Purification and characterization of a protein with fibronectin determinants and phagocytosis-enhancing activity.

A 180,000 m.w. protein isolated from human plasma increases in a dose-response fashion the percentage of human peripheral blood monocytes able to phagocytose particulate activators of the human alternative complement pathway in an otherwise protein-free medium. Because the monocyte monolayers are prepared before the introduction of the protein, the enhanced ingestion of the particulate activators is due to the interaction of the particles with the monocytes and is not due to an increase in the number of adhering monocytes. The function of the protein is specific for particulate activators and is not directed to augmenting ingestion of particles bearing IgG. The protein was purified to apparent homogeneity by sequential affinity chromatography on gelatin-Sepharose, monoclonal BD4 anti-fibronectin-Sepharose, and monoclonal AB3 antibody-Sepharose. Comparable quantities of each of the two monoclonal antibodies in solution neutralized the phagocytosis-enhancing activity in gelatin-derived starting material equally and in a dose-response fashion. Although BD4 monoclonal anti-fibronectin, which recognizes both soluble native plasma fibronectin and the protein bearing the phagocytosis-enhancing activity, defined the antigenic relatedness of the protein to fibronectin, the AB3 hybridoma antibody, which recognizes the phagocytically active protein but not soluble native fibronectin, was critical for its purification to apparent homogeneity. Ouchterlony analysis with rabbit antibody to native fibronectin reveals partial identity between native material that lacks phagocytosis-enhancing activity and the active protein, indicative of antigenic deletion from the protein. As little as 1 µg/ml of the protein increases the percentage of monocytes ingesting rabbit erythrocytes from 3% to 50%; this represents a 100-fold increase in specific activity over the gelatin affinity-derived starting material. As the isolated protein possesses a gelatin-binding site and is antigenically related to native plasma fibronectin, it seems likely that its unique antigenic and functional characteristics are latent in the native molecule.