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1.
The asparagine-linked glycopeptides (N-glycans) of a ricin-resistant mutant of baby hamster kidney (BHK) cells, RicR21, have been isolated and fractionated from a Pronase digest of disrupted cells by concanavalin A (Con A)-Sepharose chromatography, ion-exchange chromatography, and lentil lectin chromatography. The structures of all the major N-glycans have been determined by 500-MHz H NMR spectroscopy. RicR21 synthesizes only hybrid and high-mannose N-glycans. All the hybrid structures contain only three mannose residues. The major hybrid glycopeptide has the following structure: (Formula: see text). There is also about 15% of the nonfucosylated species present. Only a small amount (less than or equal to 5%) of the asialo hybrid is produced. Branched hybrid N-glycans are also present in RicR21 cells, containing two complex antenna linked beta 1----2 and beta 1----4 to the Man alpha 1----3 arm; about 70% of this species is core fucosylated. Man6GlcNAc2 glycopeptide is the most abundant (about 70%) of the high-mannose N-glycans. These studies account for the very poor ricin binding property of this mutant, as the sialic acid residues of the major hybrid N-glycan are exclusively linked alpha 2----3 to galactose and ricin is unable to bind to alpha 2----3-substituted galactosyl residues [Baenziger, J. U., & Fiete, D. (1979) J. Biol. Chem. 254, 9795-9799].  相似文献   

2.
Previous work has shown that two ricin-resistant mutants of baby hamster kidney (BHK) cells, RicR15 and RicR19, synthesize only hybrid and oligomannose-type asparagine-linked oligosaccharides [Hughes, R. C. and Mills, G. (1985) Biochem. J. 226, 487-498]. In the present report glycopeptides were released from disrupted cells by exhaustive digestion with pronase, fractionated by chromatography on concanavalin-A--Sepharose, DEAE-Sephacel and lentil-lectin--Sepharose and characterized by 500-MHz 1H-NMR spectroscopy. The major hybrid structure identified in both cell lines contains five mannose residues and the sequence NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----2 linked to the alpha 1----3 arm mannose of the core pentasaccharide. Analysis of extracts of normal or mutant cells has shown in the mutants a deficiency in alpha-mannosidase activity measured with p-nitrophenyl alpha-mannoside. This activity is swainsonine-sensitive and exhibits a pH optimum at about 6-6.5. Assays using a specific substrate for alpha-mannosidase II, a terminal processing glycosidase in conversion of penta-mannose hybrid intermediates to complex N-glycans, reveals a reduced activity in RicR15 cells. Analysis of glycopeptides obtained from cells labelled with [3H]fucose or [3H]galactose revealed a small proportion of branched complex N-glycans of normal structure in mutant cells.  相似文献   

3.
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   

4.
The purpose of the study was to examine the role of N-linked oligosaccharides in preventing combination of free alpha molecules with human chorionic gonadotropin (hCG)-beta subunit to form the intact hormone, hCG. Culture media from JEG cells incubated in the presence or absence of Swainsonine were filtered on Sephadex G-100, and free alpha was identified by radioimmunoassay. Swainsonine interferes with glycosylation by inhibiting alpha-mannosidase II, resulting in formation of hybrid structures. Approximately 50% of the free alpha molecules from Swainsonine-treated cells (Swainsonine pool 2) had an apparent molecular size that was smaller than that of free alpha from control cells. The oligosaccharides of control alpha molecules were resistant to endo-beta-N-acetylglucosaminidase H treatment. In contrast, virtually all of the Swainsonine free alpha molecules contained endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides. Swainsonine free alpha and control free alpha molecules were incubated with an excess of hCG-beta subunit, followed by chromatography on Sephadex G-100. Each fraction was assayed by radioimmunoassay for intact hCG and for alpha. Less than 10% of control free alpha molecules combined with hCG-beta. In contrast, 54% of Swainsonine alpha pool 2 and 40% of Swainsonine alpha pool 1 combined with beta to form hCG. Thus, modulation of N-linked oligosaccharide processing converted free alpha molecules to forms that can combine with hCG-beta. These results indicate that the inability of a substantial portion of control free alpha molecules to combine with hCG-beta is due to the presence of N-linked oligosaccharide structures that interfere with combination.  相似文献   

