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1.
Sulfated N-linked oligosaccharides in mammalian cells. I. Complex-type chains with sialic acids and O-sulfate esters 总被引:9,自引:0,他引:9
L Roux S Holojda G Sundblad H H Freeze A Varki 《The Journal of biological chemistry》1988,263(18):8879-8889
The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines (Freeze, H. H., and Varki, A. (1986) Biochem. Biophys. Res. Commun. 140, 967-973). Here we report improved methods to metabolically label cell lines with 35SO4 and to release sulfated N-linked oligosaccharides with peptide:N-glycosidase F as well as the partial structure of some of these novel oligosaccharides. The released 35SO4-labeled chains from Chinese hamster ovary (CHO) cells and bovine pulmonary artery endothelial cells (CPAE) were characterized by gel filtration, anion exchange and lectin affinity chromatography, and various enzymatic and chemical treatments. Each cell line contains a class of sulfated oligosaccharide chains bearing from two to six negative charges in varying combinations of O-sulfate esters and sialic acids. These molecules represent a significant proportion of both the total 35SO4 label and the total anionic N-linked oligosaccharides. They are also relatively enriched in a CHO mutant that is deficient in glycosaminoglycan chain synthesis. Lectin affinity chromatography of such molecules from CPAE cells indicates that the majority are sialylated multiantennary complex-type chains. The sulfate esters are exclusively of the primary type. Sequential exoglycosidase digestions, including beta-hexosaminidase A treatment at low pH, demonstrate that at least one-third of these sulfate esters are found in the following structure, (formula; see text) where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., Roux, L., Varki, A., and Freeze, H. H. (1988) J. Biol. Chem. 263, 8890-8896). 相似文献
2.
Complex-type N-linked oligosaccharides of gp120 from human immunodeficiency virus type 1 contain sulfated N-acetylglucosamine. 总被引:3,自引:0,他引:3
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A Shilatifard R K Merkle D E Helland J L Welles W A Haseltine R D Cummings 《Journal of virology》1993,67(2):943-952
The major envelope glycoproteins gp120 and gp41 of human immunodeficiency virus type 1, the causative agent for human AIDS, contain numerous N-linked oligosaccharides. We report here our discovery that N-acetylglucosamine residues within the complex-type N-linked oligosaccharides of both gp120 and its precursor, gp160, are sulfated. When human Molt-3 cells persistently infected with human T-cell leukemia virus IIIB were metabolically radiolabeled with 35SO4, gp160, gp120, and to some extent gp41 were radiolabeled. The 35SO4-labeled oligosaccharides were quantitatively released by N-glycanase treatment and were bound by immobilized Ricinus communis agglutinin I, a lectin that binds to terminal beta-galactosyl residues. The kinetics of release of sulfate upon acid hydrolysis from 35SO4-labeled gp120 indicate that sulfation occurs in a primary sulfate ester linkage. Methylation analysis of total glycopeptides from Molt-3 cells metabolically radiolabeled with [3H]glucosamine demonstrates that sulfation occurs at the C-6 position of N-acetylglucosamine. Fragmentation of the gp120-derived 35SO4-labeled glycopeptides by treatment with hydrazine and nitrous acid and subsequent reduction generated galactosyl-anhydromannitol-6-35SO4, which is the expected reaction product from GlcNAc-6-sulfate within a sulfated lactosamine moiety. Charge analysis of the [3H]galactose- and [3H]glucosamine-labeled glycopeptides from gp120 and gp160 indicates that approximately 14% of the complex-type N-linked oligosaccharides are sulfated. 相似文献
3.
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
4.
