Determinants of specificity of a baculovirus-expressed antibody Fab fragment that binds selectively to the activated form of integrin alpha IIb beta 3.
PAC1 is an IgM kappa murine monoclonal antibody that, like the Arg-Gly-Asp-containing ligand fibrinogen, binds to integrin alpha IIb beta 3 only on activated platelets. The unique binding properties of PAC1 may be determined by its large size, its multivalency, and by variable region sequences, including an Arg-Tyr-Asp at residues 100A-C in H-CDR3. To study the molecular determinants of PAC1 function, baculoviruses containing cloned cDNA for the Fd heavy and kappa light chains of PAC1 were used to co-infect Sf9 insect cells. Infected cells secreted a soluble, monovalent, 50-kDa Fab fragment that bound saturably to agonist-stimulated platelets but not to resting cells. Fab binding was inhibited > 85% by 10 mM EDTA, 1 mM RGDS, 1 mM fibrinogen gamma 397-411, or 12 microM fibrinogen, but not by 1 mM RGES. Compared to PAC1 IgM, a 60-fold higher molar concentration of PAC1 Fab was required for half-maximal binding to platelets or for half-maximal inhibition of fibrinogen binding. PAC1 Fab bound to an activated form of alpha IIb beta 3 expressed in Chinese hamster ovary cells, but not to the resting form of the receptor in these cells or to alpha v beta 3 in human endothelial cells. Conversion of Asp100C to Glu by site-directed mutagenesis rendered the antibody inactive, indicating that the Arg-Tyr-Asp sequence in H-CDR3 is essential for PAC1 recognition of alpha IIb beta 3. Binding of fibrinogen or PAC1 IgM to platelets induced tyrosine phosphorylation of a 140-kDa platelet protein, but binding of PAC1 Fab did not. These studies demonstrate that the specificity of PAC1 for activated alpha IIb beta 3 is determined by an integrin recognition sequence within H-CDR3. However, the strength of this binding interaction and the ability of PAC1 to trigger signaling in platelets also depend on antibody valency.
Abrams
C
C
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Deng
Y J
YJ
Steiner
B
B
O'Toole
T
T
Shattil
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics; D001666:Binding Sites, Antibody; D001792:Blood Platelets / Q000276:immunology; D004730:Endothelium, Vascular / Q000276:immunology; D005340:Fibrinogen / Q000494:pharmacology; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D066298:In Vitro Techniques; D016023:Integrins / Q000276:immunology*; D008969:Molecular Sequence Data; D009036:Moths; D009842:Oligopeptides; D019000:Phosphotyrosine; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000276:immunology*; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D016023:Integrins; D009842:Oligopeptides; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen