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. 2015 Nov 5;10(11):e0140994.
doi: 10.1371/journal.pone.0140994. eCollection 2015.

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

Annette Kaiser  1 , Barbara Langer  1   2 , Jude Przyborski  3 , David Kersting  1 , Mirko Krüger  1
Affiliations

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

Annette Kaiser et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Cloning strategy of the nucleic acid sequence encoding the PfG protein from Plasmodium falciparum: A set of four different primer combinations was applied for the amplification to cover the complete coding region of PfG protein from P. falciparum.
The primer pairs G1f# and G10#r, Gs 8f# and G9#r, G20f# and G21#r, G18f# and G2#r were employed in RT-PCR reactions with polyA+-RNA from schizont stages. Four different cDNA fragments in the range of 780 bp, 810 bp, 570 bp and 635 bp were obtained, respectively. Bold arrows in blue represent the full sequence of 2.7 kb while small black arrows with double arrowheads indicate the overlapping regions between the different fragments. Black lines represent the different cDNA fragments obtained with the sequence-specific set of primers used for the different amplificates. Downstream primers are located at the bottom of the black lines. B) Assembly reactions were performed with two fragments in each reaction from a total of four fragments. Primer combinations in particular assembly primers (assT1# and assT2#) with protruding 5’ and 3’ ends for subcloning into pET-28a expression vector were employed in the different PCR reactions (see Material and Methods within). From four amplificates two were assembled in each reaction with the primer set shown in the figure. C) Final assembly reaction of the two obtained fragments from reaction B resulted in the entire fragment of 2.7 kb.
Fig 2
Fig 2. Multiple amino acid alignment of a stimulatory Gαs-subunit from human (Gsαlpha-subunit), Dictyostelium dioscoidum, Komagatella pastoris (Pichia pastoris) and the amino acid sequence of the PfG protein spanning amino acid sequence positions from 361–911 for a better alignment.
The EngA protein from Mycoplasma mobile strain K163 was included. Conserved amino acids present in the EngA2 domain are marked in yellow. This EngA2 subfamily represents the second GTPase domain of EngA and its orthologs, which are composed of two adjacent GTPase domains. Since the sequences of the two domains are more similar to each other than to other GTPases, it is likely that an ancient gene duplication, rather than a fusion of evolutionarily distinct GTPases gave rise to this family. Although the exact function of these proteins has not been elucidated. Asterisks label amino acid identities, colons (:) and dots (.) label amino acid similarities. The alignment represents the putitative characteristic domains of the PfG protein from Plasmodium. At the C-terminus a KH-like domain (purple colour, amino acid position 805–875) of the EngA subfamily of essential bacterial GTPases with two adjacent GTPase domains is located. The EngA2 domain covers amino acid positions 730–800. The GTPase domain elements for binding ribonucleoproteins are missing. A small GTP-binding domain respresents a Ras-like protein which is similar to the DER protein in E. coli responsible for cell viability and also present in Neisseria gonorrhea and Thermotoga maritima. This EngA2 subfamily CD represents the second GTPase domain of EngA and its orthologs, which are composed of two adjacent GTPase domains. Since the sequences of the two domains are more similar to each other than to other GTPases, it is likely that an ancient gene duplication, rather than a fusion of evolutionarily distinct GTPases, gave rise to this family. Although the exact function of these proteins has not been elucidated. The GTP/Mg-binding site is marked by a blue arrow and responsible for the chemical binding of GTP and Mg2+. Five G Box motifs for GTP-binding are present within the EngA2 domain including a switch region II which is a surface loop undergoing conformational change upon GTP-binding. The G1 box motif: GXXXXGK[T/S] is a signature motif of a phosphate-binding loop. G2 box motif: T Thr is conserved throughout the superfamily, but surrounding residues are conserved within families. G3 box motif: DXXG overlaps the Switch II region, which includes the Walker B motif. G4 box has the motif: [N/T]KXD and the G5 box the motif: [C/S]A[K/L/T].
Fig 3
Fig 3. 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).
Fig 4
Fig 4. 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.
Fig 5
Fig 5. Native Blue Gel electrophoresis after expression and purification of the PfG protein under native conditions by nickel-chelate-chromatography.
Separation was performed on a 6–12% Bis-Tris gel. a lane 1 Native unstained protein marker (NativeStainTM) 20kDa = Soybean Trypsin Inhibitor, 66 kDa = Bovine Serum Albumin, 146kDa = Lactate Dehydrgenase; 242 kDa = B-phycoerythrin, 480 kDa = Apoferritin band 2; lane 2 lysate; lane 3 flowthrough; lane 4 wash fraction 1, lane 5 wash fraction 2; lane 6 eluate 1; lane 7 eluate 2. b Immunoblot characterization of the different affinity purification steps of PfG protein from P. falciparum. Native-Blue-PAGE electrophoresis was employed to separate the purified proteins. Subsequent immunoblot analysis followed using a monoclonal anti MBP (a murine anti-maltose binding protein) His antibody.
Fig 6
Fig 6. Saturation curve representing the binding of BODIPY®FL GTPΎS with the expressed PfG protein from P. falciparum.
The depicted plot shows the absolute fluorescense.of the formed PfG-Protein-BGTP complex monitored at a wavelength between 480 nm and 510 nm versus increasing protein concentrations with a crude extract of the purified expressed PfG- protein (black square) and a purified enzyme preparation (black circle) after native purificaion. A non-recombinant pET-28a vector (open triangle) and a constitutively expressed Gαs human subunit (open square) were employed as a positive and a negative control. Each point represents the mean value of three different experiments.
Fig 7
Fig 7. 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).
Fig 8
Fig 8. Costimulation of the constitutively expressed human G protein alphas long subunit in comparison to PfG from P. falciparum.
The expressed recombinant proteins were stimulated with mastoparan alone simulating the receptor in case of Gαs human (black circle) and PfG from P. falciparum (rhomb.) Costimulation was performed with mastoparan and Mg2+ for plasmodial PfG (triangle) and the constitutively expressed G alpha-s subunit (rhomb).
Fig 9
Fig 9. Absorbance versus enzyme amount: For quantification of the reaction absorbance versus enzyme concentration was plotted.
To keep linearity, the assay time was fixed for 30 min and the temperature kept at 25°C.
Fig 10
Fig 10. (a) Detection of endogenous PfG in the erythrocytic stages: A) Immunodetection of endogenous PfG by a peptide anti-PfG antibody (dilution 1:1000) in protein extracts from enriched schizonts (lane S) and trophozoites (lane T). Recombinant PfG protein expressed in E.coli (lane R) was employed as a control. (b) Fluorescence emission after binding of BODIPY FL GTPγS of endogenous, immunoprecipitated PfG in crude protein extracts of schizonts (black square), trophozoites (black triangle) versus recombinant PfG (black circle). Each point represents the mean value of three different experiments. (c) Silver stained immunprecipitated PfG protein after SDS PAGE. Lane M) Precision Plus Marker (Biorad, Munich, Germany) E) Immunoprecipitated PfG protein; Black Arrows indicate possible proteins that might form a complex with PfG. (d) Indirect immunofluorescence using anti-PfG antisera: 1) Differential Interference Contrast Microscopy 2) Anti PfG Staining (PfG) 3) Staining with DAPI (4′,6-Diamidin-2-phenylindol) 4) Merge of the two different stainings AK and DAPI 4) Overlay of both stainings.
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