Sulfated high-Mannose type oligosaccharides in cruzipain, the major cysteine proteinase of Trypanosoma cruzi

BARBOZA M. ,; DUSCHAK V.G. ,; FUKUYAMA Y.,; NONAMI H.,; ERRA-BALSELLS R; CAZZULO J.J.; COUTO A .S..

Honolulu, Hawaii

Congreso; Annual Meeting of the Society of Glycobiology; 2004

Society of Glycobiology

SULFATED HIGH-MANNOSE TYPE OLIGOSACCHARIDES IN CRUZIPAIN, THE MAJOR CYSTEINE PROTEINASE OF TRYPANOSOMA CRUZI M. Barboza a, V.G. Duschak b, Y. Fukuyamac, H. Nonamic, R. Erra-Balsellsd, J.J. Cazzulo a and A.S. Couto d   a IIB-INTECH, UNSAM-CONICET, Argentina; b Instituto Nacional de Parasitología, Dr. Mario Fatala Chabén, Ministerio de Salud, Argentina; c College of Agriculture, Ehime University, Japan; d CIHIDECAR-Departamento de Química Orgánica, FCEyN, UBA, Argentina.   Trypanosoma cruzi, the parasitic protozoan which causes Chagas disease, the American Trypanosomiasis, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual carboxy-terminal extension that contains a number of post-translational modifications and most antibodies in natural and experimental infections are directed against it. In the present work we report the presence of sulfated N-linked oligosaccharides as components of a lysosomal glycoprotein of the parasitic protozoan Trypanosoma cruzi. We took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in the C-T domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. Interestingly, the signal at m/z 990 corresponding to the sulfated core structure strongly indicated that the sulfate group is linked to a core-GlcNAc. Futhermore, by UV-MALDI-TOF mass spectrometry analysis in the positive-ion mode showed (i) a main population of high-mannose type oligosaccharides, (ii) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species and (iii) as an interesting fact, fucosylated oligosaccharides were also characterized. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain showed to be much higher than estimated before. Sulfated N-linked-oligosaccharides have been reported as components of glycoproteins from viruses, mammalian cells, birds and a few other organisms. Sulfated high-mannose type glycans have only been described as component of glycoproteins from Dictyostelium discoideum. Likewise, these glycoproteins are localized in lysosome, however, Man-6-SO4 accounts for the majority of the sulfated sugar. In Trypanosoma cruzi, sulfated structures have been described as part of glycolipids. The present study constitutes the first report on the presence of sulfated oligosaccharides in glycoproteins of T. cruzi. The finding of sulfated glycans indicates the presence of an active sulfotransferase which has not been described in T. cruzi so far. Studies to address the precise nature of the sulfate sustitution and their biological significance are currently in progress in our laboratory.