Strain-transcending neutralization of malaria parasite by antibodies against Plasmodium falciparum enolase.

BACKGROUND: Plasmodium enolase is a target for the growth neutralizing antibodies. Interestingly, the three invasive stages i.e. sporozoites, merozoites, and ookinetes express this protein on their cell surface. Polyclonal anti-Plasmodium falciparum enolase (Pfeno) antibodies disrupt traversal of ookinete through mosquito mid-gut wall as well as have inhibitory effect on parasite growth at erythrocytic stage. In a recent study, it was observed that immunization with a unique epitope of parasite enolase (EWGWS) could confer partial protection against mouse malaria. Further validation is needed for the protective potential of this unique epitope in otherwise highly conserved enolase. METHODS: In order to investigate the efficacy of growth inhibitory potential of the epitope of P falciparum enolase, a monoclonal antibody specific to EWGWS is generated. In vitro parasite growth inhibition assays and passive immunization of Plasmodium yoelii (or Plasmodium berghei) infected mice were used to assess the parasite growth neutralizing activity of the antibody. RESULTS: Screening a panel of monoclonal antibodies raised against recombinant Pfeno that were specific to EWGWS resulted in isolation of H12E1. This antibody recognized only EWGWS epitope containing enolases. H12E1 strongly inhibited parasite growth in culture. This inhibition was strain transcending. Passive infusion of this antibody in P. yoelii or P. berghei infected mice showed significant reduction in parasitemia as compared to controls (p < 0.001). Surface Plasmon Resonance measurements indicated high affinity binding of H12E1 to P. falciparum enolase (KD ~ 7.6 × 10-9M). CONCLUSIONS: A monoclonal antibody directed against EWGWS epitope of Pfeno was shown to inhibit the growth of blood stage malarial parasites. This inhibition was species/strain transcending and is likely to arise due to blockade of enolase on the surface of merozoites, functionally implicating Pfeno in invasion related events. Presence of enolase on the cell surface of merozoites and ookinetes could potentially result in inhibition of host cell invasions at erythrocytic and transmission stages in the parasite life cycle. It is suggested that antibodies against EWGWS epitope have the potential to confer dual stage, species and strain transcending protection against malaria.

Functions of tryptophan residues in EWGWS insert of Plasmodium falciparum enolase.

Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. This enzyme is thus a potential target for anti-malarial treatments. A unique feature of Pfeno is the presence of a pentapeptide insert 104 EWGWS 108. The functional role of tryptophan residues in this insert was investigated using site-directed mutagenesis. Replacement of these two Trp residues with alanines (or lysines) resulted in a near complete loss of enolase activity and dissociation of the normal dimeric form into monomers. Molecular modeling indicated that 340R forms π-cation bonds with the aromatic rings of 105W and 46Y. Mutation induced changes in the interactions among these three residues were presumably relayed to the inter-subunit interface via a coil formed by 46Y : 59Y, resulting in the disruption of a salt bridge between 11R : 425E and a π-cation interaction between 11R : 59Y. This led to a drop of ~ 4 kcal·mole-1 in the inter-subunit docking energy in the mutant, causing a ~ 103 fold decrease in affinity. Partial restoration of the inter-subunit interactions led to reformation of dimers and also restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme's active site. Since Plasmodium enolase is a moonlighting protein with multiple parasite-specific functions, it is likely that these functions may map on to the highly conserved unique insert region of this protein. ENZYMES: Enolase(EC4.2.1.11).

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite.

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.

Cloning, expression, purification and characterization of Plasmodium spp. glyceraldehyde-3-phosphate dehydrogenase.

