Synthesis and Structure of Major Glycan Classes 1/24/05

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1 Synthesis and Structure of Major Glycan Classes 1/24/05

2 Large O-linked Glycosaminoglycans and polylactosamine structures Glycoprotein N-linked and O- linked oligosaccharides Glycolipid oligosaccharides

3 The building blocks = Glucose (Glc) Hexose, = Mannose (Man) unspecified (Hex) = Galactose (Gal) = N-acetylglucosamine (GlcNAc) = N-acetylgalactosamine (GalNAc) = N-acetylhexosamine, unspecified (HexNAc) = Fucose (Fuc) = Xylose (Xyl) = Sialic acid, unspecified (Sia) = Glucuronic acid (GlcA) = Iduronic acid (IdoA) = Uronic acid, unspecified (HexA) Simplified Traditional Fucα 3 Siaα3Galβ4GlcNAcβ2Manα Fucα 6 6 Manβ4GlcNAcβ4GlcNAc 3 9Ac-Siaα6Galβ4GlcNAcβ2Manα Symbolic Representation 9Ac α3 α3 β 4 β2 α6 β 4 β2 α 6 3 α β4 α6 β4 9Ac α3 α3 β4 β4 α3 β2 β2 α6 α3 β4 α6 β4-9ac After Varki, A

4 Glycan synthesis in a cellular context

5 Overview From ER through Trans-Golgi and points inbetween

6 ER processing of N-linked glycans

7 Major Classes of N-Glycans Hybrid Complex High-Mannose (oligo-mannose) After Varki, A

8 GlcNAc Man Glc Gal Sia Fuc Biosynthesis of N-Glycans: Production of GlcNAc-P-P-Dolichol Tunicamycin Blocks - not very specific! Dolichol Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

9 GlcNAc Man Glc Gal Sia Fuc Biosynthesis of the N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER) CDG = Congenital Disorder of Glycosylation in Humans Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

10 GlcNAc Man Glc Gal Sia Fuc Biosynthesis of the N-Glycan Precursor on Lumenal Leaflet of ER Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

11 GlcNAc Man Glc Gal Sia Fuc Completion of Biosynthesis of N-Glycan Precursor on Lumenal Leaflet of ER - and Transfer to Protein Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

12 Oligosaccharyltransferase complex (OST) in the ER membrane transfers the dolichol N-glycan precursor to asparagine residues on nascently translated proteins Target sequon for N-glycosylation Necessary but not sufficient X = any amino acid except proline Rarely can be Asn-X-Cys Transfer cotranslational/immediate post-translational before folding ~2/3 of proteins have sequons ~ 2/3 sequons actually occupied (some variably) Yeast OST complex contains nine membrane-bound subunits After Varki, A

13 GlcNAc Man Glc Gal Sia Fuc ER Initial Processing of N-Glycans in the ER and Golgi Golgi Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

14 Calnexin (and Calcireticulin) function during glycoprotein folding in the endoplasmic reticulum Improperly folded proteins are re-glucosylated by glucosyltransferase which acts as sensor for improper folding 3 Glucose Residues After Varki, A

15 ER glycolipid synthesis

16 Biosynthesis of Ceramide and Glucosylceramide

17 ER glycolipid synthesis

18 Basic Glycosylphosphatidylinositol (GPI) Anchor Phospholipid After Hart, G

19 Examples of GPI-Anchored Proteins Cell surface hydrolases alkaline phosphatase acetylcholinesterase 5 nucleotidase Protozoal antigens trypanosome VSG leishmanial protease plasmodium antigens Adhesion molecules neural cell adhesion molecule heparan sulfate proteoglycan Mammalian antigens carcinoembryonic antigen Thy-1 Others scrapie prion protein folate receptor decay accelerating factor After Hart, G

20 Structure of the Basic GPI Anchor GPI-Linked Protein Etn P NH 2 = Mannose (Man) = Glucosamine = Ethanolamine = Phosphate PNH Defect Etn P Pig-A NH 2 INOSITOL P Cell Surface Membrane After Hart, G

21 Structural Analysis of the GPI Anchor Enzymatic and chemical cleavage sites are useful in identifying GPI anchored membrane proteins After Hart, G

