Glycoprotein biosynthesis in <i>Succharomyces cerevisiae</i>. Partial purification of the α-1,6-mannosyltransferase that initiates outer chain synthesis

Romero, Pedro; Sleno, B.; Herscovics, Annetté

doi:10.1093/glycob/4.2.135

Cited by 23 publications

(17 citation statements)

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“…In S. cerevisiae, two tightly related a-1,6-mannosyltransferase homologues, Och1p and Hoc1p, have been identified and characterized. Och1p has been described as a critical protein in the extension of N-linked oligosaccharide chains, and catalyses the addition of the first a-1,6-linked mannose to the core oligosaccharide (Nakanishi-Shindo et al, 1993;Romero et al, 1994;Nakayama et al, 1997). In contrast, Hoc1p has been defined as a regulator protein belonging to the M-Pol II complex, and is involved in the last step of the long a-1,6-linked backbone extension (Neiman et al, 1997;Munro, 2001;Stolz & Munro, 2002).…”

Section: Discussionmentioning

confidence: 99%

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Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

2004

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In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae α-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of the Y. lipolytica proteins, including the previously isolated YlMnn9p, in the N-glycosylation pathway, a phenotypic analysis of the disrupted strains ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9 and ΔYlmnn9ΔYloch1 was performed. Disruption of the YlMNN9, YlANL1 and YlOCH1 genes caused an increased sensitivity to SDS, compatible with a glycosylation defect, and to Calcofluor White, characteristic of cell-wall defects. Moreover, Western-blot analysis of a heterologous glycosylated protein confirmed a direct role of YlMnn9p and YlAnl1p in the N-glycosylation process. These mutant strains, ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9 and ΔYlmnn9ΔYloch1 may thus be used to establish a model for the Y. lipolytica N-linked glycosylation pathway.

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Section: Discussionmentioning

confidence: 99%

“…In this model, the formation of the mannan outer chain is initiated by the Och1p protein, a type II membrane a-1,6-mannosyltransferase that defines an early Golgi compartment (Nakanishi-Shindo et al, 1993;Romero et al, 1994;Nakayama et al, 1997). Upon arrival in the Golgi, Och1p adds a single a-1,6-mannose to all N-glycan cores.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

2004

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“…Subsequent in vitro assays using ER membrane preparations identified a catalytic activity that could generate the Man8B structure from Man 9 GlcNAc 2 , and this activity was shown to be sensitive to inhibition by dMNJ, kifunensine, and EDTA but not swainsonine or 1,4-dideoxy-1,4-imino-D-mannitol (12)(13)(14). Although the mammalian enzyme has not been purified, cloned, or biochemically characterized other than through in vitro assays in crude membrane preparations, an ER ␣-mannosidase activity producing the identical Man8B isomer in Saccharomyces cerevisiae has been purified, characterized, cloned, and expressed (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). This yeast enzyme has many catalytic characteristics in common with the enzyme identified in mammalian tissues.…”

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confidence: 99%

Identification, Expression, and Characterization of a cDNA Encoding Human Endoplasmic Reticulum Mannosidase I, the Enzyme That Catalyzes the First Mannose Trimming Step in Mammalian Asn-linked Oligosaccharide Biosynthesis