A rat osteogenic cell line (UMR 106-01) synthesizes a highly sulfated form of bone sialoprotein 总被引:2,自引:0,他引:2
R J Midura D J McQuillan K J Benham L W Fisher V C Hascall 《The Journal of biological chemistry》1990,265(9):5285-5291
The rat osteosarcoma cell line (UMR 106-01) synthesizes and secretes relatively large amounts of a sulfated glycoprotein into its culture medium (approximately 240 ng/10(6) cells/day). This glycoprotein was purified, and amino-terminal sequence analysis identified it as bone sialoprotein (BSP). [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to label the BSP. Sulfate esters were found on N- and O-linked oligosaccharides and on tyrosine residues, with about half of the total tyrosines in the BSP being sulfated. The proportion of 35S activity in tyrosine-O-sulfate (approximately 70%) was greater than that in N-linked (approximately 20%) and O-linked (approximately 10%) oligosaccharides. From the deduced amino acid sequence for rat BSP (Oldberg, A., Franzén, A., and Heineg?rd, D. (1988) J. Biol. Chem. 263, 19430-19432), the results indicate that on average approximately 12 tyrosine residues, approximately 3 N-linked, and approximately 2 O-linked oligosaccharides are sulfated/molecule. The carboxyl-terminal quarter of the BSP probably contains most, if not all, of the sulfated tyrosine residues because this region of the polypeptide contains the necessary requirements for tyrosine sulfation. Oligosaccharide analyses indicated that for every N-linked oligosaccharide on the BSP, there are also approximately 2 hexa-, approximately 5 tetra-, and approximately 2 trisaccharides O-linked to serine and threonine residues. On average, the BSP synthesized by UMR 106-01 cells would contain a total of approximately 3 N-linked and approximately 25 of the above O-linked oligosaccharides. This large number of oligosaccharides is in agreement with the known carbohydrate content (approximately 50%) of the BSP. 相似文献
5.
J C Calvo D Rodbard A Katki S Chernick M Yanagishita 《The Journal of biological chemistry》1991,266(17):11237-11244
The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 +/- 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of approximately 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of approximately 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 +/- 0.2-fold in media and 3.2 +/- 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation. 相似文献
6.
Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate 总被引:4,自引:0,他引:4
The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins. 相似文献
7.
Occurrence of sulfate in an asparagine-linked complex oligosaccharide of chicken adipose lipoprotein lipase 总被引:3,自引:0,他引:3
After adipocytes were labeled with Na2[35SO4], immunoadsorbed with immobilized antilipoprotein lipase, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, a labeled band was identified at 59,700 daltons, the molecular mass of chicken lipoprotein lipase (LPL). Excess unlabeled LPL prevented the immunoadsorption of this labeled species, hence the labeled species was determined to be LPL. Digestion of LPL with endo-beta-N-acetylglucosaminidase H (Endo H) caused a shift in mobility of LPL in SDS-PAGE with no loss of radioactivity, whereas digestion with glycopeptidase F resulted in removal of 99% of the radioactivity. Adipocytes cultured with Trans35S-label and tunicamycin produced an LPL species of 52,000 daltons, but tunicamycin abolished the incorporation of 35SO4 into LPL. This established that 35SO4 was incorporated into an N-linked oligosaccharide of LPL. Endo H digestion of pulse-chase labeled LPL revealed the presence of two complex and one high mannose-type N-linked oligosaccharides. A single 35SO4-labeled tryptic peptide was isolated by reverse phase chromatography. The amino acid sequence of the peptide established that the 35SO4 oligosaccharide is conjugated at Asn-45. Behavior of the 35SO4-labeled oligosaccharide on concanavalin A-agarose, sequential exoglycosidase digestion, and chemical analysis of the 35SO4 oligosaccharide confirms that this moiety is of the complex type. Sequential exoglycosidase digestion, thin layer chromatography of the released monosaccharides, and the use of glycosylation inhibitors established that the sulfated sugar is a core N-acetylglucosamine (GlcNAc). The data show that chicken LPL contains two complex and one high mannose N-linked oligosaccharides and that 35SO4 is incorporated into LPL on a GlcNAc residue of a complex oligosaccharide located at Asn-45. 相似文献
8.
The effect of swainsonine and castanospermine on the sulfation of the oligosaccharide chains of N-linked glycoproteins 总被引:2,自引:0,他引:2
MDCK (Madin-Darby canine kidney) cells infected with the NWS strain of influenza virus incorporate 35SO4 into complex types of oligosaccharides of the N-linked glycoproteins. On the other hand, when these virus-infected MDCK cells are incubated in the presence of swainsonine, an inhibitor of the processing mannosidase II, approximately 40-80% of the total [35S]glycopeptides were of the hybrid types of structures. Thus, these sulfated, hybrid types of glycopeptides were completely susceptible to digestion by endoglucosaminidase H, whereas the sulfated glycopeptides from infected cells incubated without swainsonine were completely resistant to endo-beta-N-acetylglucosaminidase H. When virus-infected MDCK cells were incubated in the presence of castanospermine, an inhibitor of the processing glucosidase I, the N-linked glycopeptides contained mostly oligosaccharide chains of the Glc3Man7-9GlcNAc2 types of structures, and these oligosaccharides were devoid of sulfate. Structural analysis of these abnormally processed oligosaccharides produced in the presence of swainsonine or castanospermine indicated that they differed principally in the processing of one oligosaccharide branch as indicated by the structures shown below. They also differed in that only the swainsonine-induced structures were sulfated. These data indicate that removal of glucose units and perhaps other processing steps are necessary before sulfate residues can be added. (Formula: see text). 相似文献
9.
M van de Rijn I L Weissman M Siegelman 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(5):1477-1482
The mouse lymph node specific homing receptor gp90MEL-14 is a 95-kDa molecular mass ubiquitinated cell surface molecule involved in the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. The molecule is thought to consist of a core protein to which ubiquitin side chains are covalently bound. Recently we cloned the cDNA encoding the core protein; this cDNA clone encodes for a polypeptide with an estimated molecular mass of 37 kDa. We have studied the biosynthesis of gp90MEL-14 in an effort to explain the difference in molecular mass between the core protein and the 95-kDa mature molecule. Pulse labeling experiments show a rapid synthesis of a 70-kDa precursor form that contains high-mannose N-linked oligosaccharides. On processing of the high-mannose oligosaccharides into complex N-linked oligosaccharides, the precursor matures in a single step into the 95-kDa form. Experiments using deglycosylating enzymes and inhibitors of N-linked glycosylation demonstrate that the molecular mass of deglycosylated gp90MEL-14 is 45 kDa; extensive N-linked glycosylation is responsible for the difference in molecular mass with the mature 95-kDa form. The core protein molecular weight of in vitro transcribed and translated gp90MEL-14 cDNA is consistent with the estimated molecular mass of 37 kDa, calculated from the cDNA sequence of the core protein, and 8 to 10 kDa less than the protein molecular mass of gp90MEL-14 translated in vivo in the presence of tunicamycin (45 kDa). Inasmuch as we have ruled out glycosylation as accounting for this discrepancy, this is consistent with the addition of one ubiquitin moiety to the core protein during biosynthesis. Limited proteolysis confirms the similarity between in vitro transcribed gp90MEL-14 cDNA and the tunicamycin form of gp90MEL-14. 相似文献
10.
Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells. 总被引:12,自引:0,他引:12
A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained. 相似文献
11.
Characterization of heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells in culture 总被引:4,自引:0,他引:4
Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and maintained in culture. Proteoglycans were labeled using [35S]sulfate, [3H]serine, [3H]glucosamine, or [3H]mannose as precursors. A species of heparan sulfate proteoglycan was purified using DEAE-Sephacel chromatography under dissociative conditions in the presence of detergent. The heparan sulfate proteoglycan, which constituted approximately 15% of the 35S-labeled proteoglycans in the culture medium has a similar hydrodynamic size (Kd = 0.62 on Sepharose CL-2B) and buoyant density distribution in CsCl density gradients as the low buoyant density dermatan sulfate proteoglycan synthesized by the same granulosa cells and described in the accompanying report (Yanagishita, M., and Hascall, V. C. (1983) J. Biol. Chem. 258, 12847-12856). The heparan sulfate chains (average Mr = 28,000) have an average of 0.8-0.9 sulfate groups/repeating disaccharide, of which 50% are N-sulfate, 30% are alkaline-labile O-sulfate (presumably on the 6-position of glucosamine residues), and 20% are alkaline-resistant O-sulfate groups. Alkaline borohydride treatment released both N-linked oligosaccharide-peptides containing mannose, glucosamine, and sialic acid, and O-linked oligosaccharides. Trypsin digestion of the proteoglycan generated fragments which contain (a) glycosaminoglycan-peptides with an average of 2 heparan sulfate chains/peptide; (b) clusters of O-linked oligosaccharides on peptides; and (c) N-linked oligosaccharide-peptides, which are as small as single N-linked oligosaccharides. The compositions of the O-linked and N-linked oligosaccharides and the trypsin fragments of this heparan sulfate proteoglycan were very similar to those of the low buoyant density dermatan sulfate proteoglycan synthesized by the same cells. 相似文献
12.
