On the biological role of glycoproteins

Eylar, E.H.

doi:10.1016/0022-5193(66)90179-2

Cited by 419 publications

(77 citation statements)

References 117 publications

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“…It was proposed by Eylar that membrane glycoproteins are glycosylated by different transferases than are the serum proteins (36). Recent experiments indicate that, in contrast to serum proteins, membrane glycoproteins newly mannosylated with GDPmannose, with synthetic dolichol phosphate-mannose, or with dolichol pyrophosphate-oligosaccharide-mannose are localized on the outer surface of the rough and smooth microsomal membranes and can be removed by protease and phospholipase treatment of intact vesicles (37).…”

Section: Discussionmentioning

confidence: 99%

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes.

Elhammer¹,

Svensson²,

Autuori³

et al. 1975

The Journal of Cell Biology

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The presence in the Golgi fraction ofglycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [l'C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS-gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer being most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components.

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Section: Discussionmentioning

confidence: 99%

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes.

Elhammer¹,

Svensson²,

Autuori³

et al. 1975

The Journal of Cell Biology

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“…From the composition and number of the carbohydrate chains in carboxypeptidase Y, the average chain size appears to be -13 mannoses (5)-i.e., the size ofa core unit. From these facts, one might postulate that the externalization of a mannoprotein is associated with or dependent on the addition of the outer chain and that intracellular mannoproteins lack a signal that specifies outer-chain addition (6). Although it is an attractive idea, a strong argument against this hypothesis is our recent isolation of S. cerevisiae mutants that make and secrete mannoproteins, including invertase, that appear to possess only the core oligosaccharide units (7).…”

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confidence: 99%

Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated.

Hashimoto¹,

Cohen²,

Zhang³

et al. 1981

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

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Carboxypeptidase Y, a vacuolar enzyme from Saccharomyces cerevisiae, was digested with endo-fi-N-acetyl-Dglucosaminidase H to release the four oligosaccharide chains that are linked to asparagine in the glycoprotein. The oligosaccharides were fractionated into a neutral and acidic component, and the latter proved to phosphorylated. From its gel filtration pattern, the neutral fraction was shown to be a mixture of at least four homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with eight mannoses and one N-acetylglucosamine [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345-4358]. The yeast oligosaccharide has one additional mannose unit in an al-+3 or al-*6 linkage, whereas the larger homologs appear to have two, three, and four more mannose units. One phosphorylated oligosaccharide with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and then subjected to mild acid hydrolysis. This released mannose and mannobiose that were glycosidically linked to the phosphate group, whereas complete acid hydrolysis yielded Dmannose 6-phosphate. The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with a-mannosidase, and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2. These results suggest that the mannosidase-resistant phosphorylated oligosaccharide has the structure Man--P--6aMan--aMan--6fBMan--s4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead ofmannose. A second phosphorylated oligosaccharide with a mannose/ phosphate ratio of 6.5 probably contains two phosphodiester groups, but its structure has not been investigated in detail.The yeast Saccharomyces cerevisiae has extracellular periplasmic glycoproteins (mannoproteins), such as invertase, that contain =50% mannose (1) and intracellular glycoproteins, such as carboxypeptidase Y, that contain 15% mannose (2) and are similar in some respects to mammalian glycoproteins (3). The asparagine-linked carbohydrate chains of the external invertase contain 100-150 mannose units that are differentiated into a core unit of 11 mannoses and an outer chain of 90-140 mannoses (4). From the composition and number of the carbohydrate chains in carboxypeptidase Y, the average chain size appears to be -13 mannoses (5)-i.e., the size ofa core unit. From these facts, one might postulate that the externalization of a mannoprotein is associated with or dependent on the addition of the outer chain and that intracellular mannoproteins lack a signal that specifies outer-chain addition (6). Although it is an attractive idea, a strong argument against this hypothesis is our recent isolation of S. cerevisiae mutants that make and secrete mannoproteins, including invertase, that appear to possess only the core oligosaccharide units (7).To determine whether the carbohydrate chains have any role in specifying the compartmentation of glycoproteins in yeast, ...

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“…The oligosaccharides on glycoproteins appear to play important roles in protein secretion (1), specific recognition of serum glycoproteins (2), protection of glycoproteins against proteolytic degradation (3), the insertion or proper orientation of glycoproteins in the plasma membrane (4,5), and cell adhesion and morphology (4)(5)(6). Because the transport of metabolites across cell membranes is thought to be mediated via glycoprotein carriers (7), we examined whether inhibiting protein glycosylation would alter a number of plasma membrane enzymatic and nutrient transport processes.…”

mentioning

confidence: 99%

Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.

Olden¹,

Pratt²,

Jaworski³

et al. 1979

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

168

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Using tunicamycin, we have investigated the role of glycoproteins in membrane transport. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins. Inhibition of protein glycosylation in chick embryo Iibroblasts by tunicamycin or other inhibitors of glycosylation resulted in defective transport of glucose, uridine, and amino acid analogs (a-aminoisobutyrate and cycloleucine). The defect in glucose transport is accompanied by decreased glucose metabolism, as determined by rates of CO2 and lactate production. In contrast, tunicamycin treatment did not affect other membrane-associated processes, such as secretion of fibronectin and procollagen, uptake of glucose by passive diffusion, Na+/K+ ATPase and adenylate cyclase activities, or stimulation of adenylate cyclase by prostaglandin and cholera toxin. Two glucose/glycosylation-regulated membrane proteins with apparent subunit molecular weights of 95,000 and 75,000 were induced by tunicamycin treatment. Our results indicate that glycoprotein glycosylation is required for membrane transport.The oligosaccharides on glycoproteins appear to play important roles in protein secretion (1), specific recognition of serum glycoproteins (2), protection of glycoproteins against proteolytic degradation (3), the insertion or proper orientation of glycoproteins in the plasma membrane (4, 5), and cell adhesion and morphology (4-6). Because the transport of metabolites across cell membranes is thought to be mediated via glycoprotein carriers (7), we examined whether inhibiting protein glycosylation would alter a number of plasma membrane enzymatic and nutrient transport processes.For these studies we have used tunicamycin, an inhibitor of the synthesis of N-acetylglucosaminyl pyrophosphoryl polyisoprenol (8-10). Because this reaction is required for the synthesis of the core sequence of N-glycosidically linked oligosaccharides, tunicamycin treatment results in synthesis of glycoproteins deficient in asparagine-linked oligosaccharides (10). -We present evidence that inhibition of protein glycosylation results in defective membrane transport and glucose metabolism in chick embryo fibroblasts (CEF). We demonstrate that the carbohydrate moieties of glycoproteins are required for the normal transport of metabolites across the plasma membrane and their subsequent metabolism. This effect might occur directly on carrier function or indirectly by inhibiting insertion of transport molecules into the membrane or by increasing their degradation rate (3). MATERIALS AND METHODSCell Culture. Secondary or tertiary CEF were grown for 24 hr in the presence or absence of tunicamycin (0.05 ,4g/ml or 50 nM) in 35-mm plastic tissue culture dishes (Costar) as described (11).Transport Assays. For uptake experiments, the conditioned medium was aspirated and fresh medium containing 0.05 ,g of tunicamycin per ml and the radiolabeled transport substrate was added. After incubation at 23°C for the ...

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On the biological role of glycoproteins

Cited by 419 publications

References 117 publications

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes.

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes.

Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated.

Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.

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