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1.
Peptidokeratan sulfates were retained by a concanavalin A-Sepharose column and readily eluted with 20 mM methyl alpha-D-mannoside from the column. Linkage region-enriched glycopeptides, prepared by endo-beta-galactosidase digestion of the peptidokeratan sulfates, behaved similarly to the peptidokeratan sulfates on the same affinity chromatography. Further, behaviors of both the peptidokeratan sulfates and the linkage region-enriched glycopeptides on pea lectin-Agarose columns were consistent with the results of the chromatography on concanavalin A-Sepharose. These findings confirmed the typical biantennary structure previously suggested for the linkage region on the basis of structural studies of the linkage region-enriched glycopeptides.  相似文献   

2.
The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branched structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branched complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.  相似文献   

3.
Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.  相似文献   

4.
This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.  相似文献   

5.
Herpes simplex virus type-1 glycoprotein C (gC1) contains several O-linked oligosaccharides clustered near N-linked chains, and Pronase digestion produces glycopeptides carrying both oligosaccharide types. We have taken advantage of this fact to investigate the temporal relationship between the initiation of O-linked chains and the processing of N-linked oligosaccharides. gC1 was isolated from herpes-simplex-virus-infected BHK (baby-hamster kidney) cells after short labelling periods with [3H]glucosamine, and the labelled Pronase-cleaved glycopeptides fractionated on concanavalin A-Sepharose. N-[3H]Acetylgalactosamine, mostly convertible into free N-[3H]acetylgalactosaminitol on mild alkaline-borohydride treatment, was found in glycopeptides with an affinity to concanavalin A-Sepharose corresponding to that of glycopeptides carrying Man8GlcNAc2 or larger N-linked chains. Since there is evidence that the processing of N-linked chains up to Man8GlcNAc2 involves enzymes located in the rough endoplasmic reticulum, current results strongly suggest that gC1 acquires O-linked N-acetylgalactosamine before the glycoprotein routing to the Golgi apparatus. The addition of the second sugar to the nascent O-linked chain appeared to occur after a relatively long lag time.  相似文献   

6.
Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary 'complex-type' with terminal fucose; IIA, biantennary 'complex-type' without fucose; IIB, biantennary 'complex-type' with fucose; III, a mixture of 'high-mannose' chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of 'high-mannose' type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with 'complex-type' chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three 'complex-type' chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.  相似文献   

7.
Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.  相似文献   

8.
A smooth membrane fraction of Aspergillus niger catalyzed the transfer of mannose from GDP-mannose to endogenous lipid and protein acceptors. The mannolipid was acidic, as judged by diethylaminoethyl-cellulose chromatography, and had a mobility similar to ficaprenyl phosphate on thin-layer chromatograms. Mannose transfer occurred optimally at pH 6.5 to 7.5 and required Mn(2+) for use of the protein as acceptor, but either Mn(2+) or Mg(2+) with the lipid as acceptor. Glycopeptides of the mannosylated protein ([(14)C]gly) and of an alpha-glucosidase (alpha-glu) secreted by the organism were produced by Pronase digestion and separation of the products on Sephadex G-25. Because ovalbumin has a carbohydrate composition similar to that of alpha-glu and because the carbohydrate structure of ovalbumin is known, ovalbumin glycopeptides (Ov) were similarly obtained and used as standards in determining carbohydrate structures. Oligosaccharide chains of [(14)C]gly, alpha-glu, and Ov were obtained by treatment of the respective glycopeptides with endo-beta-N-acetylglucosaminidase, reduction with NaBT(4), and concanavalin A-Sepharose chromatography. The (3)H-labeled oligosaccharides obtained were subjected to the following treatments: (i) digestion with alpha- and beta-mannosidases, (ii) Smith degradation, and (iii) acetolysis. Subsequently, changes in paper chromatographic mobilities were detected. Also, alpha-glu was permethylated, and the partially methylated alditol acetates were analyzed by gas-liquid chromatography. The resultant proposed structure shows that the oligosaccharide chain of alpha-glu is almost identical to that of an Ov chain, while [(14)C]gly has a structure which is probably the same as that of alpha-glu. It is suggested that the transferase(s) involved in [(14)C]gly synthesis in vitro may be responsible for glycosylation of secreted enzymes.  相似文献   

