Sulfated Glycosaminoglycans & Hyaluronan Chapter 11 April 13, 2004 Jeff Esko jesko@ucsd

1. Sulfated Glycosaminoglycans & HyaluronanChapter 11April 13, 2004Jeff Eskojesko@ucsd.edu

2. GLYCOSAMINOGLYCANS (GAGs) CHONDROITIN/DERMATAN SULFATE HEPARAN SULFATE P S S S S S Ser-O- S S S S -O-Ser NS NS 2S NS S N-LINKED KERATAN SULFATE CHAINS S S GLYCO- S Ac PHOSPHOLIPID S ANCHOR S O-LINKED P CHAIN GLYCO- S SPHINGOLIPIDS E t n P S HYALURONIC ACID O N N NH Ser/Thr Asn Ac 2 Asn INOSITOL P Cytosol O = GlcNAc = GlcA Ser = GalNAc = IdoA O-LINKED GlcNAc

3. Skeletal Achondroplasias Simpson-Golabi-Behmel Overgrowth Syndrome (SGBS) Hereditary Multiple Exostoses (HME) Proteoglycans and Human Disease

4. Vulval Morphogenesis in C. elegans

5. wild-type sqv sqv Mutants of C. elegans Herman and Horvitz (1999) described a set of C. elegans mutants defective in vulval development (sqv, squashed vulva) sqv mutations alter epithelial invagination

6. SQV-4 UDP-Glc Dehydrogenase SQV-1 UDP-GlcA Decarboxylase SQV-6 Xylosyltransferase SQV-7 Nucleotide sugar multi-transporter SQV-2 Galactosyltransferase II UDP- UDP- UDP- UDP- Ser UDP- UDP- n UDP- UDP- SQV-5 Chondroitin Synthase SQV-3 Galactosyltransferase I SQV-8 Glucuronosyltransferase I Gal Cytosol Golgi GlcA GalNAc SQV Proteins Encode the Enzymes Required for Chondroitin Biosynthesis Xyl Glc

7. Chondroitin Proteoglycan Xyl Gal GlcA Chondroitin is one type of glycosaminoglycan (GAG) chain GalNAc

8. sqv wild-type One theory of cellular invagination is that the adjacent epithelial cells may secrete a chondroitin sulfate proteoglycan in a polarized fashion Hydration of the matrix might cause expansion and an inward curvature of the cell layer.

9. b4 b3 b 4 b 3 b 4 b 3 b 4 b 3 b 4 b 3 GalNAc GlcA GlcNAc GlcA b4 b3 b 4 b 3 b 4 b 3 b 4 b 3 b 4 b 3 How does this apply to vertebrates? • Non-sulfated chondroitin not found in vertebrates • Instead, they make hyaluronic acid which is similar in structure

10. b4 b3 b 4 b 3 b 4 b 3 b 4 b 3 b 4 b 3 Hyaluronan (HA) n≥1000 • Abundant in skeletal tissues, synovial fluid, and skin • Synthesis is elevated in expanding tissues (morphogenesis, invasion) GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA

11. Physical Properties • 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.

12. 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 250-25,000 disaccharides (105-107 Da, ~2 µm in length)

13. HA Synthases • Three HAS genes (HAS1-3) known in vertebrates • Has2 as the primary source of HA during embryonic development • Has2-/- embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10) Nat Med. 2002 Aug;8(8):850-5 • A single HAS in Streptococcus (capsules) • Assembly process occurs differently • Virulence factor • Molecular mimicry • None detected in insects or nematodes • Homologs of genes suggest a relationship to chitin synthases (GlcNAcb1,4)n Light micrograph of Strep. mutans. From Dr. Timothy Paustian, University of Wisconsin-Madison

14. HA turnover • Eukaryotic hyaluronidase (HYAL) gene family • 3p21.3 • HYAL1, 2 and 3 • Turnover of hyaluronan in most tissues is rapid • t1/2 of ~1 day in epidermal tissues • Large hyaluronan molecules in the extracellular space interact with cell surface receptors • Fragments produced by an associated GPI anchored hyaluronidase, most likely Hyal2. • Fragments transported to lysosomes for complete degradation, most likely involving Hyal1 • HYAL2 null mice are embryonic lethal • Lysosomal storage disorder in a person with a mutation in HYAL1

