Sulfated Glycosaminoglycans & Hyaluronan

Chapter 11

April 13, 2004

Jeff Esko

jesko@ucsd.edu

GLYCOSAMINOGLYCANS (GAGs)

HEPARAN SULFATE

CHONDROITIN/DERMATANSULFATE

KERATANSULFATE

HYALURONICACID

S S SP SSer-O-

S SS

NS NSNS 2S

S-O-Ser

NH2

S

S

S SS

OSer/Thr

NAsn

N-LINKEDCHAINS

O-LINKEDCHAIN

NAsn

P

Ac

OSer

O-LINKED GlcNAc

EtnP

INOSITOL P

GLYCO-PHOSPHOLIPID

ANCHOR

GLYCO-SPHINGOLIPIDS

Ac

Cytosol

SS

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

Cytosol Golgi

Ser

SQV-3Galactosyltransferase I

SQV-2Galactosyltransferase II

SQV-8Glucuronosyltransferase I

UDP-

SQV-7Nucleotide sugarmulti-transporter

n

SQV-5Chondroitin Synthase

Glc

GlcA

Gal

Xyl

GalNAc

SQV Proteins Encode the Enzymes Required for Chondroitin Biosynthesis

UDP-

UDP- UDP-

UDP-

SQV-4UDP-Glc

Dehydrogenase

UDP-

UDP-

SQV-1UDP-GlcA

Decarboxylase

UDP-

SQV-6Xylosyltransferase

Chondroitin Proteoglycan

Chondroitin is one type of glycosaminoglycan (GAG) chainGlcA

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

GalNAc GlcA

GlcNAc GlcA

Hyaluronan (HA)

GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA

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 250-25,000 disaccharides (105-107 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. 2002 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 (GlcNAc1,4)n

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

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 4S 4S

6S 6S

GalNAc GlcA

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 SS S

S S S S SS

SS 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

HyaluronicAcid

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

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

PAPSATP

GAG SulfationSO42- SO4

2-

DTDSTout in

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 ProteoglycansSLRPs - 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/NidogenCollagen Type IV

Perlecan:Heparan Sulfate

Proteoglycan

Bamacan:Chondroitin Sulfate

Proteoglycan

Perlecan - Basement Membrane Proteoglycan

LDLreceptor-like

repeats

Lamininrepeats

Ig-like repeats Laminin &EGF motifs

467 KDa

Perlecan knockouts: Perinatal lethal chondrodysplasiaHuman mutations: Schwartz-Jampel syndrome, Silverman-Handmaker type Dyssegmental dysplasia

Glycoconj J 19, 263–267, 2003)

Heparan Sulfate Proteoglycans

6S 6S 6S

6S 6S 6S

NSNS NS

NS NSNS

NS3S

NS2S

2S

NS

2S

GlcAGlcNAc IdoA Chondroitin sulfate

4S4S 4S

4S4S

NSNS NSNS

6S6S

3SXylGalGalGlcA

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

Biosynthesis of a Heparan Sulfate Chain

NSNS NSNS

6S6S

GalGalGlcA

CopolymeraseComplex

EXT1/EXT2

GlcNAc N-deacetylaseN-sulfotransferases(NDST) (4 isozymes)

3S

GlcNH2/S3-O-sulfotransferases(3OST) (6 isozymes)

2S

Uronic acid2-O-sulfotransferase

IdoA

Epimerase

GlcNAc/S6-O-sulfotransferases(6OST) (3+ isozymes)

6S GlcNAc

EXTL3EXTL2?

Xyl

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-BindingDomain TM

C1 C2

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 unclearModulation 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

?

Signaling EventMitogenesis

FGF

Heparan sulfate

FGF

•Wnts •TGF-/BMPs •HGF•HB-EGF•Hedgehog•FGF•VEGF•Angiopoietin

Heparan Sulfate Proteoglycans: Co-receptors and Signaling Molecules

Proteoglycan Turnover

Plasma membrane

Endocytosis

Step1proteaseendoglycosidase

Step 2endoglycosidase

Step3exoglycosidasesulfatase

Golgi

HS oligosaccharide(~10 kDa)

HS oligosaccharide(~5 kDa)

HS chain Shedding

• 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)