22 Examples of C-Terminal Sequences Signaling the Addition of GPI-Anchors Protein Acetylcholinesterase (Torpedo) Alkaline Phosphatase (placenta) Decay Accelerating Factor GPI-Signal Sequence NQFLPKLLNATAC DGELSSSGTSSSKGIIFYVLFSILYLIFY TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP HETTPNKGSGTTS GTTRLLSGHTCFTLTGLLGTLVTMGLLT PARP (T. Brucei) EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF Prion Protein (hamster) QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG Thy-1 (rat) KTINVIRDKLVKC GGISLLVQNTSWLLLLLLSLSFLQATDFISI Variant Surface Glycoprotein (T. Brucei) ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF Bold AA is site of GPI attachment Sequence to right is cleaved by the transpeptidase upon Anchor addition After Hart, G

23 Golgi processing of N-linked glycans

24 GlcNAc Man Glc Gal Sia Fuc Completion of Processing of N-Glycans in ER and Golgi Final products often show microheterogeneity at each N-Glycosylation site Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

25 GlcNAc-Transferases Determine Number of Antennae of N-glycans After Varki, A

26 Some representative examples of mammalian complex-type N-glycans After Varki, A

27 Evolutionary Variations of the N-glycan Processing Pathway Yeast Slime Mold Paucimannose Plants Insects α3 N Asn α3 N Asn 6α α3 6α β 2 α4 α4 α 4 α3 N Asn N Asn α6 Vertebrates After Varki, A

28 Golgi processing of O-linked glycans

29 O-Glycosidic Linkage H HO OH OH H H H GalNAc O NAc O α H O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry) β-elimination H 2 C C O CH Ser NH 2 After Esko, J

30 Examples of O-Glycans Yeast mannoproteins α-dystroglycan Nuclear Proteins Cytoplasmic Proteins GalNAc Man Fuc GlcNAc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Mucins Notch Coagulation Factors Fibrinolytic Factors After Esko, J

31 Mucin-Type O-GalNAc Glycans α3 α3 β4 β3 β4 β3 α3 β4 β3 β6 α Ser/Thr Major vertebrate O-glycan Begins in cis-golgi by attachment of GalNAc in α- linkage to specific Ser/Thr residues Assembly is simpler than N- linked chains - no lipid intermediate is used Always involves nucleotide sugars Always occurs by addition to non-reducing terminus or by branching After Esko, J

32 Polypeptide GalNAc Transferases Regions in white, pink, red, and black represent, respectively, 0 29%, 30 69%, 70 99%, and 100% sequence identity (Hagen et al. (2003) Glycobiology 13:1R-16R). After Esko, J 12 members of mammalian ppgalnact family Estimated size of family = 24 Share structural features in active site Some have lectin (ricin) domain

33 Core 1 and Core 2 Synthesis Core 1 GalT Core 2 GlcNAcT β3 β3 β6 Ser/Thr Ser/Thr Ser/Thr After Esko, J

34 Core 3 and Core 4 Synthesis Core 3 GlcNAcT Core 4 GlcNAcT β3 β3 β6 Ser/Thr Ser/Thr Ser/Thr After Esko, J

35 Unusual Core O-Glycan Structures Core 1 Core 2 Core 3 Core 4 β3 β3 β6 β3 β3 β6 Ser/Thr Ser/Thr Ser/Thr Ser/Thr Core 5 Core 6? Core 7 Core 8 α3 β6 α6 α3 Ser/Thr Ser/Thr Ser/Thr Ser/Thr After Esko, J

36 Mucins are Heavily O-glycosylated Apomucin contain tandem repeats (8-169 amino acids) rich in proline, threonine, and serine (PTS domains) Glycosylation constitutes as much as 80% of mass and tend clustered - bottle brush Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts After Esko, J

37 Mucin Production Goblet cells in intestinal crypts Lung Epithelium After Esko, J

38 Mucins: Protective Barriers for Epithelial Cells Lubrication for epithelial surfaces Modulate infection: Receptors for bacterial adhesins Secreted mucins can act as decoys Barrier against freezing: Antifreeze glycoproteins [Ala-Ala-Thr] n 40 with Core 1 disaccharides After Esko, J

39 Questions What is the function of multiple polypeptide GalNAc transferases? Do various transferases within a family act on the same or different substrates? How is tissue specific expression of transferases regulated? How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues? After Esko, J

