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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

Transcription levels of the PfG mRNA from P. falciparum.Northern Blot analysis with cellular RNA from different developmental stages: E Trophozoite = Early Trophozoite; L Trophozoite = Late Trophozoite; Schizonts and Gametocytes. Detection of signals was performed by phoshoimage analysis using the digoxigenin–dUTP-labeled alpha-tubulin 2 gene as a constituitively expressed gene in the blood stages or the PfG cDNA from P. falciparum as molecular probe. Two stage specific controls were employed:i) The MSP1 fragment resulted in a signal of 5161bp in the blood stages and a signal of 3.8 kb in the gametocytes.
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pone.0140994.g004: Transcription levels of the PfG mRNA from P. falciparum.Northern Blot analysis with cellular RNA from different developmental stages: E Trophozoite = Early Trophozoite; L Trophozoite = Late Trophozoite; Schizonts and Gametocytes. Detection of signals was performed by phoshoimage analysis using the digoxigenin–dUTP-labeled alpha-tubulin 2 gene as a constituitively expressed gene in the blood stages or the PfG cDNA from P. falciparum as molecular probe. Two stage specific controls were employed:i) The MSP1 fragment resulted in a signal of 5161bp in the blood stages and a signal of 3.8 kb in the gametocytes.

Mentions: Next, we monitored transcription of the PfG m-RNA in different developmental stages i.e. young and late trophozoites, schizonts and gametocytes. (Fig 4). The alpha tubulin 2 gene [27] from P. falciparum (PF3D7_ 0903700) was employed as a constitutively transcribed control in RNA prepared from mixed erythrocytic stages [25]. Transcription of PfG increased approximately 12-fold in mature trophozoites and schizonts. This result prompted us to investigate transcript levels in sexual precursor stages like gametocytes where a 22-fold increase was detected. Stage specific probes were employed as controls, i. e. a 92 bp fragment from MSP-1 (merozoite surface protein1) transcribed in the erythrocytic stages and [28] and the 1223 bp probe from the Pfg377 var gene for gametocytes resulting in transcript levels of approximately 90% for both probes in each developmental stage (Fig 4).


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)

Transcription levels of the PfG mRNA from P. falciparum.Northern Blot analysis with cellular RNA from different developmental stages: E Trophozoite = Early Trophozoite; L Trophozoite = Late Trophozoite; Schizonts and Gametocytes. Detection of signals was performed by phoshoimage analysis using the digoxigenin–dUTP-labeled alpha-tubulin 2 gene as a constituitively expressed gene in the blood stages or the PfG cDNA from P. falciparum as molecular probe. Two stage specific controls were employed:i) The MSP1 fragment resulted in a signal of 5161bp in the blood stages and a signal of 3.8 kb in the gametocytes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140994.g004: Transcription levels of the PfG mRNA from P. falciparum.Northern Blot analysis with cellular RNA from different developmental stages: E Trophozoite = Early Trophozoite; L Trophozoite = Late Trophozoite; Schizonts and Gametocytes. Detection of signals was performed by phoshoimage analysis using the digoxigenin–dUTP-labeled alpha-tubulin 2 gene as a constituitively expressed gene in the blood stages or the PfG cDNA from P. falciparum as molecular probe. Two stage specific controls were employed:i) The MSP1 fragment resulted in a signal of 5161bp in the blood stages and a signal of 3.8 kb in the gametocytes.
Mentions: Next, we monitored transcription of the PfG m-RNA in different developmental stages i.e. young and late trophozoites, schizonts and gametocytes. (Fig 4). The alpha tubulin 2 gene [27] from P. falciparum (PF3D7_ 0903700) was employed as a constitutively transcribed control in RNA prepared from mixed erythrocytic stages [25]. Transcription of PfG increased approximately 12-fold in mature trophozoites and schizonts. This result prompted us to investigate transcript levels in sexual precursor stages like gametocytes where a 22-fold increase was detected. Stage specific probes were employed as controls, i. e. a 92 bp fragment from MSP-1 (merozoite surface protein1) transcribed in the erythrocytic stages and [28] and the 1223 bp probe from the Pfg377 var gene for gametocytes resulting in transcript levels of approximately 90% for both probes in each developmental stage (Fig 4).

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