The ␣1,2-mannosyltransferase gene MNT1 of the human fungal pathogen Candida albicans has been shown to be important for its adherence to various human surfaces and for virulence (Buurman, E. T., Westwater, C., Hube, B., Brown, A. P. J., Odds, F. C., and Gow, N. A. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7670 -7675). The CaMnt1p is a type II membrane protein, which is part of a family of proteins that are important for both O-and N-linked mannosylation in fungi and which represent a distinct subclass of glycosyltransferase enzymes. Here we use heterologous expression of CaMNT1 in the methylotrophic yeast Pichia pastoris to characterize the properties of the CaMnt1p enzyme as an example of this family of enzymes and to identify key amino acid residues required for coordination of the metal co-factor and for the retaining nucleophilic mechanism of the transferase reaction. We show that the enzyme can use both Mn 2؉ and Zn 2؉ as metal ion co-factors and that the reaction catalyzed is specific for ␣-methyl mannoside and ␣1,2-mannobiose acceptors. The N-terminal cytoplasmic tail, transmembrane domains, and stem regions were shown to be dispensable for activity, whereas truncations to the C-terminal catalytic domain destroyed activity without markedly affecting transcription of the truncated gene.In Saccharomyces cerevisiae, the process of glycosylation is essential for growth (1-3) affecting protein folding and stability, protection against proteolysis, intracellular trafficking, and the biophysical properties of the cell wall (4 -7). In the pathogenic fungus, Candida albicans, glycosylated outer cell wall mannoproteins form direct interactions with the host and are therefore critical for immunological reactivity, colonization, and adhesion of host tissues (8 -11). Both the protein and carbohydrate components of Candida mannoproteins have been implicated in mediating adhesion to host cells (12-17). Glycosylation is therefore important for pathogenicity and for host-fungal interactions.The structure of the O-and N-linked mannan-oligosaccharides to serine/threonine and asparagine residues, respectively, is determined by glycosyltransferases, which in fungi include enzymes encoded by the MNT and MNN gene families (18).These enzymes transfer a mannose from a GDP-mannose donor to the hydroxyl group of an oligosaccharide acceptor (19). Oligosaccharides are assembled by the sequential and concerted action of an array of glycosyltransferases as proteins pass through the secretory system. In the yeast-like fungi including S. cerevisiae and C. albicans, the outer mannose chains of N-linked glycans form extensive branched structures consisting of an ␣1,6-backbone on to which, ␣1,3-, ␣1,2-, and 1,2-mannan side chains are attached (20, 21). In contrast O-glycosylation of C. albicans involves the addition of short, linear chains formed by three or more mannose sugars (22-25). The mannan structures in C. albicans may vary in different strains and serotypes (11).Mutants with disrupted genes that function in the synthesis of ma...