Plasmodium spp. solely rely on glycolysis for their energy needs during asexual multiplication in human RBCs, making the enzymes of this pathway potential drug targets. We have cloned, over-expressed and purified Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGapdh) for its kinetic and structural characterization. ∼ 30-40 mg pure recombinant enzyme with a specific activity of 12.6 units/mg could be obtained from a liter of Escherichia coli culture. This enzyme is a homotetramer with an optimal pH ∼ 9. Kinetic measurements gave KmNAD=0.28 ± 0.3 mM and KmG3P=0.25 ± 0.03 mM. Polyclonal antibodies raised in mice showed high specificity as was evident from their non-reactivity to rabbit muscle Gapdh. Western blot of Plasmodium yoelii cell extract showed three bands at MW ∼ 27, ∼ 37 and ∼ 51 kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the ∼ 51 kDa form was present only in the soluble fraction of the extract. Subcellular distribution of Gapdh in P. yoelii was examined using differential detergent fractionation method. Each fraction was analyzed on a two-dimensional gel and visualized by Western blotting. All four subcellular fractions (i.e., cytosol, nucleus, cytoskeleton and cell membranes) examined had Gapdh associated with them. Each fraction had multiple molecular species associated with them. Such species could arise only by multiple post-translational modifications. Structural heterogeneity observed among molecular species of PyGapdh and their diverse subcellular distribution, supports the view that Gapdh is likely to have multiple non-glycolytic functions in the parasite and could be an effective target for anti-malarial chemotherapeutics.

Immunogenicity and protective potential of a Plasmodium spp. enolase peptide displayed on archaeal gas vesicle nanoparticles.

BACKGROUND: Plasmodium falciparum enolase has been shown to localize on the surface of merozoites and ookinetes. Immunization of mice with recombinant Plasmodium enolase (rPfeno) showed partial protection against malaria. Anti-rPfeno antibodies inhibited growth of the parasite in in vitro cultures and blocked ookinete invasion of mosquito midgut epithelium. It is hypothesized that parasite specific moonlighting functions (e.g. host cell invasion) may map on to unique structural elements of Pfeno. Since enolases are highly conserved between the host and the parasite, a parasite-specific epitope of enolase was displayed on novel protein nanoparticles produced by a halophilic Archaeon Halobacterium sp. NRC-1 and tested their ability to protect mice against live challenge. METHODS: By genetic engineering, a Plasmodium-enolase specific peptide sequence (104)EWGWS(108) with protective antigenic potential was inserted into the Halobacterium gas vesicle protein GvpC, a protein localized on the surface of immunogenic gas vesicle nanoparticles (GVNPs). Two groups of mice were immunized with the wild type (WT) and the insert containing recombinant (Rec) GVNPs respectively. A third group of mice was kept as un-immunized control. Antibody titres were measured against three antigens (i.e. WT-GVNPs, Rec-GVNPs and rPfeno) using ELISA. The protective potential was determined by measuring percentage parasitaemia and survival after challenge with the lethal strain Plasmodium yoelii 17XL. RESULTS: Rec-GVNP-immunized mice showed higher antibody titres against rPfeno and Rec-GVNPs, indicating that the immunized mice had produced antibodies against the parasite enolase-specific insert sequence. Challenging the un-immunized, WT-GVNP and Rec-GVNP-immunized mice with a lethal strain of mice malarial parasite showed significantly lower parasitaemia and longer survival in the Rec-GVNP-immunized group as compared to control groups. The extent of survival advantage in the Rec-GVNP-group showed positive correlation with anti-rPfeno antibody titres while the parasitaemia showed a negative correlation. These results indicate that the parasite enolase peptide insert displayed on Halobacterium GVNPs is a good candidate as a protective antigenic epitope. CONCLUSION: The work reported here showed that the parasite-specific peptide sequence is a protective antigenic epitope. Although antibody response of B-cells to the guest sequence in Rec-GVNPs was mild, significant advantage in the control of parasitaemia and survival was observed. Future efforts are needed to display multiple antigens with protective properties to improve the performance of the GVNP-based approach.

Replacement of Ser108 in Plasmodium falciparum enolase results in weak Mg(II) binding: role of a parasite-specific pentapeptide insert in stabilizing the active conformation of the enzyme.

