BMB 170 Lecture 17 Carbohydrates, Nov 21 Glycobiology • Carbohydrates • Glycosyltransferases and glycosidases • Protein glycosylation • Glycobiology of bacteria • Glycan binding and the role of glycans

First coined by Raymond Dwek (Oxford) in 1988 Online resources

• Society for glycobiology http://www.glycobiology.org/ • Consortium for functional glycomics http:// www.functionalglycomics.org/static/index.shtml • Glycosciences (database and tools) http:// www.glycosciences.de/ • French Glyco database http://glyco3d.cermav.cnrs.fr/home.php 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 https://www.ncbi.nlm.nih.gov/books/NBK310274/ Glycobiology

Functions of glycans Model of CD59 glycosylation • Intrinsic functions Structural components N-linked glycan Cell walls Extracellular matrix O-linked glycan Modifying proteins Solubility Stability

• Extrinsic functions (glycan/lectin) GPI anchor Directing trafficking Cell/cell and cell matrix adhesion Modulating signaling

Rudd et al (1997) JBC 272:7229 • 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 What is a Carbohydrate? • An aldehyde or ketone derivative of a polyhydroxy alcohol that is synthesized in living cells

• Simplest carbohydrate is a monosaccharide : (CH20)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 Representing an oligosaccharide

Taylor & Drickhamer Intro to Glycobiology Fig 1.9 Monosaccharide symbol set

a b

c

Essenals of Glycobiology Second Edion Chapter 1, Figure 5 Sugar Abbreviation Terms and Hexose Hex abbreviations Glucose Glc Galactose Gal Mannose Man • Glycosyltransferase (adds sugar) Rhamnose Rha • Glycosidase (trims sugar) Fucose Fuc • Lectin (binds sugar) Glucosamine GlcN • Epimerase (inverts –OH Galactosamine GalN to convert between sugar Mannosamine ManN isomers) N-acetylglucosamine GlcNAc • Flippase (flips between N-acetylgalactosamine GalNAc lipid leaflets) N-acetylmannosamine ManNAc • Mutarotase (flips Glucuronic acid GlcA anomeric carbon) Galacturonic acid GalA Mannuronic acid ManA N-acetylneuramic acid NeuNAc 3-deoxy-D-manno-2-octulosonic acid Kdo 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 Fischer Projection

Horizontal bonds are in front of the plane of the page. Vertical bonds are behind the plane of the page. Common D- Aldoses Common D-Ketoses Haworth Projections

• Sir Norman Haworth FRS – 1937 Nobel for carbohydrates & vitamin C • First carbon is anomeric carbon

Glucose Vitamin C (ascorbic acid) Anomers • Two stereoisomers ( designated as α and β) of a given sugar that differ only in the configuration about the carbonyl (anomeric) carbon atom Mutarotation • The α- and β-forms of glucose can be isolated separately – Pure α-glucose has a specific rotation of +112o – Pure β-glucose has a specific rotation of +18.7o • Mutarotation is the change in optical rotation as an equilibrium mixture of anomers forms – α- or β-glucose in aqueous solution slowly changes to +52.7o – 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 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 Non-reducing sugar

Disaccharides

Reducing sugar

Reducing sugar Torsion angles that define the conformaon of the glycosidic linkages φ, ψ, and ω

1→6 linkage

C1-O1 bond C6′-O1 bond φ for 1→6 ψ for 1→6

C5′-C6′ bond ω for 1→6

Essenals of Glycobiology Second Edion Chapter 2, Figure 18 Carbohydrate diversity

• Two residues – Amino acids 2 – Monosaccharides 11 • Four residues – Amino acids 256 – Monosaccharides >35,000 • Not to mention many modifications! 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 Examples of typical N- and O-linked glycans

Essenals of Glycobiology Second Edion Chapter 2, Figure 19 Sialic acids

Most Mammals but Aquatic abundant not people 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) 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) Plant Starch Amylose (30% of starch) Amylopectin (branching every 20-30 Unbranched D-glucose-α(1,4)-D-glucose residues)

• Starch is the plant form of carbohydrate ingested by humans. • α-amylase secreted by salivary glands and the pancreas hydrolyzes amylose and amylopectin. 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 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 Glycolipids

• Phosphadylinositol (PIP, PIP2, etc) • Glycosphingolipids – Forms the calyx – extracellular matrix of cells • GPI (glycosylphosphadylinositol) – Links to C-terminus of proteins as an anchor • Pepdoglycan • Lipid A Structures of representative glycosphingolipids and glycoglycerolipids

Glycosphingolipid Glycoglycerolipid

• Found in cell membranes of all organisms • Base structure for “lipid rafts” • Animals are mostly glycosphingolipid • GalCer is the most abundant in the brain Chapter 10, Figure 1 – Structure determined in 1884 – Sphingo from sphinx because the Essenals of Glycobiology Second Edion structure was hard to determine General structure of GPI anchors

R3

Essenals of Glycobiology Second Edion Chapter 11, Figure 1 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 2.4.1.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 CAZy

hp://www.cazy.org Carbohydrate-acve enzymes (CAZymes) as a funcon of the number of genes in the genomes of different organisms

Essenals of Glycobiology Second Edion Chapter 7, Figure 1 Glycosyltransferases • Discovered by Luis Leloir (1970 Nobel Prize in Chemistry for elucidang glycogen synthesis) • Generally catalyze the transfer of a monosaccharide unit from an acvated sugar phosphate to an acceptor • Nearly every glycosidic linkage is made by a disnct enzyme High mannosse N-linked glycan 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) Products

• Energy storage • Structural • Secondary metabolites • Antibiotics • Glycolipids

“Chemistry of Glycosyltransferases” Weadge & Palcic (2008) DOI: 10.1002/9780470048672.wecb213 Glycosyl donors • Phosphate activated sugars and lipids • Acceptors – Saccharides – Polysaccharides – Lipids – Proteins – Other natural products

“Chemistry of Glycosyltransferases” Weadge & Palcic (2008) DOI: 10.1002/9780470048672.wecb213 Leloir-type GTs

• Defined as those with sugar-nucleode donors • 90% of annotaons • UDP/TDP nucleodes represent 60% of the donors • Transfer to the non- reducing end of the sugar • Regiospecific • Can be inverng or retaining mechanism Possible mechanisms

Inverting mechanism

Retaining mechanism 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) Retaining mechanism not settled

• Double displacement – Nucleophilic attack by a catalytic residue on anomeric center of donor (covalent intermediate) – Attack by acceptor after deprotonation

• SN2-like (SNi) – Concerted nucleophilic attack and departure of leaving group 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) Retaining examples

Mechanism implied from the decomposition of alkyl chlorosulfites Classifying GTs

Solved structures 12 GT-C family members 26 Orphan families Structural studies of GTs

Gloster (2014) COSB 28:131 GT-A fold

• First structure (SpsA) determined in 1999 • Two ghtly associated β/α/β folds (Rossman-like) • Typically contain a DXD mof that coordinate a divalent caon or ribose • Eukaryoc GT-As oen have N- terminal transmembrane domain • Not just a GT fold

Rabbit N-Acetylglucosaminyltransferase I: Ünligil et al (2000) EMBO J 19:5269 (1foa) First GT-A fold SpsA from B. subtilis

First example of an inverting mechanism

1qgs; Charnock & Davies (1999) Biochemistry 38:6380 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) Modularity of GTs

Coutinho et al (2003) JMB 328:307