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

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 fro...

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Autores principales: Agustí, Rosalía, Couto, Alicia Susana, Campetella, Oscar Eduardo, Muchnik de Lederkremer, Rosa María
Publicado: 1998
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Acceso en línea:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01666851_v97_n1-2_p123_Agusti
http://hdl.handle.net/20.500.12110/paper_01666851_v97_n1-2_p123_Agusti
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spelling paper:paper_01666851_v97_n1-2_p123_Agusti2023-06-08T15:16:09Z Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms Agustí, Rosalía Couto, Alicia Susana Campetella, Oscar Eduardo Muchnik de Lederkremer, Rosa María Ceramide GPI anchor Trans-sialidase Trypanosoma cruzi ceramide glycosylphosphatidylinositol membrane protein sialidase anion exchange chromatography article controlled study enzyme activity nonhuman priority journal thin layer chromatography Trypanosoma cruzi trypomastigote Bacillus thuringiensis Trypanosoma Trypanosoma cruzi 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 NaBH4 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. Copyright (C) 1998 Elsevier Science B.V. Fil:Agusti, R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Couto, A.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Campetella, O. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:De Lederkremer, R.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1998 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01666851_v97_n1-2_p123_Agusti http://hdl.handle.net/20.500.12110/paper_01666851_v97_n1-2_p123_Agusti
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-134
collection Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic Ceramide
GPI anchor
Trans-sialidase
Trypanosoma cruzi
ceramide
glycosylphosphatidylinositol
membrane protein
sialidase
anion exchange chromatography
article
controlled study
enzyme activity
nonhuman
priority journal
thin layer chromatography
Trypanosoma cruzi
trypomastigote
Bacillus thuringiensis
Trypanosoma
Trypanosoma cruzi
spellingShingle Ceramide
GPI anchor
Trans-sialidase
Trypanosoma cruzi
ceramide
glycosylphosphatidylinositol
membrane protein
sialidase
anion exchange chromatography
article
controlled study
enzyme activity
nonhuman
priority journal
thin layer chromatography
Trypanosoma cruzi
trypomastigote
Bacillus thuringiensis
Trypanosoma
Trypanosoma cruzi
Agustí, Rosalía
Couto, Alicia Susana
Campetella, Oscar Eduardo
Muchnik de Lederkremer, Rosa María
Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms
topic_facet Ceramide
GPI anchor
Trans-sialidase
Trypanosoma cruzi
ceramide
glycosylphosphatidylinositol
membrane protein
sialidase
anion exchange chromatography
article
controlled study
enzyme activity
nonhuman
priority journal
thin layer chromatography
Trypanosoma cruzi
trypomastigote
Bacillus thuringiensis
Trypanosoma
Trypanosoma cruzi
description 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 NaBH4 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. Copyright (C) 1998 Elsevier Science B.V.
author Agustí, Rosalía
Couto, Alicia Susana
Campetella, Oscar Eduardo
Muchnik de Lederkremer, Rosa María
author_facet Agustí, Rosalía
Couto, Alicia Susana
Campetella, Oscar Eduardo
Muchnik de Lederkremer, Rosa María
author_sort Agustí, Rosalía
title Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms
title_short Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms
title_full Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms
title_fullStr Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms
title_full_unstemmed Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms
title_sort structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from trypanosoma cruzi metacyclic trypomastigote forms
publishDate 1998
url https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01666851_v97_n1-2_p123_Agusti
http://hdl.handle.net/20.500.12110/paper_01666851_v97_n1-2_p123_Agusti
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