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GLYCOBIOLOGY Study of structure, synthesis and biology of sugars, particularly the sugar chains that are attached to proteins and lipids Glycoconjugates Structural diversity • Glycoproteins • Glycolipids • Proteoglycans Diversity of functions • Structural / modulation • Recognition Altered glycosylation and glycosylation defects are observed in many pathological situations (inflammation, cancer, congenital disorders of glycosylation, ) 1 Post-translational modifications of proteins are important regulators of protein function : Although proteins comprise the largest fraction of a cell's dry mass, it is estimated that more than half are modified with glycans, lipids or other metabolites. Prescher and Bertozzi, 2005, Nat. Chem. Biol. 1, 13-21 2 Location of glycoconjugates in multicellular organisms ER Golgi M.E. Taylor & K. Drickamer (Introduction to Glycobiology -2003- Oxford University Press) 3 BIOSYNTHESIS OF GLYCANS : FOCUS ON THE LARGE GLYCOSYLTRANSFERASE FAMILY . Structural and Functional aspects Prof. Christelle BRETON 4 Photo credit : Barbieri, Chenu, Francillon, Vinçon, D.R. AEPI Grenoble This slide shoud always be used with the AEPI logo. Any modification of the slide has to be expressly notified to the AEPI 5 Centre de Recherches sur les Macromolécules Végétales Glycosciences 6 Molecular Glycobiology at CERMAV Structure-Function Studies of Lectins and Glycosyltransferases 7 GLYCOSYLTRANSFERASE course : Contents 1- General features • General reaction • Cellular localization & topology • Glycosylation events in the Golgi • Take home message (1) 2- Classification • Processivity / stereochemistry • Classification • GT repertoire • Take home message (2) . 3- Structure • Structural information (current picture) • GT-A fold / DxD motif • GT-B fold • Other folds • Take home message (3) 4- Reaction mechanisms • Inverting/retaining reaction mechanisms • Ordered binding in GT-A enzymes • additional difficulties • Take home message (4) 5- Future directions 8 GLYCOSYLTRANSFERASES (Leloir enzymes*) GT XDP-sugar + OH-Acceptor sugar-O-Acceptor + XDP specificity for both the donor sugar and acceptor substrates GDP-sugars UDP-sugars CMP-sugars Fucose Mannose Glucose Galactose N-acetylglucosamine N-acetylgalactosamine Glucuronic acid Xylose ... Sialic acids . * In honor of Luis L. Leloir who discovered the first nucleotide sugar (Nobel Prize in Chemistry in 1970) 9 Examples of “activated” glycosyl donors. Nucleotide-sugar donors, sugar phosphate donors, and lipid-phosphate donors. Reaction catalyzed by a β1,3-galactosyltransferase (β β3-GalT1) 90% GTs use Nucleotide Donors 10 Eukaryotes GLYCOSYLTRANSFERASES Cellular localization and topology ER, Golgi (membrane proteins ) cytosol and nucleus (soluble forms) biological fluids (soluble forms) ?? . plasma membrane (membrane proteins) Most of the complex glycans are built into the ER and GOLGI Secretory pathway 11 Topologies of glycosyltransferases in the ER and Golgi Type II membrane protein integral membrane protein . catalytic domain RE lumen stem Golgi lumen TMD Cytoplasm NH2 Typical Golgi GT using a nucleotide-sugar Cytoplasm GT using a Dolichol-P-sugar 12 Topology of Golgi glycosyltransferases stem TMD N- Catalytic domain Add-on domain (i.e. Lectin domain) N- N- . lumen Stem variable length may modulate in vivo GT activity « cleavable » Catalytic domain globular, minimal active structure, typically ~300-350 aa Breton et al. (2000) Biochimie 13 Glycosylation events in Golgi Cytoplasme Nucleotide-sugars are synthesized in the cytosol and then transported into the Golgi by specific transporters 퀀 GT (CMP-NeuAc synthesized in the nucleus) 14 Glycosylation events in Golgi GT localization ? Supramolecular organization ? N-glycans O-glycans . Brockhausen, 2006 De Graffenreid & Bertozzi, 2004 15 TAKE HOME MESSAGE (1) Although localization the are mechanisms still under of Golgi debate, enzyme both the distribution of these enzymes among the Golgi . cisternae and specific association among glycosylation enzymes can dictate the overall glycan structures (glycome) produced by a cell. 16 GLYCOSYLTRANSFERASE course : Contents 1- General features • General reaction • Cellular localization & topology • Glycosylation events in the Golgi • Take home message (1) 2- Classification • Processivity / stereochemistry • Classification • GT repertoire • Take home message (2) . 3- Structure • Structural information (current picture) • GT-A fold / DxD motif • GT-B fold • Other folds • Take home message (3) 4- Reaction mechanisms • Inverting/retaining reaction mechanisms • Ordered binding in GT-A enzymes • additional difficulties • Take home message (4) 5- Future directions 17 GLYCOSYLTRANSFERASES N PROCESSIVITY Processive enzymes : transfer multiple sugar residues to the acceptor . (polysaccharide synthases,.....) Non-processive enzymes : transfer a single sugar residue to the acceptor 18 HO OH O OH HO STEREOCHEMISTRY OF THE GLYCOSIDIC LINKAGE HO HO O OH O OH OH Retaining Glycosyltransferase Transfer of a monosaccharide from a donor to an acceptor: OH O O OH HO OH O HO . Reaction can occur with retention or inversion of configuration of the transferred sugar HO HO HO O NH O O P P O O N O O OH HO Inverting Glycosyltransferase O O OH HO HO O OH OH HO HO OH OH O OH O HO O OH OH 19 GLYCOSYLTRANSFERASES N They are primarily classified according to the type of sugar that they transfer Galactosyltransferases (α α3-GalTs, β4-GalTs, ...) Fucosyltransferases (α α2-FucTs, α3-FucTs, α6-FucTs, ...) Sialyltransferases (ST6Gal, ST3Gal, ST8Sia, ....) ......... . There is no or limited sequence homology among the different GT families or even, within one family, among members catalyzing different reactions 20 Glycosyltransferases .... CAZy classification Classification based on amino acid sequence similarity, correlates with (i) protein folding (ii) enzyme mechanism http://www.cazy.org/ October 2012 ~ 100,000 known and putative GT sequences . distributed over 90 families ▪ Two very large families (~half of total number of GT entries) inverting GT2 and retaining GT4 – considered as the ancestral families ▪ Many families are polyspecific 21 The number of GT families in CAZy is continually increasing with the discovery of new GT genes (27 families in 1997, more than 90 in 2012) New GT families are exclusively created based on the availability of at least one biochemically-characterized protein 훀ͮ member Discovery of new GT families Experimental investigation (i.e. Rumi GT90) Bioinformatics and biochemical characterization (i.e. plant XylTs, GT77) 22 GT repertoire .... A large repertoire of genes is required for glycan assembly and function in multicellular organism : 1-2% of the genes of an organism code for GTs 훀ͮ 235 GT genes 43 families ~ 80 % annotated genes The most populated families GT1 : UGTs GT7 : β4-GTs GT27 : ppGalNAcTs GT29 : SiaTs GT31 : β3-GTs 23 Plants tend to have far more GT genes than any other organism sequenced to date . ~ 460 GT genes 42 families 20 % are annotated ▪ Several highly populated GT families GT1 : UGT-family (glycosylation of secondary metabolites) GT2, 8, 31, 47 : biosynthesis of cell wall PS ▪ Only 1 plant specific GT family (GT37) 24 TAKE HOME MESSAGE (2) A huge number of putative GT genes have been identified through large genome sequencing projects (currently ~100,000) Large number of sequence-based GT families (> 90) The vast majority of these sequences (more than 90%) are �Ϟ uncharacterized open-reading frames (particularly bacterial and plant genomes) This is a challenging task for glycobiologists to assign functions to the many uncharacterized GT sequences 25 GLYCOSYLTRANSFERASE course : Contents 1- General features • General reaction • Cellular localization & topology • Glycosylation events in the Golgi • Take home message (1) 2- Classification • Processivity / stereochemistry • Classification • GT repertoire • Take home message (2) �Ϟ 3- Structure • Structural information (current picture) • GT-A fold / DxD motif • GT-B fold • Other folds • Take home message (3) 4- Reaction mechanisms • Inverting/retaining reaction mechanisms • Ordered binding in GT-A enzymes • additional difficulties • Take home message (4) 5- Future directions 8 Glycosyltransferases .... 