Cell wall glycoproteins of Candida albicans as released by different methods

Abstract

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 (PME) at pH 8.6 and 37 "C and (ii) with zymolyase after treatment with PME. Extracts were obtained from isolated and washed cell walls (i) by boiling with PME 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; an 80 kDa component released by PME 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 PME 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-P-N-acetylglucosamidase H. The various analyses revealed that the method of extraction affected the composition and size of the constituents recognized by the reagents.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen

“…Among the numerous proteins which are removed from the cell wall by ␤ME extraction of intact cells (6,8,9), the MAb anti-CH-GAPDH detected a major protein species of 33 kDa in cell wall extracts from both blastoconidia and germinated blastoconidia (Fig. 2B, lanes 2 and 3), which indicates that GAPDH is also associated with the C. albicans cell wall.…”

“…Protein and glycoprotein components of the walls were released from intact cells by treatment with ␤-mercaptoethanol (␤ME) as previously described (6,8,9). The ␤ME-extracted cells were treated with a ␤-(1,3)-glucanase complex (Zymolyase 20T) to obtain protoplasts, which were lysed, and the supernatant (cytosolic extract) was recovered following centrifugation as previously described (8,9).…”

Fluconazole resistance in Candida glabrata is associated with increased bud formation and metallothionein production

“…Soluble proteins were extracted as described previously by Casanova & Chaffin (1991) with slight modifications. C. glabrata cells grown in RPMI agar with or without FL were collected by scraping the agar plates with a sterile wire loop.…”

Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraldehyde‐3‐phosphate dehydrogenase‐invertase fusion protein

“…cerevisiae GAPDH polypeptide ( Figure 5); it should be noted that both the internal and the glycosylated external invertase polypeptides encoded by SUC2 are present in yeast cell-free extracts (Gozalbo and The electrophoretic mobility of molecular size marker proteins is indicated Hohmann, 1989;Gozalbo, 1992). Furthermore, the 90 kDa fusion protein was also the only immunoreactive protein species released from intact cells by treatment with βME (not shown), a reducing reagent that solubilizes cell wall-associated proteins (Casanova and Chaffin, 1991;Chaffin et al, 1998). Immunoblot analysis using rabbit polyclonal antibodies against yeast GAPDH resulted in the detection of the 90 kDa fusion protein in pTDH transformant cell-free extracts, as well as the yeast GAPDH polypeptide (37 kDa) in all transformants analysed (not shown).…”

“…Sedimented cells were disrupted with glass beads by vortexing at 4 • C (six times for 1 min), and after addition of 0.5 ml PBS with the protease inhibitor cocktail, the crude extract was removed from glass beads and centrifuged for 15 min at 9000 × g at 4 • C; the supernatant (cellfree extract) was used directly for invertase activity assays or Western immunoblot analysis, or alternatively kept at −20 • C. Cell wall-associated protein and mannoprotein species were solubilized from cells by β-mercaptoethanol (βME) treatment, as previously reported (Casanova and Chaffin, 1991).…”