Characteristics of the sulfation of N-linked oligosaccharides in vesicles from Dictyostelium discoideum: in vitro sulfation of lysosomal enzymes 总被引:2,自引:0,他引:2
C H Lacoste H H Freeze J A Jones A Kaplan 《Archives of biochemistry and biophysics》1989,273(2):505-515
Lysosomal enzymes from Dictyostelium discoideum contain unusual sulfated N-linked oligosaccharides, whose synthesis has been well studied in vivo. However, little is known about the properties of the pertinent sulfotransferases. To study these transferases, we have prepared a cell-free system which transfers 35SO4 from 3'-phosphoadenosine 5'-phosphosulfate to either endogenous or exogenous acceptors. We found that the 35SO4 was released from macromolecules by protein N-glycanase F to yield a mixture of anionic oligosaccharides with 1-6 negative charges. Some of the labeled molecules contained acid-stable methyl phosphodiesters but none contained phosphomoesters or acid-labile diesters. The sulfate was found in molecules with the acid stability characteristic of esters of primary alcohols. In all these ways, the products resembled those generated in vivo. We also demonstrated that a membrane-associated form of beta-hexosaminidase and the precursor of alpha-mannosidase were among the products. In addition, glycoproteins prepared from a sulfation-deficient mutant strain could act as exogenous acceptors in permeabilized vesicles. 相似文献
13.
Sulfated N-linked carbohydrate chains in porcine thyroglobulin 总被引:3,自引:0,他引:3
N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity. 相似文献
14.
Arterial lumican. Properties of a corneal-type keratan sulfate proteoglycan from bovine aorta. 总被引:1,自引:0,他引:1
J L Funderburgh M L Funderburgh M M Mann G W Conrad 《The Journal of biological chemistry》1991,266(36):24773-24777
A glycoprotein reactive with antibodies against corneal keratan sulfate proteoglycan (KSPG) was purified 300-fold from extracts of bovine aorta using DEAE ion-exchange, gel-filtration, hydrophobic interaction, and reverse-phase chromatographic separations. The intact glycoprotein was 70-80 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Deglycosylation with endo-beta-galactosidase and N-glycanase reduced the size to 48 and 37 kDa, respectively, similar to the large isoforms of corneal KSPG. N-terminal amino acid sequence of the arterial KSPG was identical with lumican, the 37B isoform of corneal KSPG, and the arterial KSPG reacted with an antibody to synthetic peptide duplicating this sequence. Arterial KSPG and corneal lumican displayed identical tryptic maps. Arterial lumican contains fucose and mannose in amounts similar to corneal KSPG, but galactose, glucosamine, and sulfate were reduced compared to KSPG from cornea. Treatment of arterial lumican with endo-beta-galactosidase released 8-9 mol of glucosamine and galactose per mol of protein as oligosaccharides. These eluted as neutral, nonsulfated oligosaccharides on high pH anion-exchange chromatography. The size of arterial lumican was not altered by glycosidases having specificity for sulfated keratan sulfate, nor was the charge of the lumican molecule altered by digestion with endo-beta-galactosidase. These data show arterial lumican to be a glycoprotein containing unsulfated lactosaminoglycan chains. Abundance of low sulfate lumican in many tissues indicates that this protein occurs predominantly as a glycoprotein rather than as the more widely studied, highly sulfated proteoglycan present in the cornea. 相似文献
15.
The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity.
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Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix. 相似文献
16.