3D structures Structural information for 39 CAZY families Only two general folds, termed GT-A and GT-B (and variants), have been observed for all structures of nucleotide-sugar-dependent GTs solved to date . GT-A fold GT-B fold 26 ~ 80 % of the current GT families are known, or predicted, to adopt a GT-A or GT-B fold (or variants) GT-A and variants 3D (37) 2, 6, 7, 8, 13, 14, 15, 27, 29, 31, 42, 43, 44, 55, 64, 78, 81, 88 GT-B and variants 1, 3, 4, 5, 9, 10, 20, 23 28, 30, 35, 41, 52, 63, 65, 68, 70, 72, 80 �Ϟ Predicted* (34) 12, 16, 17, 21, 24, 25, 32, 34, 40, 45, 49, 54, 60, 62, 67, 69, 71, 73, 74, 75, 77, 82, 84, 92 * Based on 3D-PSSM and PHYRE programs (Bennett-Lovsey et al., 2008; Kelley et al., 2000) 11, 18, 19, 33, 37, 38, 47, 56, 61, 90 Breton et al. Biochem. Soc. Symp. (2002) Breton et al. Glycobiology (2006) Breton et al. COSB (2012) 27 There is no correlation between the overall fold of the GT and its mechanism of action since both inverting ऀ͵ and retaining enzymes of both fold types (GT-A and GTB) are known. 28 GT-A fold a single domain α/β β/α α structure �Ϟ mixed β-sheet (3214657) All of the strictly metal ion-dependent GTs for which structures have been determined to date have been of this type and include a DxD Metal Binding motif (Mg2+, Mn2+) * One exception (GT14) 29 Ubiquity and structural basis of the DxD motif in the GT-A fold 眐Ϛ cation-dependent GTs (Mn2+, Mg2+,…) Variations in the DxD sequence : DVD, DID, DDD, EDD, EED, TDD, DxH, EPD, N. 30 GT-A variant GT-A like Canonical GT-A GT42 禠Ϝ mixed β-sheet (3214657) DxD motif // β-sheet (8712456) no DxD motif Cst II - sialyltransferase Campylobacter jejuni New type of fold ! 31 GT-B fold 2 Rossmann-type domain assembly α/β β/α α structure 禠Ϝ // β-sheet (3214567) The catalytic site is located in a cleft between the two domains Metal ion-independent GTs 32 GT-B variant Human α6-Fucosyltransferase (FucT-VIII) GT23 Catalytic domain 禠Ϝ SH3 domain (Cterm) Coiled coil region (N-term) Rossmann domain in the Cterm of catalytic domain 33 GT-B variant Human O-β β-GlcNAcT (OGT) GT41 • Unique and reversible modification TPR 禠Ϝ • Found on a myriad of nuclear and cytoplasmic proteins • Extensive cross-talk with phosphorylation Hart et al., (2007) Nature, 446: 1017−1022 Int-D Lazarus et al., (2011) Nature, 469:564-567 34 New folds are observed for GTs using lipid-phosphate sugar donors No Rossmann fold !! GT51 Peptidoglycan glycosyltransferase (Lovering et al., 2007, Science) 禠Ϝ Staphylococcus aureus GT domain: bacteriophage λ-lysozyme-like fold !! 35 The process of N-linked protein glycosylation 㐐ऀ Schwarz and Aebi (2011) COSB, 21:576-582 36 | N AT U R E | V O L 4 7 4 | 1 6 J U N E 2 0 1 1 GT66 㐐ऀ The first structure of a membrane-embedded GT ! 37 3D structure of PglB (C. jejuni) GT66 㐐ऀ Lizak et al., (2011) Nature, 474, 350-355. 38 TAKE HOME MESSAGE (3) The large GT family is characterized by a conserved 3D architecture (GT-A, GT-B folds and variants) The small variety of folds observed for GTs (constraint in nucleotide-sugar binding ?) is compensated by a large structural variability in the acceptor binding domain: «Functional plasticity» which allows fine-tuning with respect to the acceptor. 㐐ऀ Novel folds are expected for GTs that use a lipid-phosphate donor Virtually nothing is known about the structures and mechanisms of these large membrane-embedded GTs. The successful crystallization of PglB now opens new avenues to fill the gap. 39 GLYCOSYLTRANSFERASE course : Contents 1- General features • General reaction • Cellular localization & topology • Glycosylation events in the Golgi • Take home message (1) 2- Classification • Processivity / stereochemistry • Classification • GT repertoire • Take home message (2) 㐐ऀ 3- Structure • Structural information (current picture) • GT-A fold / DxD motif • GT-B fold • Other folds • Take home message (3) 4- Reaction mechanisms • Inverting/retaining reaction mechanisms • Ordered binding in GT-A enzymes • additional difficulties • Take home message (4) 5- Future directions 8 Reaction