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1.
Two inhibitors of glycosylation, glucosamine and tunicamycin, were utilized to examine the effect of glycosylation inhibition in mouse neuroblastoma N18 cells on the degradation of membrane glycoproteins synthesized before addition of the inhibitor. Treatment with 10 mM-glucosamine resulted in inhibition of glycosylation after 2h, as measured by [3H]fucose incorporation into acid-insoluble macromolecules, and in a decreased rate of glycoprotein degradation. However, these results were difficult to interpret since glucosamine also significantly inhibited protein synthesis, which in itself could cause the alteration in glycoprotein degradation [Hudson & Johnson (1977) Biochim. Biophys. Acta 497, 567-577]. N18 cells treated with 5 microgram of tunicamycin/ml, a more specific inhibitor of glycosylation, showed a small decrease in protein synthesis relative to its effect on glycosylation, which was inhibited by 85%. Tunicamycin-treated cells also showed a marked decrease in glycoprotein degradation in experiments with intact cells. The inhibition of glycoprotein degradation by tunicamycin was shown to be independent of alterations in cyclic AMP concentration. Polyacrylamide-gel electrophoresis of isolated membranes from N18 cells, double-labelled with [14C]fucose and [3H]fucose, revealed heterogeneous turnover rates for specific plasma-membrane glycoproteins. Comparisons of polyacrylamide gels of isolated plasma membranes from [3H]fucose-labelled control cells and [14C]fucose-labelled tunicamycin-treated cells revealed that both rapidly and slowly metabolized, although not all, membrane glycoproteins became resistant to degradation after glycosylation inhibition.  相似文献   

2.
The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.  相似文献   

3.
Four inhibitors of oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the oligosaccharide chains of the P0 and 19-kDa glycoproteins. Several different inhibitors of oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21-24 days of age) and the uptake of 14C-amino acid and [3H]fucose or [3H]mannose was measured in P0 and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H]Mannose was not found as suitable as [3H]fucose as an oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H]mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated oligosaccharide chains. Endoglycosidase H cleaved approximately 50% of the P0 labeled with [3H]fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H]fucose label on the protein, whereas one-half remained on the oligosaccharide chain of the undegraded P0, indicating that at least one-half the P0 has a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H]fucose into the oligosaccharide chains of P0 and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. P0 synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed P0 and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.  相似文献   

4.
The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7-9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 micrograms/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.  相似文献   

5.
Cells stably infected with Rous sarcoma virus were treated with tunicamycin to prevent the glycosylation of the precursor (pr92gp) to the two viral envelope glycoproteins gp85 and gp35. Pretreatment of the cells for 4 h with the antibiotic resulted in a 90% reduction in [3H]mannose incorporation into total cellular glycoproteins, intracellular viral glycoproteins, and released virus particles. Protein synthesis and virus particle formation were not significantly affected by the treatment. A new polypeptide made in the presence of the drug was identified by immunoprecipitation of pulse-labeled cell lysates with monospecific anti-gp85 and anti-gp35 sera. This polypeptide, migrating on sodium dodecyl sulfate-polyacrylamide gels as a molecule of 62,000 daltons (pr62), contained no [3H]mannose, was labeled with [S35]methionine and [3H]arginine, could not be chased into the higher-molecular-weight glycosylated form, and contained the same [3H]arginine tryptic peptides as pr92gp. The unglycosylated pr62 was still detectable 2 h after the pulse labeling of the cells. The lack of glycosylation of pr62 did not seem to reduce its stability. No clear evidence for the incorporation of this molecule or its cleavage products into viral particles could be obtained. To code for an envelope polypeptide of 62,000 daltons, only about 1,500 nucleotides or 15% of the total coding capacity of the virus are needed.  相似文献   

6.
Tunicamycin (TM), an antibiotic that inhibits the formation of N-acetylglucosamine-lipid intermediates, thereby preventing the glycosylation of newly synthesized glycoproteins, inhibits the growth of Sindbis virus and vesicular stomatitis virus in BHK cells. At 0.5 mug of TM per ml, the replication of both viruses is inhibited 99.9%. Noninfectious particles were not detected. All the viral proteins were synthesized in the presence of TM, but the glycoproteins were selectively altered in that they migrated faster than normal viral glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting defective glycosylation. Within 1 h after TM addition, [14C]glucosamine incorporation into glycoproteins was inhibited 20%, whereas [35S]methionine incorporation was unaffected. By 2 to 3 h after TM addition, glucosamine incorporation had fallen to 15% of control value, with methionine incorporation being 60% of normal. TM did not affect the growth of the nomenveloped encephalomyocarditis virus in BHK cells, demonstrating that TM is not a general inhibitor of protein synthesis. These data demonstrate that TM specifically inhibits the glycosylation of viral glycoproteins and that glycosylation may be essential for the normal assembly of enveloped viral particles.  相似文献   

