Synthesis Of Membrane Glycoproteins Research Articles

Von Willebrand Disease in Children with Glycogen Storage Disease Type Ia.

Glycogen storage disease, Type Ia (GSD-Ia) is an autosomal recessive disease, characterized by hepatomegaly, hypoglycemia, hyperuricemia, and bleeding diathesis. Easy bruising and epistaxis are common and are associated with a prolonged bleeding time as a result of impaired platelet aggregation and adhesion. Two of four of our GSD-Ia patients were evaluated because of bleeding diathesis and both were diagnosed as von Willebrand's disease (vWd) with normal platelet count, prothrombin time and activated partial thromboplastin time.Case 1: Six year old boy diagnosed as GSD-Ia when he was 3 month of age and bleeding diathesis was first recognized 5th year of his life. His bleeding time was prolonged (15 minutes). Measurement of serum factor VIII-related antigen (vWf-Ag) revealed a decrease (% 31) and ristocetin cofactor activity was abnormal (% 37).Case 2: Nine year old girl diagnosed as GSD-Ia when he was 6 month of age and bleeding diathesis was first recognized 4th year of his life. His bleeding time was prolonged (3.5 minutes). Measurement of serum factor VIII-related antigen (vWf-Ag) revealed a decrease (% 17) and ristocetin cofactor activity was abnormal (% 5.7).Frequent diagnosis of vWd among our GSD-Ia patients might suggest alterations in membrane glycoprotein synthesis. There is no definitive explanation addresses how these alterations actually cause defective platelet aggregation. Although, the presence of association between these two separate diseases might be coincidental, in literature, diagnosis of vWd among GSD-Ia patients had been reported in only one paper. We conclude that GSD-Ia patients may have a metabolically acquired form of vWd as well as an acquired intrinsic platelet defect. Further molecular studies may be necessary to clarify the etiopathogenesis of this association.

COMBINED IMPACT OF MUCOSAL DAMAGE AND OF CYSTIC FIBROSIS ON THE SMALL INTESTINAL BRUSH BORDER ENZYME ACTIVITIES

In 61 cystic fibrosis (CF) patients, the small intestinal mucosa was studied at the time of diagnosis before starting therapy. In 19 out of 61 patients, partial villous atrophy on light microscopy and shortened villi on stereomicroscopic examination were seen. On the biopsy specimens, maltase, sucrase, lactase and alkaline phosphatase activities were studied. Comparison of the enzymatic activities in CF patients having damaged mucosa and a group of patients having similar mucosal lesions of unspecified origin (UTID), reveals a significantly more pronounced decrease of the alkaline phosphatase activity (p<0.005) in the CF patients. This is in agreement with previous reported results in CF patients with normal mucosa.The abnormal mucosal findings could be due to the decreased neutralization of the gastric content delivered into the duodenum, the early inflammatory reaction present in the CF mucosa and/or to the impaired synthesis of membrane glycoproteins and enzymes secondary to the CFTR mutation.

In vitro effects of mycophenolic acid on the nucleotide pool and on the expression of adhesion molecules of human umbilical vein endothelial cells

