donohue, Samuel Tranier & Claire Dumon : Structural and functional characterization of a hypothetical new glycoside hydrolase ............................................................................................................................................................. 37

P-10 Maxime Mock-Joubert, Christine Le Narvor, David Bonnaffé & Romain Vivès : Synthesis of putative inhibitors for the human endosulfatase, H-sulf 2, a new therapeutic target in cancer and inflammatory diseases ..................................................................................................................................................................................................................................... 37

P-11 Hélène Ledru & Chrystel Lopin-Bon : Preparation of various sulfoforms of oligosaccharides for the study of proteoglycans biosynthesis .................................................................. 38

P-12 Silvia Achilli, Blanka Didak, Corinne Vivès, Michel Thépaut, Ludovic Landemarre & Franck Fieschi : C-type lectins receptors (CLRs) arrays to screen immunocompatibility and reactivity of biological sample ...................................................................................................................................................................................................................................................... 38

P-13 Alexandre Méry, Lin Shen, Albertus Viljoen, Sydney Villaume, Christophe Mariller, Stéphane Vincent, Laurent Kremer & Yann Guérardel : Le catabolisme de la paroi mycobactérienne ................................................................................................................................................................................................................................................................................... 39

P-14 Maria Dominguès, Marie Schuler, Justyna Jaszczyk, Pierre Lafite, Richard Daniellou, Isabel Ismael & Arnaud Tatibouët : OZO derived iminosugars. The one-pot Retro-Michael/Michael addition solution ....................................................................................................................................................................................................................................................... 39

P-15 Anne-Sophie Vercoutter-Edouart, Olivier Massinon, Marlène Mortuaire, Maïté Leturcq, Tony Lefebvre & Stéphane Vincent : Synthesis of glycosides restrained in a 1,4B boat conformation. Impact on the in vitro activity of the β-N-acetylhexosaminidase C (O-GlcNAcase) and the intracellular O-GlcNAcylation levels in human cell lines .............. 40

P-16 Charles Hellec, Agnès Denys, Maxime Delos, Mathieu Carpentier & Fabrice Allain : Role of heparan sulfate 3-O-sulfotransferases in cancer cell proliferation and survival .... 40

P-17 Blanka Didak, Alexiane Decout, Eric Duverger & Ludovic Landemarre : GLYcoPROFILE®: deciphering the sweet side of cells ................................................................................... 41

P-18 A. Bordes, N. Fontelle, J. Désiré, F. Lecornué, J. Guillard & Y. Blériot : Iminosugars-based macrocycles to deliver new sweet azacrowns ............................................................... 41

P-19 Irina Sadovskaya, Evgeny Vinogradov, Anneleen Cornelissen, Thierry Grard, Stéphanie Blangy, Silvia Spinelli, Eoghan Casey, Jennifer Mahony, Jean-Paul Noben, Fabio Dal Bello, Christian Cambillau & Douwe van Sinderen : Structural investigation of cell surface assossiated polysaccharides of Lactobacillus delbrueckii subsp. bulgaricus 17 as potential substrates for bacteriophage Ld17 glycerophosphodiesterase ......................................................................................................................................................................... 42

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P-20 Joana Rocha, Milène Nitenberg, Eric Maréchal, Agnès Girard-Egrot, Maryse Block & Christelle Breton : Structural aspects and membrane binding properties of MGD1, the major galactolipid synthase in plants .......................................................................................................................................................................................................................................... 42

P-21 Franck Daligault, Romain Irague, Benoît David, Diane Jouanneau, Mirjam Czjzek, Yves-Henri Sanejouand & Charles Tellier : Mutations within a water channel change the balance between transglycosylation and hydrolysis in Agarase ................................................................................................................................................................................................... 43

P-22 Marie Buchotte, Petra Hellwig, Franck Borel, Jean-Luc Ferrer, Myriam Seemann & Jean-Bernard Behr : Synthetic access to MecPP and analogues thereof using D-galactose as a chiral scaffold .............................................................................................................................................................................................................................................................. 43

P-23 Bernadette Coddeville, Yann Guérardel, Frédéric Krzewinski, Emmanuel Maes, Dounia Mouajjah, Olga Plechakova, Martine Ratajczak, Xavier Trivelli & Nao Yamakawa : PAGés platform : a tool for glycan analysis ...................................................................................................................................................................................................................................... 44

