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1.
Patricia B Ahrens 《Biology of the cell / under the auspices of the European Cell Biology Organization》1993,77(3):255-260
Summary— Human erythroleukemia (K-562) cells grown in the presence of phorbol 12,13-dibutyrate formed aggregates of cells not seen in untreated control cultures. Furthermore, the proportion of cells in aggregates and the size of the aggregates both increased dramatically in cultures treated with both phorbol ester and kifunensine, an inhibitor of asparagine-linked oligosaccharide processing. Relative to control cells, phorbol ester treated cells exhibited a greater proportion of N-linked oligosaccharides of the complex-type. Kifunensine prevented this change and caused an accumulation of Man9GlcNAc2. The enhanced aggregation of cells treated with phorbol ester plus kifunensine depended on phorbol ester concentration and was blocked by inhibitors of protein kinase C (H7, sphinganine and sangivamycin). In flow cytometry analysis, phorbol ester treated K-562 cells showed an increase in CD44, a glycoprotein involved in cell adhesion. Moreover, monoclonal antibody to CD44 augmented reaggregation of phorbol ester treated cells. The results implicate phorbol ester induction of CD44 in aggregation of K-562 cells and demonstrate that the presence of high mannose-type asparagine-linked oligosaccharides on cell glycoproteins correlates with increased aggregation of phorbol ester treated cells. 相似文献
2.
Particulate membrane preparations from K-562 [human CML (chronic-myelogenous-leukaemia)-derived] cells catalyse the transfer of [3H]galactose from UDP-[3H]-galactose and [3H]N-acetylglucosamine from UDP-[3H]N-acetylglucosamine into an endogenous product that on digestion with Pronase yields long-chain glycopeptides (mol.wt. 7000--10 000) called 'erythroglycan'. Incorporation of either labelled sugar increased up to 60 min of incubation time. The labelled erythroglycan was isolated by chromatography on Sephadex G-50 and characterized by digestion with endo-beta-galactosidase from Escherichia freundii, followed by analysis on Bio-Gel P-2 and paper chromatography. This digestion gave the following four products: (1) a disaccharide with the sequence beta GlcNAc-beta Gal; (2) a trisaccharide with the sequence betaGal-betaGlcNAc-beta Gal; (3) a larger oligosaccharide containing galactose and N-acetylglucosamine; and (4) a putative protein-linkage region. 相似文献
3.
Unique high molecular weight (M.W. 4,000-9,000) sugar chains termed erythroglycan II have been obtained from alkali/sodium borohydride digests of I-active asialoglycoprotein derived from sialoglycoprotein GP-2, which was isolated recently from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y. et al. (1983) J. Biochem. 93, 1621-1633; (1984) ibid, 95, 1193-1200). It was found that these sugar chains comprise about 40% of total alkali-labile oligosaccharides of asialo GP-2 and contain endo-beta-galactosidase (Flavobacterium keratolyticus)-resistant highly branched and heterogeneous oligosaccharides of poly-N-acetyllactosamine type which are linked O-glycosidically to the peptide backbone through N-acetylgalactosamine. Erythroglycan II also contains endo-beta-galactosidase-susceptible straight terminal polylactosaminyl side chains. A major oligosaccharide released by the enzyme cochromatographed with Gal beta 1-4GlcNAc beta 1-3Gal. Inhibitory activity of Sendai virus-mediated hemagglutination and the receptor activity for the virus were reduced significantly but not completely by the endo-beta-galactosidase. These results indicate that both linear and branched sialosylpolylactosamine sequences in erythroglycan II are important for the reception of the virus into the target cells. 相似文献
4.
Total glycopeptides from human K-562 cells, labeled metabolically with [3H]glucosamine or [3H]mannose, were prepared by extracting the cells with organic solvents to remove lipids and by digesting the residue with pronase. 3H-labeled glycopeptides were fractionated on Sephadex G-50 revealing a high molecular weight fraction (Mr = 7,000 to 11,000), comprising approximately 10% of the [3H]glucosamine and 25% of the [3H]mannose label. Digestion of this glycopeptide fraction with endo-beta-galactosidase from Escherichia freundii, specific for a repeating structure of Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3), results in the following four products as resolved by Bio-Gel P-2 gel filtration: 1) a disaccharide with the structure beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; 2) a trisaccharide with the structure beta-galactosyl leads to beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; 3) a tetrasaccharide with the sequence alpha-N-acetylneuraminyl leads to beta-galactosyl leads to beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; and 4) a larger, complex fragment which contains mannose and beta-2-deoxy-2-acetamidoglucose and which is probably the protein linkage region. In addition, visualization of radiolabeled glycoproteins by fluorography on polyacrylamide gels revealed a 105,000-dalton "Band 3"-like glycoprotein and other bands that were sensitive to endo-beta-galactosidase. These results indicate that the K-562 cell line bears a glycopeptide, erythroglycan, which has been found on erythrocytes, and that this polymer is expressed mainly in the fetal form as a linear chain. 相似文献
5.