The immunosuppressive drug mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid (MPA) selectively inhibit inosine 5′-monophosphate dehydrogenase (IMPDH), and therefore interfere with cellular guanine nucleotide biosynthesis. IMPDH is additionally involved in the synthesis of membrane glycoproteins, some of which are adhesion receptors known to play an active part in the regulation of cell–cell contacts, which are crucial in the process of recruitment and transendothelial infiltration of activated leucocytes in the transplanted organ. As a consequence, MPA leads to a reduction of cellular infiltrates in the course of transplant rejection. In the present study, the effects of MPA on human umbilical vein endothelial cells (HUVEC) are investigated at both molecular and cellular levels. In our experiments, HUVECs are treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) in order to mimic activation occurring at a rejection crisis. The dose-dependent influence of concomitant incubation with MPA (5–20 μmol/l; 48 h, 37°C, 5% CO 2) on their intracellular nucleotide profile is observed by determining the concentrations of purine and pyrimidine nucleotides, using a HPLC method based on solvent generated ion-exchange. The possibility of synergistic effects is investigated by incubating endothelial cells with mixtures of three different immunosuppressants (mycophenolic acid; cyclosporin A, 100 ng/ml; prednisolone, 1 μmol/l)—a combination commonly used after transplantation—varying the amount of MPA (5–20 μmol/l). Stimulation with TNFα does not significantly modulate the intracellular levels of nucleotides quantitated. In the presence of MPA concentrations of at least 5 μmol/l, GTP levels (68±12%) are significantly decreased compared to controls (100%). At a concentration of 20 μmol/l MPA, the GTP amount is reduced to 58±7%. In contrast to these observations, the levels of UDP and UTP are increasing significantly under coincubation with MPA concentrations greater than 5 μmol/l. At 20 μmol/l MPA, UDP and UTP are increased to 147±19% and 114±11%, respectively. All other nucleotides (CTP, ADP, ATP) reveal no significant alterations in their intracellular concentrations under the conditions applied. Incubation of TNFα-treated HUVEC monolayers, with a mixture of three immunosuppressive drugs varying the amount of MPA, show no significant differences compared with the data observed after incubation with MPA alone. In addition, the influence of MPA (10 μmol/l) on a cellular level is observed by measuring the cell surface expression of adhesion molecules on cytokine-stimulated HUVECs, using TNFα (10 ng/ml), interferon-γ (100 ng/ml), interleukin-1β (10 ng/ml) and interleukin-8 (20 ng/ml). Expression of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) was assessed by flow cytometry. Activation of endothelial cell monolayers with TNFα significantly increases the mean fluorescence intensity of VCAM-1 (361±14%) and ICAM-1 (429±47%) surface expression, compared to controls, and additionally induces E-selectin expression (2919±134%). The same tendencies, but in a lesser degree, are observed under stimulation of cells with either IFNγ or IL-1β. Incubation with a combination of TNFα and MPA leads to a significant reduction in VCAM-1 (329±13%) and E-selectin (2613±167%) expression, compared to the values obtained for HUVEC incubated with the cytokine alone. Treatment of the cells with IL-1β/MPA also reduces the expression of VCAM-1 to a level significantly lower than the level observed after stimulation with IL-1β. Incubation with MPA alone reveals no significant modulation in the expression of all surface molecules tested compared to the values of unstimulated HUVECs. The experiments show that the immunosuppressive action of MPA not only inhibits lymphocyte proliferation but also decreases the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process.

Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event.

A polyvalent antiserum which precipitates the native, folded, but not the denatured molecular forms of pig intestinal aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) was used to determine the kinetics of polypeptide folding of the two newly synthesized brush border enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a defective cotranslational high-mannose glycosylation, increased the half-time of polypeptide folding to about 12 min for aminopeptidase N as well as for sucrase-isomaltase. Short-pulse experiments suggested that fructose exerts its effect by slowing the rate of glycosylation, making this partially a posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding.

Morphological and functional changes in the enterocyte induced by fructose

In the presence of 10-50 mM-fructose, enterocytes of organ-cultured pig intestinal-mucosal explants fail to glycosylate correctly their newly synthesized microvillar enzymes, and instead degrade them [Danielsen (1989) J. Biol. Chem. 264, 13726-13729]. In the present work, this degradation was shown to occur extremely rapidly as the microvillar enzyme aminopeptidase N (EC 3.4.11.2) was hardly detectable after a 10 min pulse with [35S]methionine. The abnormal biosynthesis of membrane glycoproteins affected both the morphology and the function of the Golgi complex as well as the microvillar membrane. Thus the stack of Golgi cisternae was condensed and devoid of dilated rims, and the secretion of a non-glycosylated protein, apolipoprotein A-1, was almost completely blocked in the presence of fructose, showing that transport through the secretory pathway is disturbed even for proteins unaffected by the defective glycosylation. The microvilli of the brush-border membrane were markedly shortened (by about 40%) in the presence of fructose, and incorporation of newly made actin into the microvillar cytoskeleton was similarly decreased. By affecting membrane glycoprotein synthesis, the common dietary sugar fructose thus profoundly perturbs the exocytic membrane traffic in the enterocyte.

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins.

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.