P-24 Eva-Maria Krammer, Emmanuel Maes, Nao Yamakawa, Jérôme De Ruyck, Gérard Vergoten, Stefan Oscarson, Mohamed Touaibia, René Roy & Julie Bouckaert : New nanomolar biphenyl C-mannopyranoside ligands reveal unprecedented binding modes in the FimH adhesin of Escherichia coli ................................................................................. 44

P-25 Isabelle Bertin-Jung, Anne Robert, Sandrine Gulberti, Chrystel Lopin-Bon, Jean-Claude Jacquinet, Sylvie Fournel-Gigleux : Fluorescent screening of glyco-active compounds with GlycoFluo technology ............................................................................................................................................................................................................................................. 45

P-26 U. Alali, M. Taouai, S. Kravchenko, C. Epoune, D. Arosio, A. Bernardi & A. Siriwardena : Chemical synthesis of multivalent chemical probes and their study as modulators of multivalent glycan-protein interactions .......................................................................................................................................................................................................................................... 45

P-27 Baptiste Schindler, Loic Barnes, Abdul-Rahman Allouche, Stéphane Chambert & Isabelle Compagnon : Infrared Multiple Photon Dissociation Spectroscopy: a new powerful technique for structural characterization of carbohydrates .......................................................................................................................................................................................... 46

P-28 Amena Butt, Hanna Hlawaty, Oualid Haddad, Erwan Guyot, Christelle Laguillier-Morizot, Carole Planès, Olivier Oudar, Nathalie Charnaux & Angela Sutton : Angiogenesis in hypoxic conditions: implication of the syndecan-4 ectodomain shedding ............................................................................................................................................................................... 46

P-29 Agata Steenackers, Vanessa Dehennaut, Stéphanie Olivier-Van Stichelen, Tony Lefebvre & Ikram El Yazidi-Belkoura : Expression of OGT correlates with migration and proliferation of colon cell lines ............................................................................................................................................................................................................................................................ 47

P-30 Alexandre Guillot, Manuel Dauchez, Nicolas Belloy, Jessica Jonquet, Laurent Duca, Béatrice Romier, Pascal Maurice, Laurent Debelle, Laurent Martiny, Vincent Durlach, Sébastien Blaise & Stéphanie Baud : Impact of sialic acids on the molecular dynamic of bi-antennary and tri-antennary glycans ................................................................................ 47

P-31 Maïté Leturcq, Marlène Mortuaire, Tony Lefebvre & Anne-Sophie Vercoutter-Edouart : The MCM2-7 helicase complex is glycosylated by O-GlcNAc Transferase. Towards a new role of OGT in the regulation of DNA replication ................................................................................................................................................................................................................. 48

P-32 Estelle Gallienne, Anna Biela-Banas, Sophie Front & Olivier R. Martin : Iminosugar-Based Galactoside Mimics as Pharmacological Chaperones for Lysosomal β-Galactosidases ....................................................................................................................................................................................................................................................................................... 48

P-33 Dominique Heymann, Carmen Ruiz-Velasco, Julie Chesneau, Jacqueline Ratiskol, Corinne Sinquin & Sylvia Colliec-Jouault : Anti-Metastatic Properties of a Marine Bacterial Exopolysaccharide-Based Derivative Designed to Mimic Glycosaminoglycans ....................................................................................................................................................... 49

P-34 François Thomas, Robert Larocque, Yongtao Zhu, Mark J. McBride, Tristan Barbeyron, Mirjam Czjzek & Gurvan Michel : Deciphering the complex alginolytic system of the marine bacterium Zobellia galactanivorans ............................................................................................................................................................................................................................... 49

P-35 Xiaomeng Pang, Anne Robert, Isabelle Bertin-Jung, Tim Van Damme, Fransiska Malfait, Sandrine Gulberti & Sylvie Fournel-Gigleux : B3GALT6 mutations causes a pleiotropic form of Ehlers-Danlos syndrome (EDS) due defects in glycosaminoglycan biosynthesis ................................................................................................................................... 50

P-36 Alexandra Lipski, Sébastien Violot, Hildegard Watzlawick, Richard Haser, Ralf Mattes & Nushin Aghajari : Structural and Functional Studies of a Trehalulose Hydrolase MutA from Rhizobium sp. ..................................................................................................................................................................................................................................................................... 50

Les CO-01, 02, 06, 07, 09, 10 et 14 présenteront également leurs résultats sous forme de posters (P-37 à P-43).