7.
We have studied the biosynthesis of rat gastric mucin in stomach segments using an antiserum against rat gastric mucin specific for peptide epitopes. Pulse-chase experiments were performed with [35S]methionine, [3H]galactose, and [35S]sulfate to label mucin precursors in different stages of biosynthesis, which were analyzed after immunoprecipitation. The earliest mucin precursor that could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a 300-kDa protein. The occurrence of N-linked "high-mannose" oligosaccharides on this protein was shown by susceptibility to degradation by endo-beta-N-acetylglucosaminidase H. This precursor could be labeled with [35S]methionine and not with [3H]galactose or [35S]sulfate. The 300-kDa precursor was converted into mature mucin after extensive glycosylation and sulfation. The mature mucin but not the 300-kDa precursor was in part secreted into the medium. Specific inhibition of sulfation with sodium chlorate had no effect on rate and amount of mucin secretion. In addition, we show that two core proteins are expressed in rats, slightly varying in Mr among individual animals.  相似文献   

8.
The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [3H]mannose and [3H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [3H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with 125I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [3H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.  相似文献   

9.
The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [3H]mannose, [3H]glucosamine, and [3H]fucose incorporation by 95, 85, and 90%, respectively, while [3H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [3H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [3H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.  相似文献   

10.
We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.  相似文献   

11.
1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.  相似文献   

12.
We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 microg/mL) or tunicamycin (0.5 and 2.0 microg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.  相似文献   

13.
Fatty acid acylation of salivary mucin in rat submandibular glands   总被引:2,自引:0,他引:2  
The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [3H]palmitic acid and [3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [3H]palmitate and [3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.  相似文献   

14.
While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAcα2,3Galβ1,3GalNAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-(3)H]- or [(14)C]sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAcα2,3Galβ1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was ~2-fold stronger than that of asialo fetuin when CMP- [9-(3)H]NeuAc was generated in situ from 5'-CMP and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [(14)C]sialyl fetuin and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by generating CMP-[(14)C]- and -[9-(3)H]NeuAc through 5'-CMP; only 20.3% (14)C and 28.0% (3)H remained with the parent compounds after the sialyl exchange. The [9-(3)H]sialyl-tagged MN glycophorin A, human chorionic gonadotropin β subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.  相似文献   

15.
Secreted human bronchial mucins, directly collected from macroscopically healthy bronchial mucosa, were prepared in the presence of six proteinase inhibitors, and analysed by electron microscopy. These mucins were similar in length distribution to molecules prepared from sputum [Slayter, Lamblin, Le Treut, Galabert, Houdret, Degand & Roussel (1984) Eur. J. Biochem. 142, 209-218], although they were a little longer, their lengths ranging up to about 1,650 nm. This length corresponds to an extended mucin peptide of about 450 kDa. In order to compare these peptide lengths with the molecular size of biosynthetic precursors, an antiserum raised against trifluoromethanesulphonic acid-treated highly glycosylated regions of human bronchial mucins was used to isolate mucin precursors synthesized in explants of human bronchial mucosa during pulse-labelling with [3H]threonine or [3H]glucosamine. A main precursor labelled with [3H]threonine and with an apparent molecular mass of about 400 kDa was detected by fluorography following SDS/polyacrylamide-gel electrophoresis. This band was observed as early as 20 min; it was more intense after a 40 min chase and had disappeared after a chase period of 280 min in unlabelled medium, presumably owing to glycosylation. Much fainter bands at about 200 kDa and between 200 and 400 kDa, also labelled with [3H]threonine, were observed mainly after a 40 min chase and had disappeared after a 280 min chase. None of these bands was labelled with [3H]glucosamine, nor did they disappear after multiple treatments with immobilized lectins. After a 280 min chase, [3H]threonine-labelled material appeared in the stacking gel, which also contained [3H]glucosamine label. The results indicate that the 200-400 kDa species are mucin precursors, whose size is comparable with that obtained by electron microscopy for respiratory mucins collected directly from the macroscopically healthy bronchial mucosa.  相似文献   