5.
The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.  相似文献   

6.
Metabolism of cartilage proteins in cultured tissue sections.   总被引:4,自引:3,他引:1       下载免费PDF全文
The asparagine-linked oligosaccharides of the complex acidic-type from [3H]mannose-, [3H]glucosamine- or [3H]galactose-labelled membrane glycoproteins of BHK21 cells and Rous-sarcoma virus were analysed by gel filtration combined with extensive digestion with endo- and exo-glycosidases from bacterial and eukaryotic sources. The neutral products from the digestion with a mixture of exoglycosidases and endo-beta-N-acetylglucosaminidase D from Diplococcus pneumoniae included a series of [3H]mannose- and [3H]glucosamine-labelled neutral oligosaccharides that were all converted by digestion with eukaryotic beta-N-acetylglucosaminidases into free N-acetylglucosamine and a small oligomannosyl core containing two alpha-linked mannose residues and a third mannose residue beta-linked to N-acetylglucosamine. These studies suggested that the complex acidic-type oligosaccharides from cellular and viral membrane glycoproteins contained a common oligomannosyl core region (Man2 alpha leads to Man beta leads to GlcNAc2), with heterogeneity in the number and/or linkage of outer branch N-acetylglucosamine residues resulting in partial resistance to beta-N-acetylglucosaminidase from a bacterial source.  相似文献   

7.
A novel lysosomal alpha-mannosidase, with unique substrate specificity, has been partially purified from human spleen by chromatography through concanavalin A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. This enzyme can catalyze the hydrolysis of only 1 mannose residue, that which is alpha(1----6)-linked to the beta-linked mannose in the core of N-linked glycans, as found in the oligosaccharides Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc and Man alpha(1----6)Man beta(1----4) GlcNAc. The newly described alpha-mannosidase does not catalyze the hydrolysis of mannose residues outside of the core, even if they are alpha(1----6)-linked, and is not active on the other alpha-linked mannose in the core, which is (1----3)-linked. The narrow specificity of the novel mannosidase contrasts sharply with that of the major lysosomal alpha-mannosidase, which is able to catalyze the degradation of oligosaccharides containing diverse linkage and branching patterns of the mannose residues. Importantly, although the major mannosidase readily catalyzes the hydrolysis of the core alpha(1----3)-linked mannose, it is poorly active towards the alpha(1----6)-linked mannose, i.e. the very same mannose residue for which the newly characterized mannosidase is specific. The novel enzyme is further differentiated from the major lysosomal alpha-mannosidase by its inability to catalyze the efficient hydrolysis of the synthetic substrate p-nitrophenyl alpha-mannoside, and by the strong stimulation of its activity by Co2+ and Zn2+. Similarly to the major mannosidase, it is strongly inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, but not by deoxymannojirimycin. The presence of this novel alpha-mannosidase activity in human tissues provides the best explanation, to date, for the structures of the oligosaccharides stored in human alpha-mannosidosis. In this condition the major lysosomal alpha-mannosidase activity is severely deficient, but apparently the alpha(1----6)-mannosidase is unaffected, so that the oligosaccharide structures reflect the unique specificity of this enzyme.  相似文献   