Previous studies in many cell lines have shown that Brefeldin A (BFA) inhibits the forward movement of newly synthesized glycoconjugates by fusing the cis-, medial-, and trans-Golgi compartments with the rough endoplasmic reticulum. Studies on the oligosaccharide processing of individual glycoproteins have yielded confusing and incomplete results regarding the location of the block. Assuming that all glycoproteins with N-linked oligosaccharides follow the same endoplasmic reticulum to the Golgi pathway, a more complete picture on the location and nature of the block can be determined by analyzing N-linked oligosaccharides synthesized in the presence of BFA. In bovine pulmonary artery endothelial cells, BFA (0.1 microgram/ml) reversibly inhibits the secretion of greater than 95% of Tran35S and [3H]Man-labeled glycoproteins without affecting protein synthesis or N-linked glycosylation. In addition, BFA inhibits the synthesis and secretion of 35SO4-labeled oligosaccharides. Initial oligosaccharide trimming is uninhibited, but further processing is affected since the majority (65%) of the chains terminate only in beta-GlcNAc residues. Concomitantly, the proportion of [3H]Man-labeled N-linked anionic oligosaccharides is reduced from 60 to 20%, and the great majority of the charge is due to one sialic acid. The rate-limiting step for sialylation appears to be the branch selective addition of beta-Gal residues. The remaining charge is due to sulfate esters (0.6%) which normally account for greater than 10% of the anionic substituents. BFA also reduces the amount of phosphorylated chains by 80% and greatly diminishes further phosphodiester processing since the majority of these oligosaccharides (60%) contain a Man-6-PO4 residue in an acid-sensitive diester linkage. The addition of all polylactosamine chains, outer-branch fucose and terminal alpha-Gal residues are completely inhibited by BFA. Secretion, fucosylation, and sialylation are completely restored when BFA is removed, but the other modification steps are only partially restored. Our results indicate that addition of sulfate esters, terminal alpha-Gal residues, polylactosamine chains, outer-branch fucose residues, some initial phosphorylation, and most phosphodiester processing may occur beyond a compartment where some beta-Gal and sialic acid residues can be added. Essentially, all of the effects on oligosaccharide processing are partially or completely reversible. 相似文献
17.
We have grown polarized epithelial Madin-Darby canine kidney II (MDCK II) cells on filters in the presence of [(35)S]sulfate, [(3)H]glucosamine, or [(35)S]cysteine/[(35)S]methionine to study proteoglycan (PG) synthesis, sorting, and secretion to the apical and basolateral media. Whereas most of the [(35)S]sulfate label was recovered in basolateral PGs, the [(3)H]glucosamine label was predominantly incorporated into the glycosaminoglycan chains of apical PGs, indicating that basolateral PGs are more intensely sulfated than their apical counterparts. Expression of the PG serglycin with a green fluorescent protein tag (SG-GFP) in MDCK II cells produced a protein core secreted 85% apically, which was largely modified by chondroitin sulfate chains. Surprisingly, the 15% of secreted SG-GFP molecules recovered basolaterally were more heavily sulfated and displayed a different sulfation pattern than the apical counterpart. More detailed studies of the differential modification of apically and basolaterally secreted SG-GFP indicate that the protein cores have been designated to apical and basolateral transport platforms before pathway-specific, post-translational modifications have been completed. 相似文献
18.
