Inhibition of sulfation in glycoconjugates of Trypanosoma cruzi

FERRERO, MR; SOPRANO, LL; ACOSTA, DM; GARCIA, GA; COUTO, AS; DUSCHAK, VG

Mar del Plata

Congreso; IX Congreso de Protozoología y Enfermedades Parasitarias.; 2011

Soc. Argentina de Protozoologia

Inhibition of sulfation in glycoconjugates of Trypanosoma cruzi Maximiliano R. Ferrero1, Luciana L. Soprano1, Diana M. Acosta1, Gabriela A. García1, Alicia S. Couto2 and Vilma G. Duschak1. 1Instituto Nacional de Parasitologia “ Dr Mario Fatala Chaben”, ANLIS-Malbrán, Ministerio de Salud de La Nación, Argentina; 2CIHIDECAR, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Bs As, Argentina Sulfation reaction is widely observed from bacteria to humans and plays a key role in various biological processes such as cell communication, growth and development as well as defence. Sulfotransferases catalize the transference reaction of the sulfate group from the ubiquitous donor 3´phosphoadenosine 5`phosphosulfate (PAPS) to an acceptor group of numerous substrates. Chlorate is known to be an in vitro inhibitor of ATP-sulfurylase, the first enzyme in the biosynthesis of PAPS which is the ubiquitous co-substrate for sulfation. The mentioned drug has been used to abolish protein sulfation on tyrosine as well as on carbohydrate residues in intact cells. Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain which bears an unusual C-terminal extension containing a number of post-translational modifications, including the presence of sulfated high mannose type oligosaccharides in the unique N-glycosylation site of this domain. Parasites treated with growing concentrations of sodium chlorate (10-80 mM) during 24, 48 and 72 h showed progressive undersulfation of cruzipain, evidenced by the significant reduction in the immunerecognition by specific antisulfate antibodies from sera of mice immunized with Cz followed by absorption with desulfated cruzipain by western blot and bidimensional electrophoresis. Interestingly, the blockage of sulfates by IgGs specific purified from sera specific for cruzipain, did not alter the biological activity of cruzipain, suggesting that these groups from the C-T contribute to evade host immune system protecting the catalytic domain of the molecule. Similarly, chlorate treatment on epimastigote forms of T. cruzi determined a significant decrease of sulfated glycosphingolipids with the concomitant increase of neutral lipids which were further identified by UV-MALDI-ToF mass spectrometry analysis. Accordingly, ultrastructural analysis of chlorate treated epimastigotes showed alterations in different membranes, a decrease of dark lipids inclusions and a significant increase in reservosomes, representing typical signs of alteration in the biosynthesis of the glycosphingolipid pathway. Our findings strongly provide indirect evidence of the presence of a sulfation mechanism via PAPS. Interestingly, preliminar assays showed that undersulfation induced by chlorate treatment was capable to inhibit the infection of host cells by trypomastigotes. Supported by CONICET-ANPCYT-UBA, INP-ANLIS-Malbrán.