Functional, molecular characterisation and subcelullar localization of the three malate dehydrogenase (MDH) isozymes in Leishmania species

LEROUX, ALEJANDRO E.; FLEMING-CÁNEPA, XIMENA; ARANDA, ALEJANDRO; MAUGERI, DANTE; CAZZULO, JUAN JOSÉ; SÁNCHEZ, MARCO; NOWICKI, CRISTINA

Mendoza

Congreso; VII Congreso Argentino de Protozoología y Enfermedades Parasitarias (SAP2005); 2005

Sociedad Argentina de Protozoología

The expression of the MDH isozymes is developmentally regulated during the life cycle of Trypanosoma brucei. The cytosolic MDH (cMDH) is the only isozyme expressed in the bloodstream forms, and it seems to be even more abundant than in the procyclic trypomastigotes. In T. cruzi the presence of a cMDH could not be evidenced. On the other hand, Leishmania major contains 3 ORFs that may be encoding for potential mitochondrial MDHs in addition to the ORFs that appear to code for a putative a glycosomal and a cytosolic-type MDH. The nucleotide sequences corresponding to a putative mMDHs (LmjF34.0160), gMDH (LmjF19.0710) and cytosolic-type MDH (LmjF28.2860) were amplified by PCR and cloned into an expression vector, pET28. Upon purification, gel filtration chromatography in non-denaturing conditions showed that the molecular mass of the native gMDH corresponded to a monomeric enzyme. The three recombinant MDHs possess almost identical kinetic parameters although, when oxaloacetate was assayed as substrate the Vmax/Km ratios of the cMDH and mMDH are 40 fold higher than that obtained for the gMDH. Digitonin extractions of intact L. mexicana promastigotes, using detergent concentrations in the range of 0-5 mg/ml, confirmed the compartmentation of the three isozymes. Identical subcellular distribution was also observed in L. major promastigotes by immunoflorescence experiments. Additionally, western blotting analysis showed that the three MDHs are also present in the insect and mammalian stages of L. mexicana. However, the expression of MDHs is developmentally regulated along the leishmanial life cycle being the mMDH remarkably more abundant in the amastigotes forms. The variations in the subcellular distribution as well as in the expression levels of the MDH isozymes along the life cycle of these parasites are evidence for metabolic differences among trypanosomatids.