Stage specific gene expression and cellular localization of two isoforms of the serine hydroxymethyltransferase in the protozoan parasite Leishmania

https://doi.org/10.1016/j.molbiopara.2006.06.009Get rights and content

Abstract

Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions.

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.

References (49)

  • D.G. Capelluto et al.

    Purification and partial characterization of three isoforms of serine hydroxymethyltransferase from Crithidia fasciculata

    Mol Biochem Parasitol

    (1999)
  • D.A. Scott et al.

    Folate utilisation by Leishmania species and the identification of intracellular derivatives and folate-metabolising enzymes

    Mol Biochem Parasitol

    (1987)
  • B. Papadopoulou et al.

    Changes in folate and pterin metabolism after disruption of the Leishmania H locus short chain dehydrogenase gene

    J Biol Chem

    (1994)
  • K.N. Truscott et al.

    Mechanisms of protein import into mitochondria

    Curr Biol

    (2003)
  • A. Schluter et al.

    Expression and subcellular localization of cpn60 protein family members in Leishmania donovani

    Biochim Biophys Acta

    (2000)
  • J.A. Rey-Ladino et al.

    Leishmania major: molecular cloning, sequencing, and expression of the heat shock protein 60 gene reveals unique carboxy terminal peptide sequences

    Exp Parasitol

    (1997)
  • T.E. Ellenberger et al.

    Biochemistry and regulation of folate and methotrexate transport in Leishmania major

    J Biol Chem

    (1987)
  • S.F. Chowdhury et al.

    Novel inhibitors of leishmanial dihydrofolate reductase

    Bioorg Med Chem Lett

    (2001)
  • D. Pez et al.

    2,4-Diaminopyrimidines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase

    Bioorg Med Chem

    (2003)
  • J.K. Acharya et al.

    A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase

    J Biol Chem

    (1992)
  • M.D. Piper et al.

    Regulation of the balance of one-carbon metabolism in Saccharomyces cerevisiae

    J Biol Chem

    (2000)
  • C.L. Gelling et al.

    Identification of a novel one-carbon metabolism regulon in Saccharomyces cerevisiae

    J Biol Chem

    (2004)
  • P.J. Stover et al.

    Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene

    J Biol Chem

    (1997)
  • S. Alfadhli et al.

    Gene organization of a Plasmodium falciparum serine hydroxymethyltransferase and its functional expression in Escherichia coli

    Mol Biochem Parasitol

    (2000)
  • Cited by (21)

    • Exoproteome dynamics in Leishmania infantum

      2013, Journal of Proteomics
      Citation Excerpt :

      The blots were washed as above, incubated with ECL Plus chemiluminescent substrate (Amersham Biosciences) and exposed using the Chemidoc XRS system (Biorad). L. infantum promastigotes episomally expressing GFP fusions with either serine hydroxymethyltransferase (SHMT) [33], hypothetical protein 26 (LinJ26.2710) or monomeric GFP [34] were recovered from cRPMI medium and labeled with 1 μg/ml Hoechst dye 33342 (Sigma) during 1 h in the dark at 27 °C. The parasites were mounted on a glass slide with 1% low melting agarose and covered with a cover slip.

    • High affinity S-adenosylmethionine plasma membrane transporter of Leishmania is a member of the folate biopterin transporter (FBT) family

      2010, Journal of Biological Chemistry
      Citation Excerpt :

      Primers and Taqman probes are listed in supplemental Table S2. Real time reverse transcription-PCR for the MAT gene was performed as described previously (32) using GAPDH and actin genes as controls (supplemental Table S2). Total Leishmania proteins (30 μg) were run on 12% polyacrylamide gels and transferred onto nitrocellulose membranes as described previously (33).

    • The role of the mitochondrial glycine cleavage complex in the metabolism and virulence of the protozoan parasite Leishmania major

      2008, Journal of Biological Chemistry
      Citation 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.

    View all citing articles on Scopus
    View full text