O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc Notch Thrombospondin Factor IX Yeast mannoproteins a-dystroglycan Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc Ser/Thr GlcNAc Notch Coagulation Factors Fibrinolytic Factors Nuclear Proteins Cytoplasmic Proteins SEPARATE LECTURE Mucins ALSO: Proteoglycans, Hydroxyproline/Hydroxylysine Glycosylation After Esko, J

O-Glycosidic Linkage O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry) b-elimination GalNAc GalNAc a a Ser After Esko, J

Glycan synthesis in a cellular context Most O-Glycosylated proteins are synthesized in the secretory pathway

O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc GlcNAc

Mucin-Type O-GalNAc Glycans b4 b3 a3 Ser/Thr b6 Major extracellular vertebrate O-glycan Begins in cis-Golgi by attachment of GalNAc in a-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

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). >20 ppGalNAcT family members Share structural features in active site Some have lectin (ricin) domain After Esko, J

Core 1 and Core 2 Synthesis T (TF) Antigen Tn Core 2 GlcNAcT Core 1 GalT (cis) Ser/Thr b3 b6 a6 a3 ST6GalNAc1 (trans) Ser/Thr Sialyl Tn Antigen b3 Disialyl T After Esko, J

From: Tongzhong Ju and Richard D. Cummings

Core 3 and Core 4 Synthesis GlcNAcT Core 4 GlcNAcT b3 b3 b6 Ser/Thr Ser/Thr Ser/Thr After Esko, J

Unusual Core O-Glycan Structures Ser/Thr b3 Core 4 b6 Core 1 Core 2 Core 7 Ser/Thr a6 Core 6? b6 Core 5 a3 Core 8 After Esko, J

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 tends to be clustered - bottle brush Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts After Esko, J

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

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

Questions What is the function of multiple polypeptide GalNAc transferases? How is tissue specific expression of transferases regulated? How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues? What roles do chaperones play? After Esko, J

O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc GlcNAc

O-Fuc Two Flavors: Mono and Tetrasaccharide One of the clearest examples of glycosylation (Fringe) modulating signal transduction What other proteins carry O-Fuc and how does glycosylation modulate activity? How is glycosylation regulated? After Esko, J

O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc GlcNAc

O-Glc Pathway Shao, L. et al. Glycobiology 2002 12:763-770; doi:10.1093/glycob/cwf085

O-Glc Always a trisaccharide? Glc & Xyl (except for proteoglycans) rarely used on mammalian glycoproteins--why both here? Many of the same proteins as O-Fuc modifed, why? Role in Modulating Signaling? Regulated by enzymes or sugar nucleotide availability? After Esko, J

O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc GlcNAc

Alpha-Dystroglycan

Muscular Dystrophies associated with glycosylation of a - DG Disease Species Affected Gene Biochemical Lesion Biochemical Phenotype Walker - Warburg Syndrome Human POMT1 O Man addition to Ser/Thr Decreased protein O mannosylation Muscle Eye Brain D isease POMGnT1 Addition of GlcNac b 2 to O Man Underglycosylated a DG, uncapped O Fukuyama type MDC Fukutin Glycosyltransferase like protein DG Limb Girdle and MDC 1C Related Protein l ike Golgi protein Myodystrophy, myd Mouse MDC 1D LARGE like Golgi protein MDC, Congenital Muscular Dystrophy; POMT, Protein Mannosyltransferase; POMGnT1, Protein Mannose, N cetylglucosaminyltransferase 1

POMT1 in the ER

Figure 3: Glycans linked to Ser/Thr through Man or GalNAc in mammalian brain and muscle. Detected structures on a-DG highlighted by red checks.

