The Dual Activity Responsible for the Elongation and Branching of β-(1,3)-Glucan in the Fungal Cell Wall

beta-(1,3)-Glucan, the major fungal cell wall component, ramifies through beta-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify beta-(1,6)-branching on beta-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(C-14) glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2. and gas1. mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced beta-(1,6)-branching on the beta-(1,3)-oligomers following its beta-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear beta-(1,3)-oligomers as well as Bgl2p-catalyzed products [short beta-(1,3)-oligomers linked by a linear beta-(1,6)-linkage]. The double S. cerevisiae gas1 Delta bgl2 Delta mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual beta-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual beta-(1,3)-glucan elongating and branching activity. Our report unravels the beta-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life. IMPORTANCE The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core beta-(1,3)-glucan ramification, is poorly understood; the ramified beta-(1,3)-glucan interconnects other cell wall components. Once linear beta-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the beta-(1,3)-glucan branching. The sick phenotype of the double Scgas1 Delta bgl2 Delta mutant suggested that beta-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual beta-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall beta-(1,3)-glucan branching mechanism. The essentiality of beta-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.