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Liste des participants

Silvia ACHILLI IBS, UMR CEA-CNRS-UGA 5075 Grenoble silvia.achilli@ibs.fr Nushin AGHAJARI MMSB, UMR 5086 Lyon nushin.aghajari@ibcp.fr Urjwan ALALI LG2A, UMR 7378 Amiens awan7062@yahoo.com Fabrice ALLAIN UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq fabrice.allain@univ-lille1.fr Sylvie ARMAND Cermav, UPR CNRS 5301 Grenoble sylvie.armand@cermav.cnrs.fr Rachel AUZELY Cermav, UPR CNRS 5301 Grenoble rachel.auzely@cermav.cnrs.fr Benjamin AYELA ICOA, UMR 7311 Orléans ayela.benjamin@gmail.com Steffi BALDINI UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq steffi.baldini@gmail.com Muriel BARDOR Glyco-MEV, EA4358, UNIROUEN Rouen muriel.bardor@univ-rouen.fr Ludovic BASTIDE Elicityl Crolles Ludovic.bastide@elicityl.fr www.elicityl-oligotech.com

Stéphanie BAUD MEDyC, UMR 7369 Reims stephanie.baud@univ-reims.fr Jean-Bernard BEHR ICMR, UMR 7312 Reims jb.behr@univ-reims.fr Isabelle BERTIN-JUNG ImoPA, UMR 7365 Vandoeuvre-lès-Nancy Isabelle.bertin-jung@univ-lorraine.fr Yves BLERIOT IC2MP, UMR 7285 Poitiers yves.bleriot@univ-poitiers.fr Claire BOISSET Cermav, UPR CNRS 5301 Grenoble claire.boisset-helbert@cermav.cnrs.fr David BONNAFFÉ ICMMO, UMR 8182 Orsay david.bonnaffe@u-psud.fr Silvère BONNETElicityl Crolles silvere.bonnet@elicityl.fr www.elicityl-oligotech.com Véronique BONNET LG2A, UMR 7378 Amiens veronique.bonnet@u-picardie.fr Alexandra BORDES IC2MP, UMR 7285 Poitiers alexandra.bordes@univ-poitiers.fr Julie BOUCKAERT UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq julie.bouckaert@univ-lille1.fr

Nathalie BOURGOUGNON LBCM, Univ. Bretagne Sud Vannes nathalie.bourgougnon@univ-ubs.fr Christelle BRETON Cermav, UPR CNRS 5301 Grenoble christelle.breton@cermav.cnrs.fr Laurine BUON Cermav, UPR CNRS 5301 Grenoble laurine.buon@cermav.cnrs.fr Amena BUTT LVTS, Inserm, U1148 Bobigny amena.butt09@gmail.com Michèle CARRET Cermav, UPR CNRS 5301 Grenoble michele.carret@cermav.cnrs.fr Valérie CHAZALET Cermav, UPR CNRS 5301 Grenoble valerie.chazalet@cermav.cnrs.fr Luc CHEVRIERElicityl Crolles Luc.chevrier@elicityl.fr www.elicityl-oligotech.com Sylvia COLLIEC-JOUAULT Ifremer, Laboratoire EM3B Nantes sylvia.colliec.jouault@ifremer.fr Isabelle COMPAGNON Institut Lumière Matière, UMR 5306 Villeurbanne isabelle.compagnon@univ-lyon1.fr Marie COUTURIER BBF, UMR 1163 Marseille marie.couturier@univ-amu.fr

