The Mannosylation of Dolichol‐Diphosphate Oligosaccharides in Relation to the Formation of Oligosaccharides and Glycoproteins in Pig‐Liver Endoplasmic Reticulum

Oliver, G.J.A.; Harrison, Jeffrey K.; Hemming, Frank W.

doi:10.1111/j.1432-1033.1975.tb02367.x

Cited by 41 publications

(14 citation statements)

References 8 publications

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“…4) (Table I) produced by the particulate fractions from GDP-mannose or UDP-GlcNAc supports this concept. Lipid-linked oligosaccharides have been implicated as precursors of the glycosyl component of glycoproteins (14,20,21). The time course kinetic data ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L

Beevers¹,

Mense²

1977

Plant Physiol.

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The reserve globulins, legumin and vicilin, in the pea cotyledon are glycoproteins. Acid hydrolysis of these glycoproteins releases the amino-sugar glucosamine and neutral sugars, principally mannose (2). Similar glycosyl components occur in the glycoproteins from animal tissues (16,27). Studies with extracts from animal tissues have established that the glycosyl components in the glycoproteins are derived originally from nucleotide sugars. The glycosyl components from the nucleotide sugars are transferred to lipid (polyisoprenol) intermediates to form lipidlinked monosaccharides which serve as precursors for the formation of lipid-linked oligosaccharides. This oligosaccharidelipid serves as the carrier of the oligosaccharide chain which combines with the polypeptide to produce glycoproteins (14,17). Similar lipid intermediates have been implicated in the biosynthesis of glycoproteins in shoots of mung bean seedlings (1,7,11), cotton fibers (6), and cotyledons of Phaseolus vulgaris (5).As part of our continuing studies of protein synthesis in the Beckman L2-65B centrifuge. The supernatant from this centrifugation was discarded and the surface of the remaining pellet was washed with reaction medium. Pellets were suspended in 3 ml of reaction mixture which, unless stated otherwise, consisted of 50 mm tris-HCl (pH 7) containing 10 mm KCI, 10 mm MgCI2, and 5 mm 2-mercaptoethanol. This suspension of cellular particulates was used as the enzyme source. Corporation). After a 60-min incubation at 37 C the reaction was terminated by the addition of 0.7 ml of H20 and 2.5 ml of chloroform-methanol (2:1, v/v). After thorough mixing the phases were separated by centrifugation and the lower organic phase recovered. The upper aqueous layer and interphase were reextracted with 2.5 ml of 2:1 chloroform-methanol; the organic phase was collected and pooled with the previous extraction.These pooled 2:1 chloroform-methanol fractions were then washed twice with 2.5 ml of chloroform-methanol-water (3:48:47). At each extraction the upper phase was discarded.The recovered washed 2:1 chloroform-methanol fraction was placed in a scintillation vial, evaporated to dryness, and radioactivity determined upon the addition of scintillation fluid (8 g 2,5-diphenyloxazole [PPOt in 2 liters of toluene, 1 liter of Triton X-100).The aqueous phase and interphase remaining after chloro-

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Section: Resultsmentioning

confidence: 99%

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L

Beevers¹,

Mense²

1977

Plant Physiol.

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“…Earlier repots have indicated a preferential stimulatory effect by Mg24 and Mn2" on the transfer of radioactivity from GDP-[14CJMan2 to lipid intermediates and glycoproteins (4,12,13,15,18,21). In contrast, very little information is available on the divalent cation effect on UDP-[14C]-GlcNAc transferase activities.…”

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confidence: 99%

“…Earlier repots have indicated a preferential stimulatory effect by Mg24 and Mn2" on the transfer of radioactivity from GDP-[14CJMan2 to lipid intermediates and glycoproteins (4,12,13,15,18,21 nucleoside diphosphate sugars are given below. Incubation was at 37 C for 60 min with the reaction terminated by the addition of 0.7 ml of H20 and 2.5 ml of chloroform-methanol (2:1, v/v).…”

Section: Introductionmentioning

confidence: 99%

Membrane-associated Glycosyl Transferases in Cotyledons of Pisum sativum

Nagahashi¹,

Mense²,

Beevers³

1978

Plant Physiol.