16.
Use was made of the asialoglycoprotein receptor system in a perfused rat liver in order to study lysosomal degradation and subsequent metabolism of radioactive derivatives of asialo-ovine submaxillary mucin and asialo-alpha 1-acid glycoprotein. A trace of N-acetyl-D-[6-3H]galactosamine-labeled asialo-ovine submaxillary (4 micrograms) was completely taken up by the tissue in less than 20 min. After 3 h 24% of the radioactivity from the mucin reappeared on newly synthesized serum glycoproteins that were secreted into the perfusate. [6-3H] Galactose asialo-alpha 1-acid glycoprotein was also rapidly cleared by the liver; however, after 3 h greater than 60% of the radioactivity derived from this sugar labeled glycoprotein was secreted back into the perfusate as [3H]glucose. Rat livers perfused with 0.15 mM beta-D-galactopyranosylmethyl-p-nitrophenyltriazene lost 90% of their beta-D-galactosidase activity within 1 h while other representative glycosidases showed no change as followed by hydrolysis of p-nitrophenylglycosides. Livers pretreated with this triazene compound metabolized [3H]GalNAc asialo-ovine submaxillary mucin normally but were unable to process [3H]Gal asialo-alpha 1-acid glycoprotein as evidenced by a complete inhibition of [3H]glucose release following addition of the latter substrate. Metabolism of N-acetyl[14C]glucosamine asialo-alpha 1-acid glycoprotein was similarly inhibited by 70%. 125I-labeled asialo-alpha 1-acid glycoprotein catabolism was not affected by the chemically induced beta-D-galactosidase deficiency. Subcellular fractionation of inhibitor-treated livers accumulating radioactive carbohydrate showed the majority of the label was associated with a fraction enriched in lysosomes. Analysis of the trapped radioactivity by high resolution Bio-Gel P-4 chromatography revealed nearly intact oligosaccharides minus only the reducing N-acetylglucosamine of the chitobiose core. Direct comparison of these sugar chains with those isolated from human and canine GM1 gangliosidosis liver by silicic acid thin layer chromatography showed those isolated from rat liver to be identical to the major subset of oligosaccharides found in the human disease. In similar experiments in which the galactosyl triazene was replaced by swainsonine, an alpha-D-mannosidase inhibitor, catabolism of [14C]GlcNAc asialo-alpha 1-acid glycoprotein resulted in the accumulation of a single oligosaccharide of the structure. Man3[14C]GlcNAc1. These results demonstrate an endo-N-acetyl-beta-D-glucosaminidase is active in rat liver lysosomes.  相似文献   

17.
Two different methods were set up to detect the possible presence of lectin-like molecules with a specificity for mannose-rich glycans in the rat cerebellum. The first, affinity histochemistry, involved the isolation of a particular class of glycoproteins from the cerebella of 11-day-old rats followed by the formation of covalent complexes with horseradish peroxidase and then incubation with cerebellar slices. The second used in vitro interactions between [3H]leucine-labeled proteins, kept in solution, with insolubilized [14C]glucosamine-labeled glycoproteins. The results of both methods are compatible with the presence of lectin-like activities inhibited by high mannose concentrations, but not other sugars. However, the binding sites preferred by these molecules seem to be more than a single mannose residue.  相似文献   

18.
We recently described a 125 kd membrane glycoprotein in Saccharomyces cerevisiae which is anchored in the lipid bilayer by an inositol-containing phospholipid. We now find that when S. cerevisiae cells are metabolically labeled with [3H]myoinositol, many glycoproteins become labeled more strongly than the 125 kd protein. Myoinositol is attached to these glycoproteins as part of a phospholipid moiety which resembles glycophospholipid anchors of other organisms. Labeling of proteins with [3H]myoinositol for short times and in secretion mutants blocked at various stages of the secretory pathway shows that these phospholipid moieties can be added to proteins in the endoplasmic reticulum and that these proteins are transported to the Golgi by the regular secretory pathway. sec53, a mutant which cannot produce GDP-mannose at 37 degrees C, does not incorporate myoinositol or palmitic acid into membrane glycoproteins at this temperature, suggesting that GDP-mannose is required for the biosynthesis of these phospholipid moieties. All other secretion and glycosylation mutants tested add phospholipid moieties to proteins normally.  相似文献   

19.
The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]Na2SO4. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis was found to inhibit the process of mucin glycosylation and sulfation, which at 100 micrograms/ml lipopolysaccharide reached the optimal inhibition of 65%. The analysis of mucin secretory responses revealed that the lipopolysaccharide by first 15 min caused a 57% stimulation in sulfomucin secretion followed thereafter by inhibition, which reached maximum of 32% by 45 min. The results suggest that colonization of gastric mucosa by H. pylori may be detrimental to the process of gastric sulfomucin synthesis and secretion.  相似文献   

20.
The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.  相似文献   

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