Giuliano CUTOLO ICOA, UMR 7311 Orléans giuliano.cutolo@etu.univ-orleans.fr Samir DAHBI ICOA, UMR 7311 Orléans samir.dahbi@univ-orleans.fr Franck DALIGAULT UFIP, UMR 6286 Nantes franck.daligault@univ-nantes.fr Richard DANIELLOU ICOA, UMR 7311 Orléans richard.daniellou@univ-orleans.fr Benoît DARBLADEElicityl Crolles benoit.darblade@elicityl.fr www.elicityl-oligotech.com Clément DELANNOY UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq cl-ment.delannoy@ed.univ-lille1.fr Philippe DELANNOY UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq philippe.delannoy@univ-lille1.fr Maxime DELOS UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq maxime.delos@hotmail.fr Agnès DENYS UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq agnes.denys@univ-lille1.fr Blanka DIDAK GLYcoDiag Orléans didak@glycodiag.com

Claire DUMON LISBP Toulouse claire.dumon@insa-toulouse.fr Ikram EL YAZIDI - BELKOURA UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq ikram.el-yazidi@univ-lille1.fr Régis FAURÉ LISBP Toulouse regis.faure@insa-toulouse.fr Vincent FERRIERES ENSCR, UMR 6226 Rennes vincent.ferrieres@ensc-rennes.fr Elizabeth FICKO-BLEAN Marine Glycobiology, UMR 8227 Roscoff efickoblean@sb-roscoff.fr Etienne FLEURY IMP INSA, UMR 5223 Lyon etienne.fleury@insa-lyon.fr Sébastien FORT Cermav, UPR CNRS 5301 Grenoble sebastien.fort@cermav.cnrs.fr François FOULQUIER UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq francois.foulquier@univ-lille1.fr Frederic FRISCOURT IECB/INCIA, UMR 5287 Pessac f.friscourt@iecb.u-bordeaux.fr Estelle GALLIENNE ICOA, UMR 7311 Orléans estelle.gallienne@univ-orleans.fr

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Emilie GILLON Cermav, UPR CNRS 5301 Grenoble emilie.gillon@cermav.cnrs.fr Sébastien GOUIN CEISAM, UMR 6230 Nantes sebastien.gouin@univ-nantes.fr Cyrille GRANDJEAN UFIP, UMR 6286 Nantes cyrille.grandjean@univ-nantes.fr Marcelo GUERIN CIC BioGUNE Derio, spain mrcguerin@gmail.com Alexandre GUILLOT MEDyC, UMR 7369 Reims alexandre.guillot@univ-reims.fr Sandrine GULBERTI ImoPA, UMR 7365 Vandoeuvre-lès-Nancy sandrine.gulberti@univ-lorraine.fr Barbara GUYEZ IPBS et LISBP Toulouse barbara.guyez@ipbs.fr William HELBERT Cermav, UPR CNRS 5301 Grenoble william.helbert@cermav.cnrs.fr Charles HELLEC UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq charles.hellec@gmail.com Thomas HURTAUX UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq thomas.hurtaux@etu.univ-lille2.fr Boutros KERBAJE LIBIOS Pontcharra Sur Turdine bkerbaje@libios.fr www.libios.fr

Simon LADEVEZE BBF, UMR 1163 Marseille simon.ladeveze@univ-amu.fr Hélène LEDRU ICOA, UMR 7311 Orléans helene.ledru@etu.univ-orleans.fr Tony LEFEBVRE UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq tony.lefebvre@univ-lille1.fr Loïc LEMIEGRE ENSCR, UMR 6226 Rennes loic.lemiegre@ensc-rennes.fr Jacques LE PENDU CRCNA, UMR 892 Inserm/6299 CNRS Nantes jacques.le-pendu@univ-nantes.fr Patrice LEROUGE Glyco-MEV, EA4358, UNIROUEN Rouen patrice.lerouge@univ-rouen.fr Maïté LETURCQ UGSF, UMR 8576 Villeneuve d’Ascq maite62150@hotmail.fr Chrystel LOPIN-BON ICOA, UMR 7311 Orléans chrystel.lopin-bon@univ-orleans.fr Emmanuel MAES UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq emmanuel.maes@univ-lille1.fr Arnaud MASSELIN Cermav, UPR CNRS 5301 Grenoble arnaud.masselin@cermav.cnrs.fr Alexandre MERY UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq alexandre.mery@yahoo.fr Jean-Claude MICHALSKI UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq Jean-Claude.michalski@univ-lille1.fr

