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

Abstract

β-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using <i>Saccharomyces cerevisiae</i> as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized <i>S. cerevisiae</i> and radiolabeled substrate UDP-(¹⁴C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the <i>bgl2</i>Δ and <i>gas1</i>Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products [short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage]. The double <i>S. cerevisiae gas1</i>Δ <i>bgl2</i>Δ mutant showed a drastically sick phenotype. An <i>Sc</i>Gas1p ortholog, Gel4p from <i>Aspergillus fumigatus</i>, also showed dual β-(1,3)-glucan elongating and branching activity. Both <i>Sc</i>Gas1p and <i>A. fumigatus</i> Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.<b>IMPORTANCE</b> The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core β-(1,3)-glucan ramification, is poorly understood; the ramified β-(1,3)-glucan interconnects other cell wall components. Once linear β-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using <i>Saccharomyces cerevisiae</i> as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the β-(1,3)-glucan branching. The sick phenotype of the double <i>Scgas1</i>Δ <i>bgl2</i>Δ mutant suggested that β-(1,3)-glucan branching is essential. In addition to <i>Sc</i>Gas1p, GH72 family <i>Sc</i>Gas2p and <i>Aspergillus fumigatus</i> Gel4p, having CBM43 in their sequences, showed dual β-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall β-(1,3)-glucan branching mechanism. The essentiality of β-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.