Identification and partial characterization of an active glucosylceramide synthase in epimastigote forms of Trypanosoma cruzi.

LANDONI MALENA, ..; DUSCHAK VILMA GLADYS,; GARAVAGLIA PATRICIA; COUTO ALICIA

Honolulu, Hawaii

Congreso; Annual Meeting of the Society of Glycobiology; 2004

the Society of Glycobiology

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF AN ACTIVE GLUCOSYLCERAMIDE SYNTHASE IN TRYPANOSOMA CRUZI. Malena Landoni1, Vilma G. Duschak2, Patricia Garavaglia2 and Alicia S. Couto1 [1] CIHIDECAR, Depto de Química Orgánica, FCEyN, UBA, Argentina, [2] Instituto Nacional de Parasitología, Dr. Mario Fatala Chabén, Ministerio de Salud, Argentina..   Biosynthesis of glycosphingolipids involves the sequential action of glycosyltransferases that in mammals seem to be functionally organized in Golgi. However, the key step involves the transfer of glucose from UDP-glucose to ceramide catalyzed by a UDP-Glucose:glucosylceramide transferase (EC 2.4.1.80; glucosylceramide synthase (GCS)) to form glucosylceramide, the precursor of most higher order glycosphingolipids. In the last years, the knowledge of the polypeptide responsable of this enzymatic activity has increased a lot in animals but very little is known about its activity in plants, fungus and bacteria, and nothing in parasitic protozoa. Trypanosoma cruzi, the American trypanosome, is responsible for Chagas disease, a serious endemic illness. In epimastigote forms of T. cruzi, glycosphingolipid structures have been early detected. On the contrary, very little is known about the enzymes involved in their biosynthesis. In this work we report the presence of an active glucosylceramide transferase (GCS) in epimastigote forms of Trypanosoma cruzi, showing a preliminar characterization. The enzyme activity was determined by detection on TLC or HPLC of the NBD-GlcCer formed using NBD-labeled substrates. The enzyme activity was measured in epimastigotes homogenates at different times, concentrations of substrate, total protein amounts and temperatures. The optimal conditions of the reaction were evaluated by quantification of the NBD-glucosylceramide concentration obtained in each case. Two substrates were assayed to examine the specificity of GCS: NBD-Ceramide and NBD-DHCeramide. Interestingly, we have observed that the T. cruzi GCS is able to use both substrates but it seems to prefer NBD-DHCeramide in contrast with the mammalian enzyme that has a poor activity with the saturated compound. The efficacy of PPMP (a well-known inhibitor of GCS), tamoxifen, b-conduritol (an inhibitor of glucosylceramidase) and CHAPS on the enzyme activity was also tested. The glycosylation yield resulted only 40% in the presence of PPMP. In T. cruzi, tamoxifen increased drastically glucosylceramide synthesis (160 %). Neither the presence of CHAPS nor b-conduritol in the reaction mixture increased the synthesis of GlcCer. In order to get some insight on the subcellular location of this enzyme, we performed subcellular fractionation experiments, by differential centrifugation of epimastigote homogenates. The enriched fractions electrophoresed by SDS-PAGE, and submitted to western blot showed a cross immunoreactive band of 56 kDa with anti-human GCS (a kind gift of Drs.Marks and Pagano) in the microsomal fraction. Glycosphingolipids have demonstrated to play roles in cell growth, survival and in escape from onset of apoptosis. To study their biosynthesis in depth might provide the tools for development of novel chemotherapies for protozoan parasitic diseases.