9.
[3H]Fucose-labelled glycopeptides in the slices of liver 24h after partial hepatectomy were fractionated on Sephadex G-50. Glycopeptides from regenerating liver contained a higher proportion of lower-Mr components than did controls. Regenerating liver contained a higher proportion of glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside than did controls. Concanavalin A-bound glycopeptides from each source were entirely bound to a lentil lectin-Sepharose column. Both the concanavalin A-bound and -unbound fractions from regenerating liver were indistinguishable from the respective controls by Bio-Gel P6 column chromatography and neuraminidase digestion. These results show that fucosyl glycopeptides from regenerating liver contain a higher proportion of biantennary species with core fucose residues than do controls. Glycopeptides from regenerating livers 12h, 72h and 144h after partial hepatectomy were also examined; however, the difference was not significant. These observations suggest that the alterations in fucosyl glycopeptides may be related to rapid growth of hepatocytes 24h after partial hepatectomy. No significant difference was found in either [3H]mannose- or [3H]fucose-labelled glycoproteins from regenerating liver and from controls by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, suggesting that the alteration in glycopeptides should depend on some differences in the late stage of oligosaccharide processing.  相似文献   

10.
Baby-hamster kidney (BHK) cells were labelled metabolically by growth in media containing radioactive sugars and the asparagine-linked glycopeptides (N-glycans) obtained by Pronase digestion of disrupted cells were fractionated by chromatography on concanavalin A-Sepharose. About 2-3% of the total [3H]galactose- or [3H]fucose-labelled glycopeptides were found to be bound tightly to the lectin column and were eluted with 500 mM-methyl alpha-mannoside. Further analysis of these minor components by chromatography on Bio-Gel P4, lentil-lectin-Sepharose and DEAE-Sephacel and sensitivity to alpha-mannosidase indicates the presence in BHK-cell glycopeptides of hybrid structures of the following form: (Formula: see text) Similar structures were identified as major features of the glycoproteins of ricin-resistant mutants RicR17 and RicR19 as described previously for RicR21 cells [Hughes, Mills & Stojanovic (1983) Carbohydr. Res. 120, 215-234]. The RicR15 cell line also produces significant amounts of hybrid N-glycans. The studies show that the novel N-glycans accumulating in ricin-resistant mutants are derived by a metabolic pathway that exists to a minor extent in normal BHK cells.  相似文献   

11.
Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.  相似文献   

12.
The membrane glycoproteins E1 and E2 of Semliki Forest virus are of about equal size but can be separated from each other by affinity chromatography on a concanavalin A-Sepharose column in the presence of sodium dodecyl sulfate. The E1 protein eluted like glycopeptides containing two peripheral sugar branches composed of N-acetylglucosamine, mannose, galactose and sialic acid. The E2 eluted like glycopeptides containing only N-acetylglucosamine and mannose.  相似文献   

13.
Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.  相似文献   

14.
Glycoprotein C (gC) was purified by immunoabsorbent from herpes simplex virus type-1-infected BHK cells labeled with [14C]glucosamine for 11 h and chased for 3 h. Glycopeptides obtained by pronase digestion of gC were fractionated by Bio-Gel filtration and concanavalin A-Sepharose chromatography. Each glycopeptide fraction was analyzed for amino sugar composition by thin-layer chromatography. The majority of radioactivity was recovered as N-acetylglucosamine, but a significant amount of labeled N-acetylgalactosamine was detected and recovered preferentially in some glycopeptide species. Mild alkaline borohydride treatment of the glycopeptides resulted in the release of small degradation products which contained N-acetylgalactosaminitol as the major labeled component and a drastic reduction of N-acetylgalactosamine in the residual glycopeptides. These results demonstrated that gC carries O-glycosidically linked oligosaccharides in addition to the N-linked di- and triantennary glycans previously described (F. Serafini-Cessi, F. Dall'Olio, L. Pereira, and G. Campadelli-Fiume, J. Virol. 51:838-844, 1984). Chromatographic behavior on DEAE-Sephacel chromatography and neuraminidase digestion of O-linked oligosaccharides indicated the presence of two major sialylated species carrying one and two sialic acid residues, respectively. The characterization of a peculiar glycopeptide species supported the notion that some of the O-linked oligosaccharides are bound to a cluster of hydroxyamino acids located near an N-glycosylation site which carries one N-linked diantennary oligosaccharide.  相似文献   

