The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion

Abstract

SummaryThe first gene characterizing the clag (cytoadherence linked asexual gene) family of Plasmodium falciparum was identified on chromosome 9. The protein product (Clag9) was implicated in cytoadhesion, the binding of infected erythrocytes to host endothelial cells, but little information on the biochemical characteristics of this protein is available. Other genes related to clag9 have been identified on different chromosomes. These genes encode similar amino acid sequences, but clag9 shows least conservation. Clag9 was detected in schizonts, merozoites and ring-stage parasites after protease digestion and peptide analysis by mass spectrometry. Using antisera raised against unique regions of Clag9 and against RhopH2, a component of the RhopH high-molecular-mass protein complex of merozoites, immunofluorescence colocalized the two proteins to the apical region of merozoites. Immunoelectron microscopy co-localized Clag9 and RhopH2 exclusively to the basal bulb region of rhoptries rather than to their apical ducts. The same Clag9-specific antibodies bound the RhopH complex, and the protein was detected in the complex purified by antibodies to RhopH2. Clag9 protein was also shown to be present in ring-stage parasites, carried through from the previous cycle with the RhopH complex, in a location identical to that of RhopH2. Transcription of the clag9 gene was shown to occur at the same time as the genes for other members of the RhopH complex, rhoph2 and 3 . The results indicate that Clag9 is part of the RhopH complex and suggest that, within this complex, the protein previously designated RhopH1 is composed of more than one protein product of the clag gene family. The results cast doubt on a direct role for Clag9 in cytoadhesion; we suggest that the primary role of the RhopH complex is in remodelling the infected red blood cell after invasion by the merozoite. The complex may have multiple functions dependent on its exact composition, which may include, with respect to Clag9, a contribution to the mechanism of cytoadhesion.

Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum

“…2a, Table 3). The phosphorylation status of one of these rhoptry proteins, CLAG 3.1, that forms part of the RhopH complex proposed to have a role in erythrocyte binding 21 , formation/function of the parasitophorous vacuole 21,22 , cyto-adherence 23 and invasion 21 , was confirmed using an anti-phospho antibody raised against pS 1382 ( Fig. 2b-d).…”

“…For the majority (22) of these 27 loci, we separately transfected 3D7 parasites with tagging vectors designed to place a C-terminal tag (haemagglutinin, Myc or green fluorescent protein) in-frame with the ePK coding sequence (Supplementary Fig. S1).…”

Diversity and evolution of the rhoph1/clag multigene family of Plasmodium falciparum

“…The genes encoding RhopH1 are members of the rhoph1/clag gene family, which was originally defined by the cytoadherence linked asexual gene (clag) on chromosome 9 in P. falciparum (clag9) and consists of at least three members; clag2, 3.1, and 9 [13][14][15].…”

Characterization of a Conserved Rhoptry-Associated Leucine Zipper-Like Protein in the Malaria Parasite Plasmodium falciparum

“…The gene product of the cytoadherence-linked asexual gene 9 (clag9) (64) was used as a second rhoptry-specific marker. CLAG9 was previously localized exclusively to the bulb of the rhoptry (31). The distribution of CLAG9 predominantly overlaps with that of RALP1 (Fig.…”