Incorporation of carbohydrates into endogenous acceptors of liver microsomal fractions

Tetas, Montserrat; Chao, Helen M.; Molnár, J.

doi:10.1016/0003-9861(70)90292-4

Cited by 83 publications

(11 citation statements)

References 23 publications

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“…Evidence for such reaction sequences in mammalian systems has been obtained for mannose [1,3--5,191, glucose [2,20], N-acetylglucosamine [3,19], galactose [3,21] c) Sugar nucleotides as well as polyprenol-linked sugars serve as immediate precursors for a stepwise formation of the protein-bound oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

The Role of Dolicholmonophosphate in Glycoprotein Biosynthesis in Saccharomyces cerevisiae

Sharma

Babczinski

Lehle

et al. 1974

European Journal of Biochemistry

160

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1. In a crude particulate fraction from yeast mannose is transferred from GDP-mannose to an endogenous "lipid" fraction, the monophosphates of dolichol-14 to dolichol-18. The same membrane fraction also catalyzes the mannosylation of glycoproteins. More than 80 of the total radioactivity in the glycoprotein fraction obtained from GDP-[14C]mannose is released by /?-elimination. The small-sized radioactive products of /?-elimination are mannose, mannobiose and mannotriose.2. Ageing of the particulate fraction leads to a drastic loss of mannosyl transfer activity from GDP-[14C]mannose to dolichol monophosphate. To the same extent the amount of [14C]mannose obtained after /?-elimination of the glycoproteins decreases. The amount of radioactive mannobiose and mannotriose is, however, much less affected.3. With decreasing GDP-mannose concentrations the amount of [14C]mannose obtained after /&elimination increases as compared to the amount of radioactive mannobiose and mannotriose. The Km-value for the incorporation of mannosyl groups directly linked to serine and/or threonine, therefore, is lower than the Km-value for the transfer of subsequent mannosyl residues.4. 6. The results are consistent with the assumption that dolichol monophosphate is involved in the mannosylation of a specific position in yeast glycoproteins, i.e. in the formation of mannosyl linkages to serine and/or threonine. The subsequent mannosylation proceeds directly from GDP-mannose in a reaction obligatorily requiring Mn2+.7. After /?-elimination of the methanol-insoluble material mannosylated in the presence of dolichol-monophosphate [14C]mannose the residual insoluble radioactivity is to a large extent transformed into dialyzable material by pronase. Mannose, therefore, is transferred from dolicholmonophosphate mannose also to glycoprotein positions not involving OH-groups of serine or threonine.Glycosylated lipids play a role in the biosynthesis of glycoproteins in animals [l -51, fungi [6,7] and plants [S]. The lipophilic component in all these cases is probably dolichol monophosphate [2,4,9,10].In Succhuvomyces cerevisiue the lipophilic mannosyl acceptor has been shown by mass spectroscopy to consist of the monophosphates of the whole family of dolichols with 14 to 18 isoprene units [lo]. From -~ his 60th birthday. mannosylated dolichol monophosphates the mannosyl group is transferred by yeast membranes to glycoproteins. Preliminary evidence has been presented [7] threonine, only the mannosyl residue directly linked to Dedicated to Professor Dr 0. Hoffmann-Ostenhof on Abbreviations. Dolichol-P, dolichol monophosphate; dolichol-P-Man, dolichol-monophosphate-mannose ; GDPthat within oligomannose chains linked to serine/ Man, guanosine-diphosphate-mannose.Eur. J. Biochem. 46 (1974)

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Section: Discussionmentioning

confidence: 99%

The Role of Dolicholmonophosphate in Glycoprotein Biosynthesis in Saccharomyces cerevisiae

Sharma

Babczinski

Lehle

et al. 1974

European Journal of Biochemistry

160

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“…The majority of the incorporation of sugars into the microsomal membrane goes into glycolipids (Table VI), part of which may be the lipid-sugar acceptor proposed by Tetas et al . since it can be hydrolyzed in 0 .1 M HCl at 80°C for 20 min (23) . There is, however, a nonlipid component in the membrane which is radioactive and which is probably protein (Table V) .…”

Section: Discussionmentioning

confidence: 99%

“…Smaller amounts were removed with alcohol-ether and ether . Chloroform : methanol (2 :1, v/v) has been shown to remove the lipid-sugar from TCA precipitates after a 15-min treatment at room temperature (23) . Repeated treatment with this solvent mixture removed 74% of the labeling with N-acetylglucosamine, 43% of the labeled mannose, 41 % of the galactose-and only 15 % of the leucine-labeled material (Table V) .…”

Section: Localization Of the Radioactive Proteins On Microsomal Subfrmentioning

confidence: 99%

The Secretory Pathways of Rat Serum Glycoproteins and Albumin

Redman

Cherian

1972

The Journal of Cell Biology

147

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These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein . N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membraneattached polysomes and not into proteins from free polysomes . Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes .Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae . Albumin was mostly recovered (98%0 ) in the cisternae, with negligible amounts in the membranes . To determine whether the radioactive sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the "C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins . The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae .

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“…In experiments with GDP-Man as donor, lipid-bound mannose has been detected (10)(11)(12). Its formation was enhanced by addition of dolichol-P (6,13), and the structure of the product has been unambiguously proved to be dolichol-PMIan by Hemming's group (14).…”

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confidence: 83%

Formation of Lipid-Bound Oligosaccharides Containing Mannose. Their Role in Glycoprotein Synthesis

Behrens¹,

Carminatti²,

Staneloni³

et al. 1973

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

145

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Incubation of GDP-['4C]mannose with liver microsomes and magnesium ions led to the formation of dolichol monophosphate mannose and of other acidlabile compounds that contain oligosaccharides. Formation of these compounds was enhanced by addition of an acceptor lipid in the same fractions of DEAE-cellulose chromatography where bound dolichol is found. Alkaline treatment of the oligosaccharides, obtained by acid methanolysis, followed by paper electrophoresis, gave rise to the formation of two positively charged substances believed to be formed by deacetylation of hexosamine residues. Incubation of the oligosaccharide-containing compounds with liver microsomes and manganese ions resulted in a transfer to endogenous protein. The role of dolichol derivatives in glycoprotein synthesis is discussed.Incubation of liver microsomes with UDP-Glc and dolichol-P leads to the formation of dolichol-P-Glc and a substance believed to be dolichol-PP-oligosaccharide (1, 2). This substance, which has been referred to as glucosylated endogenous acceptor, can also be obtained by direct transfer of glucose from dolichol-P-Glc and acts as donor in oligosaccharide transfer to protein (3).The structure of the oligosaccharide portion of glucosylated endogenous acceptor is not known, but studies with the substance labeled in the glucose moiety have given some information. Measurements of molecular weight by ion exclusion gave values of 3500, and the rate of migration on paper chromatography corresponds to that of a maltooligosaccharide of about 17 units (4). The oligosaccharide, therefore, has around 20 monosaccharide units.When the oligosaccharide prepared by acid methanolysis was subjected to electrophoresis, it behaved as an uncharged substance and on treatment with alkali, it gave rise to two positively charged substances (5). These became neutral after treatment with acetic anhydride under conditions that led to N-acetylation. From these facts it was tentatively concluded that the oligosaccharide of glucosylated endogenous acceptor contains two hexosamine residues.In experiments where UDP-['4C]GlcNAc was used as donor and dolichol-P as acceptor, it was found that substances that appear to be dolichol-PP-GlcNAc (6) and dolichol-PP-N,N'-diacetylchitobiose are formed (7). The transfer of both the sugar residue and phosphate from UDP-GlcNAc to a lipid had been described by Molnar et al. (8).These findings can be related to the fact that two N-acetylglucosamine residues are present in the inner portion of the oligosaccharide of many glycoproteins (9). In these, an N,N'-diacetylchitobiose residue is linked to the amide N of asparagine and chains of mannose residues are linked to it.In experiments with GDP-Man as donor, lipid-bound mannose has been detected (10-12). Its formation was enhanced by addition of dolichol-P (6, 13), and the structure of the product has been unambiguously proved to be dolichol-PMIan by Hemming's group (14).In previous experiments it was found that, besides dolichol-P-Man, other substances are formed which,...

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Incorporation of carbohydrates into endogenous acceptors of liver microsomal fractions

Cited by 83 publications

References 23 publications

The Role of Dolicholmonophosphate in Glycoprotein Biosynthesis in Saccharomyces cerevisiae

The Role of Dolicholmonophosphate in Glycoprotein Biosynthesis in Saccharomyces cerevisiae

The Secretory Pathways of Rat Serum Glycoproteins and Albumin

Formation of Lipid-Bound Oligosaccharides Containing Mannose. Their Role in Glycoprotein Synthesis

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