R. Agusti et al., Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms, MOL BIOCH P, 97(1-2), 1998, pp. 123-131
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.