Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa.
In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.
Piétu
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INSERM U.143, Hôpital de Bicêtre, France.
Ribba
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Chérel
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Meyer
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D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D001616:beta-Galactosidase