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1.
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   

2.
Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.  相似文献   

3.
Proteomic techniques, such as HPLC coupled to tandem mass spectrometry (LC-MS/MS), have proved useful for the identification of specific glycosylation sites on glycoproteins (glycoproteomics). Glycosylation sites on glycopeptides produced by trypsinization of complex glycoprotein mixtures, however, are particularly difficult to identify both because a repertoire of glycans may be expressed at a particular glycosylation site, and because glycopeptides are usually present in relatively low abundance (2% to 5%) in peptide mixtures compared to nonglycosylated peptides. Previously reported methods to facilitate glycopeptide identification require either several pre-enrichment steps, involve complex derivatization procedures, or are restricted to a subset of all the glycan structures that are present in a glycoprotein mixture. Because the N-linked glycans expressed on tryptic glycopeptides contribute substantially to their mass, we demonstrate that size exclusion chromatography (SEC) provided a significant enrichment of N-linked glycopeptides relative to nonglycosylated peptides. The glycosylated peptides were then identified by LC-MS/MS after treatment with PNGase-F by the monoisotopic mass increase of 0.984 Da caused by the deglycosylation of the peptide. Analyses performed on human serum showed that this SEC glycopeptide isolation procedure results in at least a 3-fold increase in the total number of glycopeptides identified by LC-MS/MS, demonstrating that this simple, nonselective, rapid method is an effective tool to facilitate the identification of peptides with N-linked glycosylation sites.  相似文献   

4.
We have carried out detailed structural studies of the glycopeptides of glycoprotein gD of herpes simplex virus types 1 and 2. We first examined and compared the number of N-asparagine-linked oligosaccharides present in each glycoprotein. We found that treatment of either pgD-1 or pgD-2 with endo-β-N-acetylglucosaminidase H (Endo H) generated three polypeptides which migrated more rapidly than pgD on gradient sodium dodecyl sulfate-polyacrylamide gels. Two of the faster-migrating polypeptides were labeled with [3H]mannose, suggesting that both pgD-1 and pgD-2 contained three N-asparagine-linked oligosaccharides. Second, we characterized the [3H]mannose-labeled tryptic peptides of pgD-1 and pgD-2. We found that both glycoproteins contained three tryptic glycopeptides, termed glycopeptides 1, 2, and 3. Gel filtration studies indicated that the molecular weights of these three peptides were approximately 10,000, 3,900, and 1,800, respectively, for both pgD-1 and pgD-2. Three methods were employed to determine the size of the attached oligosaccharides. First, the [3H]mannose-labeled glycopeptides were treated with Endo H, and the released oligosaccharide was chromatographed on Bio-Gel P6. The size of this molecule was estimated to be approximately 1,200 daltons. Second, Endo H treatment of [35S]methionine-labeled glycopeptide 2 reduced the molecular size of this peptide from approximately 3,900 to approximately 2,400 daltons. Third, glycopeptide 2 isolated from the gD-like molecule formed in the presence of tunicamycin was approximately 2,200 daltons. From these experiments, the size of each N-asparagine-linked oligosaccharide was estimated to be approximately 1,400 to 1,600 daltons. Our experiments indicated that glycopeptides 2 and 3 each contained one N-asparagine-linked oligosaccharide chain. Although glycopeptide 1 was large enough to accommodate more than one oligosaccharide chain, the experiments with Endo H treatment of the glycoprotein indicated that there were only three N-asparagine-linked oligosaccharides present in pgD-1 and pgD-2. Further studies of the tryptic glycopeptides by reverse-phase high-performance liquid chromatography indicated that all of the glycopeptides were hydrophobic in nature. In the case of glycopeptide 2, we observed that when the carbohydrate was not present, the hydrophobicity of the peptide increased. The properties of the tryptic glycopeptides of pgD-1 were compared with the properties predicted from the deduced amino acid sequence of gD-1. The size and amino acid composition compared favorably for glycopeptides 1 and 2. Glycopeptide 3 appeared to be somewhat smaller than would be predicted from the deduced sequence of gD-1. It appears that all three potential glycosylation sites predicted by the amino acid sequence are utilized in gD-1 and that a similar number of glycosylation sites are present in gD-2.  相似文献   