Gonzalez

Karaveg

Nairn

et al. 1999

Journal of Biological Chemistry

137

110

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We have isolated a full-length cDNA clone encoding a human ␣1,2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asnlinked oligosaccharides. This enzyme has been proposed to regulate the timing of quality control glycoprotein degradation in the endoplasmic reticulum (ER) of eukaryotic cells. Human expressed sequence tag clones were identified by sequence similarity to mammalian and yeast oligosaccharide-processing mannosidases, and the full-length coding region of the putative mannosidase homolog was isolated by a combination of 5-rapid amplification of cDNA ends and direct polymerase chain reaction from human placental cDNA. The open reading frame predicted a 663-amino acid type II transmembrane polypeptide with a short cytoplasmic tail (47 amino acids), a single transmembrane domain (22 amino acids), and a large COOH-terminal catalytic domain (594 amino acids). Northern blots detected a transcript of ϳ2.8 kilobase pairs that was ubiquitously expressed in human tissues. Expression of an epitope-tagged fulllength form of the human mannosidase homolog in normal rat kidney cells resulted in an ER pattern of localization. When a recombinant protein, consisting of protein A fused to the COOH-terminal luminal domain of the human mannosidase homolog, was expressed in COS cells, the fusion protein was found to cleave only a single ␣1,2-mannose residue from Man 9 GlcNAc 2 to produce a unique Man 8 GlcNAc 2 isomer (Man8B). The mannose cleavage reaction required divalent cations as indicated by inhibition with EDTA or EGTA and reversal of the inhibition by the addition of Ca 2؉ . The enzyme was also sensitive to inhibition by deoxymannojirimycin and kifunensine, but not swainsonine. The results on the localization, substrate specificity, and inhibitor profiles indicate that the cDNA reported here encodes an enzyme previously designated ER mannosidase I. Enzyme reactions using a combination of human ER mannosidase I and recombinant Golgi mannosidase IA indicated that that these two enzymes are complementary in their cleavage of Man 9 GlcNAc 2 oligosaccharides to Man 5 GlcNAc 2 .The maturation of Asn-linked oligosaccharides in mammalian cells is initiated in the endoplasmic reticulum (ER) 1 through the cleavage of three glucose residues and as many as two mannose residues soon after the Glc 3 Man 9 GlcNAc 2 oligosaccharide is transferred to the nascent polypeptide chain (1, 2). For glycoproteins that are destined for secretion or transport to other intracellular compartments, additional mannose trimming occurs in the Golgi complex through the action of members of a multigene family of mannosidases that remove the remaining ␣1,2-mannose residues to yield a Man 5 GlcNAc 2 structure (2). Further maturation by the action of GlcNAc transferase I, Golgi mannosidase II, the collection of branching GlcNAc transferases, and additional glycosyltransferases results in the array of complex oligosaccharides that are found on cellular and secreted glycoproteins (1).The initial stages of mannose tr...

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“…Most of the studies on mannosyltransferases concerning the synthesis of the cell wall mannoprotein of yeast have been done using Saccharomyces cerevisiae (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47), and some of the structural genes coding for yeast mannosyltransferases have been isolated. OCH1 encodes the initiating mannosyltransferase that adds the first ␣-1,6-linked mannose unit to a Man 8 GlcNAc 2 core oligosaccharide (38,42).…”

Section: Discussionmentioning

confidence: 99%

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

Suzuki

Shibata

Suzuki

et al. 1997

Journal of Biological Chemistry

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A particulate insoluble enzyme fraction containing mannosyltransferases from Candida guilliermondii IFO 10279 strain cells was obtained as the residue after extracting a 105,000 ؋ g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Man␣133(Man␣136)Man␣132Man␣132Man, in the presence of GDP-mannose and Mn 2؉ ion at pH 6.0 gave a third type of ␤-1,2 linkage-containing mannohexaose, Man␤132Man␣133(Man␣136)Man␣132Man-␣132Man, the structure of which was identified by means of a sequential NMR assignment. The results of a substrate specificity study indicated that the ␤-1,2-mannosyltransferase requires a mannobiosyl unit, Man␣13 3Man␣13, at the nonreducing terminal site. We synthesized novel oligosaccharides using substrates possessing a nonreducing terminal ␣-1,3-linked mannose unit prepared from various yeast mannans. Further incubation of the enzymatically synthesized oligosaccharide with the enzyme fraction gave the following structure, Man␤132Man␤132Man␣133(Man␣136)Man␣13 2Man␣132Man, which has been found to correspond to antigenic factor 9. Incubation of Candida albicans serotype B mannan with the enzyme fraction gave significantly transformed mannan, which contains the third type of ␤-1,2-linked mannose units.

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Glycoprotein biosynthesis in Succharomyces cerevisiae. Partial purification of the α-1,6-mannosyltransferase that initiates outer chain synthesis

Cited by 23 publications

References 0 publications

Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

Identification, Expression, and Characterization of a cDNA Encoding Human Endoplasmic Reticulum Mannosidase I, the Enzyme That Catalyzes the First Mannose Trimming Step in Mammalian Asn-linked Oligosaccharide Biosynthesis

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

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