Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms

• Published in: Molecular and biochemical parasitology 1998-11, Vol.97 (1), p.123-131
• Main Authors: Agusti, Rosalı́a, Couto, AliciaS, Campetella, Oscar, Frasch, AlbertoC.C., de Lederkremer, RosaM
• Format: Article
• Language: English
• Subjects: Animals; Antigens, Protozoan - chemistry; Antigens, Protozoan - drug effects; Antigens, Protozoan - isolation & purification; Ceramide; Ceramides - analysis; Chromatography, Ion Exchange - methods; Chromatography, Thin Layer - methods; Glycoproteins - chemistry; Glycoproteins - drug effects; Glycoproteins - isolation & purification; glycosylphosphatidylinositol membrane anchor; Glycosylphosphatidylinositols - chemistry; Glycosylphosphatidylinositols - isolation & purification; GPI anchor; Hydrogen-Ion Concentration; Life Cycle Stages - physiology; Neuraminidase - chemistry; Neuraminidase - drug effects; Neuraminidase - isolation & purification; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Trans-sialidase; Trypanosoma cruzi; Trypanosoma cruzi - chemistry; Trypanosoma cruzi - growth & development; trypomastigotes; Type C Phospholipases - pharmacology
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(98)00137-6

Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi shed a glycoprotein, the shed acute phase antigen, that is responsible for the trans-sialidase activity. In the present work the structure of the glycosylphosphatidylinositol membrane anchor of the trans-sialidase isolated from metacyclic forms was determined. Parasites were metabolically labelled with [9, 10( n) 3H]-palmitic acid and the glycoprotein was purified by immunoprecipitation with a monoclonal antibody directed against the repetitive aminoacid sequence. Treatment of the glycoprotein with phosphatidylinositol phospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that comigrated in TLC with a standard of ceramide. No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase isolated from culture-derived trypomastigotes where both lipids were found. Chemical and chromatographic analysis showed that the lipid moiety is palmitoyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine. The glycan constituent of the glycosylphosphatidylinositol-anchor was analysed by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH 4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid. A major oligosaccharide was obtained and enzymatic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substituted by an α-galactose. A comparison between the lipid constituent of the glycosylphosphatidylinositol anchor of several proteins and their spontaneous shedding by the action of an endogenous PI-PLC was made.