Abstract
ABSTRACTβ-(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 Saccharomyces cerevisiae as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized S. cerevisiae 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 bgl2Δ and gas1Δ 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 S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual β-(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 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.
Highlights
ABSTRACT -(1,3)-Glucan, the major fungal cell wall component, ramifies through -(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly
This dual -(1,3)-glucan elongation and branching activity was seen within the members of the glycosylhydrolase 72 (GH72) family from both yeasts and molds having a carbohydrate binding module (CBM), CBM43, in their sequence, as S. cerevisiae Gas1p (ScGas1p) ortholog Gel4p from Aspergillus fumigatus showed -(1,3)-glucan elongation and branching activity
Upon thin-layer chromatography (TLC), these two peak fractions migrated with Rf values intermediary to those calculated for -(1,3)-oligotriose-tetraose and -(1,3)-oligotetraose-pentaose (Fig. 1B)
Summary
ABSTRACT -(1,3)-Glucan, the major fungal cell wall component, ramifies through -(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. The double GAS1 BGL2 deletion mutant showed a sick phenotype, and A. fumigatus GEL4 deletion is inviable (16), indicating that -(1,3)-glucan elongation branching is an essential event during fungal cell wall construction.
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