A distinct structural feature of Plasmodium falciparum enolase (Pfeno) is the presence of a five amino acid insert -104EWGWS108- that is not found in host enolases. Its conservation among apicomplexan enolases has raised the possibility of its involvement in some important physiological function(s). Deletion of this sequence is known to lower k(cat)/K(m), increase K(a) for Mg(II) and convert dimer into monomers (Vora HK, Shaik FR, Pal-Bhowmick I, Mout R & Jarori GK (2009) Arch Biochem Biophys 485, 128-138). These authors also raised the possibility of the formation of an H-bond between Ser108 and Leu49 that could stabilize the apo-Pfeno in an active closed conformation that has high affinity for Mg(II). Here, we examined the effect of replacement of Ser108 with Gly/Ala/Thr on enzyme activity, Mg(II) binding affinity, conformational states and oligomeric structure and compared it with native recombinant Pfeno. The results obtained support the view that Ser108 is likely to be involved in the formation of certain crucial H-bonds with Leu49. The presence of these interactions can stabilize apo-Pfeno in an active closed conformation similar to that of Mg(II) bound yeast enolase. As predicted, S108G/A-Pfeno variants (where Ser108-Leu49 H-bonds are likely to be disrupted) were found to exist in an open conformation and had low affinity for Mg(II). They also required Mg(II) induced conformational changes to acquire the active closed conformational state essential for catalysis. The possible physiological relevance of apo-Pfeno being in such an active state is discussed.

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.

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 pharmacodynamic 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.

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 (Δ(5)Pfeno) 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).

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

BACKGROUND: In an earlier study, it was observed that the vaccination with Plasmodium falciparum 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. METHODS: Sub-cellular localization of P. falciparum enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting. RESULTS: Enolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (>or=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. CONCLUSION: 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.

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 beta and alpha subunits, which are quite different from the human erythrocyte PFK protein. The PfPFK9 gene beta and alpha regions were cloned and expressed as His(6)- and GST-tagged proteins in Escherichia coli. Complementation of PFK-deficient E. coli and activity analysis of purified recombinant proteins confirmed that PfPFK9beta possessed catalytic activity. Monoclonal antibodies against the recombinant beta protein confirmed that the PfPFK9 protein has beta and alpha 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.

Effect of deletion of a plant like pentapeptide insert on kinetic, structural and immunological properties of enolase from Plasmodium falciparum.

Plasmodium falciparum enolase (Pfen) is of photosynthetic lineage as evident from the presence of a plant like pentapeptide insert (104)EWGWS(108) in a highly conserved surface loop of the protein. Such a unique region which is absent in human enolase, constitutes an excellent target for inhibitor design, provided its essentiality for function could be demonstrated. A deletion Pfen lacking this insert was made and the effect of this deletion on activity and structure was assessed. Deletion of insert resulted in approximately 100-fold decrease in k(cat)/K(m) and caused dissociation of dimeric form into monomers. Since the parasite enolase localizes on the merozoite surface and confers partial protection against malaria [I. Pal-Bhowmick, M. Mehta, I. Coppens, S. Sharma, G.K. Jarori, Infect. Immun. 75(11) (2007) 5500-5008], the possibility of the insert being involved in protective response was examined. Serum from Pfen vaccinated mouse which showed prolonged survival to parasite challenge had negligible reactivity against deletion protein as compared to wild type enolase. These results indicate that the insert sequence is required for the full enolase activity and may constitute the protective antigenic epitope in parasite enolase.

Structural and functional studies on Ribonuclease S, retro S and retro-inverso S peptides.

Ribonuclease S peptide and S protein offer a unique complementation system to understand the finer features of molecular recognition. In the present study the S peptide (1-16), and its retro and retro-inverso analogs have been analyzed for their structural and biological attributes. RPHPLC, CD, and NMR analyses have revealed that the physicochemical and conformational properties of the S peptide are distinct from those of its retro and retro-inverso analogs. On the functional side, while the S peptide complemented the S protein to give RNase activity, was recognized by anti-S peptide antibodies and induced T cell proliferation, neither the retro nor the retro-inverso S peptides could do so.