mechanisms of GTs In contrast to the well-characterized catalytic mechanisms used for glycosidic bond hydrolysis 㐐ऀ (glycosidases), the mechanisms for glycoside bond formation remain less clear. For a review, see Lairson et al., (2008) Ann. Rev. Biochem., 77:521-555 Breton et al., (2012) COSB 40 Inverting Glycosyltransferase Mechanism 㐐ऀ A direct-displacement SN2-like reaction via a single oxycarbenium ion Catalytic base (i.e. Asp, Glu, His) deprotonates acceptor OH From Lairson et al. (2008) Ann Rev Biochem 41 Retaining Glycosyltransferase Mechanism: currently two hypotheses SN2-like double displacement mechanism with formation of a covalently bound glycosyl-enzyme intermediate (by analogy to glycosylhydrolases) Soya et al., (2011) Glycobiology, 21 (5) From Lairson et al. (2008) Ann Rev Biochem 負Ϝ SNi-like mechanism Front-face nucleophilic attack involving hydrogen bonding between leaving group and the acceptor nucleophile Lee et al., (2011) Nature Chem. Biol., 7 42 The « closed » active conformation of glycosyltransferases Reaction mechanism for GTs of the GT-A family Ordered binding of donor and acceptor substrates, linked to a donor substrate-induced conformational change LgtC αGalT 負Ϝ Base of UDP-Gal Binding of a distorted conformation of nucleotide sugar may be important for catalysis Acceptor disaccharide Persson et al, Nature Struct. Biol., 8 (2001) 166 Ret Inv Inv 43 Additional difficulties for glycosyltransferases GT-A Loop movement GT-B domain movement close open 負Ϝ α3-GalT GT6 Superimposition of open (free enzyme) and closed (upon UDP-Gal binding) forms Gastinel et al. (2001); Boix et al., (2001) Glycogen synthase GT5 Superimposition of open state (solid) and closed conformation (transparent) Buschiazzo et al. (2004) 44 GlycosyltransferasesN still puzzling Crystallographic and modelling studies should allow for a better understanding of molecular bases responsible for substrate specificityN 負Ϝ The blood group synthases as a case of study ! 45 Blood Group A & B Structures Made by Closely Related GTs HO OH O OR HO O O OH HO OH O(H) Precursor HO O OH HO O NH O O NH O P O P O O O O HO H3C OH O HO N OH O O OHO P O P O O O HO O GTA HO HO OH NH H3C O O OH HO O HO OR OH O O O O O O OH OH OH Blood Group A Yamamoto, Hakomori (Nature, 1990) OH O OR HO N OH O HO O UDP-Gal OH O NH HO GTB 負Ϝ UDP-GalNAc HO O O HO OH Blood Group B Both enzymes adopt a GT-A fold 46 Blood group synthases GTA/GTB – Highly homologous enzymes differing at only four critical amino acids out of a total of 354 residues – Alteration of these four crucial amino acid residues converts the specificity from GTA to GTB AAAA 負Ϝ BBBB Molecular basis for nucleotide sugar specificity ? UDP-Gal vs UDP-GalNAc 47 Homology Modelling GT6 (GT-A fold) UDP-Gal vs UDP-GalNAc α3-GalNAcT α3-GalT S185 D302 S185 D302 E303 E303 負Ϝ G268 A268 M266 L266 GTA GTB Only 2 of the 4 critical residues in binding site (L266M, G268A) M266 + A268 excludes UDP-GalNAc (steric conflict) G268 accomodation of N-acetamido group GTA should utilize UDP-Gal ??? Heissigerova et al. Glycobiology, 13 (2002) 377 48 What can we learn about substrate binding and catalysis of blood group GTs ? Crystallographic studies Modelling studies Natural mutations in blood banks Mutagenesis (chimeric enzymes) 負Ϝ UDP-Gal vs UDP-GalNAc Still unclear !!! Flexibles enzymes Mutagenesis unpredictable Much work to be done to understand catalysis Monica Palcic and coll (in Alberta and Copenhagen) 49 TAKE HOME MESSAGE (4) Catalytic mechanisms are still poorly understood • Movements of loops and/or domains involved in the binding • Flexibility A better understanding of catalytic. mechanisms of GTs together with much more structural information is needed, for the rational design of specific inhibitors as well as for engineering purposes to expand the biotechnological uses of GTs. 50 Glycosyltransferases Future directions N “in vivo” “in vitro” - Functional organization in Golgi * supramolecular organisation - Spatial and temporal expression of GTs in physiological and pathological situations (Glycomics) - “Structure/function” relationships * new 3D structures * mechanism of action, inhibitors 負Ϝ - “ORFeome” of GTs * heterologous expresssion * library of acceptors Biotechnological applications - Enzymatic synthesis of bioactive oligosaccharides - Production of recombinant GP for therapeutic use - GTs and medicine (diagnostic tools, new therapies ) - GT engineering (new catalysts with improved capabilities) - Oligo/polysaccharide engineering (modification of functional properties) ..... 