Generation and characterisation of monoclonal antibodies specific to Plasmodium falciparum enolase

Background & objectives: Glycolysis is the sole source of energy for the intraerythrocytic stages ofPlasmodium falciparum, making glycolytic enzymes putative therapeutic targets. Enolase, a singlecopy gene in P. falciparum is one such enzyme whose activity is elevated ~10–15 fold in infectedRBC’s. It holds the possibility of having multiple biological functions in the parasite and hence canbe a suitable candidate for diagnostic and chemotherapeutic purposes.Methods: We have aimed at generating parasite-specific reagents in the form of monoclonalantibodies. We have raised monoclonal antibodies against the recombinant P. falciparum enolase.Results: Two IgG monoclonals were obtained with 1:1000 titre and specific for P. falciparum enolase.Apicomplexan parasites including P. falciparum enolase has a plant like pentapeptide sequence(104EWGWS108) which is uniquely different from the host counterpart. A peptide spanning thispentapeptide region (ELDGSKNEWGWSKSK) coupled to BSA was used to raise parasite-specificantibody. Four monoclonals were obtained with 1:1000 titre and of IgM isotype.Interpretation & conclusion: All the monoclonals are specific for P. falciparum enolase and one ofthem display reactivity against native P. falciparum enolase signifying this pentapeptide to be surfaceexposed and immunogeni

Magnetic resonance studies on the interaction of metal-ion and nucleotide ligands with brain hexokinase

Our previous studies have shown that one manganous ion binds tightly to bovine brain hexokinase, with a Kd =25 ± 4 μM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (εb) 3 .5 at 9 MHz and 23 °C [ Jarori, G. K. Kasturi, S. R., and Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211,258 - 2681. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E . Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme . nucleotide . Mn. Addition of glucose 6-phosphate to enzyme . ATP . Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E · ADP· Mn· Glc-6-P appears to be formed when both the products are present. β, γ-Bidentate Cr(II1)ATP has been used to elucidate the role of direct binding of Mn(I1) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(II1)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron-spin resonance studies on the binding of Mn(I1) to the enzyme in the presence of Cr(I1I)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis. Experiments carried out with hexokinase spin-labeled with 3-(2-iodo-acetamido)-2,2,5,5-tetramethyl-lpyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site

Plasmodium falciparum enolase: stage-specific expression and sub-cellular localization

<p>Abstract</p> <p>Background</p> <p>In an earlier study, it was observed that the vaccination with <it>Plasmodium falciparum </it>enolase can confer partial protection against malaria in mice. Evidence has also build up to indicate that enolases may perform several non-glycolytic functions in pathogens. Investigating the stage-specific expression and sub-cellular localization of a protein may provide insights into its moonlighting functions.</p> <p>Methods</p> <p>Sub-cellular localization of <it>P. falciparum </it>enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting.</p> <p>Results</p> <p>Enolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (≥85–90%) present in soluble fraction, while in sexual stages it was mostly associated with particulate fraction. Apart from cytosol, enolase was found to be associated with nucleus, food vacuole, cytoskeleton and plasma membrane.</p> <p>Conclusion</p> <p>Diverse localization of enolase suggests that apart from catalyzing the conversion of 2-phosphoglycericacid into phosphoenolpyruvate in glycolysis, enolase may be involved in a host of other biological functions. For instance, enolase localized on the merozoite surface may be involved in red blood cell invasion; vacuolar enolase may be involved in food vacuole formation and/or development; nuclear enolase may play a role in transcription.</p

Anti-malarial activity of geldanamycin derivatives in mice infected with Plasmodium yoelii

Background Geldanamycin (GA), a benzoquinone ansamycin antibiotic has been shown in vitro to possess anti-plasmodial activity. Pharmacological activity of this drug is attributed to its ability to inhibit PfHSP90. The parasite growth arrest has been shown to be due to drug-induced blockage of the transition from ring to trophozoite stage. To further evaluate the consequences of this pharmacodyamic feature, the anti-malarial activity of GA analogs with enhanced drug properties in a Plasmodium-infected animal model have been evaluated for their capacity to induce clearance of the parasite. In the process, a hypothesis was subsequently tested regarding the susceptibility of the cured animals to malaria reflected in an attenuated parasite load that may be evoked by a protective immune response in the host. Methods Six weeks old Swiss mice were infected with a lethal Plasmodium yoelii (17XL) strain. On appearance of clinical symptoms of malaria, these animals were treated with two different GA derivatives and the parasite load was monitored over 15-16 days. Drug-treated animals cured of the parasite were then re-challenged with a lethal dose of P. yoelii 17XL. Serum samples from GA cured mice that were re-challenged with P. yoelii 17XL were examined for the presence of antibodies against the parasite proteins using western blot analysis. Results Treatment of P. yoelii 17XL infected mice with GA derivatives showed slow recovery from clinical symptoms of the disease. Blood smears from drug treated mice indicated a dominance of ring stage parasites when compared to controls. Although, P. yoelii preferentially invades normocytes (mature rbcs), in drug-treated animals there was an increased invasion of reticulocytes. Cured animals exhibited robust protection against subsequent infection and serum samples from these animals showed antibodies against a vast majority of parasite proteins. Conclusions Treatment with GA derivatives blocked the transition from ring to trophozoite stage presumably by the inhibition of HSP90 associated functions. Persistence of parasite in ring stage leads to robust humoral immune response as well as a shift in invasion specificity from normocytes to reticulocyte. It is likely that the treatment with the water-soluble GA derivative creates an attenuated state (less virulent with altered invasion specificity) that persists in the host system, allowing it to mount a robust immune response

Food vacuole associated enolase in plasmodium undergoes multiple post-translational modifications: evidence for atypical ubiquitination.

Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV) and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs) giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV) associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS), definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub) molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1) and the C-terminal G76 of the second (Ub2). Ub2 and third ubiquitin (Ub3) were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P. yoelii. The composition of food vacuolar proteome and likely interactors of enolase are also being reported

Plant-like phosphofructokinase from Plasmodium falciparum belongs to a novel class of ATP-dependent enzymes

Malaria parasite-infected erythrocytes exhibit enhanced glucose utilisation and 6-phospho-1-fructokinase (PFK) is a key enzyme in glycolysis. Here we present the characterisation of PFK from the human malaria parasite Plasmodium falciparum. Of the two putative PFK genes on chromosome 9 (PfPFK9) and 11 (PfPFK11), only the PfPFK9 gene appeared to possess all the catalytic features appropriate for PFK activity. The deduced PfPFK proteins contain domains homologous to the plant-like pyrophosphate (PPi)-dependent PFK β and α subunits, which are quite different from the human erythrocyte PFK protein. The PfPFK9 gene β and α regions were cloned and expressed as His6- and GST-tagged proteins in Escherichia coli. Complementation of PFK-deficient E. coli and activity analysis of purified recombinant proteins confirmed that PfPFK9β possessed catalytic activity. Monoclonal antibodies against the recombinant β protein confirmed that the PfPFK9 protein has β and α domains fused into a 200 kDa protein, as opposed to the independent subunits found in plants. Despite an overall structural similarity to plant PPi-PFKs, the recombinant protein and the parasite extract exhibited only ATP-dependent enzyme activity, and none with PPi. Unlike host PFK, the Plasmodium PFK was insensitive to fructose-2,6-bisphosphate (F-2,6-bP), phosphoenolpyruvate (PEP) and citrate. A comparison of the deduced PFK proteins from several protozoan PFK genome databases implicates a unique class of ATP-dependent PFK present amongst the apicomplexan protozoans

Plasmodium falciparum enolase complements yeast enolase functions and associates with the parasite food vacuole

Plasmodium falciparum enolase (Pfeno) localizes to the cytosol, nucleus, cell membrane and cytoskeletal elements, suggesting multiple non-glycolytic functions for this protein. Our recent observation of association of enolase with the food vacuole (FV) in immuno-gold electron microscopic images of P. falciparum raised the possibility for yet another moonlighting function for this protein. Here we provide additional support for this localization by demonstrating the presence of Pfeno in purified FVs by immunoblotting. To examine the potential functional role of FV-associated Pfeno, we assessed the ability of Pfeno to complement a mutant Saccharomyces cervisiae strain deficient in enolase activity. In this strain (Tetr-Eno2), the enolase 1 gene is deleted and expression of the enolase 2 gene is under the control of a tetracycline repressible promoter. Enolase deficiency in this strain was previously shown to cause growth retardation, vacuolar fragmentation and altered expression of certain vacuolar proteins. Expression of Pfeno in the enolase-deficient yeast strain restored all three phenotypic effects. However, transformation of Tetr-eno2 with an enzymatically active, monomeric mutant form of Pfeno (Δ5Pfeno) fully restored cell growth, but only partially rescued the fragmented vacuolar phenotype, suggesting that the dimeric structure of Pfeno is required for the optimal vacuolar functions. Bioinformatic searches revealed the presence of Plasmodium orthologs of several yeast vacuolar proteins that are predicted to form complexes with Pfeno. Together, these observations raise the possibility that association of Pfeno with food vacuole in Plasmodium may have physiological function(s)

Protective Properties and Surface Localization of Plasmodium falciparum Enolase▿

The enolase protein of the human malarial parasite Plasmodium falciparum has recently been characterized. Apart from its glycolytic function, enolase has also been shown to possess antigenic properties and to be present on the cell wall of certain invasive organisms, such as Candida albicans. In order to assess whether enolase of P. falciparum is also antigenic, sera from residents of a region of Eastern India where malaria is endemic were tested against the recombinant P. falciparum enolase (r-Pfen) protein. About 96% of immune adult sera samples reacted with r-Pfen over and above the seronegative controls. Rabbit anti-r-Pfen antibodies inhibited the growth of in vitro cultures of P. falciparum. Mice immunized with r-Pfen showed protection against a challenge with the 17XL lethal strain of the mouse malarial parasite Plasmodium yoelii. The antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues. Immunofluorescence as well as electron microscopic examinations revealed localization of the enolase protein on the merozoite cell surface. These observations establish malaria enolase to be a potential protective antigen