O-Man O-Man is clearly involved in CMD What mammalian proteins (especially in the brain) are O-Man modified besides a-DG? What are the functions of fukutin and large in O-mannosylation? Why the heterogeneity in O-Man structures, what specific structures at what sites on the protein modulate specific interactions? What is relationship between O-Man and O-GalNAc? After Esko, J

O-Glycosylation Ser/Thr GalNAc Ser/Thr Man Ser/Thr Fuc Ser/Thr Glc GlcNAc O-GlcNAc and O-Glycosylation of Hydroxyproline ---SEPARATE LECTURES (Wells, West, & Hahn)

O-Glycosylation of Hyl

O-Glycosylation of Hyl Found on Collagen and Adiponectin (which has a “collagen-like” domain) Glycosylation Essential for Basement Membrane Formation in Tissues Modulates Collagen Cross-linking? Other proteins with modification?

REALLY COMPLICATED O-Glycosylation Carl Bergmann

The Glycosaminoglycans After Esko, J

Hyaluronan (HA) After Esko, J b 4 3 b4 b3 GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA Abundant in skeletal tissues, synovial fluid, skin, and vitreous Ovulation/ fertilization Angiogenesis Cell migration Macrophage stimulation Morphogenesis and differentiation After Esko, J

Karl Meyer, Columbia University Simoni, R. D. et al. J. Biol. Chem. 2002;277:e27

Physical Properties After Hascall and Laurent Gels of high viscosity, and 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. There is both a polar and a hydrophobic face for interaction with other macromolecules HA distribution HA physical properties Discuss intramoleuclar hydrogen bonds stabilize structure. System will recover after breaking these interactions. After Hascall and Laurent

After Weigel, P HA synthase(s) located in plasma membrane                                                                        HA synthase(s) located in plasma membrane Copolymerization of UDP-GlcNAc and UDP-GlcA occurs independently of a core protein HA can contain 250-25,000 disaccharides (105- 4x 107 Da, ~10 µm, the length of an erythrocyte) Half-life rate ranges from 2 weeks in synovial fluid to 5 minutes in the bloodstream After Weigel, P

Diagram of a putative metabolic scheme for hyaluronan degradation. From Stern, R.

Hyaluronan Binding Proteins Aggrecan forms link-protein stabilized complexes with HA, load bearing function After Esko, J

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

Albert Dorfman, University of Chicago Kresge, N. et al. J. Biol. Chem. 2005;280:e28

Chondroitin Sulfate After Esko, J b 4 3 b4 b3 6S 6S 6S 4S 4S 4S IdoA 6S 6S 6S b 4 3 b4 b3 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

Extracellular Matrices Epithelial cells produce basement membranes composed of proteoglycans, reticular collagens and glycoproteins Laminin Entactin/Nidogen Collagen Type IV Perlecan: Heparan Sulfate Proteoglycan Bamacan: Chondroitin Sulfate Acts as a selective barrier to the movement of cells Tissue regeneration after injury, provides a scaffolding along which regenerating cells can migrate Kidney glomerulus has an unusually thick basement membrane that acts as a molecular filter dependent on perlecan, a heparan sulfate proteoglycan Neuromuscular junction basement membrane contains the heparan sulfate proteoglycan agrin, essential for synaptogenesis After Esko, J

Aggrecan CS/KS brain, cartilage Versican CS brain, mesenchyme Secreted PGs Aggrecan CS/KS brain, cartilage Versican CS brain, mesenchyme Neurocan CS brain Brevican*CS brain Decorin CS/DS brain, connective tissue Biglycan CS/DS brain, connective tissue Testican CS/HS brain, testis Perlecan HS brain, basement membrane Dystroglycan HS brain, muscle Agrin HS brain, muscle Claustrin KS brain Glycoword

NG2-PG CS brain, cartilage Transmembrane PGs NG2-PG CS brain, cartilage RPTP zeta/beta (phosphacan) CS/KS brain, cartilage Neuroglycan C CS brain Appican CS brain, connective tissue N-syndecan HS brain SV2 KS brain GPI-anchored PGs Glypican HS brain, muscle K-glypican HS brain, kidney Cerebroglycan HS brain Glycoword