40 Golgi processing of Glycosphingolipids

41 Major Classes of Glycosphingolipids Series Designation Core Structure Lacto (LcOSe4) Galβ3GlcNAcβ3Galβ4Glcβ1Ceramide Lactoneo (LcnOSe4) Galβ4GlcNAcβ3Galβ4Glcβ1Ceramide Globo (GbOSe4) GalNAcβ3Galα4Galβ4Glcβ1Ceramide Isoglobo (GbiOSe4) GalNAcβ3Galα3Galβ4Glcβ1Ceramide Ganglio (GgOSe4) Galβ3GalNAcβ4Galβ4Glcβ1Ceramide Muco (MucOSe4) Galβ3Galβ3Galβ4Glcβ1Ceramide Gala (GalOSe2) Galα4Galβ1Ceramide Sulfatides 3-0-Sulfo-Galβ1Ceramide Different Core structures generate unique shapes and are expressed in a cell-type specific manner After Varki, A

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43 Turnover and Degradation of Glycosphingolipids Internalized from plasma membrane via endocytosis Pass through endosomes (some remodelling possible?) Terminal degradation in lysosomes - stepwise reactions by specific enzymes. Some final steps involve cleavages close to the cell membrane, and require facilitation by specific sphingolipid activator proteins (SAPs). Individual components, available for re-utilization in various pathways. At least some of glucosylceramide may remain intact and be recycled Human diseases in which specific enzymes or SAPs are genetically deficient ( storage disorders

44 Biological Roles of Glycosphingolipids Thought to be critical components of the epidermal (skin) permeability barrier Organizing role in cell membrane. Thought to associate with GPI anchors in the trans-golgi, forming rafts which target to apical domains of polarized epithelial cells May also be in glycosphingolipid enriched domains ( GEMs ) which are associated with cytosolic oncogenes and signalling molecules Physical protection against hostile environnments Binding sites for the adhesion of symbiont bacteria. Highly specific receptor targets for a variety of bacteria, toxins and viruses.

45 Biological Roles of Glycosphingolipids Specific association of certain glycosphingolipids with certain membrane receptors. Can mediate low-affinity but high specificity carbohydrate-carbohydrate interactions between different cell types. Targets for autoimmune antibodies in Guillian-Barre and Miller-Fisher syndromes following Campylobacter infections and in some patients with human myeloma Shed in large amounts by certain cancers - these are found to have a strong immunosuppressive effects, via as yet unknown mechanisms

46 Ganglioside synthetic enzyme KOs GlcCer synthase (GlcT): Early embryonic lethal, differentiation and germ layer formation okay LacCer synthase (GalT):?? GM3 synthase (SiaTI): Increased sensitivity to insulin (skeletal muscle receptor phosphorylation) GM2 synthase (GalNAcT): Male sterility, demyelinating disorder GD2 synthase (SiaTII): Ok GM2 synthase/gd2 synthase double KO: sudden death, auditory seizures, demylination

47 Sheikh, KA, et al. (1999) PNAS 96, 7532

48 Golgi processing of Glycosaminoglycans

49 After Esko, J

50 But first, an exception: Hyaluronan (HA) β4 β3 β4 β3 β4 β3 β4 β3 β4 β3 n 1000 GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA After Esko, J Abundant in skeletal tissues, synovial fluid, and skin Synthesis is elevated in expanding tissues (morphogenesis, invasion)

51 Physical Properties After Esko, J Gels of high viscosity, but a great lubricant since at high shear its viscosity drops, but remains resilient Interglycosidic H-bonding restricts rotations across glycosidic bonds Promotes rapid recovery after mechanical perturbations Hydrated matrices rich in hyaluronan expand the extracellular space, facilitating cell migration.

