Carbohydrate structure of Saccharomyces cerevisiae mnn9 mannoprotein.

Tsai, Pei-Kuo; Frevert, J.; Ballou, Clinton E.

doi:10.1016/s0021-9258(17)43167-x

Cited by 96 publications

(20 citation statements)

References 37 publications

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“…The pattern is shown in Figure 6. The structures of these oligo- (Tsai et al, 1984). "Less than 0.5 residue/100 amino acids of tryptophan, histidine, lysine, and arginine was observed, and no cysteine or methionine was detected.…”

Section: Resultsmentioning

confidence: 97%

Saccharomyces cerevisiae structural cell wall mannoprotein

Frevert¹,

Ballou²

1985

Biochemistry

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A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.

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Section: Resultsmentioning

confidence: 97%

Saccharomyces cerevisiae structural cell wall mannoprotein

Frevert¹,

Ballou²

1985

Biochemistry

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“…Byrd et al (1982) proposed that removal of the specific al,2-mannose to form the Man8GlcNAc2 intermediate is an essential step for chain elongation to occur. On the other hand, Tsai et al (1984) detected small amounts of this mannose residue in Man11GlcNAc2 oligosaccharides isolated from the S. cerevisiae mnnl/mnn9 double mutant. In subsequent studies, Trimble and showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mnsl cells.…”

Section: Introductionmentioning

confidence: 88%

Disruption of the processing α-mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae

Puccia

Grondin

Herscovics

1993

Biochemical Journal

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Processing of N-linked oligosaccharides in Saccharomyces cerevisiae begins with the removal of glucose and mannose residues from Glc3Man9GlcNAc2 to form a single isomer of Man8GlcNAc2. The importance of mannose removal for subsequent outer chain synthesis was examined in strains of S. cerevisiae disrupted in the MNS1 gene encoding a specific alpha 1,2-mannosidase responsible for Man8GlcNAc2 synthesis [Camirand, Heysen, Grondin and Herscovics (1991) J. Biol. Chem. 266, 15120-15127]. Both MNS1 transcripts of 1.85 kb and 1.7 kb were not observed in Northern blots of mns1 cells (i.e. cells containing the disrupted gene). Analysis on Bio-Gel P-6 of endo-beta-N-acetylglucosaminidase-H-sensitive oligosaccharides following a 10 min pulse with [2-3H]mannose revealed similar amounts of labelled outer chains excluded from the gel in both control and mns1 cells. H.p.l.c. of the included oligosaccharides showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mns1 cells. Analysis of [3H]mannose-labelled core oligosaccharides from immunoprecipitated CPY and invertase by h.p.l.c. showed a similar size distribution in mns1 and control cells. Invertase immunoprecipitated from [35S]methionine-labelled mns1 cells was highly glycosylated, but migrated slightly faster than that from control cells on denaturing PAGE, indicating a small difference in glycosylation. A similar difference in mobility was observed for invertase activity stain following non-denaturing gel electrophoresis. It is concluded that the alpha-mannosidase encoded by MNS1 is the only enzyme responsible for mannose removal in vivo, and that this processing step is not essential for outer chain synthesis.

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“…The a-anomeric region (4.8 < 6 < 5.5 ppm) shows nine signals in nonintegral intensity ratios (see inset in Figure 5). Comparison of these data with the 'H NMR spectra of other high-mannose oligosaccharides (Nilsen et al, 1991;Spellman et al, 1989;Vliegenthart et al, 1983; Trimble & Atkinson, 1986;Hernandez et al, 1989) indicates that the majority (~78%) of the high-mannose oligosaccharides contain a C2,C6-disubstituted a(l-*-3)mannosyl residue in the core, a structural element that is typical for yeast glycoprotein carbohydrates (Tsai et al, 1984;Hernandez et al, 1989) (see Table II, structures M7-I, M8-I, -III, and -IV, and M9-I, -II, and -III).…”

Section: Resultsmentioning

confidence: 93%

“…Purification of PreS2+S Polypeptides. PreS2+S polypeptides were produced by expression in the S. cerevisiae mnn9 mutant (Tsai et al, 1984) and purified essentially as described previously (Ellis et al, 1988;Scolnick et al, 1984;Wampler et al, 1985). A 25% (w/v) slurry of recombinant yeast cells was lysed in a Stanstead press and clarified by phase separation in a mixture of polyethylene glycol) (PEG) 3350 and dextran T500 (Ellis et al, 1988).…”

Section: Methodsmentioning

confidence: 99%

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Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast

Miller²,

Kubek³

et al. 1992

Biochemistry

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The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.

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Carbohydrate structure of Saccharomyces cerevisiae mnn9 mannoprotein.

Cited by 96 publications

References 37 publications

Saccharomyces cerevisiae structural cell wall mannoprotein

Saccharomyces cerevisiae structural cell wall mannoprotein

Disruption of the processing α-mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast

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