Transcript GAG binding proteins - Glycobiology Research and Training

Essentials of Glycobiology
April 14, 2002
Jeff Esko
Glycosaminoglycan-Binding Proteins
Types of Carbohydrate-Binding Proteins
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Glycosyltransferases and modifying enzymes
Plant Lectins: Con A, PHA, WGA, Ricin, and many others
Animal Lectins: P,C.S, and I-type, chaperone proteins
Glycosaminoglycan-binding proteins
Antibodies induced by carbohydrate antigens
Bacterial adhesins
Viral hemagglutinins
Overview
HA binding proteins
Methods for characterizing GAG-binding proteins
Conformation and sequence considerations
Examples of heparin-binding proteins
Hyaluronan (HA)
b4
b3
b4
b3
b4
b3
GlcNAc
GlcA
b4
b3
b4
GlcA
GlcNAc
b3
n≥1000
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
Synthesized at plasma membrane, extruded
from cell
Present as capsule in some bacteria
GlcA
Physical Properties
Interglycosidic H-bonding restricts rotations across glycosidic bonds
Promotes rapid recovery after mechanical perturbations
Hyaluronan (HA)
Day & Sheehan (2001) COSB 1:1617
Hyaladherens
Aggrecan
Versican
Link Protein
Neurocan
TSG-6
Brevican
CD44
LYVE-1
= Link Module
Link Module consists of
two a-helices and two
triple-stranded antiparallel b-sheets
b5
b4
b3
a2
Day and Prestwich (2001) JBC 277:4585
b2
b6
b1
a1
www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E.html
Other HA Binding Proteins
Inter-a-inhibitor
Chondroitin
Sulfate
HC-1
HC-2
Bikunin
RHAMM
SPACR, SPACRCAN, CDC37, CD38, Layilin and others
Affinity Chromatography
- High affinity
interactions have Kd
values of 10-7-10-9
and require about 1
M NaCl to elute
- Low affinity
interactions have Kd
values of 10-4 to 10-6
and elute at 0.15 0.5 M NaCl
Lo Affinity
High Affinity
Affinity Chromatography on Immobilized GAG
-Prepare column by
covalently linking
GAG to activated
resin
-Bind sample, elute
with ligand, pH,
chelators
Affinity Co-Electrophoresis
• Pour acrylamide gel
with GAG binding
protein in gel
• Electrophorese
radioactive GAG
• Binding of chains to
protein retards their
mobility
• Measure Kd and nonbinding fraction
Non-binding fraction
San Antonio & Lander (2001)Meth Mol Biol. 171:401
Filter Binding Assay
4 Mix oligosaccharides with
protein in solution
Protein
4 Collect by filtration on
nitrocellulose membranes
4 Only oligosaccharides
bound to protein stick to
filter
Oligosaccharide
4 Solution equilibrium
conditions are achieved
4 Vary salt, divalent cations,
pH, competitors
Maccarana & Lindahl (1993) Glycobiology 3:271
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
CO2-
2S
6S
CO2NS
2S
Sugar Conformation
4
Most sugars prefer the 4C1 conformation
4
IdoA which is formed by epimerization of GlcA has the
1C or 1S conformation
4
0
4
The greater conformational flexibility means that the
sulfate and carboxylates can shift position more readily
4
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-
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
Antithrombin-Heparin
KD ~ 2.5 x 10-10 M
DG ~ 13.3 kcal/mol
Antithrombin-Heparin Interactions
Thrombin
4
4
4
Antithrombin
Binding sequence
for antithrombin is a
pentasaccharide, a
minor subfraction
Need an 18-mer to
inactivate thrombin
Heparin acts as a
template
Heparin
6S
a4
NS
6S
b4 a4 a4
NS3S 2S NS
Proteoglycan
Heparan sulfate
FGF
FGF
Signaling Event
Mitogenesis
FGF-Heparin Hexasaccharide
Crystal structure shows surface
binding
119KRTGQYKLGSKTGPGQK135
FGF-2 Binding Sequence
Frequency: 4-8 sites / chain
FGF/FGF Receptor Co-crystals
Plotnikov et al. Cell 98:641 (1999)
Potential Docking Site for Heparin
Top
View
Top View with
basic residues
shaded blue
Side
View
FGF2/FGFR1
FGF
FGF1/FGFR2
FGF
FGF
Mulloy & Linhardt (2001) COSB 11:623
FGF
FGF-2 Activation Sequence
If symmetric dimer structure is correct:
FGF-2 Binding
Domain
Receptor Binding
Domain
FGF-2 Binding
Domain
Frequency: < 1 site / chain (?)
Expression of Cell Type Specific 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)