Ulf Lindahl, Uppsala University

Heparin/Heparan Sulfate IdoA GlcNAc 6 S 6 S 6 S N S N S N S 2 S N S GlcA Gal Gal Xyl 3 S 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 After Esko, J

Biosynthesis of a Heparin/Heparan Sulfate Chain GlcNAc/S 6-O-sulfotransferases (6OST) (3+ isozymes) 6 S Copolymerase Complex EXT1/EXT2 IdoA Epimerase GlcNAc EXTL3 EXTL2? 6 S 6 S Xyl GlcA Gal Gal N S N S 2 S Uronic acid 2-O-sulfotransferase N S N S 3 S GlcNH2/S 3-O-sulfotransferases (3OST) (6 isozymes) GlcNAc N-deacetylase N-sulfotransferases (NDST) (4 isozymes) After Esko, J

Heparan Sulfate Proteoglycans Chondroitin sulfate 4S GlcA GlcNAc IdoA After Esko, J

The Heparin Anti-thrombin III binding motif Lindahl, U

Heparin’s function in the mast cell appears to have nothing to do with disturbed blood coagulation. Heparin is discharged from the mast cell only in special emergency situations, such as anaphylactic shock or other inflammatory reactions. Marine mussels have no blood in the conventional sense to anticoagulate, yet the polysaccharide in the "mast cells" turns out to contain the specific antithrombin-binding pentasaccharide sequence. It also has very high anticoagulant activity. It seems reasonable to assume that the clams contain a protease inhibitor related to antithrombin (belonging to the serpin family), but for a functional reason quite unrelated to blood coagulation.

Keratan sulfate I (corneal) and II (skeletal) Figure 11.4. Keratan sulfates consist of a sulfated polylactosamine linked either to Asn or Ser/Thr residues. The actual order of the various sulfated and nonsulfated disaccharides occurs somewhat randomly along the chain. Not shown are sialic acid and fucose residues that may be present at the termini of the chains.

Golgi processing of Glycosaminoglycans

Proteoglycan Turnover Shedding by exoproteolytic activity, MMP-7 for one 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 P l a s m e b r n E d o c y t i S p 1 g 2 3 x u f G H h ( ~ k D ) 5 Mucopolysaccharidoses After Esko, J

Mucopolysaccharidoses - Lysosomal Storage diseases I H: Hurler's a-L-iduronidase corneal clouding; dwarfism; mental retardation; early mortality MPS I S: Scheie's a -L-iduronidase corneal clouding; aortic valve disease; joint stiffening; normal intelligence and life span MPS I H/S: Huler/Scheie a -L-iduronidase similar to both I-H and I-S MPS II: Hunter's L-iduronate-2-sulfatase mild and severe forms, X-linked, also a possible autosomal form, facial and physical deformities; mental retardation MPS III A: Sanfilippo(A) Heparan N-sulfatase skin, brain, lungs, heart and skeletal muscle are affected in all 4 types of MPS-III MPS III B: Sanfilippo(B) N-acetyl-a -D-glucosaminidase congestive heart failure; progressive mental retardation MPS III C: Sanfilippo(C) N-acetylCoA: a-glucosamine- N-acetyltransferase coarse facial features; organomegaly MPS III D: Sanfilippo(D) N-acetyl-a -glucosamine-6-sulfatase moderate physical deformities; progressive mental retardation MPS IV A: Morquio's(A) N-acetylgalactosamine- 6-sulfatase corneal clouding, thin enamel, aortic valve disease, skeletal abnormalities MPS IV B: Morquio's(B) ß -galactosidasemild skeletal abnormalities, normal enamel, hypoplastic odontoid, corneal clouding MPS VI Maroteaux-Lamy N-acetylgalactosamine- 4-sulfatase 3 distinct forms from mild to severe, aortic valve disease, normal intellect, corneal clouding, coarse facial features MPS VII Sly ß –glucuronidase hepatosplenomegaly, dystosis multiplex Michael W. KING, IU School of Medicine

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