The anticarcinogenic and tumor growth inhibitory activities of lymphotoxin are associated with altered membrane glycoprotein synthesis

Membrane glycoprotein synthesis was studied in non-tumorigenic and tumor cells treated with the lymphokine hormone, lymphotoxin, to define the biochemical mechanisms by which lymphotoxin prevents carcinogenesis and inhibits growth of tumor cells. Syrian hamster lymphotoxin increased the synthesis of high molecular weight (50,000–250,000) membrane glycoproteins in non-tumorigenic secondary passage NIH N Syrian hamster embryo fibroblasts and caused a decrease in the synthesis of high molecular weight glycoproteins in benzo[ a]pyrene-induced hamster tumor cells. Increased glycoprotein synthesis varied directly with lymphotoxin concentration and duration of treatment and occurred contemporaneously with lymphotoxin-initiated prevention of carcinogen-induced morphologic transformation. These lymphotoxin-stimulated fluctuations in membrane glycoprotein synthesis are evidence of specific biochemical target cell changes associated with the anticarcinogenic and tumor-inhibitory actions of this immunologic hormone.

GERM CELL‐DEPENDENT MEMBRANE GLYCOPROTEIN SYNTHESIS ON SERTOLT CELLS CULTURED IN VITRO*

Annals of the New York Academy of SciencesVolume 383, Issue 1 p. 449-450 GERM CELL-DEPENDENT MEMBRANE GLYCOPROTEIN SYNTHESIS ON SERTOLT CELLS CULTURED IN VITRO* M. Galdieri, M. Galdieri Institute of Histology and General Embryology University of Rome Rome, ItalySearch for more papers by this authorB. Zani, B. Zani Institute of Histology and General Embryology University of Rome Rome, ItalySearch for more papers by this authorL. Monaco, L. Monaco Institute of Histology and General Embryology University of Rome Rome, ItalySearch for more papers by this author M. Galdieri, M. Galdieri Institute of Histology and General Embryology University of Rome Rome, ItalySearch for more papers by this authorB. Zani, B. Zani Institute of Histology and General Embryology University of Rome Rome, ItalySearch for more papers by this authorL. Monaco, L. Monaco Institute of Histology and General Embryology University of Rome Rome, ItalySearch for more papers by this author First published: June 1982 https://doi.org/10.1111/j.1749-6632.1982.tb23194.x * Supported by CNR project “Biology of Reproduction” Grant n. 79.01176.85 and by Ford Foundation Grant n. 790.0659. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume383, Issue1The Cell Biology of the TestisJune 1982Pages 449-450 RelatedInformation

Membrane glycoprotein synthesis: cleavage of the signal sequence in the absence of glycosylation

Membrane glycoprotein synthesis: cleavage of the signal sequence in the absence of glycosylation

Biosynthesis of intestinal microvillar proteins: Nature of precursor forms of microvillar enzymes from Ca 2+-precipitated enterocyte membranes

FEBS LettersVolume 132, Issue 2 p. 197-200 Full-length articleFree Access Biosynthesis of intestinal microvillar proteins Nature of precursor forms of microvillar enzymes from Ca2+-precipitated enterocyte membranes E.Michael Danielsen, E.Michael Danielsen Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this authorHanne Skovbjerg, Hanne Skovbjerg Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this authorOve Norén, Ove Norén Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this authorHans Sjöström, Hans Sjöström Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this author E.Michael Danielsen, E.Michael Danielsen Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this authorHanne Skovbjerg, Hanne Skovbjerg Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this authorOve Norén, Ove Norén Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this authorHans Sjöström, Hans Sjöström Department of Biochemistry C, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, DenmarkSearch for more papers by this author First published: September 28, 1981 https://doi.org/10.1016/0014-5793(81)81159-3Citations: 29AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 P.W. Robbins, S.C. Hubbard, S.J. Turco, D.F. Wirth, Cell, 12, (1977), 893– 900. 2 E.M. Danielsen, H. Sjöström, O. Norén, FEBS Lett., 127, (1981), 129– 132. 3 H. Sjöström, O. Norén, L. Jeppesen, M. Staun, B. Svensson, L. Christiansen, Eur. J. Biochem., 88, (1978), 503– 511. 4 H. Sjöström, O. Norén, L. Christiansen, H. Wacker, G. Semenza, J. Biol. Chem., 255, (1980), 11332– 11338. 5 Hedeager-Sørensen, S., Norén, O., Sjöström, H. and Danielsen, E. M. (1981) in preparation. 6 H. Skovbjerg, H. Sjöström, O. Norén, Eur. J. Biochem., 114, (1981), 653– 661. 7 U.K. Laemmli, Nature, 227, (1970), 680– 685. 8 M. Bradford, Anal. Biochem., 72, (1976), 248– 254. 9 Skovbjerg, H., Danielsen, E. M., Norén, O. and Sjöström, H. (1981) submitted. 10 G. Palade, Science, 189, (1975), 347– 358. 11 H.P. Hauri, A. Quaroni, K.J. Isselbacher, Proc. Natl. Acad. Sci. USA, 77, (1980), 6629– 6633. Citing Literature Volume132, Issue2September 28, 1981Pages 197-200 ReferencesRelatedInformation