15.
After exhaustive pronase digestion, purification by gel filtration and affinity chromatography on concanavalin A, three glycopeptide fractions were obtained from rat hemopexin. Two fractions (I and II) were concanavalin A non-reactive and one (III) was concanavalin A reactive. On the basis of carbohydrate composition, methylation analysis and proton nuclear magnetic resonance spectroscopy, the primary structure of the glycan in fraction III is proposed as being a mixture of mono- and di-sialo-diantennae of the N-glycosidic, N- acetyllactosamine type. Hydrazinolysis of glycopeptides not binding to concanavalin A yielded mixtures of oligosaccharides for both fractions. These oligosaccharides were separated by HPLC; the molar composition of each of them is given. These data suggest that rat hemopexin contains, among others, a diantennary structure bearing three sialic acid residues.  相似文献   

16.
[3H]Mannose-labelled glycopeptides in the slices of livers from neonatal and 1-, 2-, 3- and 5-week-old rats were characterized by column chromatographies on Sephadex G-50 and concanavalin A-Sepharose and by endo-beta-N-acetylglucosaminidase H digestion. The proportion of complex-type glycopeptides was increased with time until 2 weeks post partum and then returned to the neonatal level. This was mainly due to the increased proportion of concanavalin A-bound (biantennary) species. These changes were accompanied by consistent changes in the activities of processing enzymes in liver microsomal fraction, especially of N-acetylglucosaminyltransferase I. Complex-type glycopeptides from neonatal and 2- and 5-week-old rat livers were further characterized by column chromatographies on Bio-Gel P-6 and DE 52 DEAE-cellulose in combination with neuraminidase digestion. No significant difference was found between concanavalin A-bound species from neonatal liver and those from liver 5 weeks post partum, most of which were sialylated. Concanavalin A-bound species 2 weeks post partum were comparatively smaller in size and less sialylated. On the other hand, there was no significant difference among concanavalin A-unbound species from the three different sources, most of which were sialylated. Since glycoproteins from regenerating rat liver also contain a higher proportion of complex-type oligosaccharides, as previously reported, such changes in N-linked oligosaccharides of glycoproteins may be related to control of the growth of liver cells.  相似文献   

17.
Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).  相似文献   

18.
Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.  相似文献   

19.
In the light of the hypothesis that the kappa light chain accumulation in massive cutaneous hyalinosis (MCH) is related to an abnormal glycosylation pattern, we analyzed the oligosaccharide structures of the cold precipitable kappa light chains excreted in the urine of an MCH patient.The MCH kappa chains contain about 25% carbohydate. Concanavalin A-Sepharose affinity chromatography of the glycopeptides obtained from pronase digests showed that the bulk of the glycopeptides (82%) did not bind to the column; 16% had a weak affinity, and 2% a strong affinity. Methylation analyses indicated that the unbound fraction consisted mainly of tetra-antennary glycopeptides, but tri-antennary and bisecting structures were also found. Most of the weakly-bound glycopeptides had a biantennary carbohydrate structure.  相似文献   

20.
Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl alpha-D-mannopyranoside and appeared to be alpha-mannan, while that which passed through the column was virtually all beta-glucan. Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the alpha-mannan contained predominantly alpha-(1 leads to 2)-linked side chains branching from an alpha-(1 leads to 6)-linked backbone at the (1 leads to 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The beta-glucan seemed to contain mainly (1 leads to 6)-linked side chains branching from a (1 leads to 3)-linked backbone at the (1 leads to 3,6)-linked glucopyranosyl residues.  相似文献   

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