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1.
Using a number of branched and unbranched oligosaccharides, glycoproteins and artificial glycoproteins bearing Gal(beta 1-4)GlcNAc-R termini as acceptors (where R represents H, oligosaccharide, oligosaccharide-protein or fatty acid-protein), the comparative rates of transfer of NeuAc by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase of embryonic chicken liver were determined. Acceptor substrates were utilized at levels approximating physiological, near the Km value of the best acceptor, desialylated alpha 1 acid glycoprotein. The sialyltransferase has a marked preference for multi-branched acceptors. From the specificity data, it is concluded that the enzyme binds at least two Gal(beta 1-4)GlcNAc termini of an acceptor molecule, and that the relative orientation of the branches is an important factor determining the rate of catalysis by the enzyme. The use of oligosaccharides as acceptors to study sialyltransferase catalyses is emphasized. Results are discussed in the context of the mode of assembly of sialoside termini of known glycoprotein structures in vivo.  相似文献   

2.
Endo-alpha-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, 13104-13112). The present report demonstrates that at least five distinct glycoproteins secreted by HepG2 cells (alpha 1-antitrypsin, transferrin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, and alpha-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with their usual complement of complex carbohydrate units, and upon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent further processing of deglucosylated N-linked oligosaccharides, Man6-8GlcNAc, but not Man9GlcNAc, were identified; the Man8GlcNAc component occurred as the characteristic isomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared to be nonselective, a differential inhibitory effect on the secretion of the various glycoproteins was noted in the presence of CST which was directly related to the number of their N-linked oligosaccharides, ranging from minimal in alpha-fetoprotein to substantial (approximately 65%) in alpha 1-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transit would be related to movement from the endoplasmic reticulum to the Golgi compartment.  相似文献   

3.
The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.  相似文献   

4.
Carbohydrate structures of HVJ (Sendai virus) glycoproteins   总被引:7,自引:0,他引:7  
The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.  相似文献   

5.
A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.  相似文献   

6.
The clearance of total rat liver secretory glycoproteins and of alpha 1-acid glycoprotein carrying no or different types of oligosaccharide side chains was studied in vivo and in the isolated perfused rat liver. In order to obtain unglycosylated or differently glycosylated forms of secreted glycoproteins, rat hepatocyte primary cultures were incubated with various inhibitors of N-glycosylation. Tunicamycin was used for the synthesis of unglycosylated (glyco)proteins, the mannosidase I inhibitor 1-deoxymannojirimycin for the synthesis of high-mannose type and the mannosidase II inhibitor swainsonine for the synthesis of hybrid-type glycoproteins. Glycoproteins carrying carbohydrate side chains of the complex type were synthesized by control hepatocytes. In vivo and in the perfused rat liver, high-mannose-type glycoproteins were cleared at the highest rate, followed by unglycosylated and hybrid-type glycoproteins. The lowest clearance rate was found for the glycoproteins with carbohydrate side chains of the complex type. For the highly glycosylated alpha 1-acid glycoprotein the differences in clearance rates were more pronounced. The following plasma half-lives were determined in vivo: complex type, 100 min; hybrid type, 15 min; unglycosylated form, 5 min; and high-mannose type less than 1 min. In the recirculating perfused liver 28% of complex-type alpha 1-acid glycoprotein, 40% of hybrid type, 47% of unglycosylated and 93% of high-mannose-type alpha 1-acid glycoprotein were removed from the perfusate within 2 h. It is concluded that N-glycosylation and processing to complex-type oligosaccharides seems to be of great importance for the circulatory life time of plasma glycoproteins.  相似文献   

