Background
Enolase (EC 4.2.1.11) catalyzes the inter-conversion of 2-phosphoglycerate and phosphoenol pyruvate during glycolysis and gluconeogenesis. For many years enolase was regarded as a soluble glycolytic enzyme, exclusively present in cytosol. However, several recent studies have shown that enolase is a multifaceted protein with diverse biological functions and sub-cellular localizations [
1,
2]. It acts as a plasminogen receptor on the cell surface of certain pathogens [
3,
4] and has been implicated in nuclear functions in protozoans [
5,
6], plants [
7] and animal cells [
8,
9]. Enolase is also involved in stress response [
10,
11] vacuolar fusion processes [
12] and molecular chaperoning functions [
13,
14].
Plasmodium falciparum is the causative agent for the most fatal forms of malaria. The asexual blood stages of this parasite, which are responsible for clinical symptoms of the disease, are bereft of functional tricarboxylicacid cycle and solely rely on glycolysis for their energy needs. The infected cells have ~50–100 fold higher glycolytic flux as compared to uninfected red blood cells (RBCs) [
15,
16]. The levels of some of the glycolytic enzymes are highly elevated and enolase is one such enzyme whose activity levels are ~15–20 fold higher in infected cells [
17]. As enolases are known to participate in a host of moonlighting functions, it is likely that it may be recruited for certain other biological functions in the parasite. As involvement of a protein in multiple functions, invariably require its recruitment to different sub-cellular compartments, examination of the sub-cellular localization of enolase in the parasite cells may provide clues to any non-glycolytic functions it may have. Since
Plasmodium yoelii cells can be obtained easily in large quantities and many of the house keeping proteins are highly homologous with
P. falciparum, this murine malarial parasite has served as a good model system for human malarial parasite. As enolases from these two organisms are ~90% homologous, antibodies raised against recombinant
P. falciparum enolase could be used to investigate sub-cellular localization of enolase in
P. yoelli.
Discussion
In this study, two different experimental approaches were employed, namely (i) biochemical sub-cellular fractionation followed by Western blot analysis and (ii)
in situ location by immunofluorescence to investigate the sub-cellular distribution of enolase in
P. yoelii. An interesting early result in this study was the observation of the presence of enolase in the particulate fraction (Figure
1). Since glycolysis occurs in cytosol and intra-erythrocytic stages of
Plasmodium are known to have high glycolytic flux [
15,
16], it was expected that major fraction of enolase will be present in cytosol. Results presented in Figure
1A suggest that ~85–90% of enolase is cytosolic and ~10–15% is associated with particulate fraction. In cases where a protein is highly abundant in a specific sub-cellular compartment (cytosol here), one needs to demonstrate that the particulate fraction is not contaminated with cytosol and/or unbroken cells. The following evidence supports the view that in case of
P. yoelii, enolase is indeed associated with various components of particulate fraction:
(i) The presence of aldolase (another glycolytic enzyme) in soluble and particulate fractions was examined. Results presented in Figure
1A showed that the aldolase was present in cytosol and completely absent (or undetectable) in particulate fraction, indicating that particulate fraction is not contaminated with cytosol or unbroken cells;
(ii) in differential detergent solubilization of cellular membrane, nuclei and cytoskeletal elements, enolase was present in all three fractions (Figure
1B). It is highly unlikely that soluble enolase will remain associated in this multi-step detergent solubilization protocol;
(iii) profiles of various enolase isoforms (in terms of their pI and relative abundances) associated with each of the sub-cellular (cytosolic, membrane, nuclear and cytoskeletal) fractions is rather unique (Figure
3A). If the presence of enolase in these fractions was due to cytosolic contamination, one would expect to observe similar profile for all the fractions; (iv) nuclear presence of enolase is also evident from immuno fluorescence assay (Figure
2). All the evidence presented here showed diverse localization of enolase where it may have different physiological (moonlighting) functions. Immunofluorescence assay performed for
in situ nuclear localization of enolase showed the presence of variable amounts of enolase in different stages of the parasite. For instance, ring stage parasite has lot more nuclear enolase as compared to late multi-nuclear schizont stage (Figure
2). Similar observations have been reported for
T. gondii [
5] and
E. tenella [
6]. Such observations of the nuclear presence of enolase, has lead to the suggestion that it may play a role in gene expression regulation. In this context, recent report about direct interaction of enolase with H3- histone, nucleosome assembly protein and indirect interaction to many other nuclear proteins (including histone acetylase like enzymes) in
P. falciparum assumes significance [
21].
P. yoelii has a single gene for enolase. Observation of five different isoforms (pI~5.9, 6.1, 6.3, 6.5 and 6.7) suggests that four of these variants arose due to post-translational modifications. Results presented here showed that forms with pI~5.9, 6.1 and 6.3 arose due to multiple phosphorylations. It is interesting to note that none of these phosphorylated forms move to the nucleus. Phosphorylation of enolase has been widely reported in bacteria [
22], plants [
23,
24] and animals [
25,
26]. However, the physiological significance of these modifications is not understood. Enolases are highly conserved proteins across the species and do not possess any signal sequences for specific sub-cellular localization. The observation (Figure
3) of sub-cellular fraction specific isoform profile would suggest that post-translational modifications may regulate sub-cellular localization of enolase. The nuclear localization signal (NLS) in a protein is usually a stretch of basic aminoacids [
27] and no such sequence is present in the enolase. It is interesting to note that the form which gets translocated to the
Plasmodium nucleus has the most basic pI (pI~6.7) among all the isoforms.
Conclusion
In summary, in this paper it was demonstrated that in P. yoelii, a small fraction of enolase is associated with cellular membranes, cytoskeletal and nuclear fractions where it is likely to have diverse moonlighting functions. Further, the enolase undergoes several posttranslational modifications, three of which are due to protein phosphorylations. The acidic forms generated due to in situ phosphorylations, are excluded from nuclear localization. Variable amounts of enolase detected in nucleus at different life cycle stages of the parasite, suggests nuclear functions for enolase. Implicit in the diverse localization of enolase, is the complexity in its biological functions, which would make this protein an interesting drug target for malaria.
Acknowledgements
We would like to thank Prof. Victor Nussenzweig, Department of Pathology, N.Y. University Medical Centre, New York, USA, for the kind gift of rabbit anti-P. falciparum aldolase antibody, Dr. G. Swarup and Ms. Nandini Ranganathan of Centre for Cellular and Molecular Biology, Hyderabad, India are acknowledged for allowing and helping us to use their confocal microscope facility.
Authors' contributions
IPB prepared the enolase, raised polyclonal antibodies and carried out biochemical fractionations and immuno fluorescence studies. HKV performed IMAC for phosphoproteome purification. GKJ was involved in design and coordination of the study, assisted in drafting the manuscript. All authors have read the manuscript and approved the final version submitted.