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In crude particulate fractions isolated from pea (Pisum sativum) cotyledons, the transfer of radioactivity from GDP-I14Cimannose to glycolipid appears to be preferentially stimulated by Mn2' while the transfer to lipidfree residue is enhanced by Mg2'. In contrast, the transfer of radioactivity from UDP-N-acetyl-I14Clglucosamine to glycolipid shows preferential stimulation by Mg2" while the transfer to lipid-free residue prefers Mn2". These results are accounted for by the differential stimulation by Mg24 and Mn24 of glycosyl transferases associated with subcellular membranes which were separated by isopycnic sucrose density centrifugation.Particulate fractions isolated from developing pea cotyledons can transfer labeled sugar from nucleoside diphosphate sugars to lipid components and to components which remain in the residue following lipid extraction (2, 17). Earlier repots have indicated a preferential stimulatory effect by Mg24 and Mn2" on the transfer of radioactivity from GDP-[14CJMan2 to lipid intermediates and glycoproteins (4,12,13,15,18,21 nucleoside diphosphate sugars are given below. Incubation was at 37 C for 60 min with the reaction terminated by the addition of 0.7 ml of H20 and 2.5 ml of chloroform-methanol (2:1, v/v). Further lipid extraction and analysis of the lipid-free residue were described previously (2).Sucrose Gradients. Membrane fractions used in linear sucrose density gradients were prepared by sedimenting particulate material from 250 to 13,000g and 13,000 to 40,000g (17). The crude pellets were either washed in GM (0.5 M sucrose + 5 mM 2-mercaptoethanol + 30 mm Tris-MES [pH 7.5]) or GM + 5 mM EDTA and pelleted at the initial force (17). Crude pellets were overlaid on identical gradients (15-60%/o sucrose, w/w) and centrifuged to equilibrium at 81,000g for 15 hr. Preparation of sucrose gradients and fractionation were described elsewhere (17).Enzyme Assays for Isolated Membrane Fractions. Inosine diphosphatase (IDPase) activity of pH 7.5 was determined as described by Leonard and VanDerWoude (14). NADH-Cyt c reductase activity was assayed spectrophotometrically by following the reduction of Cyt c at 550 nm (10).Glycosyl transferase activities were determined either by measuring incorporation of label from nucleoside diphosphate sugar into trichloroacetic acid-insoluble material or by assaying incorporation into lipid intermediates and lipid-free residues using an extraction procedure described earlier (2). Although we use the singular form of GDP-Man transferase and UDP-GlcNAc transferase when we speak of trichloroacetic acid-precipitable material, it should be kept in mind that these precipitates represent an aggregate of glycosyl transferase activities. UDP-N -acetyl-[U-14C]glucosamine (300 mCi/mmol, 10 ,uCi/4001,) and GDP-[U-'4C]mannose (179 mCi/mmol, 50 ,Ci/2 ml) were used as substrates to measure activities of UDP-GlcNAc and GDP-Man transferases. Membrane fractions (0.3 ml) were incubated with 0

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“…In recent years, it has been found that, during the N-glycosylation of proteins in the ER, a significant number of unconjugated, free oligosaccharides (free OSs) are formed (7)(8)(9). The occurrence of free OSs has been reported in a wide variety of cells (10)(11)(12)(13)(14), and their sequential processing event of free OSs in mammalian cells has also been well characterized (15)(16)(17)(18)(19). When the free OSs are formed in the lumen of the ER, they are transported out of the ER to the cytosol (15).…”

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confidence: 99%

Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

Suzuki

Yano

Sugimoto

et al. 2002

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

127

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Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-␤-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein.The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

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The Mannosylation of Dolichol‐Diphosphate Oligosaccharides in Relation to the Formation of Oligosaccharides and Glycoproteins in Pig‐Liver Endoplasmic Reticulum

Cited by 41 publications

References 8 publications

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L

Membrane-associated Glycosyl Transferases in Cotyledons of Pisum sativum

Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

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