7.
We had shown previously that all major glycoproteins of pigeon egg white contain Galalpha1-4Gal epitopes (Suzuki, N., Khoo, K. H., Chen, H. C., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, 23221-23229). We now report that Galalpha1-4Gal-bearing glycoproteins are also present in pigeon serum, lymphocytes, and liver, as probed by Western blot with Griffonia simplicifolia-I lectin (specific for terminal alpha-Gal) and anti-P1 (specific for Galalpha1-4Galbeta1-4GlcNAcbeta1-) monoclonal antibody. One of the major glycoproteins from pigeon plasma was identified as IgG (also known as IgY), which has Galalpha1-4Gal in its heavy chains. High pressure liquid chromatography, mass spectrometric (MS), and MS/MS analyses revealed that N-glycans of pigeon serum IgG included (i) high mannose-type (33.3%), (ii) disialylated biantennary complex-type (19.2%), and (iii) alpha-galactosylated complex-type N-glycans (47.5%). Bi- and tri-antennary oligosaccharides with bisecting GlcNAc and alpha1-6 Fuc on the Asn-linked GlcNAc were abundant among N-glycans possessing terminal Galalpha1-4Gal sequences. Moreover, MS/MS analysis identified Galalpha1-4Galbeta1-4Galbeta1-4GlcNAc branch terminals, which are not found in pigeon egg white glycoproteins. An additional interesting aspect is that about two-thirds of high mannose-type N-glycans from pigeon IgG were monoglucosylated. Comparison of the N-glycan structures with chicken and quail IgG indicated that the presence of high mannose-type oligosaccharides may be a characteristic of these avian IgG.  相似文献   

8.
Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.  相似文献   

9.
Urinary oligosaccharides isolated from locoweed-intoxicated sheep were separated and quantified by reversed-phase high pressure liquid chromatography of the perbenzoylated alditols. Mannose-containing oligosaccharides were elevated as early as day 3 of feeding, but maximum levels (approx. 1 mumol/ml) were not attained until after 6 weeks of feeding. The relative abundance of individual oligosaccharides changed over the course of the feeding period. Man3GlcNAc2 reached a peak on day 3 and then rapidly declined. Two isomers were shown to be present in this fraction and the relative proportions altered with the duration of locoweed treatment. The major isomer present at early time points (less than 8 days) co-eluted with synthetic Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, was digested by endo-beta-N-acetyl-glucosaminidase D, and is probably derived from the trimannosyl core of complex glycoproteins synthesized prior to locoweed treatment. Man3GlcNAc2 isolated from day 53 urine was resistant to endo-beta-N-acetylglucosaminidase D digestion but was cleaved by endo-beta-N-acetylglucosaminidase H. This isomer has the probable structure Man(alpha 1-3)Man(alpha 1-6)Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, indicative of its origin from hybrid or high-mannose glycoproteins. Man5GlcNAc2 reached a peak on day 13 and then slowly declined, whereas Man4GlcNAc2 increased concomitantly. The rapid increase in Man5GlcNAc2 can probably be attributed to the breakdown of hybrid glycans produced as a result of swainsonine inhibition of Golgi alpha-D-mannosidase II. The onset of observable clinical signs on day 38 closely correlated with the time point at which the level of Man4GlcNAc2 exceeded Man5GlcNAc2. After locoweed feeding was discontinued, the amount of urinary oligosaccharides declined rapidly and reached baseline levels within 12 days.  相似文献   

10.
We are interested in determining whether carbohydrates are important regulatory determinants in the intracellular transport and secretion of glycoproteins. In the present study, we have used swainsonine, an indolizidine alkaloid, to modify the structure of N-glycosidically linked complex oligosaccharides. By inhibiting Golgi mannosidase II, swainsonine prevents the trimming of GlcNAc(Man)5(GlcNAc)2 to GlcNAc-(Man)3(GlcNAc)2, resulting in the formation of hybrid-type oligosaccharides. We find, from pulse-chase experiments using [35S]methionine and immunoprecipitation of individual proteins from culture media, that swainsonine treatment (1 microgram/ml) accelerated the secretion of glycoproteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin) by decreasing the lag period by 10-15 min relative to untreated cultures. The enhanced secretion was specific for glycoproteins since the secretion of albumin, a nonglycoprotein, was unaffected. When alpha 1-antitrypsin was immunoprecipitated from the cell lysates, sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorographic analysis demonstrated that the conversion of the high-mannose precursor to the hybrid form in swainsonine-treated cells occurred more rapidly (by about 10 min) than the conversion to the complex form in control cells. Since both the hybrid and complex forms of alpha 1-antitrypsin are terminally sialylated by sialyltransferase in the trans-Golgi, these results suggest that swainsonine-modified glycoproteins traverse the Golgi more rapidly than their normal counterparts. Therefore, accelerated transport within this organelle may account for the decreased lag period of glycoprotein secretion in the swainsonine-treated cultures.  相似文献   

