Studies on Parasite Glycobiology

VILMA G. DUSCHAK; ALICIA S. COUTO

Dakar-Senegal

Congreso; Kinetoplastid diseases; 2006

Mangosteen Soc.

STUDIES ON  PARASITE GLYCOBIOLOGY.    Vilma G. Duschak 1 and Alicia  S. Couto 2.   1Instituto Nacional de Parasitología, Dr. Mario Fatala Chabén, Ministerio de Salud y Ambiente; 2CIHIDECAR, Depto de Química Orgánica, F.C.E.y N., UBA, Argentina. acouto@qo.fcen.uba.ar   Biochemical and molecular characterization of glycoproteins and glycolipids present in haemoparasites is required for a detailed functional analysis: their role as surface receptor molecules, their contribution to the membrane physical properties, their function as signaling molecules affecting the host´s response to parasite infection.  By this reason, we focused our work on the structural characterization of glycoconjugates, present in  Trypanosoma cruzi and Plasmodium falciparum. In the last years, we have studied the carbohydrate post-traslational modifications of cruzipain, the major cystein proteinase of T. cruzi. Using HPAEC-PAD combined with UV-MALDI-TOF mass spectrometry we have characterized the O- and N-glycosidic chains of this enzyme. Interestingly, sulfated N-glycans have been detected for the first time. In addition, preliminary results showed that sulfate-bearing glycoproteins in Trypanosomatids are antigenic for humoral immune responses, which might contribute to clarify whether these structures play a role in the control of T. cruzi infection or in pathogenesis of Chagas heart disease. On the other hand, the glycosphingolipid (GSLs) pathway has also been attracting our attention as targets for new antiparasitic drugs. The physiological functions of the GSLs have only been documented in mammalian cells whereas very little information of their roles in other systems, is available. The core structure of the majority of GSLs, glucosylceramide, GlcCer, is synthesized by the action of a UDP-glucose:ceramide glucosyltransferase (GCS, glucosylceramide synthase EC 2.4.1.80) which was originally found in animal tissues. GlcCer  is modified by a series of Golgi glycosyltransferases to produce higher order GSL structures. Since over 400 different glycolipids are derived from GlcCer, GCS is an extremely important glycosylating enzyme. In Plasmodium falciparum, we have described for the first time, the presence of an active GCS in the intraerythrocytic stages of the parasite. Two different assays, using UDP-[14C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of D,L-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown in the three intraerythrocytic stages of the parasite and the structure of some hexosylceramides was analyzed by UV-MALDI-TOF mass spectrometry. When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial GCS must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy.  In addition, we have developed similar studies on the GCS of Trypanosoma cruzi. In contrast with the plasmodial enzyme, T. cruzi enzyme glycosylates the unsaturated ceramide. The use of different drugs affecting the GSLs pathway has been tried. Interestingly, 10 mM PPMP inhibited glucosylceramide synthesis and showed 79 % of blood trypomastigote lysis.  In contrast, 10 mM tamoxifen, a known anticancer drug, although increased all glycosphingolipid synthesis, produced 90% of blood trypomastigote lysis. Purification of both parasitic enzymes has been achieved and  studies are in progress to clarify the metabolic glycosphingolipidic pathway regulation.