Purification and characterization of an 80-kilodalton membrane protein from Leishmania donovani.
Visceral leishmaniasis is caused by the protozoan parasite Leishmania donovani. We previously described the development of 16 monoclonal antibodies specific for L. donovani. The epitope recognized by one of these monoclonal antibodies, D13, is present at high density on nearly all isolates of L. donovani and, along with two other monoclonal antibodies, has been used to develop a sensitive and specific competitive assay for serodiagnosis of visceral leishmaniasis. In this report, we characterize the antigens recognized by D13 by immunoprecipitation of [35S]methionine-labeled promastigotes as two proteins (apparent molecular mass, 72 and 80 kilodaltons). Pulse-chase studies showed no evidence of a precursor-product relationship for the two proteins. We purified the 80-kilodalton protein (p80) to homogeneity by detergent solubilization of promastigote membranes, immunoaffinity chromatography, and ion-exchange chromatography. The epitope on p80 recognized by D13 was completely destroyed by proteolysis but was not affected by periodic acid treatment. P80 did not bind to the radioiodinated lectins concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Its apparent molecular mass was not affected by tunicamycin. Thus, it does not appear to be glycosylated. This protein is highly immunogenic and may prove useful for immunoprophylaxis and serodiagnosis of visceral leishmaniasis.
White
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Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
McMahon-Pratt
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000032:analysis; D019286:Endopeptidase K; D007893:Leishmania donovani / Q000032:analysis*; D008562:Membrane Glycoproteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008715:Methionine; D008970:Molecular Weight; D011233:Precipitin Tests; D011975:Receptors, Mitogen / Q000032:analysis; D012697:Serine Endopeptidases
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D008562:Membrane Glycoproteins; D011975:Receptors, Mitogen; D008715:Methionine; D012697:Serine Endopeptidases; D019286:Endopeptidase K