RESUMEN
Trypomastigote forms of Trypanosoma cruzi were metabolically labeled with [14C]-ethanolamine and [3H]-palmitic acid. Lipids shed to the culture medium were analyzed and compared with the parasite components. Phosphatidylcholine and lysophosphatidylcholine accounted for 53% of the total incorporated precursor. Interestingly, phosphatidylethanolamine and its lyso derivative lysophosphatidylethanolamine, although present in significant amounts in the parasites, could not be detected in the shed material. Shed lipids were highly enriched in the desaturated fatty acids C16:1 and C18:1 when compared to the total fatty acid pool isolated from the parasites.
Asunto(s)
Lípidos/análisis , Trypanosoma cruzi/química , Animales , Cromatografía en Capa Delgada , Medios de Cultivo , Etanolaminas , Ácidos Grasos no Esterificados/análisis , Ácidos Grasos Insaturados/análisis , Metabolismo de los Lípidos , Lisofosfatidilcolinas/análisis , Ácido Palmítico , Fosfatidilcolinas/análisis , Trypanosoma cruzi/metabolismoRESUMEN
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.
Asunto(s)
Antígenos de Protozoos/química , Glicoproteínas/química , Glicosilfosfatidilinositoles/química , Neuraminidasa/química , Trypanosoma cruzi/química , Animales , Antígenos de Protozoos/efectos de los fármacos , Antígenos de Protozoos/aislamiento & purificación , Ceramidas/análisis , Cromatografía por Intercambio Iónico/métodos , Cromatografía en Capa Delgada/métodos , Glicoproteínas/efectos de los fármacos , Glicoproteínas/aislamiento & purificación , Glicosilfosfatidilinositoles/aislamiento & purificación , Concentración de Iones de Hidrógeno , Estadios del Ciclo de Vida/fisiología , Neuraminidasa/efectos de los fármacos , Neuraminidasa/aislamiento & purificación , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Trypanosoma cruzi/crecimiento & desarrollo , Fosfolipasas de Tipo C/farmacologíaRESUMEN
The trans-sialidase from the trypomastigote stage of Trypanosoma cruzi was metabolically labeled with [3H]-palmitic acid and purified by immunoprecipitation with a monoclonal antibody. The action of PI-PLC on the immunoprecipitate released a lipid that was analyzed by TLC. Lyso-1-O-hexadecylglycerol and N-palmitoyl-sphinganine were obtained in a 1:3 ratio. A comparison with the GPI anchors present in the different stages of T. cruzi was made.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Lípidos de la Membrana/metabolismo , Neuraminidasa/metabolismo , Trypanosoma cruzi/enzimología , Animales , Cromatografía en Capa Delgada , Ratones , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Especificidad por Sustrato , Fosfolipasas de Tipo C/metabolismoRESUMEN
Trypomastigotes were metabolically labeled with [3H]-palmitic acid or [3H]-galactose and labeled components were detected in the culture medium. Thin layer chromatography of the shed material showed several lipids in the [3H]-palmitic acid labeled sample while the sugar was mainly incorporated into macromolecules. The material incorporated with the lipidic precursor was fractionated by DEAE-Sephadex (acetate form) and the amount of radioactivity was ten times higher in the acidic lipids than in the neutral lipids. When acidic lipids were further separated by Unisil, 73% of the radioactivity was recovered in the less polar fraction. Different patterns were obtained on comparison of the shed components with the lipids remaining in the parasite.