The exchange of Arg-Gly-Asp (RGD) and Arg-Tyr-Asp (RYD) binding sequences in a recombinant murine Fab fragment specific for the integrin alpha IIb beta 3 does not alter integrin recognition.
The murine monoclonal antibody OPG2 is an excellent paradigm of natural RGD ligands and binds specifically to alpha IIb beta 3 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 kappa 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 AP7 Fd segments, by exchange of Asp105 with Glu. Neither the free Fd segments nor the free kappa chains of OPG2 or AP7 can bind to alpha IIb beta 3. The AP7 Fab fragment, like the parent OPG2 Fab, binds strongly to purified alpha IIb beta 3 but weakly, if at all, to purified alpha V beta 3. The affinity of OPG2 and AP7 Fab fragments for gel-filtered platelets, whether nonstimulated or activated by 0.2 microM 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 alpha IIb beta 3 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 alpha IIb beta 3 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 alpha IIb beta 3.
Kunicki
T J
TJ
Roon Research Center for Arteriosclerosis and Thrombosis, Scripps Research Institute, La Jolla, California 92037, USA.
Ely
K R
KR
Kunicki
T C
TC
Tomiyama
Y
Y
Annis
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera
D007140:Immunoglobulin Fab Fragments; D016023:Integrins; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid