Biosynthesis of the Core Region of Yeast Mannoproteins. Formation of a Glucosylated Dolichol-Bound Oligosaccharide Precursor, Its Transfer to Protein and Subsequent Modification

Lehle, Ludwig

doi:10.1111/j.1432-1033.1980.tb04832.x

Cited by 117 publications

(35 citation statements)

References 49 publications

(11 reference statements)

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“…It has been reported that in animal tissues (2,3), yeasts (4)(5)(6), and probably also in insects (7) and plants (8), the oligosaccharide transferred from the lipid derivative to proteins is composed of two N-acetylglucosamine, nine mannose, and three glucose residues. A report by Lehle suggests that in yeast the oligosaccharides containing two or three glucose residues are equally transferred to protein (9). However, no evidence was presented indicating that the oligosaccharides used in the assay had the same specific activity.…”

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confidence: 94%

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Parodi¹,

Allué²,

Cazzulo³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Cells of the insect parasite Crithidia fasciculata incubated with ['4C]glucose were found to possess only one lipidbound oligosaccharide with solubility in chloroform/methanol/ water mixtures and net charge similar to the charges of dolichol pyrophosphate derivatives. The saccharide moiety could be released from lipid by mild acid hydrolysis. Several enzymatic and chemical treatments of the oligosaccharide indicated that the latter had the structure Mana-+Mana---Mana--[Mana-+ Mana-+Man(al--6)]Man--GlcNAc(l1--4)GlcNAc. Two labeled oligosaccharides were liberated from proteins by a sequential treatment with a protease and endo-13-N-acetylglucosaminidase H. One ofthe protein-bound oligosaccharides had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard. These were the largest oligosaccharides obtained even after short labeling periods. It is suggested that glycosylation ofproteins in the protozoan Crithidiafasciculata does not involve glucosylated lipid-bound oligosaccharides as intermediates. It has become evident in recent years that glycosylation of asparagine residues in eukaryotic cell proteins involves dolichol pyrophosphate (DolPP)-bound oligosaccharides as intermediates (1). It has been reported that in animal tissues (2, 3), yeasts (4-6), and probably also in insects (7) and plants (8), the oligosaccharide transferred from the lipid derivative to proteins is composed of two N-acetylglucosamine, nine mannose, and three glucose residues. A report by Lehle suggests that in yeast the oligosaccharides containing two or three glucose residues are equally transferred to protein (9). However, no evidence was presented indicating that the oligosaccharides used in the assay had the same specific activity.The protein-bound oligosaccharides are then processed by loss of some of their monosaccharide constituents and addition ofother residues directly from the respective sugar nucleotides. We here report evidence suggesting that in the protozoan Crithidta fasciculata, an insect obligate parasite, the oligosaccharide transferred to protein contains two N-acetylglucosamine and seven mannose residues and that the oligosaccharide may be processed once bound to protein.MATERIALS AND METHODS Materials.[14C]Glucose (284 Ci/mol; 1 Ci = 3.7 X 1010 becquerels) was from New England Nuclear. Jack bean a-mannosidase type III and Streptomyces griseus protease type VI were purchased from Sigma and endo-,B3N-acetylglucosaminidase H (endo H), from Miles. Isolation of Lipid-Bound Oligosaccharides. C. fasciculata cells (Anopheles isolate ATCC 11745) were grown in the medium described by Bacchi et aL (10) without agar at 28°C. Six hundred milliliters of culture containing about 2 g of cells (logarithmic phase) were cooled on ice and centrifuged at 2500 X g for 10 min at 4°C. The cell pellet was resuspended in 30 ml of ice-cold minimal Eagle's medium without glucose but containing 5...

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confidence: 94%

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Parodi¹,

Allué²,

Cazzulo³

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…Besides plants, UDP-glucose:sterol glucosyltransferase activity was determined by in vitro enzyme assays in Saccharomyces cerevisiae (45)(46)(47)(48), Candida bogoriensis (49), other fungi (50), and Physarum polycephalum (51,11). Although the lipid composition of S. cerevisiae was studied in detail during the last decades, there are only rare reports on the actual isolation of sterol glucoside (SG) 1 from this yeast (8,9).…”

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confidence: 99%

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Warnecke¹,

Erdmann²,

Fahl³

et al. 1999

Journal of Biological Chemistry

122

135

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Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose: sterol glucosyltransferase (EC 2.4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 ‫؍‬ YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-␤-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-␤-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.Sterol glycosides are widespread membrane lipids, occurring in all plants, several algae (1-3), some fungi (4 -9), slime molds (10 -12), Dictyostelium (13), a few bacteria (14 -19), and even animals (20 -23). The knowledge base for sterol glycosides is rather limited compared with free sterols and sterol esters, where the synthesis, transport, and functions have been studied extensively in animals (24 -29), plants, (30 -35), and yeast (36 -40). The basis for studies on the functions of sterol glycosides is the assumption that free sterols and sterol glycosides differ physiologically. It is obvious that the attachment of a glycosyl moiety to the sterol backbone alters the physical properties of this lipid. As a result, there are changes in the properties of membranes containing different proportions of free sterols and sterol glycosides. Such changes have been studied with artificial membranes in terms of membrane fluidity, permeability, hydration, and phase behavior (41)(42)(43)(44). However, we still do not know how free sterols and sterol glycosides differ physiologically in biological membranes and why many eukaryotic organisms synthesize sterol glycosides. One of the main reasons for our limited knowledge in this field is the lack of a genetic approach. The objective of the present work was the isolation and characterization of sterol glycosyltransferase genes from eukaryotic organisms. We expect that genetic manipulation of these genes will facilitate the elucidation of sterol glycoside functions in these organisms.The predominating sugar moiety in sterol glycosides is glucose. Besides plants...

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“…In contrast, mutations in genes that accumulate lipid-linked core oligosaccharides in which five or more mannose residues have been attached or that are defective in the later processing reactions, including ALG3 (7,14), ALG5 (7,15), ALG6 (7,16), ALG8 (17), ALG9 (18), and ALG10 (19), show little or no growth phenotype. Truncated oligosaccharides are transferred by OST to proteins in vivo (11,20) and in vitro (21)(22)(23), though the full-length, glucosylated Glc 3 Man 9 GlcNAc 2 is the preferred substrate (21,24). Although the transfer of truncated oligosac-charide structures is tolerated, mutations that block OST activity are lethal (25), demonstrating that N-linked glycosylation is an essential function.…”

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The Yeast ALG11 Gene Specifies Addition of the Terminal α1,2-Man to the Man5GlcNAc2-PP-dolicholN-Glycosylation Intermediate Formed on the Cytosolic Side of the Endoplasmic Reticulum

Cipollo

Trimble

Hee

et al. 2001

Journal of Biological Chemistry

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The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). Here, we describe alg11, a new yeast glycosylation mutant that is defective in the last step of the synthesis of the Man 5 GlcNAc 2 -PP-dolichol core oligosaccharide on the cytosolic face of the ER. A deletion of the ALG11 gene leads to poor growth and temperature-sensitive lethality. In an alg11 lesion, both Man 3 GlcNAc 2 -PP-dolichol and Man 4 GlcNAc 2 -PP-dolichol are translocated into the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm ␣1,2-linked Man residues. The former are elongated to mannan, whereas the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1-kDa membrane protein that by indirect immunofluorescence resides in the ER. The Alg11 protein is highly conserved, with homologs in fission yeast, worms, flies, and plants. In addition to these Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. All of these Alg11-related proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif.

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Biosynthesis of the Core Region of Yeast Mannoproteins. Formation of a Glucosylated Dolichol-Bound Oligosaccharide Precursor, Its Transfer to Protein and Subsequent Modification

Cited by 117 publications

References 49 publications

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

The Yeast ALG11 Gene Specifies Addition of the Terminal α1,2-Man to the Man5GlcNAc2-PP-dolicholN-Glycosylation Intermediate Formed on the Cytosolic Side of the Endoplasmic Reticulum

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