Transcript GAG binding proteins - Glycobiology Research and Training

Glycosaminoglycan-Binding
Proteins
Lecture 25, Chapter 29
May 11, 2004
Jeff Esko
Types of Glycan-Binding Proteins
• Glycosyltransferases and modifying enzymes
• Antibodies induced by carbohydrate antigens
• Animal Lectins: P,C,S,R,L and I-type (Lectures 1923)
• Plant Lectins: Con A, PHA, WGA, Ricin, and
many others (Lecture 24)
• Glycosaminoglycan-binding proteins (Lecture 25)
• Bacterial adhesins and Viral hemagglutinins
(Lecture 26)
Lectin - term usually restricted to proteins that
share primary sequence homologies
Glycosaminoglycans (GAG)
b4
b3
b4
b3
b4
b3
b4
b3
b4
b3
GlcNAc GlcA
Hyaluronan
IdoA
4S
b4
b3
4S
b4
b3
4S
b4
b3
4S
b4
b3
4S
b4
4S
b3
2S
GalNAc GlcA
Chondroitin/Dermatan Sulfate
a4
b4
GlcNAc GlcA
a4
b4
a4
b4
6S
a4
NS
Heparin/Heparan Sulfate
b4
6S
IdoA
a4
NS3S
a4
2S
NS
GAG Binding Proteins
• Hyaluronan-binding proteins have a binding motif
called the Link module
• Chondroitin sulfate binds to many proteins, but
with low affinity, no apparent fold
• Hundreds of heparin binding proteins exist and
do not generally sort into families of genes
related through a common fold
• Dermatan sulfate binds to many of the same
proteins as heparin
Hyaluronan (HA)
b4
b3
b4
b3
b4
b3
b4
b3
b4
b3
n≥1000
GlcNAc GlcA
• Synthesized at plasma membrane, extruded from cell
• Abundant in skeletal tissues, synovial fluid, skin, elevated in
expanding tissues (morphogenesis, invasion)
• Interesting biophysical properties (hydration, viscous
solutions, resiliency)
• Present as capsule in some bacteria
Hyaluronan (HA)
Bent, helical, relatively
stiff structures
Day & Sheehan (2001) COSB 1:1617
Fragments are potent
signaling molecules
Aggrecan and CD44:
Hyaluronan Binding Proteins
Hyaluronan-Binding Proteins (HABPs)
Aggrecan
Versican
Link Protein
Neurocan
TSG-6
Brevican
CD44
Aggrecan Family
Link protein
CD44
TSG-6
LYVE-1
LYVE-1
= Link Module
Tissue architecture, stability
Stabilizes aggrecan-HA aggregates
Cell adhesion
Inflammation
Clearance
SS: b = b-sheet a = a-helix
• Members deduced by sequence homologies
• Note position of four conserved Cys residues, plus other
amino acids in consensus sequence
The two a-helices and
two triple-stranded antiparallel b-sheets make
up the Link Module
b5
b4
b3
a2
Day and Prestwich (2001) JBC 277:4585
b2
b6
b1
a1
• Binding site is actually generated by folding of different
segments of the chain, bringing key residues into
proximity
• Notice positively charged residues and aromatics
www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E.html
Heparin Binding Proteins
GAG partner
Oligosaccharide
Hyaluronan
Heparin/
heparan sulfate
a4
6S
Dermatan sulfate
a4
b4
NS
6S
a4
b3
a4
a4
NS3S
b4
Heparin/
heparan sulfate
Heparin/
heparan sulfate
b3
a4
b4
NS
6S
a4
NS
4S
2S
4S
Antithrombin
NS
a4
a4
NS
a3
2S
6S
Aggrecan
a4
NS
a4
b4
2S
Protein
2S
a4
2S
b4
6S
a4
NS
a3
2S
FGF-2
4S
2S
b4
2S
Lipoprotein
lipase
Heparin
cofactor II
Conformational Considerations
GAG chains assume
helical configurations,
which causes charged
residues to alternate
across the helix
NS and 2S groups are
on the same side
COO- locations depend
on whether its GlcA or
IdoA
6S
NS
a4
a4
2S
6S
NS
a4
a4
2S
6S
a4
a4
NS
2S
6S
NS
NS
CO2
6S
CO2
-
B
2S
2S
-
NS
a4
= GlcA
= GlcNAc
A
a4
2S
6S
NS
a4
2S
Sugar Conformation

Most sugars prefer the 4C1 conformation

IdoA which is formed by epimerization of GlcA has the 1C4 or 1S0
conformation

The greater conformational flexibility means that the sulfate and
carboxylates can shift position more readily

