title: Monoclonal antibodies identify residues 199-216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1.
abstract: Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. danny.tuckwell@man.ac.uk
authors.forename: D S
authors.initials: DS
authors.lastname: Tuckwell
authors.forename: L
authors.initials: L
authors.lastname: Smith
authors.forename: M
authors.initials: M
authors.lastname: Korda
authors.forename: J A
authors.initials: JA
authors.lastname: Askari
authors.forename: S
authors.initials: S
authors.lastname: Santoso
authors.forename: M J
authors.initials: MJ
authors.lastname: Barnes
authors.forename: R W
authors.initials: RW
authors.lastname: Farndale
authors.forename: M J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001665:Binding Sites; D002412:Cations; D003094:Collagen / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D038981:Receptors, Collagen; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D002412:Cations; D000939:Epitopes; D016023:Integrins; D008024:Ligands; D038981:Receptors, Collagen; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D013912:Threonine; D008345:Manganese; D003094:Collagen; D005982:Glutathione Transferase; D008274:Magnesium; D000409:Alanine