Presentation on theme: "Sulfated Glycosaminoglycans & Hyaluronan Chapter 11 April 13, 2004 Jeff Esko"— Presentation transcript:
Sulfated Glycosaminoglycans & Hyaluronan Chapter 11 April 13, 2004 Jeff Esko
GLYCOSAMINOGLYCANS (GAGs) HEPARAN SULFATE CHONDROITIN/DERMATAN SULFATE KERATAN SULFATE HYALURONIC ACID SSS P S Ser-O- SSS NS 2S S -O-Ser NH 2 S S S S S O Ser/Thr N Asn N-LINKED CHAINS O-LINKED CHAIN N Asn P Ac O Ser O-LINKED GlcNAc Etn P INOSITOL P GLYCO- PHOSPHOLIPID ANCHOR GLYCO- SPHINGOLIPIDS Ac Cytosol S S S = GlcNAc = GalNAc = GlcA = IdoA S
Proteoglycans and Human Disease Simpson-Golabi-Behmel Overgrowth Syndrome (SGBS) Hereditary Multiple Exostoses (HME) Skeletal Achondroplasias
Vulval Morphogenesis in C. elegans
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
CytosolGolgi Ser SQV-3 Galactosyltransferase I SQV-2 Galactosyltransferase II SQV-8 Glucuronosyltransferase I UDP- SQV-7 Nucleotide sugar multi-transporter n SQV-5 Chondroitin Synthase Glc GlcA Gal Xyl GalNAc SQV Proteins Encode the Enzymes Required for Chondroitin Biosynthesis UDP- SQV-4 UDP-Glc Dehydrogenase UDP- SQV-1 UDP-GlcA Decarboxylase UDP- SQV-6 Xylosyltransferase
Chondroitin Proteoglycan Chondroitin is one type of glycosaminoglycan (GAG) chain GlcA Gal Xyl GalNAc
wild-type sqv 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.
How does this apply to vertebrates? Non-sulfated chondroitin not found in vertebrates Instead, they make hyaluronic acid which is similar in structure GalNAcGlcA GlcNAcGlcA
Hyaluronan (HA) GlcNAcGlcAGlcNAcGlcAGlcNAcGlcAGlcNAcGlcAGlcNAcGlcA n≥1000 Abundant in skeletal tissues, synovial fluid, and skin Synthesis is elevated in expanding tissues (morphogenesis, invasion)
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.
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)
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 Aug;8(8):850-5 Light micrograph of Strep. mutans. From Dr. Timothy Paustian, University of Wisconsin-Madison 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 (GlcNAc 1,4) n
HA turnover Eukaryotic hyaluronidase (HYAL) gene family –3p21.3 –HYAL1, 2 and 3 Turnover of hyaluronan in most tissues is rapid –t 1/2 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
Hyaluronan Binding Proteins
Lectican Family This family of proteoglycans consist of a group of homologous core proteins Have in common an hyaluronic acid binding motif Chondroitin sulfate
Chondroitin Sulfate 4S 6S GalNAcGlcA 6S 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 The chains are easily characterized using bacterial chondroitinases which degrade the chain to disaccharides An epimerase can flip the stereochemistry of D-GlcA to L-IdoA (Dermatan Sulfate) IdoA
Chondroitin Sulfate Proteoglycan S S S S S S S S S S S S S S S S S S
Cartilage - Proteoglycan Aggregates Aggrecan: Large chondroitin sulfate proteoglycan present in cartilage and other connective tissues Core protein ~400 kDa ~100 chondroitin sulfate chains of ~20 kDa Hyaluronic Acid Aggrecan 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
Diseases - Achondroplasias GeneMiceHuman AggrecanCartilage Matrix Deficiency (CMD) ? Sulfate Transporter (DTDST) -Atelosteogenesis Type II Achondrogeneis Type Ib Diastrophic dysplasia (DTD) PAPS synthetase BrachymorphismSpondylo- epimetaphyseal dysplasia PAPS ATP GAG SulfationSO 4 2- DTDST outin
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 GeneKnockouts 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 Proteoglycan
NSNSNSNS 6S6S 3S XylGal GlcA GlcNAcIdoA 2S 6S Heparan Sulfate 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
Glypicans GPI anchored proteins 6 members Membrane Heparan Sulfate Proteoglycans Syndecans Type I Membrane Proteins 4 members
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 PKC , 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 Cell-Binding Domain TM C1C2 V
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 GeneMiceHuman EXT1/EXT2Embryonic lethal (null) Heterozygotes develop rib exostoses Hereditary Multiple Exostoses NDST1Perinatal lethal (null) Various developmental defects (forebrain, lung) Tissue specific knockouts have various physiological alterations ? NDST2Viable, mucosal mast cell deficiency? GlcA C5 EpimerasePerinatal 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 Mucopolysaccharidoses
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)