Stage specific gene expression and cellular localization of two isoforms of the serine hydroxymethyltransferase in the protozoan parasite Leishmania
Introduction
The protozoan parasite Leishmania is responsible for various clinical manifestations ranging from self-healing cutaneous lesions to potentially deadly visceral infections in tropical and sub-tropical parts of the world [1], [2]. The first line of treatment against Leishmania is based on pentavalent antimonials but the prevalence of resistance in endemic regions [3] is warranting the discovery of novel targets for the design of alternative drugs. The folate biosynthesis pathway has proved to be useful for the development of therapeutic agents for chemotherapy and for the treatment of rheumatoid arthritis [4], [5]. However, despite several intriguing peculiarities in the Leishmania folate metabolism, no drugs targeting the folate pathway were found, as yet, to be effective against Leishmania infections (reviewed in Refs. [6], [7]).
Leishmania is auxotroph for folates and must import these metabolites from an exogenous source [6], [7]. The proteins responsible for their uptake correspond to a novel class of transport membrane proteins [8], [9], [10]. Few other proteins and enzymes of the Leishmania folate pathway have been studied and these include the bifunctional dihydrofolate reductase-thymidylate synthase DHFR-TS [11], [12] and the folyl polyglutamate synthetase [13], [14]. The completion of the sequencing and annotation of the genome of several Leishmania species ([15], http://www.genedb.org) and their analyses have highlighted the presence of several proteins implicated in folate metabolism [7]. One of these, the serine hydroxymethyltransferase (SHMT) will be describe here.
Serine hydroxymethyltransferase (SHMT) is an important pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyses the reversible conversion of serine and tetrahydrofolate (THF) into glycine and 5,10-methyleneTHF [16]. Along with the glycine-cleavage complex, SHMT generates one carbon units that are transferred onto folate coenzymes for the synthesis of thymidine (dTMP) and methionine in Leishmania and also of purines in several other organisms [7], [17]. Multiple isoforms of SHMT, usually cytosolic and mitochondrial forms, are found in most eukaryotic organisms including mammals, plants, yeast and protozoa. Some exceptions can be found, however. For example, three isoforms are found in the cytosol, the mitochondrion and the glycosome of Crithidia fasciculata [18] while Trypanosoma cruzi epimastigotes carry only one version of this enzyme [19]. SHMT activity has been reported in Leishmania species and interestingly the activity was found to be higher in the amastigote stage [20]. Despite the fact that SHMT plays a number of vital functions in every cells (glycine and serine homeostasis, one carbon donor), little information is available on Leishmania SHMT isoforms.
Leishmania major genome sequence revealed the presence of two SHMT genes with homology to bacterial and eukaryotic SHMTs. In this paper, we have characterized these two proteins, determined their cellular localization, their expression throughout the life cycle, and the phenotype associated with the overexpression of SHMT in L. major.
Section snippets
Cell line and culture conditions
L. major Friedlin promastigotes cells were grown in SDM-79 (folic acid concentration of 15 μM) or M199 (folic acid concentration of 23 nM) both supplemented with 10% heat-inactivated foetal bovine serum and 5 μg/ml hemin. All experiments were done using parasites in their logarithmic growth phase. Growth inhibition assays were performed as described [21]. L. major promastigotes were transfected with expression vectors by electroporation as previously described [22].
DNA and protein sequence analysis
DNA sequencing was done with an
In silico analysis of SHMT isoforms in L. major and other kinetoplastid parasites
The genome of L. major is completely sequenced [15] and annotated (http://www.genedb.org) enabling us to find two SHMT coding genes located on chromosomes 14 and 28. The first one (LmjF28.2370) encodes a protein of 480 amino acids with a predicted molecular weight of 53.1 kDa (SHMT-L), while the second one (LmjF14.1320) encodes a shorter protein of 465 amino acids with a predicted molecular weight of 50.6 kDa (SHMT-S). The protein sequence of the two isoforms were aligned (Fig. 1) and showed 59%
Discussion
The genome sequence of L. major [15] has revealed the presence of two SHMT isoforms. Cellular localisation experiments (Fig. 2, Fig. 3) indicated that the short version is localised in the cytosol and the longer version is imported in the mitochondrion. Mitochondrial localisation of SHMT-L was supported by: (i) the presence (Fig. 1) and functionality (Fig. 3) of a N-terminal sequence predicting mitochondrial localisation; (ii) the intracellular localisation consistent with mitochondrial
Acknowledgments
We thank Dr. Andy Bognar, University of Toronto, for useful discussions on folate metabolism. This work was funded in part by CIHR group and operating grants to MO, DG and AF were the recipient of an IRSC/FRSQ studentship and post-doctoral fellowship (STP-53924) from the “Programme Stratégique de Formation en Résistance”. MO is a Burroughs Wellcome Fund Scholar in Molecular Parasitology and holds a Canada Research Chair in Antimicrobial resistance.
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2008, Journal of Biological ChemistryCitation Excerpt :However, consistent with the later withdrawal of this claim (52), nitricoxide synthase activity was not found in preparations of recombinant Leishmania GCVP protein (data not shown). The metabolic role of the GCC can be seen as somewhat redundant with that of SHMT, which occurs in both cytoplasmic and mitochondrial compartments in forms encoded by distinct genes (16). This may explain why in two standard Leishmania culture media the gcvP– mutant grew and differentiated normally.