A Putative Non-Canonical Ras-Like GTPase from P. falciparum: Chemical Properties and Characterization of the Protein.

Annette Kaiser,David Kersting,Barbara Langer,Jude Przyborski,Mirko Krüger,Gordon Langsley

doi:10.1371/journal.pone.0140994

Abstract

During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts. Key cellular mechanisms involving G-protein coupled signal transduction chains are assumed to act at these interfaces. Heterotrimeric G-proteins are absent in Plasmodium. We here describe the first cloning and expression of a putative, non-canonical Ras-like G protein (acronym PfG) from Plasmodium. PfG reveals an open reading frame of 2736 bp encoding a protein of 912 amino acids with a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation. Most notably, PfG has GTP binding capacity and GTPase activity due to an EngA2 domain present in small Ras-like GTPases in a variety of Bacillus species and Mycobacteria. By contrast, plasmodial PfG is divergent from any human alpha-subunit. PfG was expressed in E. coli as a histidine-tagged fusion protein and was stable only for 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and subsequent separation by Blue Native PAGE. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5’O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan stimulated GTP binding in the presence of Mg2+. GTPase activity was determined colorimetrically. Activity expressed as absolute fluorescence was 50% higher for the human paralogue than the activity of the parasitic enzyme. The PfG protein is expressed in the erythrocytic stages and binds GTP after immunoprecipitation. Immunofluorescence using specific antiserum suggests that PfG localizes to the parasite cytosol. The current data suggest that the putitative, Ras-like G-protein might be involved in a non-canonical signaling pathway in Plasmodium. Research on the function of PfG with respect to pathogenesis and antimalarial chemotherapy is currently under way.

Highlights

Knowledge of pathways regulated by cyclic nucleotides in Plasmodium is still rudimentary [1]
Based on a bioinformatics screen with conserved amino acid sequences of the G-alpha-s subunit (Gs)-protein alphasubunit from various organisms i.e. Dictyostelium dioscoideum, Mycoplasma, and Saccharomyces cerevisiae we identified an open reading frame (ORF) of 912 amino acids encoding a putative GTP-binding protein (PFG) present in the Plasmodium database (PlasmoDB identifier PF3D7_ 0313500)
Total cellular RNA was isolated from schizont stages of P. falciparum in vitro and mRNA was employed

Summary

Introduction

Knowledge of pathways regulated by cyclic nucleotides in Plasmodium is still rudimentary [1]. These results are in contrast to previous findings which demonstrated the occurrence of heterotrimeric G-proteins in Plasmodium by western blot analysis with cholera and pertussis toxins employing antibodies directed against epitopes of different human Galpha subunits [37].

Results

Conclusion