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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

Association and dissociation of BODIPY FL GTPγS with PfG from Plasmodium.Part A Absolute Fluorescense of binding of 50 nmol BODIPY FL GTPγS to the plasmodial, PfG-protein (black line). The increase in fluorescence was monitored until saturation over a time interval of 600 s. Part B At the arrow at t = 200 s, 20 μM unlabeled GTPγS was added and the decrease in fluorescence was monitored. The dissociation of BODIPY FL GTPγS was fit with single exponential functions and the half life value was determined i.e. t1/2 = 9 s suggesting a low affinity for the binding of BODIPY FL GTP. b Monitoring GTP-binding of the P. falciparum G-protein in a time course experiment: Binding assay with different protein concentrations of PfG from Plasmodium green line 62.5 μg, red line 16,5 μg; the reaction was antagonized with unlabeled GTP after 100 s blue line (62,5 μg purified G-protein), purple line (16,5 μg purified-G-protein), yellow line (8,0 μg G-protein).
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pone.0140994.g007: Association and dissociation of BODIPY FL GTPγS with PfG from Plasmodium.Part A Absolute Fluorescense of binding of 50 nmol BODIPY FL GTPγS to the plasmodial, PfG-protein (black line). The increase in fluorescence was monitored until saturation over a time interval of 600 s. Part B At the arrow at t = 200 s, 20 μM unlabeled GTPγS was added and the decrease in fluorescence was monitored. The dissociation of BODIPY FL GTPγS was fit with single exponential functions and the half life value was determined i.e. t1/2 = 9 s suggesting a low affinity for the binding of BODIPY FL GTP. b Monitoring GTP-binding of the P. falciparum G-protein in a time course experiment: Binding assay with different protein concentrations of PfG from Plasmodium green line 62.5 μg, red line 16,5 μg; the reaction was antagonized with unlabeled GTP after 100 s blue line (62,5 μg purified G-protein), purple line (16,5 μg purified-G-protein), yellow line (8,0 μg G-protein).

Mentions: Association/dissociation was performed with 20 μM unlabeled GTPΎS. In a first experiment the unlabeled GTPγS was added at t = 0 s and an immediate decrease in fluorescence followed which was monitored in a Perkin Elmer LS45 spectrometer (Fig 7A). The absolute fluorescense value (180) was already lower after supplementation with 20μM unlabeled GTP after 0 s in comparison to 230 of the untreated control (Fig 7A). The unlabeled nucleotide competed directly with the fluorescense labeled GTP for the binding site of the PfG protein. In a second experiment the assay was supplemented with 20μM unlabeled GTP after 200 s and the exponential decay was monitored caused by dissociation of the labeled GTP from the PfG binding site (Fig 6A). By contrast, the control experiment showed a hyperbolic saturation curve without supplementation of unlabeled GTP (Fig 7A). This result is consistent with the determined half lives i.e.t1/2 = 71,14 s for the untreated control and the decreased half life t1/2 = 9 s for the antagonized reaction. Next, kinetic parameters of the reaction were determined. In this experiment the binding of BODIPY®FL GTPΎS to PfG was monitored over 600s under different substrate concentrations (Fig 6B). PfG had a determined Km for BODIPY®FL GTPΎS of 1.77 ± 0.36 nmol. By contrast, the human Gα0 and Gαi1 subunits had a Km of 14±8 nmol and 87±22 nmol, respectively, demonstrating a high affinity for the nucleotide which is typical for a heterotrimeric G protein. Commitment to and completion of sexual development are essential for malaria parasites. It has been recently shown that a cascade of DNA-binding proteins is responsible for sexual commitment and development in Plasmodium [47]. These DNA-binding proteins belong to a group of transcription factors which are highly conserved in apicomplexan protists and amenable to environmental sensing. It will be an important issue in the future to delineate the function of the PfG protein in this context.


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)

Association and dissociation of BODIPY FL GTPγS with PfG from Plasmodium.Part A Absolute Fluorescense of binding of 50 nmol BODIPY FL GTPγS to the plasmodial, PfG-protein (black line). The increase in fluorescence was monitored until saturation over a time interval of 600 s. Part B At the arrow at t = 200 s, 20 μM unlabeled GTPγS was added and the decrease in fluorescence was monitored. The dissociation of BODIPY FL GTPγS was fit with single exponential functions and the half life value was determined i.e. t1/2 = 9 s suggesting a low affinity for the binding of BODIPY FL GTP. b Monitoring GTP-binding of the P. falciparum G-protein in a time course experiment: Binding assay with different protein concentrations of PfG from Plasmodium green line 62.5 μg, red line 16,5 μg; the reaction was antagonized with unlabeled GTP after 100 s blue line (62,5 μg purified G-protein), purple line (16,5 μg purified-G-protein), yellow line (8,0 μg G-protein).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140994.g007: Association and dissociation of BODIPY FL GTPγS with PfG from Plasmodium.Part A Absolute Fluorescense of binding of 50 nmol BODIPY FL GTPγS to the plasmodial, PfG-protein (black line). The increase in fluorescence was monitored until saturation over a time interval of 600 s. Part B At the arrow at t = 200 s, 20 μM unlabeled GTPγS was added and the decrease in fluorescence was monitored. The dissociation of BODIPY FL GTPγS was fit with single exponential functions and the half life value was determined i.e. t1/2 = 9 s suggesting a low affinity for the binding of BODIPY FL GTP. b Monitoring GTP-binding of the P. falciparum G-protein in a time course experiment: Binding assay with different protein concentrations of PfG from Plasmodium green line 62.5 μg, red line 16,5 μg; the reaction was antagonized with unlabeled GTP after 100 s blue line (62,5 μg purified G-protein), purple line (16,5 μg purified-G-protein), yellow line (8,0 μg G-protein).
Mentions: Association/dissociation was performed with 20 μM unlabeled GTPΎS. In a first experiment the unlabeled GTPγS was added at t = 0 s and an immediate decrease in fluorescence followed which was monitored in a Perkin Elmer LS45 spectrometer (Fig 7A). The absolute fluorescense value (180) was already lower after supplementation with 20μM unlabeled GTP after 0 s in comparison to 230 of the untreated control (Fig 7A). The unlabeled nucleotide competed directly with the fluorescense labeled GTP for the binding site of the PfG protein. In a second experiment the assay was supplemented with 20μM unlabeled GTP after 200 s and the exponential decay was monitored caused by dissociation of the labeled GTP from the PfG binding site (Fig 6A). By contrast, the control experiment showed a hyperbolic saturation curve without supplementation of unlabeled GTP (Fig 7A). This result is consistent with the determined half lives i.e.t1/2 = 71,14 s for the untreated control and the decreased half life t1/2 = 9 s for the antagonized reaction. Next, kinetic parameters of the reaction were determined. In this experiment the binding of BODIPY®FL GTPΎS to PfG was monitored over 600s under different substrate concentrations (Fig 6B). PfG had a determined Km for BODIPY®FL GTPΎS of 1.77 ± 0.36 nmol. By contrast, the human Gα0 and Gαi1 subunits had a Km of 14±8 nmol and 87±22 nmol, respectively, demonstrating a high affinity for the nucleotide which is typical for a heterotrimeric G protein. Commitment to and completion of sexual development are essential for malaria parasites. It has been recently shown that a cascade of DNA-binding proteins is responsible for sexual commitment and development in Plasmodium [47]. These DNA-binding proteins belong to a group of transcription factors which are highly conserved in apicomplexan protists and amenable to environmental sensing. It will be an important issue in the future to delineate the function of the PfG protein in this context.

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