Genetic and Biochemical Characterization of GH72 glucanosyltransferases shows they function in attaching glycoproteins into the fungal cell wall

Stephen Free Jie Ao BIbekananda Kar
Dept. of Biological Sciences, SUNY University at Buffalo, Buffalo, New York, USA

The Neurospora crassa genome encodes five GH72 family glucanosyltransferases, and four of these enzymes (GEL-1, GEL-2, GEL-3, and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the roles of these four glucanosyltransferases in cell wall biogenesis and demonstrated that the enzymes are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple glucanosyltransferases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2, and GEL-5 is required for the growth of the vegetative hyphae, while the GEL-1. GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of glucanosyltransferase-deficient mutants demonstrated that the incorporation of cell wall glycoproteins was severely compromised. Interestingly, we found that the mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 glucanosyltransferases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall. We further demonstrated tht the enzymes are capable of cross-linking cell wall glycoproteins and cell wall glucans.