Apical expression of three RhopH1/Clag proteins as components of the Plasmodium falciparum RhopH complex

The Plasmodium falciparum high molecular mass rhoptry protein ('PfRhopH') complex is important for parasite growth and comprises three distinct gene products: RhopH1, RhopH2 and RhopH3. We have previously shown that P. falciparum RhopH1 is encoded by either PFC0110w (clag3.2) or PFC0120w (clag3.1), members of the previously-named clag (cytoadherence-linked asexual gene) multigene family. In this report, we have further characterized rhoph1/clag members in terms of gene structure, transcription and protein expression. The cDNA sequences for all five rhoph1/clag members were determined, confirming previous in silico predictions of intron-exon boundaries. All member genes were transcribed in HB3 and 3D7 parasite lines, but clag3.2 was not transcribed in Dd2 parasites. The peak abundance of transcripts for all genes was observed during the late schizont stage. Antisera specific to Clag2 and Clag3.1 localized these proteins to the apical end of merozoites in segmented schizonts, and both proteins are found to be components of the PfRhopH complex. PfRhopH complex that was immunoprecipitated with anti-Clag9 antibody contained neither Clag2 nor Clag3.1, thereby suggesting that PfRhopH complexes contain only individual rhoph1/clag gene products. Since the PfRhopH complex binds the erythrocyte surface, and RhopH2 and RhopH3 are encoded by single copy genes, the RhopH1/Clag proteins may serve to confer some degree of specificity to the roles of the individual complexes.