Transcript GAG binding proteins - University of California, San Diego

Sulfated Glycosaminoglycans & Hyaluronan
Chapter 11
April 13, 2004
Jeff Esko
[email protected]
GLYCOSAMINOGLYCANS
(GAGs)
HEPARAN SULFATE
S
S
S
CHONDROITIN/DERMATAN
SULFATE
P
S
S
Ser-O-
S
S
S
S
-O-Ser
NS
NS 2S NS
KERATAN
SULFATE
GLYCOPHOSPHOLIPID
ANCHOR
GLYCOSPHINGOLIPIDS
O-LINKED
CHAIN
S
S
S
N-LINKED
CHAINS
Ac
S
S
P
S
Etn
P
S
Ac
NH2
INOSITOL
P
O
Ser/Thr
N
Asn
N
Asn
Cytosol
= GlcNAc
= GalNAc
S
= GlcA
= IdoA
O
Ser
O-LINKED GlcNAc
HYALURONIC
ACID
Proteoglycans and Human Disease
Skeletal Achondroplasias
Simpson-Golabi-Behmel
Overgrowth Syndrome (SGBS)
Hereditary Multiple Exostoses (HME)
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 mutations alter epithelial invagination
sqv
SQV Proteins Encode the Enzymes Required
for Chondroitin Biosynthesis
SQV-4
SQV-1
UDP-Glc
Dehydrogenase
UDP-GlcA
Decarboxylase
SQV-6
SQV-7
Xylosyltransferase
Nucleotide sugar
multi-transporter
UDP-
SQV-2
Galactosyltransferase II
UDPUDPUDP-
UDP-
Ser
UDPn
UDP-
UDP-
SQV-5
Xyl
Gal
GlcA
GalNAc
Glc
SQV-3
Chondroitin Synthase
Cytosol
Golgi
Galactosyltransferase I
SQV-8
Glucuronosyltransferase I
Chondroitin Proteoglycan
Xyl
Gal
GlcA
GalNAc
Chondroitin is one type of glycosaminoglycan (GAG) chain
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?
b4
b3
b4
b3
b4
b3
b4
b3
b4
b3
GalNAc GlcA
 Non-sulfated chondroitin not found in vertebrates
 Instead, they make hyaluronic acid which is similar in
structure
b4
b3
GlcNAc GlcA
b4
b3
b4
b3
b4
b3
b4
b3
Hyaluronan (HA)
b4
b3
b4
b3
b4
b3
b4
b3
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)
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
 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
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
IdoA
b4
4S
b3
b4
4S
b3
6S
b4
b3
6S
b4
b3
6S
b4
b3
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
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
Aggrecan
 Core protein ~400 kDa
 ~100 chondroitin sulfate
chains of ~20 kDa
Hyaluronic
Acid
 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
Aggrecan
Mice
Human
Cartilage Matrix
Deficiency (CMD)
?
-
Atelosteogenesis
Type II
Sulfate
Transporter
(DTDST)
Achondrogeneis
Type Ib
Diastrophic
dysplasia (DTD)
PAPS
synthetase
SO42out
Brachymorphism
SO42-
DTDST
in
ATP
PAPS
Spondyloepimetaphyseal
dysplasia
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
Bamacan:
Chondroitin Sulfate
Proteoglycan
Perlecan:
Heparan Sulfate
Proteoglycan
Collagen Type IV
Laminin
Entactin/Nidogen
Perlecan - Basement Membrane Proteoglycan
467 KDa
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
6S
6S
6S
NS 2S NS 2S NS
6S
NS
6S
6S
NS 2S NS
3S
NS
4S
GlcNAc
GlcA
IdoA
NS
NS
4S
4S
4S
Chondroitin sulfate
4S
Heparan Sulfate
6S
NS
NS
3S
IdoA
6S
6S
GlcNAc
NS 2S NS
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
Biosynthesis of a Heparan Sulfate Chain
GlcNAc/S
6-O-sulfotransferases
Epimerase (6OST) (3+ isozymes)
Copolymerase
Complex
EXT1/EXT2
6S
NS
GlcNAc N-deacetylase
N-sulfotransferases
(NDST) (4 isozymes)
NS
3S
IdoA 6S
6S
NS 2S NS
Uronic acid
2-O-sulfotransferase
GlcNH2/S
3-O-sulfotransferases
(3OST) (6 isozymes)
EXTL3
EXTL2?
GlcNAc
GlcA Gal Gal Xyl
Membrane Heparan Sulfate Proteoglycans
Syndecans
Type I Membrane Proteins
4 members
Glypicans
GPI anchored proteins
6 members
Syndecans
Cell-Binding
Domain
TM
V
C1
C2
•
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
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
EXT1/EXT2
NDST1
Mice
Embryonic lethal (null)
Human
Heterozygotes develop rib exostoses
Hereditary Multiple
Exostoses
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-Osulfotransferase
Perinatal lethal (null) Renal
agenesis,eye and skeletal defects
?
Heparan Sulfate Proteoglycans: Co-receptors and Signaling Molecules
•Wnts
•TGF-b/BMPs
•HGF
•HB-EGF
•Hedgehog
•FGF
•VEGF
•Angiopoietin
Heparan sulfate
FGF
FGF
Signaling Event
Mitogenesis
Proteoglycan Turnover
• Shedding by exoproteolytic
activity, MMP-7 for one
HS chain
Shedding
• 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
Plasma membrane
Endocytosis
Step1
protease
endoglycosidase
Golgi
HS oligosaccharide
(~10 kDa)
Step 2
endoglycosidase
HS oligosaccharide
(~5 kDa)
Step3
exoglycosidase
sulfatase
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)