title: Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi. Identification of sulfated high-mannose type oligosaccharides.
abstract: Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report 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 this 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. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. 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 was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.
authors.affiliation: Instituto de Investigaciones Biotecnológicas-INTECH, Universidad Nacional de Gral. San Martin, Buenos Aires, Argentina.
authors.forename: Mariana
authors.initials: M
authors.lastname: Barboza
authors.forename: Vilma G
authors.initials: VG
authors.lastname: Duschak
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fukuyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nonami
authors.forename: Rosa
authors.initials: R
authors.lastname: Erra-Balsells
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Cazzulo
authors.forename: Alicia S
authors.initials: AS
authors.lastname: Couto
mesh_terms: D000818:Animals; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000737:chemistry / Q000378:metabolism; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D025942:Oligosaccharides, Branched-Chain / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D025942:Oligosaccharides, Branched-Chain; D015800:Protozoan Proteins; D005643:Fucose; D003546:Cysteine Endopeptidases; C063214:cruzipain; D008358:Mannose