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

Kaiser A, Langer B, Przyborski J, Kersting D, Krüger M - PLoS ONE (2015)

Bottom Line: Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Centre, Institute for Pharmacogenetics, University Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Phylogram pinpointing the phylogenetic origin of the EngA2 domain in the PfG protein from Plasmodium: The closest phylogenetic relationship is evident between Plasmodium (10) PfG and Mycoplasma mobile strain K173 (1) with a determined difference in tree distance of 0.245.Both are closely related to the EngA2 domain from Bacillus subtilis (3) with a difference in tree distance of 0.0027 in comparison to Mycoplasma mobile (1). The next closest relationship of the EngA domain is to Streptomyces scabieii (6) (tree difference:0.18442) and to Mycobacterium tuberculosis (8) (tree difference:0.19355) rather than the Enterobacteriaceae Salmonella enteritica (8) and Klebsiella pneumonia (4) and Serratia marcescens (9) with tree distances of 0.02918, 0.03408 and 0.02918, respectively. Less related are the EngA2 domains present in Pseudomonas putida (tree difference:0.086057) (5) and Pseudomonas fluorescens (tree difference:0.06617).
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pone.0140994.g003: Phylogram pinpointing the phylogenetic origin of the EngA2 domain in the PfG protein from Plasmodium: The closest phylogenetic relationship is evident between Plasmodium (10) PfG and Mycoplasma mobile strain K173 (1) with a determined difference in tree distance of 0.245.Both are closely related to the EngA2 domain from Bacillus subtilis (3) with a difference in tree distance of 0.0027 in comparison to Mycoplasma mobile (1). The next closest relationship of the EngA domain is to Streptomyces scabieii (6) (tree difference:0.18442) and to Mycobacterium tuberculosis (8) (tree difference:0.19355) rather than the Enterobacteriaceae Salmonella enteritica (8) and Klebsiella pneumonia (4) and Serratia marcescens (9) with tree distances of 0.02918, 0.03408 and 0.02918, respectively. Less related are the EngA2 domains present in Pseudomonas putida (tree difference:0.086057) (5) and Pseudomonas fluorescens (tree difference:0.06617).

Mentions: The obtained data prompted us to generate a phylogram to identify the closest phylogenetic relationship of the EngA domain present in PfG to other species.The phylogram depicted in Fig 3 shows a close relationship with a tree difference of 0.245 to Mycoplasma mobile, a pathogenic gram positive Eubacterium and to Bacillus subtilis with a tree difference of 0.0027, respectively. Mycoplasma mobile is one of the fastest smooth gliding bacteria. In sum, the PfG protein cannot be classified within mammalian Gα-subunits. In a search within the PlasmoDB database we were unable to discover a G-protein ß subunit. Instead, we identified a G-beta repeat domain containing protein (PFIT-1122200). There was no database entry encoding a G-protein gamma subunit. 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 G-alpha subunits [37].


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

Kaiser A, Langer B, Przyborski J, Kersting D, Krüger M - PLoS ONE (2015)

Phylogram pinpointing the phylogenetic origin of the EngA2 domain in the PfG protein from Plasmodium: The closest phylogenetic relationship is evident between Plasmodium (10) PfG and Mycoplasma mobile strain K173 (1) with a determined difference in tree distance of 0.245.Both are closely related to the EngA2 domain from Bacillus subtilis (3) with a difference in tree distance of 0.0027 in comparison to Mycoplasma mobile (1). The next closest relationship of the EngA domain is to Streptomyces scabieii (6) (tree difference:0.18442) and to Mycobacterium tuberculosis (8) (tree difference:0.19355) rather than the Enterobacteriaceae Salmonella enteritica (8) and Klebsiella pneumonia (4) and Serratia marcescens (9) with tree distances of 0.02918, 0.03408 and 0.02918, respectively. Less related are the EngA2 domains present in Pseudomonas putida (tree difference:0.086057) (5) and Pseudomonas fluorescens (tree difference:0.06617).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4634863&req=5

pone.0140994.g003: Phylogram pinpointing the phylogenetic origin of the EngA2 domain in the PfG protein from Plasmodium: The closest phylogenetic relationship is evident between Plasmodium (10) PfG and Mycoplasma mobile strain K173 (1) with a determined difference in tree distance of 0.245.Both are closely related to the EngA2 domain from Bacillus subtilis (3) with a difference in tree distance of 0.0027 in comparison to Mycoplasma mobile (1). The next closest relationship of the EngA domain is to Streptomyces scabieii (6) (tree difference:0.18442) and to Mycobacterium tuberculosis (8) (tree difference:0.19355) rather than the Enterobacteriaceae Salmonella enteritica (8) and Klebsiella pneumonia (4) and Serratia marcescens (9) with tree distances of 0.02918, 0.03408 and 0.02918, respectively. Less related are the EngA2 domains present in Pseudomonas putida (tree difference:0.086057) (5) and Pseudomonas fluorescens (tree difference:0.06617).
Mentions: The obtained data prompted us to generate a phylogram to identify the closest phylogenetic relationship of the EngA domain present in PfG to other species.The phylogram depicted in Fig 3 shows a close relationship with a tree difference of 0.245 to Mycoplasma mobile, a pathogenic gram positive Eubacterium and to Bacillus subtilis with a tree difference of 0.0027, respectively. Mycoplasma mobile is one of the fastest smooth gliding bacteria. In sum, the PfG protein cannot be classified within mammalian Gα-subunits. In a search within the PlasmoDB database we were unable to discover a G-protein ß subunit. Instead, we identified a G-beta repeat domain containing protein (PFIT-1122200). There was no database entry encoding a G-protein gamma subunit. 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 G-alpha subunits [37].

Bottom Line: Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Centre, Institute for Pharmacogenetics, University Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus