Glycobiology. BMB 170 Lecture 17 Carbohydrates, Nov 21

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1 Glycobiology Carbohydrates Glycosyltransferases and glycosidases Protein glycosylation Glycobiology of bacteria Glycan binding and the role of glycans BMB 170 Lecture 17 Carbohydrates, Nov 21 First coined by Raymond Dwek (Oxford) in 1988

2 Online resources Society for glycobiology Consortium for functional glycomics Glycosciences (database and tools) French Glyco database

3 Carbohydrates Primary energy store in biosphere Caltech Clemons lab Hsieh-Wilson lab Mazmanian lab Jensen lab Not to mention proteomics studies Introduction to Glycobiology Maureen Taylor and Kurt Drickamer Essentials of Glycobiology Edited by Varki et al

4 Glycobiology Functions of glycans Intrinsic functions Structural components Cell walls Extracellular matrix Modifying proteins Solubility Stability Extrinsic functions (glycan/lectin) Directing trafficking Cell/cell and cell matrix adhesion Modulating signaling Model of CD59 glycosylation N-linked glycan O-linked glycan GPI anchor Rudd et al (1997) JBC 272:7229

5 Numbers Majority of the 64 approved protein products are glycosylated ~500 candidates in clinical development are glycosylated Intentional engineering of protein glycosylation can lead to improved efficacy Vaccines Bacteria and virus H. influenzae type b Cancer Therapies Sugar based Heparin anti-coagulant Number one selling drug in the world Anti-selectin therapies Inhibitors Protein drugs Erythropoietin hormone that boosts red blood cell production EPO Amgen s billion dollar drug Chorionic gonadotropin fertility drug From the urine of pregnant women

6 An aldehyde or ketone derivative of a polyhydroxy alcohol that is synthesized in living cells Simplest carbohydrate is a monosaccharide : (CH 2 0) n = carbon hydrate Saccharide is derived from the Greek sakcharon, meaning sugar Monosaccharides have 3 to 7 carbons Either aldehyde or ketone group and hydroxyl (OH) groups on nearly every carbon When n=3 the simplest or smallest biologically important carbohydrates are formed polyhydroxy ketone ketose polyhydroxy aldehyde - aldose Mono & disaccharides end in the suffix -ose Carbohydrates are commonly classified on the basis of their size Monosaccharides Oligosaccharides Polysaccharides What is a Carbohydrate?

7 Representing an oligosaccharide Taylor & Drickhamer Intro to Glycobiology Fig 1.9

8 Monosaccharide symbol set a b c Essen%als of Glycobiology Second Edi7on Chapter 1, Figure 5

9 Terms and abbreviations Glycosyltransferase (adds sugar) Glycosidase (trims sugar) Lectin (binds sugar) Epimerase (inverts OH to convert between sugar isomers) Flippase (flips between lipid leaflets) Mutarotase (flips anomeric carbon) Sugar Hexose Glucose Galactose Mannose Rhamnose Fucose Glucosamine Galactosamine Mannosamine N-acetylglucosamine N-acetylgalactosamine N-acetylmannosamine Glucuronic acid Galacturonic acid Mannuronic acid N-acetylneuramic acid Abbreviation Hex Glc Gal Man Rha Fuc GlcN GalN ManN GlcNAc GalNAc ManNAc GlcA GalA ManA NeuNAc 3-deoxy-D-manno-2-octulosonic acid Kdo

10 Fischer Projections Using these rules the distinguishing features of the 3D structure of stereoisomers can easily and accurately represented with 2D drawings Emil Fischer (Nobel 1902) established the molecular structures (stereochemistry) of many sugars and purines

11 Fischer Projection Horizontal bonds are in front of the plane of the page. Vertical bonds are behind the plane of the page.

