RESUMEN
Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N-linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase(R), oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. Treatment of trophozoites with 12 microM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.
Asunto(s)
Eritrocitos/parasitología , Glicoproteínas/biosíntesis , Plasmodium falciparum/fisiología , Proteínas Protozoarias/biosíntesis , Amidohidrolasas , Animales , Radioisótopos de Carbono , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía en Papel , Cromatografía en Capa Delgada , Cicloheximida/farmacología , Electroforesis en Gel de Poliacrilamida , Glucosa/metabolismo , Glicoproteínas/aislamiento & purificación , Humanos , Cinética , Malaria Falciparum/sangre , Manosa/metabolismo , Metionina/metabolismo , Peso Molecular , Oligosacáridos/química , Oligosacáridos/aislamiento & purificación , Parasitemia/sangre , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/aislamiento & purificación , Radioisótopos de Azufre , Tunicamicina/farmacologíaRESUMEN
Tc-85, an 85-kDa surface glycoprotein specific for the trypomastigote stage of Trypanosoma cruzi, has been implicated in the invasion of host cells by the parasite. Radioactive palmitic acid was incorporated into Tc-85 immunoprecipitated from the culture medium with the H1A10 monoclonal antibody, suggesting that shedding occurs with Tc-85 bearing its GPI anchor. In contrast to the glycoprotein remaining in the parasites, the glycosylphosphatidylinositol moiety in shed Tc-85 is resistant to phosphatidylinositol phospholipase C and becomes susceptible to the enzyme following alkali treatment. An alkylglycerol was identified by thin layer chromatography of an ether extract after the enzymatic reaction. Resistance to cleavage by phospholipase C is due to fatty acid esterification of the inositol residue in shed Tc-85. This is the first example of inositol modification in anchors from a glycoprotein of Trypanosoma cruzi.
Asunto(s)
Glicoproteínas/química , Glicosilfosfatidilinositoles/análisis , Proteínas de la Membrana/química , Proteínas Protozoarias/química , Trypanosoma cruzi/química , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Cromatografía en Capa Delgada , Pruebas de PrecipitinaRESUMEN
A hydrophobic fraction isolated from trypomastigotes of Trypanosoma cruzi is being characterized using immunological and chemical techniques. The lipopeptidophosphoglycan (LPPG) was identified in this fraction since it gave a positive reaction with anti-LPPG rabbit serum and had similar structural features such as the presence of ceramide as the lipid moiety, furanoic galactose, and a glycan moiety consistent with that obtained from an authentic sample of epimastigote LPPG, as judged by thin-layer chromatography. Furthermore, the hydrophobic fraction contained other glycolipids with different structural features. The lipid moiety of these compounds is alkylglycerol rather than a ceramide, the carbohydrate chain appears to be less complex than that in LPPG and no reactivity was observed towards an anti-LPPG serum.
Asunto(s)
Peptidoglicano/química , Fosfolípidos/química , Proteínas Protozoarias/química , Trypanosoma cruzi/química , Animales , Secuencia de Carbohidratos , Cromatografía en Capa Delgada , Glicoconjugados/química , Datos de Secuencia MolecularRESUMEN
A hydrophobic fraction isolated from trypomastigotes of Trypanosoma cruzi is being characterized using immunological and chemical techniques. The lipopeptidophosphoglycan (LPPG) was identified in this fraction since it gave a positive reaction with anti-LPPG rabbit serum and had similar structural features such as the presence of ceramide as the lipid moiety, furanoic galactose, and a glycan moiety consistent with that obtained from an authentic sample of epimastigote LPPG, as judged by thin-layer chromatography. Furthermore, the hydrophobic fraction contained other glycolipids with different structural features. The lipid moiety of these compounds is alkylglycerol rather than a ceramide, the carbohydrate chain appears to be less complex than that in LPPG and no reactivity was observed towards an anti-LPPG serum
Asunto(s)
Animales , Conejos , Glicoconjugados/química , Peptidoglicano/química , Fosfolípidos/química , Trypanosoma cruzi/química , Western Blotting , Secuencia de Carbohidratos , Glicoproteínas/inmunología , Glicoproteínas/química , Glicoconjugados/inmunología , Datos de Secuencia Molecular , Peptidoglicano/inmunología , Fosfolípidos/inmunología , Trypanosoma cruzi/inmunologíaRESUMEN
The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol-specific phospholipase C (PtdIns-specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non-N-acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry. The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns-specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 micrograms carbohydrate caused 60% inhibition of the binding of anti-CRD antibodies raised against the soluble form of variant surface glycoprotein.
Asunto(s)
Glucolípidos/análisis , Peptidoglicano/aislamiento & purificación , Fosfatidilinositoles/análisis , Fosfolípidos/aislamiento & purificación , Trypanosoma cruzi/análisis , Animales , Ceramidas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Glicosilfosfatidilinositoles , Oxidación-Reducción , Peptidoglicano/análisis , Fosfolípidos/análisis , Fosfolipasas de Tipo CRESUMEN
The structure of the carbohydrate moiety of the lipopeptidophosphoglycan from Trypanosoma cruzi was studied by 13C NMR spectroscopy and by methylation analysis of the original and of an acid-degraded sample. An oligosaccharide, consisting of 2-O-substituted and 6-O-substituted mannoses, which is linked to the ceramide, was separated by partial acid hydrolysis from an external chain that contained 3-O-substituted mannopyranosyl residues. beta-D-Galactofuranosyl terminal units are attached to position 3 of (1----2)-linked mannopyranose. Besides the previously reported monosaccharide components (mannose, galactose, glucose and glucosamine), ribose was identified in a partial acid hydrolysate of the lipopeptidophosphoglycan. The last three sugars are minor components and their organization into the overall structure of the lipopeptidophosphoglycan has not been determined.
Asunto(s)
Peptidoglicano/análisis , Fosfolípidos/análisis , Trypanosoma cruzi/análisis , Animales , Fenómenos Químicos , Química , Glicósidos/análisis , Hidrólisis , Espectroscopía de Resonancia Magnética , Metilación , Oligosacáridos/análisis , Rotación Óptica , Ribosa/análisisRESUMEN
The lipopeptidophosphoglycan (LPPG) of Trypanosoma cruzi was examined by 31P NMR spectroscopy. The main signal occurred at +0.67 ppm and corresponds to phosphodiester bonds. The presence of a signal at -25.06 indicates the existence of P-C linkages. At pH 10.5 signals which correspond to phosphate monoesters have been also obtained. Experiments on the stability of the phosphate bonds indicate that most of the monoester phosphates in LPPG are generated by alkaline hydrolysis of phosphodiester bonds. Aminoacid analysis of a LPPG hydrolysate revealed two pre-aspartic acid peaks with elution times coincident with those for authentic samples of 2-amino-3-phosphono-propionic acid and 2-aminoethyl-phosphonic acid in a 2.5:1 ratio. The main aminoacids in LPPG are Gly, Ser, Glu, Ala, Asp and Thr. Approximately half of the total aminoacids are released under saponification conditions indicating that part of the aminoacids in LPPG are bound via ester bonds.