Characterization of β-1,2-Mannosyltransferase in Candida guilliermondii and Its Utilization in the Synthesis of Novel Oligosaccharides

Suzuki, Akifumi; Shibata, Nobuyuki; Suzuki, Mikiko; Saitoh, Fumie; Oyamada, Hiroko; Kobayashi, Hidemitsu; Suzuki, Shigeo; Okawa, Yoshio

doi:10.1074/jbc.272.27.16822

Cited by 17 publications

(8 citation statements)

References 46 publications

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“…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)(23)(24)(25).…”

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confidence: 99%

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

Thomson

Bates

Yamazaki³

et al. 2000

Journal of Biological Chemistry

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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...

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confidence: 99%

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

Thomson

Bates

Yamazaki³

et al. 2000

Journal of Biological Chemistry

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“…This type of linkage has not been found in mammalian cells and, to our knowledge, has been reported to occur in only a few organisms, mostly pathogens: in glycoproteins of Candida spp., Salmonella enterica serovar Thompson, and Pichia pastoris (17,21,32) and in reserve oligosaccharides of Leishmania spp. (24) and of the thermophilic bacterium Truepera radiovitrix (our unpublished results).…”

Section: Discussionmentioning

confidence: 99%

A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di- myo -Inositol Phosphate as the Mannosyl Acceptor

et al. 2009

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In addition to di-myo-inositol-1,3-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2-(O-␤-D-mannosyl)-di-myo-inositol-1,3-phosphate (MDIP) and 2-(O-␤-D-mannosyl-1,2-O-␤-D-mannosyl)-di-myo-inositol-1,3-phosphate (MMDIP), two newly identified ␤-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima, and the assignment was confirmed by functional expression in Escherichia coli. Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus, and Archaeoglobus profundus. MDIP synthase of T. maritima had maximal activity at 95°C and apparent K m values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the L-inositol moiety was synthesized and used as a substrate for MDIP synthase. This ␤-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria, it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales. To our knowledge, this is the first ␤-1,2-mannosyltransferase characterized thus far.Thermotoga maritima was first isolated from hot marine sediments on Vulcano Island, Italy, being able to grow between 55°C and 90°C (14). This strictly anaerobic bacterium ferments a variety of simple and complex carbohydrates to acetate, hydrogen, and CO 2 (10). In line with these metabolic traits, a substantial percentage of the genes annotated in the genome of this hyperthermophile are allocated to the metabolism of mono-and polysaccharides (8, 23). Therefore, T. maritima has been pointed out as a source of glycoside hydrolases with potential industrial relevance, namely, in processes of conversion of biomass into biofuels (3, 34).Like many other hyperthermophiles isolated from marine environments, Thermotoga maritima is slightly halophilic (optimum NaCl concentration of 2.7%, wt/vol) and has developed biochemical strategies to counterbalance the external osmotic pressure. The accumulation of low-molecular-mass organic compounds in the cytoplasm is the most common osmoadaptation mechanism, which enables a rapid response to fluctuations in the salinity of the external medium. Interestingly, the organic solutes encountered in organisms adapted to thrive in hot environments are clearly different from those ...

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“…Subsets of eukaryotes may have Man-T that have specialized functions. Of interest because of the novelty of the linkage they catalyze are, for example, the presumed Dol-P-Man-requiring C-mannosyltransferase, and the five P-Man-T activities involved in modifying cell wall mannan of the fungal genus Candida [69], which includes the human pathogen C. albicans. In Saccharomyces cereuisiae, the genes for the Man-T that add additional a-linked Man to proteinbound GPI anchors in the Golgi may yet be found in the MnnIp or KtrIp families.…”

Section: "Missing" Eukaryotic Man-tmentioning

confidence: 99%

Mannosyltransferases

Ernst

Hart

Sînaÿ

2000

Carbohydrates in Chemistry and Biology

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Characterization of β-1,2-Mannosyltransferase in Candida guilliermondii and Its Utilization in the Synthesis of Novel Oligosaccharides

Cited by 17 publications

References 46 publications

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

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

A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di- myo -Inositol Phosphate as the Mannosyl Acceptor

Mannosyltransferases

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