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 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.

Function of Disintegrin-like/Cysteine-rich Domains of Atrolysin A

“…This disintegrin has an IC 50 value for inhibition of platelet aggregation similar to that observed for RGD-containing disintegrins (14). In another example, using a murine Fab fragment specific for the integrin ␣ IIb ␤ 3 , Kunicki and colleagues (40) demonstrated that the cognate RGD sequence could be exchanged with RYD without an alteration in integrin recognition. These data suggest that some limited diversity in this sequence region may be tolerated and still give rise to a ligand with reasonable potency for inhibiting aggregation.…”

A Molecular Basis for Affinity Modulation of Fab Ligand Binding to Integrin αIIbβ3

“…AP7 and PAC1.1 Fd and chain cDNAs were prepared as described previously (6,7). An additional AP7 Fd construct was generated in which the oligonucleotide sequence (CATCAC) 3 was inserted just upstream of the TGA stop codon.…”

“…1 (AP7.1, AP7.2, and AP7.3) were generated by splice overlap extension polymerase chain reaction (7). AP7 Fd cDNA (7) was used as a template to generate AP7.1, AP7.2, and AP7.3 Fd cDNAs.…”

“…Cloning and Analysis of Recombinant Baculoviruses-Recombinant viruses were cloned by infection of Sf9 cells (Invitrogen) (2 ϫ 10 6 in 2 ml of complete Grace's medium) seeded in T25 culture flasks, as described (6,7). The sequence of each recombinant clone was confirmed prior to its use, using Sequenase version 2.0 (U.S. Biochemical Corp.).…”

“…To address the molecular basis of conformation-sensitive binding of an RGD ligand, we exploited the binding properties of two well characterized RGD ligands, AP7 and PAC1.1, which are the RGD-containing analogs of the previously described RYD-containing antibodies, OPG2 and PAC1, respectively (6,7). The characterization of PAC1.1 was accomplished in this study.…”