The exchange of Arg-Gly-Asp (RGD) and Arg-Tyr-Asp (RYD) binding sequences in a recombinant murine fab fragment specific for the integrin αIIbβ3 does not alter integrin recognition

Abstract

The murine monoclonal antibody OPG2 is an excellent paradigm of natural RGD ligands and binds specifically to αIIbβ3 integrin. A reactive Arg103-Tyr104-Asp105 (RYD) tripeptide is located in an extended loop, the third complementarity-determining region of the heavy chain (H3). When compared to other RGD ligands, the RYD tripeptide of OPG2 is unique, in that the side chains are fixed in a stable orientation that we have defined by x-ray crystallography. In this study, we express OPG2 H chain segments (Fd) and K chains as components of active, Fab heterodimers by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing cDNA specific for each protein. Recombinant AP7 Fd segments are generated from the parent OPG2 Fd segments by replacement of Tyr104 with Gly, while recombinant AP7E Fd segments are produced from APT Fd segments, by exchange of Asp105 with Glu. Neither the free Fd segments nor the free K chains of OPG2 or AP7 can bind to αIIbβ3. The AP7 Fab fragment, like the parent OPG2 Fab, binds strongly to purified αIIbβ3 but weakly, if at all, to purified αvβ3. The affinity of OPG2 and AP7 Fab fragments for gel-filtered platelets, whether nonstimulated or activated by 0.2 μM phorbol 12-myristate 13-acetate, is identical. As with other natural RGD ligands, the binding of recombinant OPG2 Fab or AP7 Fab fragments to purified αIIbβ3 or to gel-filtered platelets is completely inhibited by the peptide RGDW or by addition of EDTA. AP7E Fab fragments do not bind at all to either purified αIIbβ3 or platelets. Our results demonstrate, for the first time within a natural protein ligand, that the tripeptides RGD and RYD exhibit equivalent binding capacity and specificity for the integrin αIIbβ3.