Lymphocyte function-associated antigen 1 (LFA-1) contains sulfated N-linked oligosaccharides 总被引:3,自引:0,他引:3
The murine lymphocyte function-associated antigen 1 (LFA-1) is a glycoprotein heterodimer consisting of an Mr 180,000 alpha-chain and an Mr 95,000 beta-chain. Although LFA-1 has been studied extensively in the past few years due to its involvement in various antigen-specific T lymphocyte responses, virtually nothing is known about its glycosylation. In this report, we have analyzed the oligosaccharide moieties of the murine LFA-1 molecule. Utilizing a T lymphoma cell line, EL-4, it was found that [35S] sulfate, [3H]glucosamine, [3H]mannose, and [3H]fucose were incorporated into both the alpha- and beta-chains of LFA-1. Isolated alpha- and beta-chains from anti-LFA-1 immunoprecipitates of [3H]glucosamine-labeled NP-40 lysates were subjected to tryptic-chymotryptic digestion, and the resulting glycopeptides were fractionated by reverse-phase high performance liquid chromatography. Five major [3H]glucosamine-labeled glycopeptides were generated by this procedure from each of the two polypeptide chains. Treatment of the individual glycopeptides with almond emulsin peptide:N-glycosidase or Endo F demonstrated that the [3H]glucosamine label existed almost entirely in N-linked oligosaccharide structures (Mr 5000 to 10,000). By using similar techniques, the majority of the [35S]sulfate moieties were also found covalently bound to N-linked oligosaccharides. In addition, both [35S]sulfate-labeled alpha- and beta-chains were susceptible to Keratanase and endo-beta-galactosidase digestions, indicating the presence of sulfated N-acetyllactosamine sequences. The expression of [35S]sulfate-labeled LFA-1 on various cell types was also examined. LFA-1 was found to be sulfated only on thymocytes and splenic T cells, but not on macrophages, splenic B, or bone marrow cells. 相似文献
19.
P Bratt M M Anderson B M?nsson-Rahemtulla J W Stevens C Zhou F Rahemtulla 《The International journal of biochemistry》1992,24(10):1573-1583
1. We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight and the molecular size of the core protein was estimated to be greater than 200 kDa. 3. The glycosaminoglycan chains were susceptible to chondroitinase ABC and N-linked oligosaccharides were present on the protein core of the molecule. 4. Immunological studies provided evidence that a hyaluronic acid binding region was present in the core protein of versican. 5. The overall structure was similar to that of versican isolated from bovine sclera. 6. Decorin had a molecular weight of 102 kDa and its glycosaminoglycan chain was completely digested by specific glycosidases. 7. The partially deglycosylated core protein had a molecular weight of 55 kDa and N-linked oligosaccharides were present on the molecule. 相似文献
20.
L K Price H U Choi L Rosenberg E R Stanley 《The Journal of biological chemistry》1992,267(4):2190-2199
Colony stimulating factor-1 (CSF-1) is a homodimeric glycoprotein that humorally regulates the proliferation and differentiation of mononuclear phagocytic cells and locally regulates cells of the female reproductive tract. Alternative splicing of the human CSF-1 mRNA leads to alternative expression of the CSF-1 homodimer as a secreted glycoprotein or as a membrane-spanning molecule with cell surface biological activity. In the present study, analysis of immunoaffinity-purified CSF-1 from mouse L929 cell medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that CSF-1 is predominantly secreted as highly sulfated species of 375- and 250-kDa with a smaller amount of a 100-kDa species. Analysis by gel filtration in 4 M guanidine HCI buffer, indicated that, in contrast to the 100-kDa species, the highly sulfated species exhibit anomalously high molecular weights and self-association on SDS-PAGE similar to the dermatan sulfate proteoglycan, biglycan. The three predominant CSF-1 species were shown to be an 80-kDa homodimer, an 80-kDa/50-kDa heterodimer, and a 50-kDa homodimer. The 80-kDa subunit contained a single 18-kDa chondroitin sulfate chain that was absent from the 50-kDa subunit. Furthermore, treatment of the 80- and 50-kDa subunits, synthesized in the presence of tunicamycin, with chondroitinase ABC, neuraminidase, and endo-alpha-N-acetyl galactosaminidase reduced their apparent molecular masses to 60 and 25 kDa, respectively. These results are consistent with intracellular proteolytic cleavage of the 80-kDa chondroitin sulfate containing subunits from the membrane spanning CSF-1 precursor at a point carboxyl-terminal to the single consensus sequence for glycosaminoglycan addition and cleavage of the 50-kDa glycoprotein subunit at a position aminoterminal to this site. The predominance of the proteoglycan form of secreted CSF-1, which represents only 3-4% of the total trichloroacetic acid-precipitable counts released from 35SO4(2-)-labeled L cells, has important implications for regulation by this growth factor. 相似文献