The Clag family is comprised of gigantic membrane proteins, which are found in all vertebrate parasitic apicomplexan lineages except the basal Cryptosporidium. These proteins have been implicated in many aspects of parasite biology. Phylogenetic analysis suggests that there is one ancient lineage of the Clag family typified by the rhoptry neck protein Ron2, which is conserved across the “crown group” apicomplexans (Click here for a detailed review on apicomplexan adhesion modules and surface proteins). This protein localizes to the rhoptry neck and is delivered to the host cell during invasion. Once delivered to the host cell, Ron2 along with few other parasite secreted rhoptry neck proteins creates a “receptor” complex on the host cell surface. This receptor complex recognizes the APPLE domain protein Ama1 delivered via the micronemes and anchored on the parasite cell surface. This interaction then leads to the formation of the moving junction which is a hallmark of the apicomplexan invasion process. Indeed the phyletic patterns of the Ron2 and the APPLE domain proteins precisely match suggesting that they emerged coevally as part of the invasion strategy used by crown group apicomplexans. From these ancestral Ron2 proteins of the moving junction complex were derived in the Plasmodium lineage the RopH1 or Clag proteins. There are 5 paralogs of the Clag/RopH1 protein in Plasmodium falciparum, several of which have been previously shown to be exported via the rhoptries into the host cell. Each Clag associates with two other proteins RhopH2 and RhopH3 to give rise to multiple (up to 5) RhopH complexes that apparently differ in the type of Clag they possess. Phylogenetic analysis of the Clag family suggests that the RhopH1 (i.e. Clags proper) are a Plasmodium-specific clade that branched off from the Ron2s before the divergence of the P.vivax, P.yoelii/chabaudi and P.falciparum lineages. Even in the common ancestor of these lineages the Clags proper had already split into two distinct lineages: 1) The group-1 Clags which are typified by the Clag9. These are thus far found only in a single copy in each species of Plasmodium studied to date. 2) The group-2 Clags typified by Clag2, Clag3.1/2, and Clag8. These appear to undergo lineage specific expansions in some species. Thus, all the P. falciparum Clags in group-2 are part of lineage specific expansion of 4 paralogs with Clag3.1 and Clag3.2 being alternatively expressed genes encoding different versions of the Clag3 gene. Similarly, in P. vivax there is duplication with two distinct group-2 Clags. In conclusion, it appears that the RhopH complexes are likely to be of distinct groups depending on the group of Clag they contain (e.g. Clag9 type RhopH or Clag2/8/3.1/2 type RhopH).
Early studies on the Clag9 protein implicated it in the binding of infected erythrocytes to the endothelial receptor CD36. Subsequently, the RhopH complex has been shown to be important for successful growth of P.falciparum in the host; however, the exact role of this Plasmodium-specific offshoot of the Ron2 lineage has been shrouded in mystery. They do not appear to form part of the moving junction complex with the Rons, but form distinct complexes on the erythrocyte membrane. This was the state of affairs until an unexpected turn of events connected the Clags (or at least Clag3) to an arduous quest that has been spearheaded by Sanjay Desai for almost two decades. Sanjay Desai’s work has been central to the identification of a key nutrient uptake mechanism for the parasite at the host cell membrane. This is believed to occur via the plasmodial surface anion channel (PSAC). However, its molecular basis had remained unknown. Due to the technological and methodological advances in the Desai lab this channel activity was finally mapped to single parasite locus on chromosome 3, which turned out to be none other than the gene encoding Clag3. This suggested that the membrane spanning segments of Clag3 might very well constitute the structural elements of this anion channel. Previously, a mutant version of the channel, which conferred resistance to Plasmodium against the broad spectrum actinobacterial peptide inhibitor of serine and cysteine peptidases, leupeptin (N-Acetyl-leucyl-leucyl-arginine aldehyde) had been identified. Sequencing revealed that this A1210T mutation mapped to the main conserved TM helix we had predicted at the C-terminus of the protein. This conserved TM helix has a distinctive glycine in most of the Clags suggesting that it might adopt a pi-helix conformation that might be critical for channel activity. Treatment with the peptidase pronase E significantly reduced sorbitol uptake via this channel. Mapping of the peptidase site revealed that it cleaved the protein in the predicted exposed region (which hypervariable among Clags, probably due to positive selection), just upstream of this conserved helix. This supports our prediction based on sequence analysis that the helix is likely to form a transmembrane channel in conjunction with the further N-terminal TM regions. Indeed, the further N-terminal helices also show an atypical composition for TM regions suggesting that this might contribute to the formation of a peculiar intramembrane structure that typifies the channel.
This leads to the question as to whether there might be general implications for the whole Clag family (i.e. including the Ron2s). The TM regions are not particularly conserved between the Clags proper and the Ron2s. This suggests that they might not have similar roles as far as channel activity goes. However, they do share extensive similarity in their N-terminal alpha-super helical region. This, coupled with their shared tendency to translocate to the host membrane suggests that they might still possess similar functions. This extended helical region might act as a scaffold for assembly of the Ron complex in the formation of the moving junction, whereas in the case of the Clags it might contribute to the formation of the RhopH complex via interaction with the other RhopH proteins. Thus it appears that the original principles in assembly of the Ron complex were reused for the assembly of a distinct host membrane complex in the Plasmodium lineages. It is conceivable that the ancestral Ron complex also performed special roles in reorganizing the host membrane during moving junction formation via its TM helices. This may have indeed been reused in the phenomenon of nutrient uptake by the Clags. In light of this it would be interesting to study if the Toxoplasma-specific paralogs of Ron2 have been adapted for a similar function. It would also be of interest to understand how the innovation of the Ron2 complex made the invasion mechanism of the crown group apicomplexans different from that of the more basal forms like Cryptosporidium. For complete details on this study, click here.