52 HA synthase(s) located in plasma membrane, spans membrane 12 times Copolymerization of UDP-GlcNAc and UDP-GlcA occurs independently of a core protein HA can contain ,000 disaccharides ( Da, ~2 µm in length) After Esko, J

53 After Esko, J Hyaluronan Binding Proteins

54 Chondroitin Sulfate IdoA 6S 6S 6S β4 β3 β4 β3 β4 β3 β4 β3 β4 β3 4S 4S 4S GalNAc GlcA Non-sulfated chondroitin is rare in vertebrates, but multiple types of sulfated chondroitins are known (A, B, C, D, etc) Multiple sulfotransferases decorate the chain An epimerase can flip the stereochemistry of D-GlcA to L-IdoA (Dermatan Sulfate) The chains are easily characterized using bacterial chondroitinases which degrade the chain to disaccharides After Esko, J

55 Cartilage - Proteoglycan Aggregates Aggrecan Aggrecan: Large chondroitin sulfate proteoglycan present in cartilage and other connective tissues Core protein ~400 kda Hyaluronic Acid ~100 chondroitin sulfate chains of ~20 kda High charge density creates osmotic pressure that draws water into the tissue (sponge) Absorbs high compressive loads, yet resilient Forms aggregates with hyaluronic acid (HA) After Esko, J

56 Extracellular Matrices Epithelial cells produce basement membranes composed of heparan sulfate proteoglycans, reticular collagens and glycoproteins Bamacan: Chondroitin Sulfate Proteoglycan Perlecan: Heparan Sulfate Proteoglycan Collagen Type IV After Esko, J Laminin Entactin/Nidogen

57 Heparan Sulfate Proteoglycans 6S 6S 6S NS2SNS2SNS 6S 6S 6S NS NS2SNS 3S NS NS NS 4S 4S 4S 4S 4S GlcNAc GlcA IdoA Chondroitin sulfate After Esko, J

58 Heparan Sulfate 6S IdoA 6S 6S GlcNAc NS NS 3S NS 2S NS GlcA Gal Gal Xyl After Esko, J Characterization of heparan sulfate is based on different criteria - GlcNAc vs GlcNS - 3-O-Sulfo and 6-O-sulfo groups -IdoA vs GlcA Heparinases degrade chain into disaccharide units Nitrous acid degrades chains at GlcNS Disaccharides characterized by HPLC or mass spectrometry

59 Biosynthesis of a Heparan Sulfate Chain Copolymerase Complex EXT1/EXT2 Epimerase 6S IdoA GlcNAc/S 6-O-sulfotransferases (6OST) (3 + isozymes) 6S 6S EXTL3 EXTL2? GlcNAc NS NS 3S NS 2S NS GlcA Gal Gal Xyl GlcNAc N-deacetylase N-sulfotransferases (NDST) (4 isozymes) Uronic acid 2-O-sulfotransferase GlcNH 2 /S 3-O-sulfotransferases (3OST) (6 isozymes) After Esko, J

60 Proteoglycan Turnover Shedding by exoproteolytic activity, MMP-7 for one HS chain Shedding Endosulfatase recently discovered that removes sulfate groups on proteoglycans at cell surface: remodeling Heparanase (endohexosaminidase) clips at certain sites in the chain. Outside cells, it plays a role in cell invasion processes Inside cells it s the first step towards complete degradation in lysosomes by exoglycosidases and sulfatases Golgi HS oligosaccharide (~10 kda) HS oligosaccharide (~5 kda) Endocytosis Plasma membrane Step1 protease endoglycosidase Step 2 endoglycosidase Step3 exoglycosidase sulfatase Mucopolysaccharidoses After Esko, J

61 GAG Summary Proteoglycans contain glycosaminoglycans: chondroitin sulfate, dermatan sulfate, or heparan sulfate Heparan sulfate, Chondroitin, and dermatan sulfate proteoglycans are found in the matrix and play structural roles in cartilage, bone and soft tissues and are found at the cell surface and play roles in cell adhesion and signaling during development Proteoglycans in the extracellular matrix can also act as a reservoir of growth factors, protect growth factors from degradation, and facilitate the formation of gradients Human diseases in proteoglycan assembly are rare Degradation of these compounds is also important (MPS) After Esko, J

62 Overview

63 General Principles Glycan synthesis is compartmentalized within cells Precursors begin as lipid-linked species on the cytoplasmic face of the ER, requiring that substrates be flipped for further processsing Addition of N-linked glycans generally occurs cotranslationally ER glycosylation may have evolved as a method for quality control, Golgi modifications are layered on top of this well-conserved function Within the Golgi and distal secretory pathway, specific glycan structures can impart targeting information The assembly-line model holds well for N-linked proteins but may not be as applicable to glycolipid and GAG synthesis