The glycosylphosphatidylinositol anchor of the trypomastigote-specific Tc-85 glycoprotein from Trypanosoma cruzi. Metabolic-labeling and structural studies.
The Tc-85 glycoprotein, specific for the infective stage of Trypanosoma cruzi, is anchored via glycosylphosphatidylinositol. The protein was purified from parasites, labeled metabolically with palmitic acid, by immunoprecipitation with the H1A10 monoclonal antibody or by affinity column chromatography on wheat germ agglutinin. Antisera to the soluble form of the variant surface glycoprotein of Trypanosoma brucei brucei cross-reacted with Tc-85 when the immunoprecipitate was analysed by Western blotting. The reaction was intensified upon previous incubation of the glycoprotein with phosphatidylinositol-specific phospholipase C. Such recognition was abolished when the cyclic phosphate was opened by mild acid treatment. The lipid cleaved by phospholipase C digestion, was identified as 1-O-hexadecylglycerol by reverse-phase thin-layer chromatography. The glycan core was deaminated and chemically labeled by reduction with NaB3H4. The labeled glycoprotein was exhaustively treated with pronase and dephosphorylated with 50% HF. Although microheterogeneity of the oligosaccharide moiety was apparent, by thin layer chromatography, a main spot coincident with Man(alpha 1-2) Man(alpha 1-6) Man(alpha 1-4) anhydromannitol was shown, consistent with the conserved core structure of all glycosylphosphatidylinositol anchors analysed to date.
Couto
A S
AS
Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
De Lederkremer
R M
RM
Colli
W
W
Alves
M J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D008055:Lipids / Q000032:analysis; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011233:Precipitin Tests; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000737:chemistry*; D014909:Wheat Germ Agglutinins
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D008055:Lipids; D010169:Palmitic Acids; D015800:Protozoan Proteins; D014909:Wheat Germ Agglutinins; D019308:Palmitic Acid