Characterization of a soluble protein of Coccidiodes immitis with activity as an immunodiffusion-complement fixation antigen.
A 48-kilodalton (kDa) electrophoretically distinct antigen from Coccidioides immitis mycelial- and spherule-endospore-phase filtrates was previously associated by immunoblotting with the immunodiffusion band that corresponds to complement-fixing activity (ID-CF). To characterize this antigen and its precursor, both mycelial- and spherule-endospore-phase filtrates were fractionated by size exclusion chromatography, lectin affinity chromatography, and nondenaturing electrophoresis. By size exclusion chromatography, most of the protein and carbohydrate of the crude filtrates eluted in a peak of average molecular size less than 30 kDa, although other components were detected. ID-CF activity was associated with the component at a relative mobility of 110 kDa. Fractions containing the ID band that corresponded to tube precipitin activity occurred from 200 to 40 kDa. The appearance of the 48-kDa band in denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) specifically coincided with the fractions containing ID-CF activity. Nondenaturing PAGE of filtrates showed silver-stainable and immunoblot-reactive bands in the region of 110 kDa. Prior treatment with pronase destroyed this electrophoretically separable antigen, whereas periodate had no effect. Trypsin did not affect the 110-kDa band in unheated or unreduced antigen. Mycelial filtrates were chromatographed on lentil lectin or concanavalin A-Sepharose 4B to deplete them of glucose- or mannose-containing carbohydrate. The effluent fraction contained ID-CF activity and, upon denaturing electrophoresis, the 48-kDa antigen. The 110-kDa protein represents the ID-CF antigen which is heat labile and denatured to a 48-kDa band by sodium dodecyl sulfate-PAGE.
Zimmer
B L
BL
Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616.
Pappagianis
D
D
D000946:Antigens, Fungal / Q000302:isolation & purification*; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003045:Coccidioides / Q000032:analysis*; D003168:Complement Fixation Tests; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000302:isolation & purification*; D007106:Immune Sera; D005779:Immunodiffusion
D000946:Antigens, Fungal; D002241:Carbohydrates; D005656:Fungal Proteins; D007106:Immune Sera