Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.

Abstract

A monoclonal antibody (mAb), sis 1, generated against human c-sis-encoded platelet-derived growth factor (PDGF) BB, was shown by enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to recognize human PDGF BB and human platelet PDGF AB but not the human PDGF AA. This monoclonal antibody potently inhibited PDGF receptor-binding and mitogenic activities of both human PDGF BB and PDGF AB but had no effect on PDGF AA. Finally, we demonstrated that an immunoaffinity-purified anti-c-sis peptide antibody (anti-V4) which also blocked binding of PDGF BB to its cognate receptor and competed with mAb sis 1 for binding to PDGF BB. All of these results suggest that mAb sis 1 recognizes an epitope of the c-sis gene product, PDGF BB, that spatially overlaps the V4 surface domain of PDGF BB, immunochemically localizing a region of PDGF BB critical for PDGF receptor binding and activation.

A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity.

“…Each nondimerizing mutant gene product, as represented by A133, was detected only by anti-sis C (Fig. 2), consistent with the known impaired recognition of reduced forms of the v-sis/PDGF B product by either anti-PDGF or MAb sis 1 (8,21).…”

“…We have recently described a neutralizing monoclonal antibody, MAb sis 1, directed against PDGF B. Immunochemical studies have mapped its recognition site to the amino-terminal half of the v-sis/PDGF B product's transforming domain (21). The availability of our series of v-sis deletion mutants made it (8) possess potent neutralizing activity but do not readily detect the fully reduced v-sis/ PDGF B product.…”

“…In contrast, biologic activity correlated with the extent of dimer formation for analogous deletions throughout the rest of the molecule. Using site-specific antisera directed against predicted v-six peptides, we have recently shown that residues 131 to 154 represent a surface domain of PDGF BB (21). Moreover, v-sis/PDGF BB recognition by a neutralizing monoclonal antibody, MAb sis 1, has been immunochemically mapped to this domain, since peptide antisera recognizing the surface domain compete for MAb sis 1 recognition of PDGF BB (21).…”

“…Native human PDGF, isolated from platelets, was used to generate anti-PDGF polyclonal serum (15,27). A neutralizing monoclonal antibody directed against the PDGF B homodimer designated MAb sis 1 has been reported (21). Antibodies recognizing either the a or ,B PDGF receptor were recently described (24).…”

“…Recently, we used immunochemical approaches to localize a PDGF B surface subdomain and the site of interaction with a monoclonal antibody that neutralizes PDGF mitogenic activity and blocks ligand-receptor interaction (21). The present studies were undertaken in an effort to combine molecular genetic approaches with available immunologic probes to map the subdomain within the v-sis/PDGF B molecule critical to PDGF receptor interaction.…”