Epitopes in α8ß1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

ABSTRACT

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W241-loop on the top-face of the ß-propeller. Loop-tips sufficiently close to W241 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W334 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W334 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W334 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W241 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W334 and an top-loop", and "solely W241", accounting for 82% of published RGD-integrin-mAbs.