Maxime MOCK-JOUBERT ICMMO, UMR 8182 Orsay maxime.mock@gmail.com Solange MORERA I2BC, UMR 9198 Gif sur Yvette solange.morera@i2bc.paris-saclay.fr Dounia MOUAJJAH UGSF, UMR Lille1-CNRS 8576 Villeneuve d’Ascq dounia.mouajjah@gmail.com Claire MOULIS LISBP Toulouse moulis@insa-toulouse.fr Laurence MULARD UCB, Institut Pasteur Paris laurence.mulard@pasteur.fr Jérôme NIGOU IPBS, UMR 5089 Toulouse jerome.nigou@ipbs.fr Milène NITENBERG Cermav, UPR CNRS 5301 Grenoble milene.nitenberg@cermav.cnrs.fr Mehdi OMRI LG2A, UMR 7378 Amiens mehdi.omri@u-picardie.fr Xiaomeng PANG ImoPA, UMR 7365 Vandoeuvre-lès-Nancy xiaomengpangnancy@gmail.com Corinne PAU-ROBLOT BioPI, EA3900, Univ. Picardie Amiens corinne.pau-roblot@u-picardie.fr Jérôme PELLOUX BioPI, EA3900, Univ. Picardie Amiens jerome.pelloux@u-picardie.fr Serge PEREZ DPM, UMR UGA-CNRS 5063 Grenoble spsergeperez@gmail.com

Cédric PEYROT ICOA, UMR 7311 Orléans cedric.peyrot@univ-orleans.fr Jacques PRANDI IPBS, UMR 5089 Toulouse jacques.prandi@ipbs.fr Cédric PRZYBYLSKI IPCM, UMR 8232 Paris cedric.przybylski@upmc.fr Yves QUENEAU ICBMS, UMR 5246 Villeurbanne yves.queneau@insa-lyon.fr Françoise QUIGNARD ICGM, UMR 5253 Montpellier quignard@enscm.fr Mialy RANDRIANTSOA Elicityl Crolles Mialy.randriantsoa@elicityl.fr www.elicityl-oligotech.com Caroline RÉMOND UMR, INRA-URCA FARE Reims caroline.remond@univ-reims.fr Olivier RENAUDET DCM, UMR UGA-CNRS 5250 Grenoble olivier.renaudet@univ-grenoble-alpes.fr Emeline RICHARD Cermav, UPR CNRS 5301 Grenoble emeline.richard@cermav.cnrs.fr Anne ROBERT ImoPA, UMR 7365 Vandoeuvre-lès-Nancy anne.robert@univ-lorraine.fr Catherine RONIN Siamed’Xpress Gardanne catherine.ronin@siamedxpress.com Irina SADOVSKAYA Equipe BPA, Univ. Littoral-Côte d’Opale Boulogne-sur-mer Irina.Sadovskaya@univ-littoral.fr

Baptiste SCHINDLER Institut Lumière Matière, UMR 5306 Villeurbanne baptiste.schindler@univ-lyon1.fr Marie SCHULER ICOA, UMR 7311 Orléans marie.schuler@univ-orleans.fr Arnaud TATIBOUET ICOA, UMR 7311 Orléans arnaud.tatibouet@univ-orleans.fr Charles TELLIER UFIP, UMR 6286 Nantes charles.tellier@univ-nantes.fr François THOMAS LBI2M, UMR 8227 Roscoff francois.thomas@sb-roscoff.fr Annabelle VARROT Cermav, UPR CNRS 5301 Grenoble annabelle.varrot@cermav.cnrs.fr Anne-Sophie VERCOUTTER-EDOUART UGSF, UMR Lille1-CNRS 8576 Villeneuve d'Ascq anne-sophie.vercoutter@univ-lille1.fr Romain VIVES IBS, UMR CEA-CNRS-UGA 5075 Grenoble romain.vives@ibs.fr Joanne XIE PPSM, UMR 8531 Cachan joanne.xie@ens-cachan.fr Nao YAMAKAWA UGSF, UMR Lille1-CNRS 8576 Villeneuve d'Ascq nao.yamakawa@univ-lille1.fr Agata ZYKWINSKA Ifremer, Laboratoire EM3B Nantes Agata.Zykwinska@ifremer.fr

