Abstract
Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed alpha/beta structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.
Highlights
The eukaryotic protozoa of the genus Leishmania are the causative agents of several human diseases referred to as leishmaniases
Expression and Purification of Recombinant L. mexicana GDP-mannose Pyrophosphorylase—The L. mexicana gene encoding GDP-mannose pyrophosphorylase (GDP-MP) was subcloned into the expression vector pMALc2T, containing an N-terminal maltose-binding protein (MBP) tag, and expressed in E. coli
The recombinant fusion protein was initially purified on amylose resin, and the MBP tag was subsequently removed by hydrolysis with thrombin
Summary
Parasite and Bacterial Cultures—Leishmania mexicana promastigotes were grown at 26 °C in M199 medium supplemented with 10% fetal bovine serum. Size Exclusion Chromatography (SEC)—Following ion-exchange chromatography, purified GDP-MP was dialyzed against three changes of 100 column volumes of 20 mM Tris, pH 7.5, 0.5 M NaCl, 1 mM DTT. Western Blot Analysis—Parasite and bacterial lysates, as well as enzyme-containing fractions, were analyzed by SDS-PAGE on 10% acrylamide gels, and immunoblot analysis was performed as described [18] using rabbit antibodies to histidine-tagged GDP-MP followed by horseradish peroxidase-conjugated sheep anti-rabbit or anti-mouse IgG (Chemicon). The assay was carried out at 30 °C for 30 min in a 100-l reaction containing 50 mM Tris (pH 7.5), 8 mM MgCl2, 100 M GTP, 100 M mannose 1-phosphate, 1 mM DTT, 0.1 units mlϪ1 inorganic pyrophosphatase (Sigma), and 0.1 g of recombinant GDP-MP. Activity was expressed as change in OD650/0.1 g of enzyme/30 min
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