Abstract
Integrin alpha 2 beta 1 is a cell surface adhesion receptor for collagen and echovirus 1. Here we localized the epitopes for anti-alpha 2 monoclonal antibodies using interspecies (human/bovine) alpha 2 chimeras with different lengths of human alpha 2 sequence on the amino-terminal side and site-directed mutagenesis. The antibodies that block the collagen and/or echovirus 1 binding to human alpha 2 beta 1 (6F1, RMAC11, 12F1, and AA10) recognizes a small region (residues 173-259) within the I domain. Asp-160 and Arg-242 are critical for binding of the two other function-inhibiting antibodies, P1H5 and 5E8, respectively. Notably, mutations of Asp-151 and Asp-254 block the binding of alpha 2 beta 1 to collagen. These data suggest that the I domain (residues 140-359) is critically involved in the ligand/receptor interactions, and collagen and echovirus 1 binding sites are adjacent or overlapping within the I domain. The sequence of the residues 173-259 of alpha 2 overlap with the peptide sequences (M11 and M20) that derive from von Willebrand factor A1 and A3 domains (homologous to the alpha 2 I domain) and block von Willebrand factor/collagen interaction, suggesting that the epitope region of alpha 2 (residues 173-259) may really be involved in ligand recognition.
Highlights
RMAC111, 2F1, and AA10) recognizesasmallregion within the I domain.hp-160 and Arg242 are critical for binding of the two other functioninhibiting antibodies, P1H5 and 5E8,respectively
1 a receptor for echovirus 1,and anti-a2 mAbs 5E8, 12F1, and binding sites are adjacent or overlapping within the IAAlO block echovirus 1binding to cells [17, 18](Table I). a2pl domain.The sequence of the residues 173-259 of a2 over- has been shown to be involved in cell-cell interaction (19, lap with the peptide sequences (M11 and M20) that de- 20), probably by interacting with a3pl[21]
Homologybe- domain is involved in the interactioonf a 2 p l with collagen and tween human andbovine a2 subunit maturpeeptide sequences echovirus 1, since 6F1, RMAC11, 12F1, and AAlO have been is 86% (Fig. 1)
Summary
Some anti-a2 mono- sites in the a2 subunbiyt mapping epitopes for function-inhibclonal antibodies (mAbs)‘(5E8,6F1, RMAC11, and PlH5) have iting mAbs using interspecies a 2 chimeras expressed on Chibeen reported to inhibit the interactionbetween a2pl andex- nese hamster ovary (CHO) cells. Endothelial Cells-To facilitate mapping epitopes for anti-human a2 mAbs, bovine a2 cDNA was cloned and sequenced.
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