Essentials of Glycobiology April 14, 2002 Jeff Esko

1
Essentials of Glycobiology April 14,
2002Jeff Esko

• Glycosaminoglycan-Binding Proteins

2
Types of Carbohydrate-Binding Proteins

• 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

3
Overview

• HA binding proteins
• Methods for characterizing GAG-binding proteins
• Conformation and sequence considerations
• Examples of heparin-binding proteins

4
(No Transcript)
5
Hyaluronan (HA)
n1000
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
GlcNAc
GlcA
Synthesized at plasma membrane, extruded from
cell Present as capsule in some bacteria
6
Physical Properties
Interglycosidic H-bonding restricts rotations
across glycosidic bonds Promotes rapid recovery
after mechanical perturbations
7
Hyaluronan (HA)
Day Sheehan (2001) COSB 11617
8
Hyaladherens
9
(No Transcript)
10
(No Transcript)
11
Link Module consists of two a-helices and two
triple-stranded anti-parallel b-sheets
b5
b4
b3
a1
b2
b6
a2
b1
Day and Prestwich (2001) JBC 2774585
12
(No Transcript)
13
www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E
.html
14
Other HA Binding Proteins
Inter-a-inhibitor
Chondroitin Sulfate
HC-1
HC-2
Bikunin
RHAMM
SPACR, SPACRCAN, CDC37, CD38, Layilin and others
15
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
NaCl
16
Affinity Chromatography on Immobilized GAG
-Prepare column by covalently linking GAG to
activated resin -Bind sample, elute with ligand,
pH, chelators
17
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 non-binding fraction

Non-binding fraction
San Antonio Lander (2001)Meth Mol Biol. 171401
18
Filter Binding Assay

• Mix oligosaccharides with protein in solution
• Collect by filtration on nitrocellulose membranes
• Only oligosaccharides bound to protein stick to
  filter
• Solution equilibrium conditions are achieved
• Vary salt, divalent cations, pH, competitors

Maccarana Lindahl (1993) Glycobiology 3271
19
(No Transcript)
20
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
21
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

22
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-

23
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 18543
24
Antithrombin-Heparin
KD 2.5 x 10-10 M DG 13.3 kcal/mol
25
Antithrombin-Heparin Interactions
Thrombin
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
6S
a4
b4
a4
a4
NS
NS3S
NS
2S
26
Proteoglycan
Heparan sulfate
FGF
FGF
27
FGF-Heparin Hexasaccharide

• Crystal structure shows surface binding
• 119KRTGQYKLGSKTGPGQK135

28
FGF-2 Binding Sequence
Frequency 4-8 sites / chain
29
FGF/FGF Receptor Co-crystals
Plotnikov et al. Cell 98641 (1999)
30
Potential Docking Site for Heparin
Top View
Top View with basic residues shaded blue
Side View
31
FGF2/FGFR1
FGF1/FGFR2
FGF
FGF
FGF
FGF
Mulloy Linhardt (2001) COSB 11623
32
FGF-2 Activation Sequence
If symmetric dimer structure is correct
Receptor Binding Domain
FGF-2 Binding Domain
FGF-2 Binding Domain
Frequency lt 1 site / chain (?)
33
Expression of Cell Type Specific Heparan Sulfates
- FGF FR1
Heparinase
FGF-2 plus AP-tagged 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. 14137 (2000)