A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis

Cells infected with a temperature-sensitive mutant (ts-26) of Rauscher murine leukemia virus (R-MuLV) or with wild-type virus were labeled with 35S-methionine, and cell extracts were examined for radioactive polypeptides which could be precipitated by monospecific antisera to viral proteins. When shifted from permissive (31 degrees C) to nonpermissive (39 degrees C) temperature, cells infected with ts-26 rapidly begin to accumulate gPr90enr, the glycoprotein precursor to the membrane envelope glycoprotein gp70 and to the membrane-associated protein p15E. Simultaneously, formation of these mature virion proteins ceases. In addition, lactoperoxidase-catalyzed surface labeling with 125I--iodine indicates that the plasma membrane of cells infected with ts-26 becomes depleted of gp70 antigens at 39 degrees C. Nevertheless, at 39 degrees C these cells release defective MuLVs which lack gp70 and p15E but contain an outer membrane. The released particles also contain an aberrantly processed form of the major virion core protein p30, and many of these virion cores have an unusual immature crescent shape. It has previously been reported that cells infected with the ts-26 mutant of R-MuLV process a 65,000 dalton precursor (Pr65gag) of the virion core proteins more slowly at 39 degrees C than do cells infected with wild-type virus (Stephenson, Tronick and Aaronson, 1975). Although we have confirmed these results, this effect is relatively small and it is known that various alterations of MuLV assembly can lead secondarily to inhibited processing of Pr65gag. We propose that the ts-26 mutant has a primary temperature-sensitive defect in membrane glycoprotein synthesis and that this change causes pleiotropic effects on core morphogenesis.

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins

Endo-β-N-acetylglucosaminidase H (endo H) is an enzyme which acts on asparagine- and lipid-linked oligosaccharides containing five or more mannose residues. Complex oligosaccharides and glycopeptides are completely resistant to the action of the enzyme. We have carried out pulse-chase experiments with 35S-methionine and 3H-mannose in uninfected cells and in cells infected with Sindbis virus and vesicular stomatitis virus (VSV). In each case, the labeled materials were analyzed for sensitivity to endo H by polyacrylamide gel electrophoresis and gel filtration. We find that endo H releases all the labeled mannose from pulse-labeled proteins. Initially, the released material is nearly identical in size to the endo H cleavage product derived from lipid-linked oligosaccharides present in the same cells. During chase periods, 35S-methionine and 3H-mannose protein becomes increasingly resistant to the enzyme. Moreover, the 3H-mannose-labeled material released from the protein during chase periods is smaller in size than the oligosaccharide from the lipid. On the basis of these results and results from other laboratories, we propose that during glycosylation of asparagine residues, a common oligosaccharide is transferred from the lipid carrier to protein and is subsequently processed to yield the so-called “high mannose” and “complex” oligosaccharides. Since, on the basis of present evidence, the lipid-linked oligosaccharide contains two N-acetylglucosamine, 8–12 mannose and 1–2 glucose molecules, it seems probable that the carbohydrate-processing systems remove half or more of the mannose and all of the glucose residues at sites destined to become complex glycopeptides. Removal of mannose and glucose residues may also occur at sites destined to become mature high mannose glycopeptides.

Energy metabolism and T-cell-mediated cytolysis. II. Selective inhibition of cytolysis by 2-deoxy-D-glucose.

