Expression and size heterogeneity of a 63 kilodalton membrane glycoprotein during growth and transformation of Leishmania mexicana amazonensis.
Our previous work by immunoprecipitation with a specific monoclonal antibody showed multiple, closely apposed electrophoretic bands of a major surface antigen specific to the promastigote stage of Leishmania mexicana amazonensis. Here, we analyzed the antigen during growth and transformation of this parasite with particular emphasis on the origin of the multiple bands. Immunobinding assays revealed the presence of the antigen throughout all phases of growth of cloned and uncloned promastigotes in various media for different number of generations. More antigen is expressed by promastigotes grown in Medium 199 plus fetal bovine serum than those in serum-supplemented Schneider's medium or a defined medium; however, this is clone-dependent. Purified monoclonal antibody coupled to Affi-Gel 10 gave a high capacity of antigen binding, resolving four electrophoretic bands of 60-66 kDa. A 63 kDa membrane protein, representing one of the four bands, became predominant after [35S]methionine label and chase. Pretreatment of promastigotes with 10 micrograms ml-1 tunicamycin reduces the antigen to a single band of 54 kDa. Treatment of the antigen bound to the affinity gel with endoglycosidase-H produces similar, but less complete effect. These results indicate glycosylation of this antigen with asparagine-linked oligosaccharides, which appears to account at least in part for its expression as multiple, closely apposed bands during biosynthesis. Binding of fluorescein isothiocyanate-labeled 6H12 monoclonal IgG or Fab to the promastigotes showed an even distribution of the antigen over the cell surface and its capping upon the addition of rabbit anti-mouse IgG. Additional hybridomas prepared against amastigotes yielded monoclonal antibodies which recognized surface antigens common to both stages of the parasite.
D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis*; D007163:Immunosorbent Techniques; D007894:Leishmania mexicana / Q000254:growth & development* / Q000276:immunology; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D006023:Glycoproteins; D009844:Oligosaccharides; D014415:Tunicamycin