12 Common D- Aldoses

13 Common D-Ketoses

14 Haworth Projections Sir Norman Haworth FRS 1937 Nobel for carbohydrates & vitamin C First carbon is anomeric carbon Glucose Vitamin C (ascorbic acid)

15 Anomers Two stereoisomers ( designated as α and β) of a given sugar that differ only in the configuration about the carbonyl (anomeric) carbon atom

16 Mutarotation The α- and β-forms of glucose can be isolated separately Pure α-glucose has a specific rotation of +112 o Pure β-glucose has a specific rotation of o Mutarotation is the change in optical rotation as an equilibrium mixture of anomers forms α- or β-glucose in aqueous solution slowly changes to o It does not matter whether one starts with pure α- or β-glucose Results in an equilibrium mixture of 36% α-glucose and 64% β- glucose More stable β-glucose form predominates A very small amount of the open-chain form exists in this equilibrium

17 Glycosidic bond formation Glycosidic bond - The bond between the anomeric carbon of a carbohydrate and some other group or molecule Taylor & Drickhamer Intro to Glycobiology Fig 1.8, 10 &11

18 Non-reducing sugar Disaccharides Reducing sugar Reducing sugar

19 Torsion angles that define the conforma6on of the glycosidic linkages φ, ψ, and ω 1 6 linkage C1-O1 bond φ for 1 6 C6 -O1 bond ψ for 1 6 C5 -C6 bond ω for 1 6 Essen%als of Glycobiology Second Edi7on Chapter 2, Figure 18

20 Two residues Amino acids 2 Carbohydrate diversity Monosaccharides 11 Four residues Amino acids 256 Monosaccharides >35,000 Not to mention many modifications!

21 Oligosaccharide A linear or branched carbohydrate usually from two to six(?) monosaccharide units - in practice this generally just means a large complex unit The most common disaccharides Sucrose Lactose Maltose

22 Examples of typical N- and O-linked glycans Essen%als of Glycobiology Second Edi7on Chapter 2, Figure 19

23 Sialic acids Most abundant Mammals but not people Aquatic organisms and bacteria Sialic acids comprise ~50 sugars derived from neuraminic acid or deaminoneuraminc acid Found as terminal sugars on glycoconjugates Linked to tumor markers and virulence factors in bacteria Lack of Neu5Gc is uniquely human We have a mutated form of CMP-sialic acid hydroxylase (one of a few clear genetic differences between us and chimps that leads to a global biochemical affect) Chou..Varki (1998) PNAS 95:11751 Münster-Kühnel et al (2004) Glycobiology 14:43R (Review)

24 Homopolysaccharides As high as 20,000 Kilodaltons Storage forms of glucose Starch (Plants) Amylose (unbranched) Amylopectin (branched) Glycogen (Animals) Structural fibers Cellulose (Plants) Chitin (Arthropods) Glycosaminoglycans (Animals)

25 Plant Starch Amylose (30% of starch) Unbranched D-glucose-α(1,4)-D-glucose Amylopectin (branching every residues) Starch is the plant form of carbohydrate ingested by humans. α-amylase secreted by salivary glands and the pancreas hydrolyzes amylose and amylopectin.

26 Glycogen Principal storage form of glucose in animal cells Stored as granules in liver (10% w/w) and muscle Glucose units linked by α-1,4 and α-1,6-glycosidic bonds Branches every 8-10 residues Very compact for efficient storage

27 Cellulose D-glucose-β(1,4)-D-glucose Alternates up and down Most abundant organic molecule on earth Mammals lack cellulases Ding & Himmel J Agric Food Chem (2006) 54:597

28 Glycolipids Phospha7dylinositol (PIP, PIP 2, etc) Glycosphingolipids Forms the calyx extracellular matrix of cells GPI (glycosylphospha7dylinositol) Links to C-terminus of proteins as an anchor Pep7doglycan Lipid A

29 Structures of representative glycosphingolipids and glycoglycerolipids Glycosphingolipid Glycoglycerolipid Chapter 10, Figure 1 Essen%als of Glycobiology Second Edi7on Found in cell membranes of all organisms Base structure for lipid rafts Animals are mostly glycosphingolipid GalCer is the most abundant in the brain Structure determined in 1884 Sphingo from sphinx because the structure was hard to determine

30 General structure of GPI anchors R3 Essen%als of Glycobiology Second Edi7on Chapter 11, Figure 1

31 Glycosyltransferases CAZy defined The biosynthesis of di-, oligo- and polysaccharides involves the action of hundreds of different GTs (EC 2.4.x.y) catalyse the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds classified as either retaining or inverting enzymes according to the stereochemistry of the substrates and reaction products IUBMB enzyme classifications do not indicate the intrinsic structural features of the enzymes, nor do they adequately accommodate enzymes which act on several distinct substrates. The CAZy database proposes the classification of GTs using nucleotide diphospho-sugar, nucleotide monophospho-sugars and sugar phosphates (EC x) and related proteins distinct sequence-based the same 3-dimensional fold is expected to occur within each of the families some families turn out to have similar three-dimensional structures polyspecificity (enzymes with different donor and/or acceptor found in the same family) is common among GT families making precise functional predictions often unreliable or inaccurate

