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 α-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.

Fig. 1.

Growth pattern of the wild-type and the och-1 mutant colonies. The wild type, the och-1 knockout mutant, and the och-1 knockout mutant that was transformed with the wild-type copy of the och-1 gene (knock-in) were grown in Vogel's medium with 2% sucrose agar in plates and slants for a period of 6 to 10 days.

Fig. 2.

Differential interference contrast (DIC) microscopy images of the growing edge of the colony from wild-type cells and och-1 mutant cells at 40× resolution (bar represents 10 μm).

Fig. 3.

Representations of the N-linked oligosaccharides from S. cerevisiae (left structure) and N. crassa (right structure). In S. cerevisiae, Och1p adds the initial α-1,6-mannose of a long α-1,6-mannose chain to the N-linked oligosaccharide (GlcNac 2/mannose 8 structure). Side branches containing 2 α-1,2-mannoses and a terminal α-1,3-mannose are added to the mannoses in the α-1,6-chain. In N. crassa, OCH-1 adds the initial α-1,6-mannose of a short chain of α-1,6-mannoses to the N-linked oligosaccharide (GlcNac 2/mannose 8 structure). Approximately 30% of the mannoses in the chain have side branches containing two α-1,2-mannoses and two galactofuranoses.

Fig. 4.

Proteomic analysis of cell wall proteins and secreted proteins. Lane 1, och-1 mutant cell wall proteins from 2 mg of cell wall; lane 2, och-1 mutant secreted proteins (20 μg); lane 3, wild-type cell wall proteins from 2 mg of cell wall; lane 4, wild-type secreted proteins (20 μg).

Fig. 5.

Coomassie brilliant blue dye assay of cell wall protein. Wild-type and och-1 mutant cell walls were incubated in the presence of Coomassie blue, and the amounts of Coomassie blue absorbed by the cell wall were determined.

Fig. 6.

Secretion of ACW-1 is elevated in the och-1 mutant. Western blot analysis of protein using antibody directed against ACW-1. Lane 1, wild-type cytosolic fraction (30 μg of cytosolic protein); lane 2, och-1 cytosolic fraction (30 μg of cytosolic protein); lane 3, wild-type secreted protein fraction (normalized to the amount of secreted protein from 30 μg of cytosolic protein); lane 4, och-1 secreted protein fraction (normalized to the amount of secreted protein from 30 μg of cytosolic protein); lane 5, wild-type secreted protein fraction (normalized to the amount of secreted protein from 500 μg of cytosolic protein); lane 6, och-1 secreted protein fraction (normalized to the amount of secreted protein from 500 μg of cytosolic protein).

Fig. 7.

Secretion of GEL-1 is elevated in the och-1 mutant. Western blot analysis using antibody directed against GEL-1. Lane 1, wild-type cytosolic fraction (30 μg of cytosolic protein); lane 2, och-1 cytosolic fraction (30 μg of cytosolic protein); lane 3, wild-type secreted protein fraction (normalized to the amount of secreted protein from 30 μg of cytosolic protein); lane 4, och-1 secreted protein fraction (normalized to the amount of secreted protein from 30 μg of cytosolic protein).

Fig. 8.

The covalent incorporation of ACW-1 into the cell wall is reduced in the och-1 mutant. Western blot analysis using antibody directed against the cell wall protein ACW-1. Lane 1, wild-type cytosolic fraction (30 μg of cytosolic protein); lane 2, och-1 cytosolic fraction (30 μg of cytosolic protein); lane 3, wild-type SDS-soluble cell wall protein fraction (normalized to 30 μg of cytosolic protein); lane 4, och-1 secreted protein fraction (normalized to 30 μg of cytosolic protein).