Molecular Organization and Cross-linking Analysis of the Plasmodium falciparum Erythrocyte Binding Proteins Rhop-H and SERA

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The 120 kDa serine-rich antigen (SERA) of Plasmodium falciparum and the high molecular weight rhoptry protein complex (Rhop-H) bind to erythrocytes and are co-eluted by high salt. Their interaction with the host erythrocyte may be of crucial importance to merozoite invasion and parasitophorous vacuole membrane (PVM) formation. We investigated the timing of protein release into the SCS. The release of Rhop-H and SERA into schizont SCS was detected two hours after removal of biosynthetic label with maximum levels detected at twenty-five hours. Optimal protein binding to erythrocytes was obtained with SCS collected at twenty-five hours. Both proteins were detected in the SCS of schizont stages by immunoprecipitation, but not in ring and trophozoite stages. The identity of SERA was confirmed using different antibodies specific to SERA and no immunological cross-reactivity was detected between SERA and Rhop-H. Affi-blue gel chromatography of SCS for enrichment of both proteins for erythrocyte binding and analysis by immunoblotting showed SERA and Rhop-H in the same fractions. The topological distribution of the proteins on the erythrocyte surface was investigated by chemical cross-linking analysis using DTSSP. SERA and Rhop-H proteins bound at a distance of approximately 12Å on the mouse erythrocyte surface with no other associated proteins. Similar results were obtained using cross-linked schizont extracts precipitated by specific antibodies and analyzed on two-dimensional (2-D) SDS-PAGE gels. Sedimentation analysis demonstrated that SERA and Rhop-H do not form a complex in vivo, and both types of proteins possess different sedimentation rates. We conclude that prior association of SERA with Rhop-H is not required for erythrocyte binding.