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