Glucosylceramide synthase in Trypanosoma cruzi, a key enzyme of the glycosphingolipid pathway

T.A. PIÑERO; M. LANDONI; L.L. SOPRANO; M.I. ESTEVA; V.G: DUSCHAK; A.S. COUTO

Buzios, Rio de Janeiro, Brasil

Congreso; XIII International Congress of Protistology; XXV Annual Meeting of the Brazilian Society of Protozoology; XXXVI Annual Meeting on Basic Research in Chagas’Disease; 2009

Sphingolipids are important components of eukaryotic cells, many of which function as bioactive signaling molecules. Although for mammalian cells most metabolic products have been largely studied and a lot of the enzymes involved have been well-characterized, the panorama in parasitic protozoa is practically unknown. The core structure of the glycosphingolipids, glucosylceramide, is synthesized by a UDP-glucose: ceramide glucosyltransferase (GCS). Recently, we have shown the presence of this active enzyme in the intraerythrocytic stages of P. falciparum. In this work we report the characterization of an active GCS in different strains of epimastigote forms of T. cruzi. Purification of GCS was achieved via a 5 step protocol involving a Green-dye agarose affinity column. This procedure led to a protein with an apparent MW of 70 kDa whose specific activity was proven using NBD-Ceramide and UDP-Glucose as substrates. Alternatively, active GCS was purified in a single step by immunoaffinity chromatography using a specific polyclonal anti-T. cruzi GCS mouse antibody.  In order to get some insight on the subcellular location of this enzyme, western blot developed with antihuman GCS showed that the enzyme was present in the enriched membrane fraction obtained by subcelullar fractionation after parasite grinding with silicon carbide. In addition, immunoelectron microscopy techniques showed label not only in the Golgi apparatus as expected, but also in microdomains all around the parasite membrane. The effect of PPMP (D,L-threo-phenyl-2-palmitoylamino-3-morpholine-1-propanol) on culture epimastigote forms of T. cruzi showed a significant reduction on parasite growth. Similar results were obtained on blood trypomastigote forms. In addition, glycosphingolipids obtained from PPMP-treated parasites, showed a significant decrease of the synthesized glucosylceramide (60%) indicating a possible association between both events. Differences between this key enzyme of the glycosphingolipid pathway and the mammalian enzyme would allow to consider T. cruzi GCS as a new chemotherapeutic target.