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 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 alpha-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.
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CIHIDECAR (CONICET), Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Couto
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Campetella
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Frasch
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de Lederkremer
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D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D002518:Ceramides / Q000032:analysis; D002852:Chromatography, Ion Exchange / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D006023:Glycoproteins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008018:Life Cycle Stages / Q000502:physiology; D009439:Neuraminidase / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D014349:Trypanosoma cruzi / Q000737:chemistry* / Q000254:growth & development; D010738:Type C Phospholipases / Q000494:pharmacology
D000953:Antigens, Protozoan; D002518:Ceramides; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C069715:trans-sialidase; D009439:Neuraminidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase