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1.
Andrea Hoffmann Manfred Nimtz Ulrich Wurster † Harald S. Conradt 《Journal of neurochemistry》1994,63(6):2185-2196
Abstract: The carbohydrate structures of β-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn29 and Asn56 ). Enzymatically released N -glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N -acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N -acetylglucosamine residues of nearly all structures were found to be α1,6-fucosylated. Peripheral fucose (about 20%α1,3-linked to N -acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N -acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N -acetylglucosamine only were present. At least 20 different N -glycans were identified. Analysis of the site-specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N -acetylglucosamine, as well as terminal N -acetylglucosamine and galactose residues, and significant amounts of N -acetylneuraminic acid in α2,3 linkage), this protein can be classified as "brain-type" glycosylated. 相似文献
2.
Yasuda J Eguchi H Fujiwara N Ookawara T Kojima S Yamaguchi Y Nishimura M Fujimoto J Suzuki K 《Biochemical and biophysical research communications》2006,342(1):127-134
The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, spontaneously develops hepatitis as the result of abnormal copper accumulation in liver. The findings of this study show that copper, hydrogen peroxide, and lipid peroxides accumulate to drastically high levels in LEC rat serum in acute hepatitis but not chronic hepatitis. The effect of these reactive oxygen species (ROS) on oligosaccharides of glycoproteins in the LEC rat serum was examined. Lectin blot and lectin ELISA analyses showed that sialic acid and galactose residues of serum glycoproteins including transferrin were decreased in acute hepatitis. Further analyses of oligosaccharide structures of transferrin demonstrated that di-sialylated and asialo-agalacto biantennary sugar chains, but not tri-sialylated sugar chain, exist on transferrin in the acute hepatitis rats. In addition, treatment of non-hepatitis rat serum with copper ions and hydrogen peroxide decreased tri-sialylated sugar chain of the normal transferrin and increased di-sialylated and asialo-agalacto biantennary sugar chains. This is the first evidence to show that ROS result in the cleavage of oligosaccharides of glycoproteins in vivo, and indicate this cleavage of oligosaccharides may contribute the development of acute hepatitis. 相似文献
3.
J W Webb K Jiang B L Gillece-Castro A L Tarentino T H Plummer J C Byrd S J Fisher A L Burlingame 《Analytical biochemistry》1988,169(2):337-349
We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples. 相似文献
4.
Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding. 相似文献
5.
N P Arbatski? A O Zheltova D V Iurtov V A Derevitskaia N K Kochetkov 《Bioorganicheskaia khimiia》1989,15(12):1641-1648
The structure and heterogeneity of carbohydrate chains of hemagglutinin (HA) and neuraminidase (NA), the surface glycoproteins of influenza virus A/Krasnodar/101/59 (H2N2), were investigated. Hemagglutinin was reduced with beta-mercaptoethanol and its heavy (HA1) and light (HA2) chains were separated by gel chromatography. Amino acid and sugar composition of HA1, HA2 and NA was elucidated. The carbohydrate chains of the glycoproteins were cleaved off by the alkaline LiBH4 treatment and oligosaccharides were reduced with NaB[3H]4. They were fractionated by subsequent two-step HPLC on Ultrasphere-C8 and Zorbax-NH2 columns with simultaneous identification using nonlabelled oligosaccharides of known structures. Some of the major oligosaccharides isolated from HA1, HA2 and NA were thus identified as high mannose chains, containing 5-9 mannose residues, and complex chains, first of all biantennary chains having or not having bisecting N-acetylglucosamine and/or fucose residues. The approach which has been developed enables one to study the structure and heterogeneity of carbohydrate chains starting from one nmole of a desialylated N-glycoprotein. 相似文献
6.
Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast. 总被引:3,自引:0,他引:3
C C Ip W J Miller D J Kubek A M Strang H van Halbeek S J Piesecki J A Alhadeff 《Biochemistry》1992,31(1):285-295
The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins. 相似文献
7.
Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine beta-hydroxylase 总被引:2,自引:0,他引:2
R K Margolis J Finne T Krusius R U Margolis 《Archives of biochemistry and biophysics》1984,228(2):443-449
Dopamine beta-hydroxylase present in the soluble matrix of bovine adrenal medullary chromaffin granules contains biantennary complex oligosaccharides and high-mannose oligosaccharides in a molar ratio of approximately 2:1. The high-mannose oligosaccharides contain an average of six mannose residues. The largest biantennary oligosaccharides (40% of the total) have two complete peripheral branches consisting of sialic acid-galactose-N-acetylglucosamine, but an equal proportion lack sialic acid on one branch and the remainder lack N-acetylglucosamine and/or galactose. Affinity chromatography on lentil lectin-agarose demonstrated that 84% of the dopamine beta-hydroxylase biantennary oligosaccharides are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. Based on carbohydrate concentration and the proportions of biantennary and high-mannose oligosaccharides, it would appear that the four dopamine beta-hydroxylase subunits of Mr congruent to 75,000 are not identical with respect to their oligosaccharide moieties. In chromaffin granule membranes, high-mannose and biantennary oligosaccharides comprise 20 and 35%, respectively, of the glycoprotein carbohydrate. Almost 40% is present in the form of large complex oligosaccharides with three or more antennas, less than 3% of which have both a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Chromaffin granule membranes also contain a small proportion (approximately 6%) of O-glycosidically linked glycoprotein oligosaccharides which are predominantly monosialyl derivatives of galactosyl-N-acetylgalactosamine. The ratio of N-acetyl- to N-glycolylneuraminic acid in dopamine beta-hydroxylase and the glycoproteins of chromaffin granule membranes is approximately 1.5:1, which is within the same range as that previously found in membrane gangliosides and in the chromogranins isolated from the soluble granule matrix. 相似文献
8.
Differential effects of oncogenic transformation on N-linked oligosaccharide processing at individual glycosylation sites of viral glycoproteins 总被引:5,自引:0,他引:5
S C Hubbard 《The Journal of biological chemistry》1987,262(34):16403-16411
Hamster sarcoma virus (HSV) transformation of Nil-8 fibroblasts is associated with an increase in the average size of N-acetyllactosamine (complex) type N-linked glycans due to an increase in both the average number of branches/chain and in the fraction of N-linked glycans containing poly(GlcNAc(beta 1,3) Gal-(beta 1,4)) (polylactosaminylglycan) chains. Analysis of glycopeptides from the envelope glycoproteins of Sindbis virus and vesicular stomatitis virus (VSV) grown in Nil-8 and Nil/HSV cells indicated that the transformation-associated shift to larger N-linked oligosaccharides selectively affects some glycosylation sites far more than others. Glycosylation of the Sindbis virus glycoproteins and of Asn-179 of VSV G was similar in Nil-8 and Nil/HSV cells; oligosaccharide processing generally did not proceed beyond the biantennary complex stage. In contrast, Asn-336 of VSV G carried primarily biantennary complex glycans in Nil-8-grown virus (ratio, triantennary, and larger to biantennary complex glycans (tri+/bi) = 0.5) but more highly branched structures in Nil/HSV-grown virus (tri+/bi = 8.1). All of the triantennary or larger oligosaccharides from Asn-336 of Nil/HSV-grown VSV G bound to leukoagglutinating phytohemagglutinin-agarose, indicating the presence of a branch attached to the Man3GlcNAc2 core via a beta 1,6-linked GlcNAc residue and suggesting that increased UDP-GlcNAc:alpha-D-mannoside beta 1,6-N-acetylglucosaminyl transferase V (GlcNAc transferase V) activity accompanied transformation. At least 20% of these leukoagglutinating phytohemagglutinin-binding oligosaccharides were sensitive to an enzyme specific for polylactosaminylglycan chains, Escherichia freundii endo-beta-galactosidase. 相似文献
9.
Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins 总被引:1,自引:0,他引:1
S E Zamze D A Ashford E W Wooten T W Rademacher R A Dwek 《The Journal of biological chemistry》1991,266(30):20244-20261
The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa. 相似文献
10.
S Naik J E Oates A Dell G W Taylor P M Dey J B Pridham 《Biochemical and biophysical research communications》1985,132(1):1-7
A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N-acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - alpha-galactosidase I, an enzyme lectin from Vicia faba. The latter is shown to contain high mannose carbohydrate chains. 相似文献
11.
Structures of the carbohydrate moieties of human prostatic acid phosphatase elucidated by H1 nuclear magnetic resonance spectroscopy 总被引:1,自引:0,他引:1
The structures of the oligosaccharides comprising the carbohydrate moieties of human prostatic acid phosphatase were elucidated by 1H NMR spectroscopy. Homogeneous enzyme was digested with Pronase P, and three asparagine-linked carbohydrate moieties were obtained upon fractionation of the digest using a concanavalin A-Sepharose affinity column. One fraction did not bind to the column, while the portion that did bind was separated into two fractions by elution with two concentrations of mannose. The high-resolution 1H NMR spectra for the three fractions were recorded at 470 MHz. From these data, the structures were deduced to be high mannose, partially sialylated and fucosylated biantennary complex, and fucosylated, partially sialylated triantennary complex oligosaccharides. No O-linked carbohydrate moiety was detected, although the possible presence of small O-linked oligosaccharides cannot be completely discounted from these data. 相似文献
12.
Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion. 总被引:2,自引:0,他引:2
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P. A. Schindler C. A. Settineri X. Collet C. J. Fielding A. L. Burlingame 《Protein science : a publication of the Protein Society》1995,4(4):791-803
Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272. 相似文献
13.
14.
Carbohydrate-structure-dependent recognition of desialylated serum glycoproteins in the liver and leucocytes. Two complementary systems. 总被引:2,自引:1,他引:1
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Oligosaccharides with four different types of branching were prepared from purified human transferrin, alpha 2-macroglobulin, caeruloplasmin and alpha 1-acid glycoprotein and labelled with NaBH3 3H. Binding of these oligosaccharides to rat liver plasma membrane, rat leucocytes, pig liver plasma membranes and pig leucocyte plasma membranes was investigated. A striking dependence of binding on oligosaccharide branching was observed. The values of apparent association constants Ka at 4 degrees C vary from 10(6) M-1 (biantennary structure) to 10(9) M-1 (tetra-antennary structure) in the liver, whereas in the leucocytes the Ka values were found to be of reversed order, from 1.8 X 10(9) M-1 for biantennary to 2.2 X 10(6) M-1 for tetra-antennary structures. The binding is completely inhibited by 150 mM-D-galactose, but 150 mM-D-mannose has almost no effect on binding. Leucocyte plasma membranes bind preferentially 125I-asialoglycoproteins with biantennary oligosaccharides, thus completing the specificity pattern of the hepatic recognition system for desialylated glycoproteins. Possible physiological roles of these two complementary recognition systems under normal and pathological conditions are discussed. 相似文献
15.
Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin 总被引:18,自引:0,他引:18
Laminin, a high molecular weight (1,000,000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chromatography using the alpha-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrate by weight. Methylation analysis revealed that the oligosaccharides of laminin contained bi- and triantennary chains, the blood group I structure, and repeating sequences of 3Gal beta 1,4GlcNAc beta 1 units. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretarded and a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl alpha-D-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing alpha-D-galactopyranosyl unit. The Con A reactive oligosaccharides were subjected to sequential digestion with endo- and exoglycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complex and biantennary complex structures containing poly(lactosaminyl) repeating units. The poly(lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly(lactosamine) structures. All sialic acid is present as N-acetylneuraminic acid linked alpha 2,3 to galactose. 相似文献
16.
Indravathamma Poola 《Carbohydrate polymers》1989,10(4):281-288
Rice seeds contain a 2-acetamido-2-deoxy-
-glucose-specific lectin. It has an Mr of 36 000 and is composed of two identical, non-covalently bound subunits of Mr 18 000. Each subunit consists of two disulfide-linked polypeptide chains of Mrs 10 000 and 8000. The lectin activity is highly stable to several chemical denaturants and heat treatment. The lectin interacts with glycoproteins, which have either clustered O-linked oligosaccharides or N-linked oligosaccharides. The N-linked glycoproteins include high
-mannose, hybrid and complex biantennary structures. 相似文献
17.
