| The fungal cell wall has a complex, laminate structure composed primarily of polymeric, mixed glucans and chitin, as well as a minor fraction of proteins and glycoproteins. To study this proteinaceous component, an antiserum (NcCW) has been raised against a cell wall extract from the filamentous fungus, Neurospora crassa. Immuno-gold labelling confirmed that this NcCW antiserum binds primarily to the cell envelope and cleavage with endo-proteinases and chemical reagents confirmed that the reacting antigens are proteinaceous. Western blot analysis revealed that five prominent proteins and more than 20 minor proteins are recognized by the NcCW antiserum. Extractions of the cell wall fraction with detergents, urea, sodium hydroxide, hydrochloric acid and trifluoromethanesulfonic acid suggest that several of the antigens are covalently linked to the cell wall through an O-glycosyl linkage. A comparison of these multi-species profiles of NcCW antiserum binding and lectin-binding supports the conclusion that this antiserum primarily recognizes polypeptides. The NcCW antiserum was utilized to identified 6 cDNA clones, encoding putative cell wall-associated proteins, from an expression library. A similar approach using antiserum raised against Schizophyllum commune cell walls (ScCW) resulted in the identification of 14 clones, encoding cell wall-associated and novel proteins, and provides evidence that the ScCW antiserum mainly recognized proteins.; Complementary approaches, namely, bioinformatic and proteomic-based methods, were also employed to further identify wall-associated proteins of N. crassa. To characterize the surface proteins, intact hyphae were treated with trypsin and the tryptic fragments were compared to proteins released by chemical treatments from purified cell walls. To obtain amino acid sequence information to allow protein identification, proteins extracted with (i) urea plus 2-mercaptoethanol, from purified walls, were separated by two-dimensional polyacrylamide gel electrophoresis (2-D SDS PAGE) before mass spectrometric analysis, whereas, (ii) the trypsin extracts, from intact cells, were directly examined by on-line liquid chromatography tandem mass spectrometry (CapLC-MS/MS). Furthermore, proteins secreted into the growth media and released by Novozyme(TM) treatments from purified cell walls were identified by mass spectrometry. The proteins identified in the datasets included homologs of yeast wall proteins, glycolytic enzymes, mitochondrial proteins, heat shock proteins, translation factors and novel proteins. |
