EXPRESSION OF WILD-TYPE AND MUTATED RABBIT OSTEOPONTIN IN ESCHERICHIA-COLI, AND THEIR EFFECTS ON ADHESION AND MIGRATION OF P388D1 CELLS

Recombinant wild-type rabbit osteopontin (rOP) and the protein with an aspartate-to-glutamate transposition induced by a point mutation in t he rabbit OP cDNA within the Gly-Arg-Gly-Asp-Ser (GRGDS) sequence were expressed in Escherichia coli and purified to homogeneity. P388D1 cel ls bound rOP in a saturable manner. rOP induced adhesion and haptotaxi s of P388D1 cells, whereas mutated rabbit OP (rOPmut) did not. Anti-rO P IgG F(ab')(2) and synthetic GRGDS peptide inhibited rOP-mediated adh esion and haptotaxis of P388D1 cells. Fibronectin (FN)mediated adhesio n of P388D1 cells was markedly inhibited in the presence of fluid-phas e rOP. Adhesion of P388D1 cells to rOP was significantly inhibited by anti-[alpha-subunits of VLA4 (alpha(4)) and VLA5 (alpha(5))] monoclona l antibodies (mAbs), but not by anti-[alpha-subunit of vitronectin (VN ) receptor (alpha V) or Mac-1 (alpha(M))] mAb. Adhesion of P388D1 cell s to FN and VN was significantly inhibited by anti-alpha(V) mAb but no t anti-alpha(4), -alpha(5), or -alpha(M) mAb. Haptotaxis of P388D1 cel ls to rOP was significantly inhibited by anti-alpha(V) mAb, but not by anti-alpha(4), -alpha(5), and alpha(M) mAbs, whereas that to FN showe d no inhibition with all three mAbs. Haptotaxis of P388D1 cells to VN was significantly inhibited by anti-alpha(5) and -alpha(V) mAbs but no t by anti-alpha(4) and -alpha(M) mAbs. Similar features of inhibition of adhesion and haptotaxis of P388D1 cells to human OP were observed b y mAbs. rOP had no chemotactic effect on P388D1 cells. Significant pol ymorphonuclear leucocyte migration was observed 3-12 h after intraderm al injection of rOP into rabbits.