The effect of the hexose analogue 2-deoxy-D-glucose (2-DG) on T-cell-mediated cytolysis has been investigated. 2-DG inhibited cytolysis in glucose-free medium but not in medium containing equimolar concentrations of glucose. This inhibition was reversible and quantitatively competitive with glucose. Among other natural sugars examined, only mannose competed effectively with 2-DG and reversed the inhibition of cytolysis, whereas sodium pyruvate, fructose, galactose, fucose, and glucosamine were without effect. Mannose and glucose were equally effective in competing with 2-DG on a molar basis. When other glucose analogues such as 5-thio-D-glucose (5-SH-G) and 3-O-methylglucose were investigated under the same conditions, no inhibition of cytolysis was observed however, 5-SH-G (but not 3-O-methylglucose) was able to reverse the inhibitory effect of 2-DG in a competitive fashion. Taken together with the data presented in the accompanying paper, these findings provide strong evidence that 2-DG inhibits T-cell-mediated cytolysis by a mechanism that is unrelated to energy production. The possibility that inhibition is related to interference with membrane glycoprotein synthesis is discussed.

Characterization of human malignant melanoma cell lines

We have established conditions for the study of membrane glycoprotein synthesis and turnover in cultured human malignant melanoma cell lines using the labeled precursor [3H]glucosamine. Uptake of label increased parallel with cell growth, reaching a steady state in resting cultures. Fifteen to 30% of incorporated label can be released from the cells by trypsin treatment depending on the conditions of exposure to the enzyme, and about 50% of the incorporated label is spontaneously shed from the cells within 96 hr of incubation. Labeling in exhausted medium gave a 5- to 8-fold increase in uptake which was inhibited by addition of glucose (2 mg per ml) into the culture medium. The percentage of trypsin-releasable material was identical in fresh and exhausted medium; however, the percentage shed was less in cells initially labeled in exhausted medium. These data provide background information for further studies on the antigenic composition of the glycoproteins of cultured melanoma cells.

The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins.

Membrane preparations from hen oviduct catalyze the transfer of mannose from GDP-mannose into three components: mannosyl phosphoryl polyisoprenol, oligosaccharide-lipid, and glycoprotein. Eivence that mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of both oligosaccharide-lipid and glycoproteins was previously reported (Waechter, C.J., Lucas, J.J., and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7570-7579). In this study the oligosaccharide-lipid has been isolated, and the oligosaccharide has been partially characterized. Based on paper chromatography the oligosaccharide chain contains 7 to 9 glycose units. The glycose at the reducing terminus is N-acetylglucosamine, whereas mannose is found at the nonreducing end. When UDP-N-acetyl[14C]glucosamine is incubated with oviduct membranes in the absence of GDP-mannose, a 14C-labeled chitobiosyl lipid, but little oligosaccharide-lipid is synthesized. When GDP-mannose is also present in the incubation mixture an oligosaccharide-lipid is formed containing N-acetyl[14C]glucosaminyl residues. This oligosaccharide-lipid is chromatographically identical with the [14C]mannose-containing oligosaccharide-lipid isolated in the earlier study cited above. When the N-acetyl[14C]glucosamine-oligosaccharide released from the oligosaccharide-lipid by mild acid is treated with partially purified alpha-mannosidase the major radioactive product is [14C]chitobiose. Evidence that the [14C]mannose-containing oligosaccharide-lipid serves as an oligosaccharide donor for glycoprotein synthesis was obtained by incubation of partially purified oligosaccharide-lipid with the membranes. The products of this incubation were shown to be glycoproteins on the basis of their sensitivity to pronase, as determined by both gel filtration and paper electrophoresis. Similar experiments, using oligosaccharide-lipid doubly labeled with [14C]mannose and N-acetyl[3H]glucosamine, provided evidence that the oligosaccharide chain of the oligosaccharide-lipid is transferred en bloc to glycoprotein s.

Membrane glycoprotein biosynthesis: changes in levels of glycosyl transferases in fibroblasts transformed by oncogenic viruses.

AbstractFibroblasts transformed by oncogenic viruses were found to have greater enzymic activities of four membrane glycoprotein:glycosyl transferases on a cell or protein basis then two non‐transformed fibroblastic lines. These enzymes are responsible for the synthesis of membrane glycoproteins; each of the four transferases studied, the polypeptide:N‐acetylgalactosaminyl, glycoprotein:galactosyl, fetuin:fucosyl and PSM:fucosyl transferases, was more than twice as active in the transformed cell lines using both endogenous and added receptor. The most pronounced differences occurred with the doubly (SV‐PY‐3T3) transformed fibroblasts in all cases; with the N‐acetylgalactosaminyl and galactosyl transferases the increase was 8–16 fold over the non‐transformed cells. It was demonstrated that these results do not arise from a changed level of glycosidase activities.