Gain- and loss-of-function mutants confirm the importance of apical residues to the primary interaction of human glycoprotein VI with collagen.
BACKGROUND
By site-directed mutagenesis of recombinant receptor fragments, we have previously identified residue lysine59 of the platelet collagen receptor glycoprotein VI (GPVI) as being critical for its interaction with the synthetic ligand collagen-related peptide (CRP) and the inhibitory phage antibody 10B12. Lysine59 is proposed to lie on the apical surface of the receptor near the linker joining the two immunoglobulin (Ig)-like extracellular domains. Recently, others have postulated the involvement of a portion of the first domain distant from the interdomain hinge as being involved in an extended collagen-binding site.


OBJECTIVE
To extend our knowledge of the primary collagen-binding site of GPVI, a number of neighboring residues on the apical surface of recombinant soluble GPVI were mutated to alanine and binding of these mutants, as well as the lysine59 mutant, to fibrillar collagen was measured.


RESULTS
Binding of recombinant GPVI to collagen, like CRP, was dramatically reduced by the mutation of residue lysine59 to glutamate. Remarkably, the mutation of residues arginine60 in domain one and arginine166 in domain two, individually to alanine, which had no significant affect on CRP binding, reduced binding of recombinant GPVI to collagen. Mutation of the residue lysine41 to alanine dramatically increased binding to both CRP and collagen. This mutation abolished 10B12 binding, confirming its position in the epitope of our inhibitory phage antibody.


CONCLUSIONS
Residues lysine59, arginine60, and arginine166, from both Ig-like domains of GPVI, are critical for collagen binding by the receptor. This provides additional evidence for a basic patch on the apical surface of the receptor as the primary collagen-binding site of GPVI.
O'Connor
M N
MN
Department of Haematology, Division of Transfusion Medicine, University of Cambridge, Cambridge, UK.
Smethurst
P A
PA
Farndale
R W
RW
Ouwehand
W H
WH
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D003094:Collagen / Q000737:chemistry* / Q000235:genetics; D018604:Epitope Mapping; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D002352:Carrier Proteins; D007136:Immunoglobulins; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C435908:collagen-related peptide; C098341:platelet membrane glycoprotein VI; D003094:Collagen