S J Swiedler J H Freed A L Tarentino T H Plummer G W Hart 《The Journal of biological chemistry》1985,260(7):4046-4054
The influence of peptide structure of endogenous cell-surface glycoproteins on the branching and sialylation of their asparagine-linked oligosaccharides was evaluated in a murine B cell lymphoma, AKTB-1b. This cell line simultaneously synthesizes two classes of major histocompatibility antigens that, within each class, share a high degree of amino acid sequence homology and possess potential N-linked glycosylation sites at invariant positions. [3H]Mannose-labeled oligosaccharides were released from each of 11 purified glycosylation sites by the almond peptide:N-glycosidase and analyzed by a variety of chromatographic procedures and glycosidase treatments. The data indicate: 1) a unique distribution of oligosaccharide structures is present at each glycosylation site; 2) each site-specific oligosaccharide pattern is highly reproducible, independent of the number of in vivo tumor passages. The heavy chain of the class I antigens, H-2Kk and H-2Dk contain two and three sites, respectively, in which biantennary structures predominate. However, each site varies with respect to the extent of sialylation and the proportions of more highly branched structures present. The class II antigens, I-Ak and I-Ek, each contain an alpha-chain site toward the N terminus and a single beta-chain site where the overall extent of sialylation is similar, yet the distributions of antennary structures are dramatically different for each. The alpha-chains of each class II antigen also contain a more C-terminal underglycosylated site where sialylation and branching are reduced to differing degrees depending upon the site. The influence of peptide structure on oligosaccharide microheterogeneity is manifest at two levels. First, the overall distributions of oligosaccharides at corresponding sites on structurally related glycoproteins are similar. Second, the specific "fingerprint" of sialylation and branching patterns at a particular site are reproducibly unique. These data suggest that subtle changes in peptide structure are reflected in the extent of sialylation and branching of oligosaccharides found at corresponding glycosylation sites of structurally related glycoproteins. 相似文献
18.
Harazono A Kawasaki N Itoh S Hashii N Ishii-Watabe A Kawanishi T Hayakawa T 《Analytical biochemistry》2006,348(2):259-268
Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc. 相似文献
19.
Pang PC Tissot B Drobnis EZ Sutovsky P Morris HR Clark GF Dell A 《The Journal of biological chemistry》2007,282(50):36593-36602
Human sperm lack major histocompatibility class I molecules, making them susceptible to lysis by natural killer (NK) cells. Major histocompatibility class I negative tumor cells block NK cell lysis by expressing sufficient amounts of bisecting type N-glycans on their surfaces. Therefore, sperm could employ the same strategy to evade NK cell lysis. The total N-glycans derived from sperm were sequenced using ultrasensitive mass spectrometric and conventional approaches. Three major classes of N-glycans were detected, (i) high mannose, (ii) biantennary bisecting type, and (iii) biantennary, triantennary, and tetraantennary oligosaccharides terminated with Lewisx and Lewisy sequences. Immunostaining of normal sperm showed that glycoproteins bearing Lewisy sequences are localized to the acrosome and not the plasma membrane. In contrast, defective sperm showed distinct surface labeling with anti-Lewisy antibody. The substantial expression of high mannose and complex type N-glycans terminated with Lewisx and Lewisy sequences suggests that sperm glycoproteins are highly decorated with ligands for DC-SIGN. Based on previous studies, the addition of such carbohydrate signals should inhibit antigen-specific responses directed against sperm glycoproteins in both the male and female reproductive systems. Thus, the major N-glycans of human sperm are associated with the inhibition of both innate and adaptive immune responses. These results provide more support for the eutherian fetoembryonic defense system hypothesis that links the expression of carbohydrate functional groups to the protection of gametes and the developing human in utero. This study also highlights the usefulness of glycomic profiling for revealing potential physiological functions of glycans expressed in specific cell types. 相似文献
20.
Kenji Mori Masanao Matsui Yusuke Sumiki 《Bioscience, biotechnology, and biochemistry》2013,77(1):72-73
Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M) was proved to act on complex type biantennary oligosaccharides of glycoproteins by using dansylated asparagine-linked and pyridylaminated oligosaccharides, as the substrate. The enzyme could act on both asialo- and sialo-biantennary oligosaccharides. This is the only endo-β-N-acetylglucosaminidase known to act on sialo glycans, though their activity for them was weak. The enzyme could liberate complex type biantennary oligosaccharides from native human asialotransferrin, which was ascertained by a combination of the pyridylaminated method and HPLC. The enzyme had substrate specificity for high-mannose type oligosaccharides different from those of the endo-β-N-acetylglucosaminidases of other microorganisms: ovalbumin glycopeptide-IV was a better substrate for Endo-M than glycopeptide-V. The enzyme could act on complex type triantennary oligosaccharides of dansylated glycopeptide prepared from calf fetuin. The enzyme had various novel specificities in regard to activities on complex type and high-mannose type oligosaccharides in glycoproteins. 相似文献