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.

Sub-cellular localization and post-translational modifications of the Plasmodium yoelii enolase suggest moonlighting functions.

BACKGROUND: Enolase (2-Phospho-D-glycerate hydrolase; EC 4.2.1.11) is one of the glycolytic enzymes, whose levels are highly elevated in malaria parasite infected red blood cells. In several organisms, enolases have been shown to have diverse non glycolytic (moonlighting) biological functions. As functional diversity of a protein would require diverse sub-cellular localization, the possibility of involvement of Plasmodium enolase in moonlighting functions was examined by investigating its sub-cellular distribution in the murine malarial parasite, Plasmodium yoelii. METHODS: Cellular extracts of P. yoelii were fractionated in to soluble (cytosolic) and particulate (membranes, nuclear and cytoskeletal) fractions and were analysed by one and two-dimensional gel electrophoresis. These were probed by Western blotting using antibodies raised against recombinant Plasmodium falciparum enolase. Immunofluorescence assay was used for in situ localization. Fe+3 based metal affinity chromatography was used to isolate the phospho-proteome fraction from P. yoelii extracts. RESULTS: Apart from the expected presence of enolase in cytosol, this enzyme was also found to be associated with membranes, nuclei and cytoskeletal fractions. Nuclear presence was also confirmed by in situ immunofluorescence. Five different post translationally modified isoforms of enolase could be identified, of which at least three were due to the phosphorylation of the native form. in situ phosphorylation of enolase was also evident from the presence of enolase in purified phosphor-proteome of P. yoelii. Different sub-cellular fractions showed different isoform profiles. CONCLUSION: Association of enolase with nuclei, cell membranes and cytoskeletal elements suggests non-glycolytic functions for this enzyme in P. yoelii. Sub-cellular fraction specific isoform profiles indicate the importance of post-translational modifications in diverse localization of enolase in P. yoelii. Further, it is suggested that post-translational modifications of enolase may govern the recruitment of enolase for non-glycolytic functions.

Differential susceptibility of Plasmodium falciparum versus yeast and mammalian enolases to dissociation into active monomers.

In the past, several unsuccessful attempts have been made to dissociate homodimeric enolases into their active monomeric forms. The main objective of these studies had been to understand whether intersubunit interactions are essential for the catalytic and structural stability of enolases. Further motivation to investigate the properties of monomeric enolase has arisen from several recent reports on the involvement of enolase in diverse nonglycolytic (moonlighting) functions, where it may occur in monomeric form. Here, we report successful dissociation of dimeric enolases from Plasmodium falciparum, yeast and rabbit muscle into active and isolatable monomers. Dimeric enolases could be dissociated into monomers by high concentrations ( approximately 250 mm) of imidazole and/or hydrogen ions. Two forms were separated using Superdex-75 gel filtration chromatography. A detailed comparison of the kinetic and structural properties of monomeric and dimeric forms of recombinant P. falciparum enolase showed differences in specific activity, salt-induced inhibition and inactivation, thermal stability, etc. Furthermore, we found that enolases from the three species differ in their dimer dissociation profiles. Specifically, on challenge with imidazole, Mg(II) protected the enolases of yeast and rabbit muscle but not of P. falciparum from dissociation. The observed differential stability of the P. falciparum enolase dimer interface with respect to mammalian enolases could be exploited to selectively dissociate the dimeric parasite enzyme into its catalytically inefficient, thermally unstable monomeric form. Thus enolase could be a novel therapeutic target for malaria.

Generation and characterisation of monoclonal antibodies specific to Plasmodium falciparum enolase.

