Functional Characterization of the Candida albicans MNT1Mannosyltransferase Expressed Heterologously in Pichia pastoris

Abstract

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...

Structure of Kre2p/Mnt1p

“…Kre2p/Mnt1p requires manganese for optimal activity (3,8). Substitution of the manganese for Mg 2ϩ , Ca 2ϩ , or Ni 2ϩ has been found to impair catalysis.…”

“…(2), a GT family, which so far includes nine S. cerevisiae proteins (1) and five from the human pathogen Candida albicans (3,4). There are no homologs in humans or other higher eukaryotes.…”

Molecular and Phenotypic Analysis ofCaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

“…The structure of the outer chains of N-linked oligosaccharides on glycoproteins in C. albicans is similar, although not identical, to the structure of the outer chains of N-linked oligosaccharides on glycoproteins in S. cerevisiae (53). Genes related to S. cerevisiae mannosyltransferase genes have been identified as functional homologues (50,(54)(55)(56) and by their sequence sim-* Corresponding author. Mailing address:…”

Isolation of Sporothrix schenckii MNT1 and the biochemical and functional characterization of the encoded α1,2-mannosyltransferase activity

“…The recombinant enzyme showed an absolute requirement of Mn 2+ for activity, at an optimal concentration of 15 mM, similar to ScKre2 and ScKtr1, which utilize only this metal ion as cofactor (Romero et al, 1997), whereas CaMnt1 can also utilize to a lesser extent Zn 2+ and Co 2+ (Thomson et al, 2000).…”

“…In C. albicans, the MNT1 gene family has five members with more specialized functions: CaMnt1 and CaMnt2 have redundant activities in the addition of the second and third mannose residues to O-linked mannans Buurman et al, 1998;Thomson et al, 2000), both CaMnt4 and CaMnt5 participate in the synthesis of N-linked mannan outer chains, and CaMnt3 and CaMnt5 have redundant participation in the synthesis of the phosphomannan moiety (Mora-Montes et al, 2010a). Furthermore, this gene family is important for proper cell wall composition, virulence and interaction with immune cells (Mora-Montes et al, 2010a).…”