Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques.
Candida albicans ATCC 26555 blastoconidia and blastoconidia bearing germ tubes were metabolically labelled by incubating the cells with 14C-labelled protein hydrolysate and were subsequently tagged with biotin. Double-labelled (radioactive and biotinylated) cell wall proteins and glycoproteins were extracted from intact cells of both growth forms by treatment with 2-mercaptoethanol (beta ME) and with beta-glucanases (Zymolyase) after treatment with beta ME. The beta ME- and Zymolyase-extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotted (immunoblotted) to nitrocellulose paper. Polyacrylamide gels were stained with Coomassie blue and processed for fluorography. Western blot analysis was performed either with peroxidase conjugated-concanavalin A (ConA) or Extravidin. Blotted proteins were also reacted with polyclonal antibodies and monoclonal antibodies against mannoprotein components from mycelial cell walls of the ATCC 26555 strain. Labelling with biotin allowed identification of a complex array of cell wall protein and glycoprotein components within a very wide molecular mass range (from 650 to 13 kDa). These appeared to be genuine cell wall components. Biotinylated high-molecular-mass glycoproteins that were not stained with Coomassie blue or that appeared as poorly resolved polydisperse bands by indirect ConA-peroxidase staining of Western blots were detected as sharply defined bands following reaction with the Extravidin-peroxidase conjugate. Biotinylated molecules retained unaltered reactivities against ConA, polyclonal antibodies, and monoclonal antibodies.
Casanova
M
M
Sección Departamental de Microbiología, Facultad de Farmacia, Universitat de València, Spain.
Lopez-Ribot
J L
JL
Martinez
J P
JP
Sentandreu
R
R
D000946:Antigens, Fungal / Q000737:chemistry; D000954:Antigens, Surface / Q000737:chemistry; D001345:Autoradiography; D001710:Biotin / Q000737:chemistry*; D015153:Blotting, Western; D002176:Candida albicans / Q000737:chemistry* / Q000276:immunology; D002473:Cell Wall / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D012995:Solubility; D013194:Staining and Labeling
D000946:Antigens, Fungal; D000954:Antigens, Surface; D005656:Fungal Proteins; D006023:Glycoproteins; D008623:Mercaptoethanol; D001710:Biotin; D004693:Glucan Endo-1,3-beta-D-Glucosidase