The Essentials of Nucleotide Sugars Wells

Sugar Nucleotides (high energy donors) in Mammals What mammalian sugar is missing?

General Principles for Synthesis 1: initiated by kinase reaction, consumes 2 NTP example: Glc  Glc-6-PGlc-1-PUDP-Glc 2: multiple ways (epimerization, decarboxylation, etc.) example: UDP-GlcNAcUDP-GalNAc ; UDP-GlcA UDP-Xyl 3: nucleotide-exchange reaction example Gal-1-P +UDP-Glc  Glc-1-P + UDP-Gal

Glucose is only Sugar Needed for Synthesis of All Sugar Nucleotides Note: No salvage Pathway for GlcA! Key Control Points: Early step enzymes that are inhibited by end-product Sugar nucleotide

Dolichol phosphate: The “other” high energy sugar donor Chapter 8, Figure 2 Essentials of Glycobiology Second Edition Dol-P-Man N-link, O-Man, C-Man, GPI anchors Dol-P-Glc N-linked biosynthesis Dol-P-GlcNAc N-linked biosynthesis

Really complex O-glycans: Sulfated glycosaminoglycans and hyaluronic acid Tiemeyer with significant input from Carl Bergmann (CCRC) and Jeffrey Esko (UCSD)

The glycospace of animal glycans

The Glycosaminoglycans

Keratan sulfate I (corneal) and II (skeletal) Keratan sulfates consist of a sulfated polylactosamine extended on either Asn or Ser/Thr linked glycan. 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.

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

Vulval Morphogenesis in C. elegans The vulva is a passageway that accepts sperm from males and expels eggs from the gonad

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

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

Chondroitin Proteoglycan (non-sulfated) Xyl Gal GlcA Chondroitin is one type of glycosaminoglycan (GAG) chain GalNAc

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

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

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

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

After Hascall and Laurent Physical Properties 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

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

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

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

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

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

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

Chondroitin Sulfate 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

CS types, not mutually exclusive Taken from Essentials of Glycobiology, 1st edition

Chondroitin Sulfate Proteoglycan

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 Aggrecan Hyaluronic Acid

Diseases - Achondroplasias Gene Mice Human Aggrecan Cartilage Matrix Deficiency (CMD) ? Sulfate Transporter (DTDST) - Atelosteogenesis Type II Achondrogeneis Type Ib Diastrophic dysplasia (DTD) PAPS synthetase Brachymorphism Spondylo- epimetaphyseal dysplasia PAPS ATP SO42- DTDST out in GAG Sulfation

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

Interstitial Proteoglycans SLRPs - Small Leucine Rich Proteoglycans Gene Knockouts Decorin (single CS chain) Fragile skin Thin dermis Increased resistance to Borrelia burgdorfei Biglycan (two CS chains) Decrease in bone mass and dentin mineralization Fewer osteoblasts Mild muscular dystrophy Fibromodulin (keratan sulfate Ectopic tendon ossification Lumican (keratan sulfate Corneal opacification

Extracellular Matrices Epithelial cells produce basement membranes composed of heparan sulfate 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

Perlecan - Basement Membrane Proteoglycan 467 KDa muscle development and differentiation appear to be normal and the nerve terminals are normally formed Neuromuscular junction of the mutant mouse muscles appear normal. Acetylcholine esterase is completely absent at the neuromuscular junctions, although it is synthesized normally. LDL receptor-like repeats Laminin repeats Ig-like repeats Laminin & EGF motifs Perlecan knockouts: Perinatal lethal chondrodysplasia Human mutations: Schwartz-Jampel syndrome, Silverman-Handmaker type Dyssegmental dysplasia Glycoconj J 19, 263–267, 2003)

Heparan Sulfate Proteoglycans Chondroitin sulfate 4S GlcA GlcNAc IdoA

Heparan Sulfate 6 S 6 S 6 S N S N S N S 2 S N S 3 S 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

Ulf Lindahl, Uppsala University

Biosynthesis of a 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 GlcA Gal Gal Xyl 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)

The Heparin Anti-thrombin III binding motif Heparin vs. Heparan, tomato vs. tomahto? Heparin is the most highly sulfated form of heparan found in nature. Still, signficant microheterogeneity imparts important function. Anti-thrombin III binding to Heparin is periodate sensitive, but other heparin and heparan activities are not. The Heparin Anti-thrombin III binding motif Lindahl, U

Despite heparin’s ability to disturb blood coagulation, it’s normal function in the mast cells that make it is not clear. Heparin is discharged from the mast cell only in special emergency situations, such as anaphylactic shock. Marine mussels have no blood in the conventional sense to anticoagulate (no vasculature), yet the polysaccharide in their "mast cells" contains the specific antithrombin-binding pentasaccharide sequence and is very high in anticoagulant activity. It seems reasonable to assume that mussels express a protease inhibitor related to antithrombin (ie: belonging to the serpin family), but for a functional reason quite unrelated to blood coagulation.

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

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

Syndecans Cell-Binding Domain TM C1 C2 V 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 Transgenic expression of syndecan-1 uncovered a physiological control of feeding behavior by syndecan-3 (Cell 2001, 106:105-116) Bacterial infection seems to depend on syndecan-1 (Nature 2001, 411:98-102)

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 Underlying mechanism unclear Modulation of growth factor(s)?

Biosynthetic Knockouts Gene Mice Human EXT1/EXT2 Embryonic lethal (null) Heterozygotes develop rib exostoses Hereditary Multiple Exostoses NDST1 Perinatal lethal (null) Various developmental defects (forebrain, lung) Tissue specific knockouts have various physiological alterations ? NDST2 Viable, mucosal mast cell deficiency GlcA C5 Epimerase Perinatal lethal (null) Renal agenesis, lung defects, and skeletal malformations Uronosyl 2-O-sulfotransferase Perinatal lethal (null) Renal agenesis,eye and skeletal defects

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

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) b-galactosidase: mild 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 b-glucuronidase: hepatosplenomegaly, dystosis multiplex Michael W. KING, IU School of Medicine

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, chondroitin 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 Regulated degradation of these compounds is also important (MPS)