BACKGROUND AND OBJECTIVES: Glycolysis is the sole source of energy for the intraerythrocytic stages of Plasmodium falciparum, making glycolytic enzymes putative therapeutic targets. Enolase, a single copy gene in P. falciparum is one such enzyme whose activity is elevated approximately 10-15 fold in infected RBC's. It holds the possibility of having multiple biological functions in the parasite and hence can be a suitable candidate for diagnostic and chemotherapeutic purposes. METHODS: We have aimed at generating parasite-specific reagents in the form of monoclonal antibodies. 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 this pentapeptide region (ELDGSKNEWGWSKSK) coupled to BSA was used to raise parasite-specific antibody. Four monoclonals were obtained with 1:1000 titre and of IgM isotype. INTERPRETATION AND CONCLUSION: All the monoclonals are specific for P. falciparum enolase and one of them display reactivity against native P. falciparum enolase signifying this pentapeptide to be surface exposed and immunogenic.

Conformations of nucleotides bound to wild type and Y78F mutant yeast guanylate kinase: proton two-dimensional transferred NOESY measurements.

Wild type and Y78F mutant yeast guanylate kinase (GKy) were studied to investigate the effects of a site-directed mutation on bound substrate conformations. Previously published work showed that Y78 is involved in GMP binding and that the Y78F mutant has 30-fold weaker GMP binding and 2 orders of magnitude less activity, than the wild type. Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) and guanosine conformations of guanosine 5'-monophosphate (GMP) bound to wild type and Y78F mutant yeast guanylate kinase in the complexes GKy x Mg(II)ATP, GKy x Mg(II)ADP, GKy x GMP, and GKy x Mg(II)ADP x [U-13C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations. For adenyl nucleotides in wild type complexes, all glycosidic torsion angles, chi, were 54 +/- 5 degrees. In Y78F mutant complexes, adenyl nucleotide glycosidic torsion angles were 55 +/- 5 degrees (GKy x MgATP) and 49 +/- 5 degrees (GKy x MgADP). Thus, the adenyl nucleotides bind similarly for both the wild type and Y78F mutant complexes. However, in the fully constrained, two-substrate complexes, GKy x Mg(II)ADP x [U-13C]GMP, the guanyl glycosidic torsion angle, chi, is 50 +/- 5 degrees with the wild type and 83 +/- 5 degrees with the Y78F mutant. This difference suggests that an unfavorable torsion may be a large part of the mechanism for significantly weaker GMP binding to reaction complexes of the Y78F mutant.

Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase.

We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities (approximately 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at approximately 50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean +/- SD mass of 51396 +/- 16 Da, which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was approximately 100 kDa, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of approximately 30 U.mg(-1) and K(m2PGA) = 0.041 +/- 0.004 mm. The Michaelis constant for the reverse reaction (K(mPEP)) is 0.25 +/- 0.03 mm. pH-dependent activity measurements gave a maximum at pH 7.4-7.6 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na+, whereas K+ has a slight activating effect. The cofactor Mg2+ has an apparent activation constant of 0.18 +/-0.02 mm. However, at higher concentrations, it has an inhibitory effect. Polyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite, Plasmodium yoelii, which shares 90% identity with the P. falciparum protein.

The Raman detection of peptide tyrosine phosphorylation.

Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylation of tyrosine amino acid residues in peptides. Four peptides are investigated, with sequences derived from the human protein-tyrosine kinase, p60c-src, with Y-216, Y-419, and Y-530 phosphorylation sites. Although the spectra of the four peptides are quite different, tyrosine phosphorylation is found to invariably induce the collapse of a doublet at 820-850cm(-1) and the attenuation of a peak around 1205cm(-1). Moreover, amide III band shifts suggest that tyrosine phosphorylation may promote beta sheet formation, particularly in peptides that lack phenylalanine residues. The degree of tyrosine phosphorylation in peptide mixtures is determined using DCDR combined with partial least squares multivariate calibration with a 2% root mean standard error of prediction.