56

Oligosaccharides & Polysaccharides

Free & functionalized glycans Glycoconjugates

Extraction from biomass Biosynthesis by fermentation

Lectins

Customized products & services

Analysis

R&D contracts

Glycoproducts for life sciences

c o n t a c t @ e l i c i t y l . f r p h + 3 3 4 7 6 4 0 7 1 6 1 f a x + 3 3 4 7 6 5 5 9 9 5 0 E l i c i t y l S A 7 4 6 a v e n u e A m b r o i s e C r o i z a t F - 3 8 9 2 0 - C r o l l e s - F r a n c e w w w . e l i c i t y l - o l i g o t e c h . c o m

57

Siamed'Xpress innove dans les dosages sanguins en dépistant précocément les troubles de la thyroïde , évocateurs des expositions aux polluants

L’entreprisevientdevalidersestestsdedépistageprécocedestroublesdelathyroïdesur plus de 1300 patients. Elle souhaite à présent porter au marché les premiersdosages harmonisés compatibles avec la Santé connectée et développer différentspartenariatsstratégiques.

SiaMed’Xpressdéveloppedesprotéineshumainesdont leglycoprofil hypersialyléestpour lapremière fois,en toutpoint identiqueàcellesqui sontprésentesdansnotrecirculation. Les biomarqueurs ainsi développés peuvent avantageusement servir àconstruiredesdosagesplusprécis,devaleurdiagnostiqueprécoce sur labased’unecalibrationmassiquerépondantauxnouvellesnormesinternationales.

Les avancées technologiques développées par SiaMed’Xpress au cours de ses 5dernières années sur l’ingénierie des protéines glycosylées ont également permis definaliser plusieurs preuves de concept sur des biomolécules d’usage thérapeutiquemajeuretd’engagerdifférentsprojetspartenariaux.

Siamed'Xpress s'adresse à Wiseed

pour financer l'accès au marché de ses dosages https://www.wiseed.com/fr/startups/siamed-xpress

Editeurs C. Breton, S. Armand & M. Carret, Cermav, 2016

© Logo Kawthar Bouchemal

Illustration couverture M. Carret d’après photo J.L. Rigaux

Impression Impression et Ressources en Imagerie Scientique (IRIS)

Grenoble INP, 1025 rue de la Piscine, 38402 Saint Martin d’Hères

60

Lundi 23 mai Mardi 24 mai Mercredi 25 mai Jeudi 26 mai Vendredi 27 mai8h45

CI-02 F. Foulquier CI-09 J. Nigou CI-13 M. Bardor CI-20 C. Przybylski

CO-1 S. Baldini (Prix BF-AV) CO-09 E. Richard CO-13 E. Ficko-BleanCO-2 G. Cutolo CO-10 J. Xie CO-14 C. Pau-Roblot

9h45CI-03 F. Friscourt CI-10 L. Mulard CI-14 J. Pelloux CI-21 D. Bonnaffé

10h45CI-04 C. Grandjean CI-11 M. E. Guerin CI-15 C. Rémond

CO-3 A. Varrot CO-11 T. Hurtaux CO-15 C. PeyrotCO-4 S. Gouin CO-12 R. Fauré CO-16 I. Compagnon

11h45CI-05 O. Renaudet CI-12 Y. Blériot CI-16 E. Fleury

12h15

14h30CI-06 C. Moulis

CO-5 C. DumonCO-6 M. Omri

15h30et CI-07 F. Quignard

16h00 : Pause café 16h00 : Pause café16h30

CI-08 N. Bourgougnon CI-17 R. Daniellou (Prix GFG)

CO-7 A. Zykwinska - CO-8 V. Bonnet

17h30CEREMONIE D'OUVERTURE CI-19 S. Pérez

17h45CI-01 J. Le Pendu

18h30Duo1 S. Morera & Y. Queneau

19h00Apéritif d'accueil

19h30 Dîner de gala

Soirée dansante

P r o g r a m m e

(randonnée, visites)Après-midi libre

Session POSTERS (nos impairs)

Duo2 S. Dahbi & I. Bertin-Jung

CI-18 C. Ronin

Session POSTERS (nos pairs)

ASSEMBLEE GENERALE GFG

Montage des posters

ACCUEIL

9h15

Dîner

Déjeuner

10h15 : Pause café

11h15

15h00

17h00

CEREMONIE DE CLOTURE