Computational annotation of typical apicomplexan proteins and biochemical studies of highly conserved plasmodium falciparum acyl CoA binding proteins

Abstract

The phylum Apicomplexa is an obligate parasitic clade of alveolate eukaryotes surviving within a wide range of animals from molluscs to mammals. The apicomplexan parasites include Plasmodium and Toxoplasma gondii, recently a number of apicomplexan parasites have been sequenced. But these apicomplexan genomes have a large number of hypothetical proteins. There was an alternate hypothesis that many apicomplexan proteins may not find orthologs in BLAST searches using standard BLOSUM matrices because of unusual amino acid composition. In my group, a novel series of substitution matrices was developed for P. falciparum in particular and for apicomplexan proteins in general to address this issue. In the present work, ApicoAlign (http://www.cdfd.org.in/apicoalign/) was developed to provide public access to this novel series of matrices. Using these parasite specific substitution matrices, some probable functions (kinase/protease) were predicted for 1374 hypothetical proteins of 15 apicomplexan parasites. A detailed study of these predicted kinases and proteases revealed the conservation of motifs and critical amino acid residues. There are few apicomplexan proteins like acyl CoA binding proteins (ACBPs) which are very well conserved across all the eukaryotes. This very high level of sequence conservation of ACBPs occurs despite the fact that most of the P. falciparum proteins have diverged from their eukaryotic orthologs. This fact was interesting to further study these proteins and my initial hypothesis was that these proteins either should have multiple roles/functions and/or be very crucial for P. falciparum. In order to further study these proteins, all the four P. falciparum ACBPs were successfully cloned, expressed and purified and characterized using isoelectric focusing, fluorescence quenching and isothermal titration calorimetry. It was also demonstrated that Pf ACBPs can bind to RBC ghosts by flow cytometry and to various lipids using protein lipid overlay assay.