Characterization of the recombinant Candida albicans beta-1,2-mannosyltransferase that initiates the beta-mannosylation of cell wall phosphopeptidomannan.

摘要

The presence of beta-mannosides in their cell walls confers specific features on the pathogenic yeasts Candida albicans and Candida glabrata compared with non-pathogenic yeasts. In the present study, we investigated the enzymatic properties of Bmt1 (beta-mannosyltransferase 1), a member of the recently identified beta-mannosyltransferase family, from C. albicans. A recombinant soluble enzyme lacking the N-terminal region was expressed as a secreted protein from the methylotrophic yeast Pichia pastoris. In parallel, functionalized natural oligosaccharides isolated from Saccharomyces cerevisiae and a C. albicans mutant strain, as well as synthetic alpha-oligomannosides, were prepared and used as potential acceptor substrates. Bmt1p preferentially utilizes substrates containing linear chains of alpha-1,2-linked mannotriose or mannotetraose. The recombinant enzyme consecuti-vely transfers two mannosyl units on to these acceptors, leading to the production of alpha-mannosidase-resistant oligomannosides. NMR experiments further confirmed the presence of a terminal betaMan (beta-1,2-linked mannose) unit in the first enzyme product. In the future, a better understanding of specific beta-1,2-mannosyltransferase molecular requirements will help the design of new potential antifungal drugs.Registry Number/Name of Substance 0 (Mannans). 0 (Phosphopeptides). 0 (Recombinant Proteins). 0 (phosphopeptidomannan). EC 2-4-1 (Mannosyltransferases). EC 2-4-1 (beta-1,2-mannosyltransferase IV). PHA4727WTP (Mannose).