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)