Plasmodium falciparum: analysis of protein-protein interactions of the 140/130/110-kDa rhoptry protein complex using antibody and mouse erythrocyte binding assays.

The high-molecular-weight rhoptry proteins of Plasmodium falciparum exist in a multiprotein complex consisting of proteins of 140, 130, and 110 kDa. The complex of rhoptry proteins binds to human and mouse erythrocyte membranes in association with a 120-kDa SERA protein. These proteins are believed to participate in the process of erythrocyte invasion. We have used six different antibodies (polyclonal and monoclonal) known to precipitate the high-molecular-weight rhoptry protein complex (HMWC) to analyze the structural relationship of proteins within the complex. Limited proteolysis of immune complexes (IC) immobilized on Sepharose beads (protein "footprinting") and binding of SV8 protease generated peptides to intact mouse erythrocytes was performed. The 140-kDa polypeptide was more susceptible to protease digestion followed by the 130- and 110-kDa polypeptides. The susceptibility of the 140-kDa polypeptide to protease digestion was independent of the type of precipitating antibody. We identified a 120-kDa protein as the major proteolytic fragment of the 140-kDa protein. SV8 protease generated peptide fragments derived from the 110- and 130-kDa proteins contained putative mouse erythrocyte binding domains. Immunoprecipitation of SV8-generated peptides gave peptide profiles similar to those obtained with protein "footprinting". Additional experiments performed to investigate the stability of the HMWC using chaotropic and lyotropic agents demonstrated that the HMWC was stable to perturbatory reagents known to disaggregate macromolecular complexes. Solubilization of schizonts with 6 M urea and 4 M MgCl2 followed by IC formation led to differential precipitation of the 110-kDa polypeptide, while solubilization with 3 M KCl resulted in the differential precipitation of the 140- and 130-kDa polypeptides, suggesting that both proteins may be in direct association. Treatment of immobilized IC with different perturbatory agents including 6 M urea, 3 M KCl, 4 M MgCl2, or 2% SDS from an insoluble matrix resulted in the elution of the intact complex. The mouse erythrocyte binding property of the HMWC is conserved among different geographical isolates of P. falciparum. The results provide insights concerning the mechanism of protein-protein interaction within the complex.