Identification of an active glucosyl-ceramide transferase in epimastigote forms of Trypanosoma cruzi

M. LANDONI,; VG. DUSCHAK; A.S. COUTO.

Bariloche, Río Negro, Argentina.

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2003

Biosynthesis of glycosphingolipids involves the sequential action of glycosyltransferases. The key step involves the transfer of glucose from UDP-glucose to ceramide catalyzed by a UDP-Glucose:glucosylceramide transferase (glucosyl ceramide synthase, GCS) with special characteristics. This work shows the partial characterization of the glucosyl ceramide transferase from epimastigote forms of Trypanosoma cruzi. The enzymatic assay was performed by adding a fluorescent ceramide (BODIPY or NBD analogues) pre-coupled to BSA, UDP-glucose and b-NAD to tubes pre-coated with dipalmitoylphosphatidylcholine. Parasite lysates used as the enzyme source (50-100 mg protein) in Hepes buffer pH 7.4 were added and the reaction was incubated at 37ºC. The mixture was extracted with Chloroform: methanol and analysed by TLC in different solvents. Two strains (Tulahuen 2 and CL Brenner) were assayed using different concentrations of the fluorescent ceramides. The optimal incubation time was 30 min for Tul 2 and 120 min for CL strain. Different protein concentration of lysates were tried and the effect of the use of different detergents was compared. T.cruzi GCS uses saturated and unsaturated ceramides as substrates at variance to malarial GCS that presents specificity for the saturated one. Another component, with lower Rf was detected. An inositolphosphoceramide structure, already reported in T. cruzi, was evidenced after Phosphatidylinositolphospolipase C treatment.