Greater binding possibilities and induced fit
Do Consensus Sequences Exist?
 Generally, GAG binding proteins contain clustered Lysine and
Arginine residues
 In 1989, Cardin and Weintraub proposed a consensus sequence
for heparin binding proteins, B = basic residue
-XBBXBX-
-XBBBXXBX-
 Spacing would place basic residues on the same
face of an a-helix (3.4 residues/turn) or a b-strand
(alternating faces)
 It turns out that most binding proteins do not fit
this pattern and binding site is composed of
positive residues contributed by different
segments of the protein
Antithrombin
•
•
•
•
Antithrombin, a serpin (serine protease inhibitor)
Inactivates proteases involved in coagulation (Factors IIa and Xa)
Blocks coagulation
Antithrombin deficiency results in thrombosis (clot formation)
• Heparin binds to antithrombin, alters its conformation, and
enhances rate of inactivation of Xa and IIa by a factor of 104
• Only need a heparin pentasaccharide to activate
OSO3O
O
OH
O
O
NAc
OSO3-
±OSO3-
COO-
O
O
OH
OH
OSO3
-
O
NHSO3-
COO
OH
O
O
O
OSO3-
OH
O
NHSO3-
Antithrombin-Heparin
KD ~ 2.5 x 10-10 M
DG ~ 13.3 kcal/mol
Heparin-Antithrombin
D
A
 Binding site for heparin is in a cleft formed by two helices
 Binding is oriented, with pentasaccharide in cleft and
flanking chain to the non-reducing side extending up and
over the protein
 An 18-mer is actually needed to inactivate thrombin, so it
acts like a template to approximate antithrombin-thrombin
 Interaction with thrombin does not require specific
oligosaccharide sequence (low affinity)
Jin et al. (1997) PNAS 94:14683
D
3.6 kcal
2.1 kcal
D
E
E
1.8 kcal
F
G
6.9 kcal
P
H
Contribution of Individual Groups to Affinity
• Blue numbers refer to kcal binding
deduced by altering the glycan
groups
• Red numbers refer to kcal binding
deduced by mutating amino acids
1.8
2.8
1.7
0.4
0
5.1
3.7
3.6
2.1
Atha et al. (1985) Biochemistry 24:6723
6.9
Protein
GAG partner
Aggrecan
Hyaluronan
Antithrombin
Heparin/
heparan sulfate
FGF-2
Lipoprotein lipase
Oligosaccharide
a4
6S
a4
b4
NS
6S
a4
b3
a4
a4
NS3S
b4
Heparin/
heparan sulfate
Heparin/
heparan sulfate
b3
a4
b4
NS
6S
NS
a4
NS
a4
NS
a4
2S
2S
a4
6S
NS
a4
2S
a4
2S
6S
NS
a4
2S
Heparin versus Heparan Sulfate
The difference between heparin and heparan sulfate is
quantitative not qualitative
Characteristic
Sulfate/hexosamine
Heparan sulfate
0.8 - 1.8
Heparin
1.8-2.4
GlcN N-sulfates
IdoA content
40-60%
30-50%
•5%
8
70%
•
Solubility in 2 M KAc at
ph 5.7, 4ÞC
Yes
No
Site of synthesis
Size
Virtually all cells
10-70 kDa
Mast cells
10-12 kDa
Binding to Antithrombin 0-0.3%
~30%
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
FGF-Heparin Hexasaccharide
 Crystal structure shows surface
binding
119KRTGQYKLGSKTGPGQK135
FGF/FGF Receptor Co-crystals
Plotnikov et al. Cell 98:641 (1999)
FGF2/FGFR1
Heparin
FGF
FGF
Mulloy & Linhardt (2001) COSB 11:623
• Symmetric structure
• Heparin interacts with both
ligands and receptors
• Two heparin
oligosaccharides present
in crystal
Potential Docking Site for Heparin
Top
View
Top View with
basic residues
shaded blue
Side
View
FGF-2 Activation Sequence
If symmetric dimer structure is correct:
b4
a4
NS
b4
6S
6S
a4
a4
a4
a4
a4
a4
a4
a4
a4
NS 2S NS
2S NS
2S
NS
2S
NS
Receptor Binding
Domain
FGF-2 Binding
Domain
= GlcNAc
6S
= GlcA
= IdoA
b4
a4
NS 2S
FGF-2 Binding
Domain
Expression of “Active” Heparan Sulfates
- FGF + FR1
Heparinase
FGF-2
plus APtagged
receptor
FGF alone
Locate all HS by antibody staining
K= keratinocytes, BM = basement membrane, V = blood vessel, FR1-AP = alkaline
phosphatase fusion to FGF receptor-1, 3G10 = monoclonal antibody to heparinase treated HS
Chang et al. FASEB J. 14:137 (2000)
FGF2/FGFR1
FGF
FGF1/FGFR2
FGF
FGF
FGF
Its never simple!
Mulloy & Linhardt (2001) COSB 11:623
FMDV
Depression that
defines binding
site for heparin is
made up of
segments from all
three major
capsid proteins
Fry et al. (1999) Embo J 18:543
GAG partner
Oligosaccharide
Hyaluronan
Heparin/
heparan sulfate
a4
6S
Dermatan sulfate
a4
b4
NS
6S
a4
b3
a4
a4
NS3S
b4
Heparin/
heparan sulfate
Heparin/
heparan sulfate
b3
a4
b4
NS
6S
a4
NS
4S
2S
4S
Antithrombin
NS
a4
a4
NS
a3
2S
6S
Aggrecan
a4
NS
a4
b4
2S
Protein
2S
a4
2S
b4
6S
a4
NS
a3
2S
FGF-2
4S
2S
b4
2S
Lipoprotein
lipase
Heparin
cofactor II