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 19-23)
• 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
GlcNAc GlcA
Hyaluronan
GalNAc GlcA
4S 4S4S4S 4S 4S
2S
IdoA
Chondroitin/Dermatan Sulfate
6S 6S
NS NS3S 2S NS
GlcNAc GlcA
IdoA
Heparin/Heparan Sulfate
Glycosaminoglycans (GAG)
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)
GlcNAc GlcA
n≥1000
• 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)
Day & Sheehan (2001) COSB 1:1617
Bent, helical, relatively stiff structures
Fragments are potent signaling molecules
Aggrecan and CD44:Hyaluronan Binding Proteins
Aggrecan
Versican
NeurocanLink Protein
BrevicanTSG-6
CD44 LYVE-1 = Link Module
Hyaluronan-Binding Proteins (HABPs)
Aggrecan Family Tissue architecture, stabilityLink protein Stabilizes aggrecan-HA aggregatesCD44 Cell adhesionTSG-6 InflammationLYVE-1 Clearance
• Members deduced by sequence homologies
• Note position of four conserved Cys residues, plus other amino acids in consensus sequence
SS: b = -sheet a = -helix
The two -helices and two triple-stranded anti-parallel -sheets make up the Link Module
54
1
3
6
2 1
2
Day and Prestwich (2001) JBC 277:4585
www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E.html
• Binding site is actually generated by folding of different segments of the chain, bringing key residues into proximity
• Notice positively charged residues and aromatics
GAG partner Oligosaccharide Protein
Hyaluronan Aggrecan
Heparin/heparan sulfate
Antithrombin
Heparin/heparan sulfate
FGF-2
Heparin/heparan sulfate
Lipoproteinlipase
Dermatan sulfate Heparincofactor II
4S 4S 4S
2S 2S 2S
NS NS 2S
6S 6S 6S
NS NS NS2S 2S 2S
6S 6S
NS NS3S 2S NS
Heparin Binding Proteins
A
B
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
NS
6S
2S
NS 2S
CO2-
CO2-
6S
NS
6S
NS
6S
NS2S 2S 2S
6S
NS
6S
NS2S 2S
= GlcNAc = GlcA
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
O O-OSO3OHHO
HOOCOHOHOHOOC
O-OHOOH
HOHOOC
OSO3GlcA 4C1 IdoA 1C4 IdoA 2S0
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 -helix (3.4 residues/turn) or a -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
OO
OOH
NHSO3-
OSO3-
OCOO-
OH
OSO3-
OO
OOSO3
-
NHSO3-
±OSO3-
O
COO-
OH
OH
OOH
NAc
OSO3-
OO
Antithrombin-Heparin
KD ~ 2.5 x 10-10 M G ~ 13.3 kcal/mol
Heparin-Antithrombin
A
D
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)
D
E
P
D
E
F
G
H
6.9 kcal
1.8 kcal
3.6 kcal2.1 kcal
Jin et al. (1997) PNAS 94:14683
Contribution of Individual Groups to Affinity
3.7
0.4
0 5.1
1.7
2.8
Atha et al. (1985) Biochemistry 24:6723
• Blue numbers refer to kcal binding deduced by altering the glycan groups
• Red numbers refer to kcal binding deduced by mutating amino acids
3.6 2.1 6.9
1.8
Protein GAG partner Oligosaccharide
Aggrecan Hyaluronan
Antithrombin Heparin/heparan sulfate
FGF-2 Heparin/heparan sulfate
Lipoprotein lipase Heparin/heparan sulfate
NS NS 2S
6S 6S 6S
NS NS NS2S 2S 2S
6S 6S
NS NS3S 2S NS
Heparin versus Heparan Sulfate
Characteristic Heparan sulfate Heparin
Sulfate/hexosamine 0.8 - 1.8 1.8-2.4
GlcN N-sulfates 40-60% ≥85%
IdoA content 30-50% ≥70%
Solubility in 2 M KAc atph 5.7, 4˚C
Yes No
Site of synthesis Virtually all cells Mast cells
Size 10-70 kDa 10-12 kDa
Binding to Antithrombin 0-0.3% ~30%
The difference between heparin and heparan sulfate is quantitative not qualitative
6S
NS NS NS2S
6S
Signaling EventMitogenesis
FGF
Heparan sulfate
FGF
•Wnts •TGF-/BMPs •HGF•HB-EGF•Hedgehog•FGF•VEGF•Angiopoietin
Heparan Sulfate Proteoglycans: Co-receptors and Signaling Molecules
FGF-Heparin Hexasaccharide
Crystal structure shows surface binding
119KRTGQYKLGSKTGPGQK135
FGF/FGF Receptor Co-crystals
Plotnikov et al. Cell 98:641 (1999)
FGF2/FGFR1
FGF FGF
Mulloy & Linhardt (2001) COSB 11:623
• Symmetric structure
• Heparin interacts with both ligands and receptors
• Two heparin oligosaccharides present in crystal
Heparin
Potential Docking Site for Heparin
Top View
Top View with basic residues shaded blue
Side View
FGF-2 Activation Sequence
FGF-2 BindingDomain
FGF-2 BindingDomain
Receptor BindingDomain
If symmetric dimer structure is correct:
NS NS 2S
6S 6S 6S
NS NS NS2S 2S 2S
NS NS 2S
= GlcA = IdoA= GlcNAc
Expression of “Active” Heparan Sulfates
FGF alone
FGF-2 plus AP-tagged receptor
- FGF + FR1 Heparinase
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 FGF1/FGFR2
FGFFGF
FGF FGF
Mulloy & Linhardt (2001) COSB 11:623
Its never simple!
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 Protein
Hyaluronan Aggrecan
Heparin/heparan sulfate
Antithrombin
Heparin/heparan sulfate
FGF-2
Heparin/heparan sulfate
Lipoproteinlipase
Dermatan sulfate Heparincofactor II
4S 4S 4S
2S 2S 2S
NS NS 2S
6S 6S 6S
NS NS NS2S 2S 2S
6S 6S
NS NS3S 2S NS