32 CAZy hlp://

33 Carbohydrate-ac6ve enzymes (CAZymes) as a func6on of the number of genes in the genomes of different organisms Essen%als of Glycobiology Second Edi7on Chapter 7, Figure 1

34 Glycosyltransferases Discovered by Luis Leloir (1970 Nobel Prize in Chemistry for elucida7ng glycogen synthesis) Generally catalyze the transfer of a monosaccharide unit from an ac7vated sugar phosphate to an acceptor Nearly every glycosidic linkage is made by a dis7nct enzyme High mannosse N-linked glycan

35 Glycosyltransferases ~1-2% of genes (in all organisms including viruses) Humans have 230 and C. elegans have ~240 Plants have lots (Arabidopsis has ~450 and Populus has more than 800) Some genomes that are obligate parasites have few (Mycoplasma have none) ~32,000 ORFs recognized 91 families (GT-2 and GT-4 account for ~50%) 2 major folds GT-A GT-B GT-C (?) generally defined as other (non-nucleotide substrates)

36 Products Energy storage Structural Secondary metabolites Antibiotics Glycolipids Chemistry of Glycosyltransferases Weadge & Palcic (2008) DOI: / wecb213

37 Glycosyl donors Phosphate activated sugars and lipids Acceptors Saccharides Polysaccharides Lipids Proteins Other natural products Chemistry of Glycosyltransferases Weadge & Palcic (2008) DOI: / wecb213

38 Leloir-type GTs Defined as those with sugar-nucleo7de donors 90% of annota7ons UDP/TDP nucleo7des represent 60% of the donors Transfer to the nonreducing end of the sugar Regiospecific Can be inver7ng or retaining mechanism

39 Possible mechanisms Inverting mechanism Retaining mechanism

40 Inverting Mechanism Single displacement mechanism Nucleophilic attack of the acceptor on the anomeric center of the donor Requires catalytic amino acid as general base Oxocarbenium-ion transition state α-2,3-sialyltransferase Cst-I from Cj: Chiu et al (2007) Biochemistry 46:7196 (2p2v)

41 Retaining mechanism not settled Double displacement Nucleophilic attack by a catalytic residue on anomeric center of donor (covalent intermediate) Attack by acceptor after deprotonation S N 2-like (S N i) Concerted nucleophilic attack and departure of leaving group

42 LgtC Catalyzes step in lipooligosaccharide synthesis α-1,4-galactosyltransferase bi-bi kinetic mechanism Mechanism not clear, maybe no catalytic amino acid Persson et al (2001) Nat Struct Biol 8:166 (1ga8)

43 Retaining examples Mechanism implied from the decomposition of alkyl chlorosulfites

44 Classifying GTs Solved structures 12 GT-C family members 26 Orphan families

45 Gloster (2014) COSB 28:131 Structural studies of GTs

46 GT-A fold First structure (SpsA) determined in 1999 Two 7ghtly associated β/α/β folds (Rossman-like) Typically contain a DXD mo7f that coordinate a divalent ca7on or ribose Eukaryo7c GT-As oeen have N- terminal transmembrane domain Not just a GT fold Rabbit N-Acetylglucosaminyltransferase I: Ünligil et al (2000) EMBO J 19:5269 (1foa)

47 First GT-A fold SpsA from B. subtilis First example of an inverting mechanism 1qgs; Charnock & Davies (1999) Biochemistry 38:6380

48 GT-B Fold First structure reported in 1994 for β- glucosyltransferase from phage T4 Also two β/α/β folds (Rossman-like) that face each other Each associated with the donor and acceptor substrate binding sites Also other non GT enzymes (e.g. UDP GlcNAc 2- epimerase) Vrielink et al (1994) EMBO J 13:3413 (1bgt)

49 Modularity of GTs Coutinho et al (2003) JMB 328:307