The Neurospora crassa dfg5 and dcw1 Genes Encode α-1,6-Mannanases That Function in the Incorporation of Glycoproteins into the Cell Wall

Abstract

The covalent cross-linking of cell wall proteins into the cell wall glucan/chitin matrix is an important step in the biogenesis of the fungal cell wall. We demonstrate that the Neurospora crassa DFG5 (NCU03770) and DCW1 (NCU08127) enzymes function in vivo to cross-link glycoproteins into the cell wall. Mutants lacking DFG5 or DCW1 release slightly elevated levels of cell wall proteins into their growth medium. Mutants lacking both DFG5 and DCW1 have substantially reduced levels of cell wall proteins in their cell walls and release large amounts of known cell wall proteins into the medium. DFG5 and DCW1 are members of the GH76 family of glycosyl hydrolases, which have specificity to recognize and cleave α-1,6-mannans. A model for incorporation of glycoproteins into the cell wall through the α-1,6-mannan core of the N-linked galactomannan is presented. In this model, DFG5 and DCW1 recognize the N-linked galactomannan present on glycoproteins and cross-link it into the cell wall glucan/chitin matrix.

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

“…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.…”

“…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.…”

“…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.…”

“…We recently showed that in Neurospora crassa, two functionally redundant cell wall ␣-1,6-mannanases, DCW-1 and DFG-5, function in the cross-linking of cell wall proteins into the cell wall glucan-chitin matrix (18). These two enzymes belong to carbohydrate-active enzymes (CAZy) glycosyl hydrolase (GH) family 76, which contains endo-␣-1,6-mannanases.…”

Avoiding the Ends: Internal Epitope Tagging of Proteins Using Transposon Tn7

“…Dcw1 and its paralog Dfg5 are important in cell-wall structure and integrity in S. cerevisiae and other fungi (Kitagaki et al 2002(Kitagaki et al , 2004Gonzalez et al 2010;Maddi et al 2012). Traditional methods of N-or C-terminally tagging Dcw1 are expected to fail due to interference with protein localization because Dcw1 localization at the cell membrane requires an N-terminal secretory signal sequence and a C-terminal signal sequence for addition of a glycosylphosphatidylinositol (GPI) anchor.…”