11.
N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146). Twenty-five major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Galalpha(1,4)Galbeta(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. N-Acetylneuraminic acids were alpha(2,6)-linked only to beta-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.  相似文献   

12.
To investigate the molecular basis of the differential ability of human, equine, and guinea pig alpha 2-macroglobulins to inhibit hemagglutination and infectivity of a human influenza virus, A/Memphis/102/72 (H3N2), the structures of oligosaccharides released from the three glycoproteins by hydrazinolysis were analyzed comparatively. Approximately seven to eight sugar chains were released from each subunit of two potent inhibitors (equine and guinea pig alpha 2-macroglobulins) and a weak inhibitor (human alpha 2-macroglobulin). More than 70% of the oligosaccharides contained sialic acids in all three cases. Structural analysis of these sialo-oligosaccharides revealed that all of the three glycoproteins contain biantennary oligosaccharides with one and two sialic acids as major sugar chains (70-80% of total sugar chains). Four percent of the biantennary oligosaccharides from equine sample, 10% of those from guinea pig, and 24% of those from human contain a fucosylated trimannosyl core. No triantennary oligosaccharide was detected in equine alpha 2-macroglobulin. However, human and guinea pig alpha 2-macroglobulins contain both fucosylated and nonfucosylated triantennary oligosaccharides. All sialic acid residues occur as the Sia alpha 2----6Gal group. The one unique feature of the carbohydrate groups of equine and guinea pig alpha 2-macroglobulins was the presence of 4-O-Ac-Neu5Ac as 30-50% of the total sialic acids, while human alpha 2-macroglobulin contained only Neu 5Ac. However, 4-O-Ac-Neu5Ac is not responsible for the potent inhibition of influenza virus infection and hemagglutination as will be described in the accompanying paper.  相似文献   

13.
Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.  相似文献   

14.
Endogenous ligands for the hepatic lectin which is specific for mannose and N-acetylglucosamine (mannan-binding protein, MBP) were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that serum glycoproteins, alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-acid glycoprotein, and a lysosomal enzyme, beta-glucuronidase were the major constituents of the endogenous ligands. These endogenous ligands consisted of high mannose-type oligosaccharides of Man9GlcNAc2 and Man8GlcNAc2, and had rapid turnover rates with an average half-life of 45 min, indicating that they were mainly composed of biosynthetic intermediates of glycoproteins. In view of the identification of the endogenous ligands as the biosynthetic intermediates of glycoproteins, the possible functions of the intracellular lectin are discussed in relation to the intracellular transport of glycoproteins.  相似文献   

15.
The hepatitis B surface antigen, which constitutes the currently available vaccine, is the empty envelope of the hepatitis B virus. We investigated the carbohydrate structures of the envelope glycoproteins. The intact oligosaccharides were enzymatically released from the coat glycoproteins using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and isolated by gel permeation chromatography. Cesium ion liquid secondary ion mass spectra of the intact, underivatized oligosaccharides showed molecular weights of 1932, 2078, and 2223. The mixture included partially and totally sialylated structures, a fraction (approximately 8%) of which were substituted with a single terminal fucose residue; no desialylated oligosaccharides were detected. The reducing termini of the oligomers were derivatized by reduction of the Schiff base formed using p-aminobenzoic acid ethyl ester, and fragmentation patterns identical to those produced from standard biantennary complex oligosaccharides were obtained. Methylation linkage analysis of the oligosaccharides showed that the carbohydrate composition and the mannose branching patterns also resembled those of a biantennary oligosaccharide. The results of this study indicate that glycosylation of the hepatitis B surface antigen, which takes place in the liver, is typical of other serum glycoproteins made in the liver; and this analytical strategy, including cesium ion liquid secondary ion mass spectrometry, is an effective approach for the structural analysis of complex carbohydrates available in only the 1-10 micrograms sample size range.  相似文献   

