α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

Abstract

The enzyme ␣-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.The fungal cell wall is an important organelle that protects the cell from various environmental stresses. It is a dynamic structure that interacts with the environment and is modified to accommodate growth, cell division, and development. Fungal cell walls have been shown to contain ␤-1,3-glucan, ␣-1,3-glucan, ␤-1,6-glucan, mixed ␤-1,3/␤-1,4-glucans, chitin, and mannan/galactomannan (6, 19). These polysaccharide polymers constitute 80 to 85% of the cell wall mass, while glycoproteins constitute the remaining 15 to 20% (6). The cell wall glycoproteins are required for vital functions, like structural support, signal transduction, biofilm formation, and cell wall biosynthesis. In the case of pathogenic fungi, the cell wall is critical for the invasion of host tissues (8). Because of their accessibility and the crucial functions they perform, cell wall proteins could be important targets for the development of antifungal therapeutics.The glucan and chitin cell wall polymers are synthesized by enzyme complexes (glucan synthases and chitin synthases) that are associated with the plasma membrane. Glucan and chitin are vectorially passed into the cell wall space during synthesis and cross-linked together in the cell wall space. The mannan and galactomannan present in the cell wall are found as glycoconjugates on cell wall proteins. Mannosylation of cell wall proteins occurs in the endoplasmic reticulum (ER) and Golgi apparatus at O-linked and N-linked glycosylation sites. In Saccharomyces cerevisiae, mannosylation of N-linked glycosylation is initiated by the addition of an ␣-1,6-linked mannose ...

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

“…The bacterial enzymes function in the degradation of the S. cerevisiae, C. albicans, and Schizosaccharomyces pombe outer chain mannans (32). We propose that the fungal enzymes function as both hydrolases and transferases (transglycosylases) in cross-linking N-linked oligosaccharides into the cell wall (18,19). Kitagaki et al (31,36) previously characterized S. cerevisiae mutants affected in Dcw1p and Dfg5p.…”

“…A Coomassie brilliant blue G250 dye binding assay was used to assess the amounts of protein within mutant and wild-type cell walls (18,19). The assay used cell wall material, isolated as described above, that had been resuspended at a concentration of 2 mg/ml in water and subjected to sonication to disperse the cell wall material.…”

“…5, the cell walls from the D/D (outer chain mannan-deficient) mutant contained less protein than the wild-type cell walls. Although the dye binding assay does not give a linear relationship between the amount of dye bound and the amount of cell wall used (18,19), the amount of protein in the mutant cell wall relative to the wild-type cell wall can be estimated by looking at the amounts of wild-type and mutant cell walls needed to absorb the same amount of dye. Based on that criterion, the mutant cell wall appeared to have approximately half as much protein per milligram of cell wall as the wild-type cell wall.…”

“…Given the previously identified role of the mannanases and outer chain mannans in the incorporation of proteins into the N. crassa cell wall (18,19), we wanted to determine whether the cell walls of our C. albicans mutants had a reduced amount of protein. As one way to examine the amount of cell wall protein present in mutant and wild-type cell walls, we used a Coomassie brilliant blue dye binding assay (18,19). In this assay, increasing amounts of purified cell wall material are incubated with a set amount of dye, and the dye is allowed to bind to the protein in the purified cell walls.…”

“…These two enzymes belong to carbohydrate-active enzymes (CAZy) glycosyl hydrolase (GH) family 76, which contains endo-␣-1,6-mannanases. We have also shown that N-linked galactomannan (structurally related to the N-linked outer chain mannan of C. albicans) is required for cross-linking cell wall proteins into the cell wall (19). The N. crassa research showed that the ␣-1,6-mannan backbone of the N-linked galactomannan is the important structural feature needed for the incorporation of proteins into the cell wall.…”