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

Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi s hed a glycoprotein, the shed acute phase antigen, that is responsible for t he trans-sialidase activity. In the present work the structure of the glyco sylphosphatidylinositol membrane anchor of the trans-sialidase isolated fro m metacyclic forms was determined. Parasites were metabolically labelled wi th [9, 10(n)H-3]-palmitic acid and the glycoprotein was purified by immunop recipitation with a monoclonal antibody directed against the repetitive ami noacid sequence. Treatment of the glycoprotein with phosphatidylinositol ph ospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that co migrated in TLC with a standard of ceramide. No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase is olated from culture-derived trypomastigotes where both lipids were found. C hemical and chromatographic analysis showed that the lipid moiety is palmit oyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine. Th e glycan constituent of the glycosylphosphatidylinositol-anchor was analyse d by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid. A major oligosaccharide was obtained and enzyma tic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substitu ted by an alpha-galactose. A comparison between the lipid constituent of th e glycosylphosphatidylinositol anchor of several proteins and their spontan eous shedding by the action of an endogenous PI-PLC was made. (C) 1998 Publ ished by Elsevier Science B.V. All rights reserved.