16.
The carbohydrate chains of the respiratory-mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive straight-phase high-performance liquid chromatography (HPLC) on a silica-based alkylamine column, and reverse-phase HPLC on a silica-based octadecyl column, respectively. The structures of the oligosaccharidealditols were determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are: (formula; see text) 23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631-642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643-654]. The sialic acid residue was found to occur either in alpha (2----3)- or alpha (2----6)-linkage to a galactosyl residue or in alpha (2----6)-linkage to GalNAc-ol.  相似文献   

17.
Three phosphate-containing sialyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration chromatography, ion-exchange chromatography and paper chromatography. Studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, phosphate determination, ion-exchange chromatography and glycosidase and phosphatase treatments indicated the following three structures for the compounds isolated: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc(alpha)-P. These sialyloligosaccharide 1-phosphates represent a novel class of oligosaccharides. Their oligosaccharide chains are identical with the common sialyloligosaccharide end groups of glycoproteins and glycolipids. The excretion of these compounds in normal human urine may indicate the existence of a novel, as yet unrevealed pathway in the metabolism of complex carbohydrates.  相似文献   

18.
《The Journal of cell biology》1984,98(5):1720-1729
1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse- chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1- antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1- antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha- mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N- linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.  相似文献   

19.
It has been proposed that in rat and murine tissues glucosidase II (GII) is formed by two subunits, GIIalpha and GIIbeta, respectively, responsible for the catalytic activity and the retention of the enzyme in the endoplasmic reticulum (ER). To test this proposal we disrupted genes (gls2alpha(+) and gls2beta(+)) encoding GIIalpha and GIIbeta homologs in Schizosaccharomyces pombe. Both mutant cells (gls2alpha and gls2beta) were completely devoid of GII activity in cell-free assays. Nevertheless, N-oligosaccharides formed in intact gls2alpha cells were identified as Glc(2)Man(9)GlcNAc(2) and Glc(2)Man(8)GlcNAc(2), whereas gls2beta cells formed, in addition, small amounts of Glc(1)Man(9)GlcNAc(2). It is suggested that this last compound was formed by GIIalpha transiently present in the ER. Monoglucosylated oligosaccharides facilitated glycoprotein folding in S. pombe as mutants, in which formation of monoglucosylated glycoproteins was completely (gls2alpha) or severely (gls2beta and UDP-Glc:glycoprotein:glucosyltransferase null) diminished, showed ER accumulation of misfolded glycoproteins when grown in the absence of exogenous stress as revealed by (a) induction of binding protein-encoding mRNA and (b) accumulation of glycoproteins bearing ER-specific oligosaccharides. Moreover, the same as in mammalian cell systems, formation of monoglucosylated oligosaccharides decreased the folding rate and increased the folding efficiency of glycoproteins as pulse-chase experiments revealed that carboxypeptidase Y arrived at a higher rate but in decreased amounts to the vacuoles of gls2alpha than to those of wild type cells.  相似文献   

20.
Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were homogenized and microvillar membranes prepared by calcium precipitation and differential centrifugation. Oligosaccharides present on the microvillar glycoproteins were identified by lectin blotting before and after in vitro and in situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins expressed a structurally restricted range of terminal sugars and oligosaccharide linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid N-linked oligosaccharides, some with alpha1-6 linked fucose as a core residue. Complex-type N-linked oligosaccharides on eight glycoproteins all possessed terminal N-acetylglucosamine, and alpha- and beta-linked N-acetylgalactosamine. Eight glycoproteins expressed O-linked oligosaccharides all containing N-acetylgalactosamine with or without further substitutions of fucose and/or galactose. Galactosebeta1-3/4/6N-acetylglucosamine-, sialic acidalpha2-3/6galactose-, fucosealpha1-2galactose- and galactosealpha1-3galactose- were not detected. Terminal alpha-linked N-acetylgalactosamine residues on N-linked oligosaccharides are described for the first time in insects. The nature and function of these midgut glycoproteins have yet to be identified, but the oligosaccharide side chains are candidate receptors for ookinete binding and candidate targets for transmission blocking strategies.  相似文献   

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