Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes.
We have used pulse-chase immunoprecipitations methods to study early post-translational processing of CBI-gp, a lysosomal membrane glycoprotein expressed by African trypanosomes, Rap67, a polyclonal antibody to CBI-gp, immunoprecipitated a 100-kDa glycoprotein, gp100, from both bloodstream forms (BF) and procyclic forms (PF) of Trypanosoma brucei gambiense immediately after a 5-min pulse with radiomethionine. N-Glycanase digestion released a 67-kDa core protein, p67, from gp100 of both life cycle forms V8 protease digestion of p67 from BF and PF yielded 13 identical methionyl peptides, suggesting that gp100 from both life cycle forms have very similar or identical p67 core molecules. In BF, gp 100 carried both endoglycosidase H (EndoH)-resistant and EndoH-sensitive, N-linked oligosaccharides immediately after labeling. In PF, all the N-linked sugars on gp100 were EndoH sensitive. In BF, gp100 chased progressively into slower migrating 150-180-kDa components that obtained the CBI epitope, traveled to the cell surface where they could be biotinylated, and were proteolytically processed. The increase in mass of gp100 during chase in BF resulted from an elongation of N-linked oligosaccharides. Maturation of gp100 into 150-180-kDa CBI-gp was inhibited if BF were chased in the presence of glucosidase inhibitors castanospermine or deoxynojirimycin. In PF, gp100 did not increase in mass, could not be biotinylated on the cell surface, and was not proetolyzed during extended chases. Cryoimmunoelectron microscopy revealed that the antigens detected by rap67 are abundant in lysosomes and endosomes in both BF and PF. Thus, BF and PF express very similar or identical lysosomal membrane glycoproteins but process and transport them in very different ways.
Kelley
R J
RJ
Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.
Brickman
M J
MJ
Balber
A E
AE
D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000378:metabolism; D001693:Biological Transport, Active; D001710:Biotin; D006031:Glycosylation; D008247:Lysosomes / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000276:immunology / Q000378:metabolism*; D014347:Trypanosoma brucei gambiense / Q000254:growth & development* / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D001710:Biotin