8.
We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.  相似文献   

9.
The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.  相似文献   

10.
The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.  相似文献   

11.
Recombinant human tissue plasminogen activator (rt-PA), produced by expression in Chinese hamster ovary cells, is a fibrin-specific plasminogen activator which has been approved for clinical use in the treatment of myocardial infarction. In this study, the structures of the Asn-linked oligosaccharides of Chinese hamster ovary-expressed rt-PA have been elucidated. High mannose and hybrid oligosaccharides were released from the protein by endoglycosidase H digestion, whereas N-acetyllactosamine-type ("complex") oligosaccharides were released by peptide:N-glycosidase F digestion. The oligosaccharides were fractionated by gel permeation chromatography and anion exchange high performance liquid chromatography (HPLC), and their structures were analyzed by composition and methylation analysis, high pH anion exchange chromatography, fast atom bombardment-mass spectrometry (FAB-MS), and 500-MHz 1H NMR spectroscopy. High mannose oligosaccharides were found to account for 38% of the total carbohydrate content of rt-PA and consisted of Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2 in the ratio 1.8:1.7:1. Two hybrid oligosaccharides were identified and accounted for 3% of the carbohydrate of rt-PA. The N-acetyllactosamine-type oligosaccharides were found to comprise diantennary (34% of total carbohydrate), 2,4-branched triantennary (11%), 2,6-branched triantennary (9%), and tetraantennary (5%) structures. Sialylation of these oligosaccharides was by alpha (2----3) linkages to galactose. Most (greater than 90%) of the N-acetyllactosamine-type structures contained fucose alpha (1----6) linked to the Asn-linked N-acetylglucosamine residue. The distribution of oligosaccharide structures at individual glycosylation sites (Asn residues 117, 184, and 448) was also determined. rt-PA exists as two variants that differ by the presence (type I) or absence (type II) of carbohydrate at Asn-184. Tryptic glycopeptides were isolated by reversed phase high performance liquid chromatography and treated with peptide:N-glycosidase F. The oligosaccharides released from each glycosylation site were analyzed by high pH anion exchange chromatography. By this analysis, Asn-117 was demonstrated to carry exclusively high mannose oligosaccharides. When glycosylated, Asn-184 carried diantennary, 2,4-branched triantennary, 2,6-branched triantennary, and tetraantennary N- acetyllactosamine oligosaccharides in the ratio 9.0:4.5:1.4:1. Asn- 448 carried the same types of oligosaccharides, but in the ratio 7.5:1.6:2.1:1. The distributions of Asn-linked oligosaccharides at positions 117 and 448 were found not to be affected by the presence or absence of carbohydrate at position 184. The relevance of the  相似文献   

12.
The N-glycans of human serum transferrin produced in Trichopulsia ni cells were analyzed to examine N-linked oligosaccharide processing in insect cells. Metabolic radiolabeling of the intra- and extracellular protein fractions revealed the presence of multiple transferrin glycoforms with molecular weights lower than that observed for native human transferrin. Consequently, the N-glycan structures of transferrin in the culture medium were determined using three-dimensional high performance liquid chromatography. The attached oligosaccharides included high mannose, paucimannosidic, and hybrid structures with over 50% of these structures containing one fucose, alpha(1,6)-, or two fucoses, alpha(1,6)- and alpha(1,3)-, linked to the Asn-linked N-acetylglucosamine. Neither sialic acid nor galactose was detected on any of the N-glycans. However, when transferrin was coexpressed with beta(1,4)-galactosyltransferase three additional galactose-containing hybrid oligosaccharides were obtained. The galactose attachments were exclusive to the alpha(1, 3)-mannose branch and the structures varied by the presence of zero, one, or two attached fucose residues. Furthermore, the presence of the galactosyltransferase appeared to reduce the number of paucimannosidic structures, which suggests that galactose attachment inhibits the ability of hexosaminidase activity to remove the terminal N-acetylglucosamine. The ability to promote galactosylation and reduce paucimannosidic N-glycans suggests that the oligosaccharide processing pathway in insect cells may be manipulated to mimic more closely that of mammalian cells.  相似文献   