15. Hyaluronan Binding Proteins

16. Chondroitin sulfate Lectican Family This family of proteoglycans consist of a group of homologous core proteins Have in common an hyaluronic acid binding motif

17. b4 b3 b 4 b 3 b 4 b 3 b 4 b 3 b 4 b 3 Chondroitin Sulfate IdoA 6S 6S 6S 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

18. S S S S S S S S S S S S S S S S S S Chondroitin Sulfate Proteoglycan

19. Aggrecan Hyaluronic Acid Cartilage - Proteoglycan Aggregates • Forms aggregates with hyaluronic acid (HA) • 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

20. ATP SO42- SO42- PAPS GAG Sulfation out DTDST in Diseases - Achondroplasias

21. Extracellular Matrices • Cells are surrounded by an extracellular matrix • Fibroblasts and other connective tissue cells produce a fibrillar matrix (ground substance) composed of small interstitial proteoglycans, fibrillar collagens, and glycoproteins

22. Interstitial ProteoglycansSLRPs - Small Leucine Rich Proteoglycans

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

24. Perlecan knockouts: Perinatal lethal chondrodysplasia Human mutations: Schwartz-Jampel syndrome, Silverman-Handmaker type Dyssegmental dysplasia Glycoconj J 19, 263–267, 2003) Perlecan - Basement Membrane Proteoglycan 467 KDa Ig-like repeats LDL receptor-like repeats Laminin repeats Laminin & EGF motifs

25. 6S 6S 6S NS 2S NS 2S NS 6S 6S 6S NS NS 2S NS 3S NS NS NS 4S GlcA 4S 4S 4S 4S GlcNAc IdoA Chondroitin sulfate Heparan Sulfate Proteoglycans

26. 6 6 S S 6 S N S N S N S 2 S N S 3 S Heparan Sulfate IdoA GlcNAc GlcA Gal Gal Xyl • 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

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

28. Membrane Heparan Sulfate Proteoglycans Syndecans Type I Membrane Proteins 4 members Glypicans GPI anchored proteins 6 members

29. Cell-Binding Domain TM V C1 C2 Syndecans • Syndecan cytoplasmic domains composed of two regions that are conserved among the syndecans (C1 and C2) and a variable region (V) • C2 domain is a binding site for PDZ domains in cytoskeletal proteins (e.g., syntenin) and signaling molecules (e.g., CASK) • These domains are also phosphorylated on tyrosine by PKCa, which may regulate binding • Syndecan core protein can initiate downstream signaling when it participates as an adhesion receptor • Having said all this, syndecan-1 and syndecan-4 knockouts have mild phenotypes. Not clear about syndecan-2 and -3

30. Underlying mechanism unclear Modulation of growth factor(s)? Glypicans • Glypicans have a large globular domain with the 2-3 heparan sulfate chains lying between this and the membrane • Glypicans can initiate downstream signaling and participate as an adhesion receptors in vitro. • Knockout of glypican-1 is unremarkable - redundancy? • Knockout of glypican-3 has remarkable phenotype, identical to Simpson-Golabi-Behmel Syndrome

31. Biosynthetic Knockouts

32. Wnts • TGF-/BMPs • HGF • HB-EGF • Hedgehog • FGF • VEGF • Angiopoietin Signaling Event Mitogenesis Heparan Sulfate Proteoglycans: Co-receptors and Signaling Molecules Heparan sulfate FGF FGF

33. S h e d d i n g H S c h a i n P l a s m a m e m b r a n e E n d o c y t o s i s S t e p 1 p r o t e a s e e n d o g l y c o s i d a s e G o l g i H S o l i g o s a c c h a r i d e S t e p 2 ( ~ 1 0 k D a ) e n d o g l y c o s i d a s e H S o l i g o s a c c h a r i d e ( ~ 5 k D a ) S t e p 3 e x o g l y c o s i d a s e s u l f a t a s e 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 Mucopolysaccharidoses

34. Summary • Proteoglycans contain glycosaminoglycans: chondroitin sulfate, dermatan sulfate, or heparan sulfate • Chondroitin and dermatan sulfate proteoglycans are found in the matrix and play structural roles in cartilage, bone and soft tissues • Tissue architecture • Heparan sulfate and dermatan sulfate proteoglycans are found at the cell surface and play roles in cell adhesion and signaling during development • Growth control, positive and negative • 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)