Cell wall glycoproteins of Candida albicans as released by different methods

Different methods of extraction frequently used in other studies were used to release glycoproteins from both intact cells and isolated cell walls of yeast and hyphal forms of Candida albicans. Extracts were obtained from whole cells by treatment (i) with 2-mercaptoethanol (beta ME) at pH 8.6 and 37 C degrees and (ii) with zymolyase after treatment with beta ME. Extracts were obtained from isolated and washed cell walls (i) by boiling with beta ME and sodium dodecyl sulphate (SDS), (ii) by boiling with SDS and (iii) by treatment with zymolyase after SDS. The extracts were separated by SDS-polyacrylamide gel electrophoresis and analysed by Western blotting with four reagents. Analysis with concanavalin A (ConA) revealed different glycoprotein populations depending on the treatment. Three possible germ-tube-specific constituents were observed; and 80 kDa component released by beta ME from both intact cells and cell walls, and 47 kDa and 43 kDa moieties released by zymolyase only from intact cells. MAb 4C12, specific for the protein portion of a large germ tube constituent, recognized polydisperse material which just entered the gel in beta ME extracts and in the region extending up from 200 kDa to near the top of the gel in zymolyase extracts. MAb 24.17, specific for a carbohydrate determinant of yeast phase cells, reacted with disperse material in the region from the top of the gel to one-third to two-thirds the distance to the 220 kDa mass marker. Antiserum specific for the serotype A determinant of mannan reacted with large disperse component(s) migrating in the region from the top of the gel to about two-thirds the distance to the 220 kDa mass marker and with a 180 kDa component. The components recognized by MAb 4C12, but not those recognised by MAb 24.17 and serotype A antiserum, were effected by treatment with endo-beta-N-acetylglucosamidase H. The various analyses revealed that the method of extraction affected the composition and size of the constituents recognized by the reagents.