13.
Five cell lines of ricin-resistant BHK cells have been assayed for gross carbohydrate analysis of cellular glycoproteins, for the activities of several glycosidases and of specific glycosyl transferases active in assembly of N-glycans of glycoproteins. The latter enzymes include sialyl transferase using asialofetuin as glycosyl acceptor, fucosyl transferases using asialofetuin and asialoagalactofetuin acceptors, galactosyl transferases using ovalbumin, ovomucoid and N-acetylglucosamine as acceptors and N-acetylglucosaminyl transferases using ovalbumin and glycopeptides as acceptors. Cell line RicR14, binding less ricin than normal BHK cells, contains reduced amounts of sialic acid, galactose and N-acetylglucosamine in cellular glycoproteins and lacks almost completely N-acetylglucosamine transferase I, an essential enzyme in assembly of ricin-binding carbohydrate sequences of N-glycans. These cells also contain reduced levels of N-acetylglucosamine transferase II active on a product of N-acetylglucosamine transferase I action. Sialyl transferase activity is severely depressed while fucose-(alpha 1 leads to 6)-N-acetylglucosamine fucosyl transferase activity is increased. Cell lines RicR15, 17, 19 and 21 showed partial deficiencies in galactosyl and N-acetylglucosaminyl transferases. A hypothesis is put forward to account for the different carbohydrate compositions and ricin binding properties of glycoproteins synthesised by these cells in terms of the determined enzyme defects, the normal level of sialyl transferases detected in RicR15 and RicR21 cells and the elevated levels of sialyl and fucosyl transferases detected in RicR17 and 19 cells. None of the above changes in glycosyl transfer reactions in the RicR cell lines are due to enhanced glycosidase or sugar nucleotidase activities in the mutant cells.  相似文献   

14.
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   

15.
Structures of the sugar chains of mouse immunoglobulin G   总被引:2,自引:0,他引:2  
The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.  相似文献   

16.
N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146). Twenty-five major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Galalpha(1,4)Galbeta(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. N-Acetylneuraminic acids were alpha(2,6)-linked only to beta-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.  相似文献   

17.
Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell surface glycoproteins that are involved in various leukocyte functions. The asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialdase-treated acidic oligosaccharides were fractionated by chromatography on lectin columns followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that Leu-CAMs contain mainly high mannose type and high molecular weight complex type sugar chains. The latter sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc beta 1----groups together with the Gal beta 1----4GlcNAc group in their outer-chain moieties. In addition to these sugar chains, a small amount of monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs. Furthermore, analysis of the asparagine-linked sugar chains released from the beta-subunit of Leu-CAMs by a series of lectin chromatography showed that subunit-specific glycosylation is not observed between the alpha- and beta-subunits of Leu-CAMs.  相似文献   

18.
The carbohydrate moieties of human urinary ribonuclease UL   总被引:2,自引:0,他引:2  
Ribonuclease UL purified from pooled human urine contains approximately 20.7% of neutral sugar and 7.8% of aminosugar. All sugars were quantitatively released as oligosaccharides on hydrazinolysis. The oligosaccharides were converted to tritium-labeled oligosaccharides on reduction with NaB3H4. The radioactive oligosaccharide fraction was separated into a neutral and an acidic fraction on paper electrophoresis. All oligosaccharides in the acidic fraction could be converted to neutral oligosaccharides with the release of one sialic acid residue by sialidase digestion. Both fractions were shown to be mixtures of more than fourteen oligosaccharides by gel permeation chromatography. Structural studies on these oligosaccharides involving sequential exoglycosidase digestion in combination with methylation analysis revealed that ribonuclease UL contains sialylated and non-sialylated mono, bi-, tri-, and tetraantennary complex type sugar chains with N-acetyllactosamine outer chains, and tri- and tetraantennary complex type sugar chains with various numbers of Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains. An important finding was that all sialic acid residues in the acidic oligosaccharides only occur as the Sia alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 group. Both fucosylated and non-fucosylated trimannosyl cores were found among the asparagine-linked sugar chains of ribonuclease UL.  相似文献   

19.
The carbohydrate chains of the respiratory-mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive straight-phase high-performance liquid chromatography (HPLC) on a silica-based alkylamine column, and reverse-phase HPLC on a silica-based octadecyl column, respectively. The structures of the oligosaccharidealditols were determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are: (formula; see text) 23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631-642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643-654]. The sialic acid residue was found to occur either in alpha (2----3)- or alpha (2----6)-linkage to a galactosyl residue or in alpha (2----6)-linkage to GalNAc-ol.  相似文献   

20.
The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.  相似文献   

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