51 THE END 負Ϝ Database development at Cermav : Glyco3D 負Ϝ CERMAV - GT team Christelle Breton Anne Imberty Aline Thomas Olivier Lerouxel Valerie Chazalet Post-doc and PhDs Magali Audry Gaelle Batot Peter Both Sara Fasmer Hansen Charlotte Jeanneau Joana Rocha Collaborations �ҕ Eric Maréchal (CEA-Grenoble) Emmanuel Bettler (IBCP Lyon) Jan Mucha (Bratislava) Soren B. Engelsen (Univ. Copenhagen) Monica Palcic & Ole Hindsagaul (CRL- Copenhagen) Funding French ANR CEE Bioinformatics strategy Arabidopsis proteome 30690 sequences Use of several remote homology detection methods to search the « test Arabidopsis proteome » for new candidate GT genes 1- Removal of large sequences (>1000 aa) 29457 sequences Test Arabidopsis proteome 2- Removal of sequences with a cTP or mTP (TargetP) 21755 sequences HMMSearch (1D level) PSI-BLAST + Structural overlap (2D level) �ҕ ProFit (3D level) 3- Removal of proteins with no TMD (TMHMM) 5666 sequences 4- Removal of CAZy accessions (460 seqs) > 150 new candidate sequences + Chemometrics Hansen et al. (2009) J. Proteome Res. Hansen et al., (2010) Mol. Biosystems Test Arabidopsis proteome (5315 protein sequences) Evaluation of candidate GT sequences (~150)… Close examination of each sequence Annotation in databases Blast and Psi-BLAST TMHMM Phyre (fold recognition) Hydrophobic Cluster Analysis method (HCA) �ҕ ~ 20 strong candidate genes* harbouring a clear GT signature * Distantly related to GT4, GT14, GT23, GT65 In any case, the validation step requires the critical expertise of the biologist The populations of the various GT families show wide variations, with two large families, the inverting GT2 and retaining GT4, accounting for about half of the total number of GT entries, and 犐ҕ which can be considered as the most ancestral families for which enzymes stereochemistries have evolved (Martinez-Fleites et al., 2006). of both Glycosyltransferases …. CAZy Large differences among GT families number of sequences GT2 (> 13,000 seqs) GT4 (> 10,000 seqs) GT63 (1 seq) 50% of total seqs 8 families have > 1000 sequences 13 families have < 20 sequences 犐ҕ origin and function GT2 Cellulose synthase, chitin synthase, Dol-P-Man/Glc synthase GlcTs, GalTs, GalfTs, GnTs, RhaT, ... animal, plant, yeast, bacteria,Q GT27 pp-α α-GalNAcTs animal (mucin-type glycosylation) MOLECULAR GLYCOBIOLOGY at CERMAV Group Leader A. Imberty GLYCOSYLTRANSFERASES and biosynthesis of glycans - Structure-function studies and engineering of GTs - Biogenesis of PCW polysaccharides Specific犐recognition of carbohydrates by LECTINS ҕ - Lectins and bacterial infections - Development of anti-adhesive drugs - Lectins and biodiversity New tools in Glycosciences - Functionalized surfaces, gold nanoparticles - Glyco-bioinformatics - Modelling sulfated polysaccharides A better understanding of catalytic mechanisms of GTs together with much more structural information is needed, however, for the rational design of specific inhibitors as well as for engineering purposes to expand the biotechnological uses of GTs. the limiting factor in developing carbohydrate-based compounds for clinical application was the high cost and complexity of producing glycans in large quantities. the use of metabolically engineered bacteria that overexpress heterologous GTs has become a powerful method for the large-scale production of complex glycans at low �Ϣ cost (Endo and Koizumi, 2000; Fierfort and Samain, 2008). Glycobiology research will greatly benefit from these major advances in cost-effective technologies for carbohydrate synthesis. Catalytic mechanisms are still poorly understood Movements of loops and/or domains involved in the binding