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Abstract 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. yoelli. 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.

We have cloned, over-expressed and purified enolase fromPlasmodium falciparumstrain NF54 inEscherichia coliin 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 (≈ 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at≈ 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≈ 100 kDa, indicating thatP. falciparumenolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of≈ 30 U·mg−1 andKm2PGA = 0.041 ± 0.004 mm. The Michaelis constant for the reverse reaction (KmPEP) 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 recombinantP. falciparumenolase 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 theP. falciparumprotein. [ABSTRACT FROM AUTHOR]

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.