Addition of asparagine-linked oligosaccharides to nascent murine leukemia virus (MuLV)-encoded membrane glycoproteins was inhibited either completely by tunicamycin or specifically at Asn-X-Thr glycosylation sites by incorporation of the threonine analogue beta-hydroxynorvaline. In conditions of partial analogue substitution, a series of subglycosylated components is formed which are related by a constant apparent Mr difference when assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The total number of asparagine-linked oligosaccharides is then estimated by dividing the measured apparent Mr of one oligosaccharide into the total apparent Mr difference between the complete glycoprotein and the polypeptide chain that is synthesized in cells incubated with tunicamycin. Correct results were obtained using glycoproteins with known numbers of oligosaccharides. Our analyses indicate that the gp70 membrane envelope glycoproteins of certain ecotropic MuLVs contain seven oligosaccharides, whereas the GIX+ antigen-containing variant gp70 contains one fewer Asn-X-Thr-linked oligosaccharide. The membrane glycoprotein encoded by the gag gene of Friend MuLV contains only one asparagine-linked oligosaccharide. Similarly, the gp55 membrane glycoprotein encoded by Friend erythroleukemia virus contains four asparagine-linked oligosaccharides. Pulse-chase and cell surface iodination analyses indicate that MuLV membrane envelope glycoprotein processing by partial proteolysis and transport to the cell surface can be efficiently blocked by structural perturbations caused by incorporation of different amino acid analogues or by loss of oligosaccharides. Our data also suggest that loss of oligosaccharides may expose new antigenic sites in viral membrane glycoproteins and increase their susceptibility to intracellular proteolysis. 相似文献
6.
Carbohydrate structure of glycoprotein 52 encoded by the polycythemia-inducing strain of Friend spleen focus-forming virus 总被引:1,自引:0,他引:1
The primary envelope (env) gene product of the polycythemia-inducing variant of Friend spleen focus-forming virus (F-SFFVP), representing a glycoprotein with an apparent Mr of 52,000 (gp52), was isolated from F-SFFVP-infected normal rat kidney cells metabolically labeled with [2-3H]mannose in the presence or absence of glucose. Structures of the oligosaccharides present were determined by micromethylation analysis, acetolysis, and digestion with exoglycosidases. Gp52 radiolabeled in the presence of glucose contains solely oligomannosidic glycans comprising 6 to 9 mannose residues (Man6-9GlcNAc2), some of which carry additional glucose. The structures of the glycans found reflect the typical intermediates of oligosaccharide processing. The glycosylation of gp52 isolated from glucose-deprived cells (-Glc), however, is characterized by increased amounts of Man5-7 species comprising other structural isomers. Only gp52 (-Glc) glycans are, in part, further processed yielding incomplete complex-type oligosaccharides. Our results demonstrate that the limited post-translational processing of the primary F-SFFVP env gene product is neither due to aberrant trimming of its oligomannosidic glycans nor due to transfer of immature lipid-linked oligosaccharide-intermediates as observed in glucose-starved cells. 相似文献
7.
The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells 总被引:12,自引:0,他引:12
B P Peters R J Hartle R F Krzesicki T G Kroll F Perini J E Balun I J Goldstein R W Ruddon 《The Journal of biological chemistry》1985,260(27):14732-14742
Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium. 相似文献
8.
Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function 总被引:21,自引:0,他引:21
A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required. 相似文献
9.
alpha-Glucosidase II-deficient cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing. 总被引:4,自引:0,他引:4
The kinetics of N-linked oligosaccharide processing and the structures of the processing intermediates have been examined in normal parental BW5147 mouse lymphoma cells and the alpha-glucosidase II-deficient PHAR2.7 mutant cells. The mutant cells accumulated glucosylated intermediates but were able to deglucosylate and process about 40% of their oligosaccharides to complex-type. This processing was not due to residual alpha-glucosidase II activity since the alpha-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butyl-DNJ did not prevent it. Parent cells also showed alpha-glucosidase II-independent processing in the presence of DNJ and N-butyl-DNJ. Membrane preparations from both parent and mutant cells had endo alpha-mannosidase activity, that is, split Glc1,2Man9GlcNAc to Glc1,2Man plus Man8GlcNAc, indicating that this was a candidate for an alternate route to complex oligosaccharide formation in the mutant cells. A balance study in which the cellular glycoproteins, intracellular water soluble saccharides, and saccharides secreted into the medium were isolated and analyzed from [2-3H]mannose-labeled mutant cells showed that the cells formed the di- and trisaccharides Glc1Man and Glc2Man in amounts equivalent to the deglucosylated oligosaccharides found in the cellular glycoproteins. This result shows unequivocally that the alpha-glucosidase II-deficient mutant cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing. 相似文献
10.
Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells 总被引:2,自引:4,他引:2
Joseph F. Poduslo 《Journal of neurochemistry》1985,44(4):1194-1206
The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined. 相似文献
11.
Human transferrin receptor contains O-linked oligosaccharides 总被引:2,自引:0,他引:2
We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types. 相似文献
12.
An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures. 相似文献
13.
Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor 总被引:41,自引:0,他引:41
R D Cummings S Kornfeld W J Schneider K K Hobgood H Tolleshaug M S Brown J L Goldstein 《The Journal of biological chemistry》1983,258(24):15261-15273
In human fibroblasts, the receptor for low density lipoprotein (LDL) is synthesized as a precursor of apparent Mr = 120,000 which is converted to a mature form of apparent Mr = 160,000, as determined by migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels (Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724). The current paper describes the relationship of N- and O-glycosylation to this post-translational modification. Oligosaccharides were analyzed from precursor and mature forms of LDL receptors that had been immunoprecipitated from cells grown in media containing radioactive sugars. In human epidermoid carcinoma A-431 cells, the receptor precursor appears to contain one N-linked high mannose oligosaccharide and approximately 6-9 N-acetylgalactosamine residues linked O-glycosidically to Ser/Thr residues. In the mature receptor, the O-linked oligosaccharides are mono- and disialylated species having the core structure of galactose leads to N-acetylgalactosamine leads to Ser/Thr. The single N-linked oligosaccharide of the mature receptor can either be a tri- or tetraantennary complex-type species. Similar results were obtained with normal human fibroblast receptor except that the O-linked oligosaccharides on the precursor are neutral disaccharides, of which one component is GalNAc and the N-linked complex type unit on the mature receptor is less branched. Since the addition of GalNAc residues to Ser/Thr residues precedes the conversion of N-linked high mannose-type oligosaccharides to complex-type structures, the transfer of N-acetylgalactosamine must occur prior to the entry of glycoproteins into the region of the Golgi containing the processing enzyme alpha-mannosidase I. We also studied the receptor from tunicamycin-treated cells and after treatment with neuraminidase. In addition, we analyzed the receptor synthesized by a lectin-resistant clone of Chinese hamster ovary cells that is deficient in adding galactose residues to both N- and O-linked oligosaccharides. These studies suggest that the apparent differences in molecular weight between the precursor and mature forms of the LDL receptor are largely, if not entirely, due to the addition of sialic acid and galactose residues to the O-linked GalNAc residues.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
14.
We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases. 相似文献
15.
We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Isolation and some structural analyses of a proteodermatan sulphate from calf skin. 总被引:1,自引:0,他引:1
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A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000). 相似文献
17.
The K-562 cell line is a culture of human leukemia stem cells originally derived from a patient with chronic myelogenous leukemia in blast crisis. We have applied a sensitive method capable of detecting subtle differences in charge-associated and noncharge-related cell surface properties between closely related cell populations to K-562 cells from different sources and having different histories. The method consists of isotopically labeling aliquots of each of two cell populations to be compared with 51Cr-chromate and mixing the labeled cells with an excess of unlabeled cells with which they are to be compared. The mixtures are subjected to countercurrent distribution in either a charge-sensitive or a noncharge-sensitive dextran-poly(ethylene glycol) aqueous two-phase system. The distribution curves are analyzed for total cells (in terms of electronic counts) and labeled cells (in terms of cpm). Alterations in relative specific activities through the distribution curves are indicative of differences in surface properties between such cell populations. Using this method we have found surface differences, both charge-associated and noncharge-related, between any two K-562 cell sublines examined. Interestingly, whereas we observed differences among K-562 sublines, we never witnessed a change in surface properties of the respective sublines. The differences among the sublines examined remained unaltered for more than 40 passages in our hands. It thus appears likely that the event(s) leading to an altered K-562 cell surface, detectable by partitioning, does not occur gradually. 相似文献
18.
Sulfated N-linked oligosaccharides in mammalian cells. II. Identification of glycosaminoglycan-like chains attached to complex-type glycans 总被引:3,自引:0,他引:3
G Sundblad S Holojda L Roux A Varki H H Freeze 《The Journal of biological chemistry》1988,263(18):8890-8896
In the preceding paper (Roux, L., Holojda, S., Sundblad, G., Freeze, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889) we described the metabolic labeling and isolation of sulfated N-linked oligosaccharides from mammalian cell lines. All cell lines studied contained a class of sulfated sialylated complex-type chains with 2-6 negative charges. In this paper, we show that bovine pulmonary arterial endothelial (CPAE) and human erythroleukemia (K562) cell lines also contain a class of more highly charged sulfated but less sialylated oligosaccharides. These molecules were further characterized by ion exchange chromatography and various enzymatic and chemical treatments. In both cell lines they contained greater than 6 negative charges, but those from K562 were even more highly charged than those from CPAE. Nitrous acid, heparinase, and heparitinase degradation of K562 oligosaccharides released 88, 64, and 78%, respectively, of 35S label. Combined digestion with the two enzymes resulted in 87% release. The corresponding values for CPAE were 48, 25, and 50% (60% for the two enzymes together). Chondroitinase ABC (or AC) digestion of K562 and CPAE oligosaccharides released 10 and 5%, respectively. About 30% of the 35S-labeled oligosaccharides from CPAE were sensitive to endo-beta-galactosidase, indicating that poly-N-acetyl-lactosamine structures were present on some chains. Highly charged [3H]mannose-labeled sulfated oligosaccharides from CPAE cells became neutral after treatment with heparinase/heparitinase but were resistant to Pronase, further proving that glycosaminoglycan (GAG)-like chains were directly attached to N-linked oligosaccharides. Such neutralized oligosaccharides did not bind to concanavalin A-Sepharose, but some interacted with phytohemagglutinin L4, indicating that they were bi-, tri-, or tetra-antennary complex-type chains. Thus, K562 and CPAE cells contain different types of GAG chains directly attached to asparagine-linked oligosaccharides. Such molecules were not found in many other cell lines that synthesize the more typical O-linked GAG chains. This suggests that the occurrence of these novel N-linked chains is not a random event resulting from accidental initiation of GAG chain synthesis on N-linked intermediates in the Golgi apparatus. 相似文献
19.
We have previously described that the N-glycosylation process was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides. The relationship between oligosaccharide-P-P-dolichol and its metabolic products (glycoproteins, oligosaccharide-phosphates and neutral oligosaccharides) was investigated by analysing the structure of the oligosaccharide moieties and the kinetic behaviour of the various species in pulse and pulse/chase experiments. For these studies, a glycosylation mutant of Chinese hamster ovary cells (B3F7) which does not synthesize mannosylphosphoryldolichol was utilized. Evidence was obtained for the presence of two pools of oligosaccharide-P-P-dolichol which have different fates. One pool is not glucosylated, is rapidly labelled and immediately chased by mannose, and generates the oligosaccharide-phosphate species. The second pool is glucosylated, exhibits a lag time (5-10 min) prior to being labelled, and is utilized in the glycosylation of proteins and in the production of neutral oligosaccharides. We postulate that the cleavage of non-glycosylated lipid intermediates generating oligosaccharide-phosphates represents a 'bypass' in the dolichol cycle which allows direct regeneration of dolichyl phosphate. The other metabolic fate of non-glucosylated oligosaccharide-lipids, glucosylation, results in their use as effective substrates for the glycosylation of proteins or in the generation of neutral oligosaccharides. 相似文献
20.
We have used a Chinese hamster ovary cell line deficient in N-acetylglucosaminyltransferase 1 activity (Lec1) to study the effects of altered asparagine-linked oligosaccharides on the structure, biosynthesis, and function of glucose transporter protein. Immunoblots of membranes of Lec1 cells show a glucose transporter protein of Mr 40,000, whereas membranes of wild-type (WT) cells contain a broadly migrating Mr 55,000 form similar to that observed in several other mammalian tissues. The total content of immunoreactive glucose transporters in Lec1 cells is 3.5-fold greater than that of WT cells. Digestion with endoglycosidases, treatment with inhibitors of glycosylation, and interactions with agarose-bound lectins demonstrate that glucose transporters of both cell lines derive from a similar Mr 38,000 core polypeptide and that both contain asparagine-linked oligosaccharide. Transporters in Lec1 cells contain primarily "undecorated" but "trimmed" mannose-type asparagine-linked oligosaccharides, while the protein in WT cells contains a mixture of "decorated" and "trimmed" asparagine-linked oligosaccharides. Biosynthetic and turnover studies demonstrate that Lec1 cells, in contrast to WT cells, are unable fully to process the core asparagine-linked oligosaccharides of maturing glucose transporters. When radiolabeled in methionine-deficient medium both Lec1 and WT cells show similar rates of synthesis and turnover of glucose transporter proteins. It should be noted, however, that starvation for a critical amino acid may alter the ability of the cell to synthesize or degrade proteins. The abilities of Lec1 and WT cells to transport hexoses and to interact with the inhibitor cytochalasin B are very similar. The results indicate that, although altered asparagine-linked glycosylation can affect the content and biogenesis of glucose transporters, these changes do not greatly modify cellular hexose uptake. The possibility that alterations in asparagine-linked glycosylation may change the cell surface localization or acquisition of a "functional conformation" of the glucose transporter is also suggested. 相似文献