5.
Plasma desorption mass spectrometry (PD-MS) was used to assess the molecular weight heterogeneity of glycopeptides (6-12 amino acids) from each of the three N-linked glycosylation sites of bovine fetuin (R.G. Spiro (1962) J. Biol. Chem. 237, 382-388). The glycopeptides were purified by a combination of anion exchange chromatography and reverse-phase HPLC. Since no detectable fragmentation was observed in the PD-MS of these asialoglycopeptides, the observation of multiple molecular ions could be attributed to either carbohydrate or peptide heterogeneity. Assignment of molecular ions, within 3 to 5 amu of the theoretical mass, of glycopeptides from each glycosylation site was made from amino acid composition, peptide sequence around the glycosylation sites, and previously reported triantennary oligosaccharide structures (B. Nilsson, N.E. Nordén, and S. Svensson (1979) J. Biol. Chem. 254, 4545-4553). Ion groups differing in mass by one N-acetyllactosamine unit were observed in glycopeptides from the Asn-Asp and Asn-Cys sites, localizing these previously observed biantennary oligosaccharide structures (R.R. Townsend, M.R. Hardy, T.C. Wong, and Y.C. Lee (1986) Biochemistry 25, 5716-5725; S. Takasaki and A. Kobata (1986) Biochemistry 25, 5709-5715) to these two sites. The presence of biantennary oligosaccharides at the Asn-Asp sites could be substantiated using 1H NMR but were not detected in the Asn-Cys glycopeptides. PD-MS was also implemented in the purification protocol for these glycopeptides and proved to be useful in assessing purity of chromatographic fractions which were mixtures of glycopeptides displaying both carbohydrate and peptide heterogeneity. A preparation scheme was developed to obtain molecular ions of desialylated glycopeptides by PD-MS.  相似文献   

6.
Electrophoretically homogeneous type 1 (GP-C1 and GP-C2), type 2 (GP-C3a and GP-C3b,) and type 3 (GP-D1, and GP-D2) glycopeptides fromAspergillus niger glucoamylase II (Manjunath and Raghavendra Rao, preceding paper) were separately treated with alkaline borohydride. The (\-eliminated oligosaccharides were subjected to single and sequential digestion with specific glycosidases and the products analysed by gas liquid chromatography. The studies revealed that carbohydrate moieties were present as mannose, Man-Man-, and trisaccharide structures, namely, (a) GIc-Man-Man-, (b) Gal-Man-Man, (c) Man-Man-Man-, (d) GlcNAc-Man-Man-, and (e) Xyl-Man-Man. None of the glycopeptides contained all the trisaccharide structures (a) to (e). Type 1 glycopeptide contained structures (a), (b) and (c); type 2, (a) and (d) and type 3, (a), (b) and (e). The number of carbohydrate units (mono-, di-and trisaccharides) present in the major glycopeptides was determined and tentative structures for the glycopeptides proposed. Carbohydrate units appeared to occur in clusters of 4 to 7 in each glycopeptide, a structure unique to the carbohydrate moiety inAspergillus niger glucoamylase. Based on carbohydrate analysis and yields of glycopeptide, the number of units of each type of glycopeptide present in glucoamylase II was tentatively calculated to give two of type Man:Glc:Gal = 12–15:l:l, one of type Man:Glc:GlcN = 10-l1:1:2 and one of type Man :GIc :Gal:Xyl = 4–8:0.1:0.5-0.8:0.3-1 glycopeptides.  相似文献   

7.
Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.  相似文献   

8.
Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.  相似文献   

9.
Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.  相似文献   

10.
A chromatographic method was developed for quantitative analysis of site-specific microheterogeneity of the two N-linked glycosylation sites in recombinant human interferon-γ produced from Chinese hamster ovary (CHO) cell culture. After the interferon-γ was harvested by affinity chromatography, the tryptic digestion was carried out. The two glycopeptide pools, isolated from reversed-phase chromatography of tryptic digestion of interferon-γ, were subjected to further separation by hydrophilic interaction chromatography. Each peak in the chromatograms was identified by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF–MS). The overall elution order of the glycopeptides was the following: neutral glycopeptides, monosialylated glycopeptides, bisialylated glycopeptides, trisialylated glycopeptide and tetrasialylated glycopeptides. Based on the integrated peak area for each compound in the chromatograms, the percentage for each glycan was utilized to quantify the glycosylation pattern of the interferon-γ. Finally, sialylation and antennarity structure percentages at the two glycosylation sites were chosen as the quality indicators in process monitoring of interferon-γ production from a serum-free suspension-batch CHO culture.  相似文献   

11.
Endo β-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N′ -diacetylchitobiose, N,N′ -diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase)mixture readily leaved high mannose and complex oligosaccharides (neutral and sialyated) with common core α1–6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core α1–6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides. However, it was similar to Endo H when reduced ovalbumin oligosaccharides were used as substrates, consistent with the recently isolated Endo F subfraction F1 being similar to Endo H [Trimble, R. B. and Tarentino, a. L. (1991). J. Biol. Chem. 266, 1646]. Results obtained in this study suggest that the complex oligosaccharides cleaving enzymes F2 and F3 show high specificity towards peptide free oligosaccharides with the core α1-6 linked fucose, unlike the glycopeptide substrates. Therefore PNGase free Endo F1, F2 and F3 mixtures should be useful in the functional evaluation of the oligosaccharides in glycoproteins.  相似文献   

12.
A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.  相似文献   

13.
High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.  相似文献   

14.
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   

15.
In this report we describe an on-column method for glycopeptide enrichment with cellulose as a solid-phase extraction material. The method was developed using tryptic digests of several standard glycoproteins and validated with more complex standard protein digest mixtures. Glycopeptides of different masses containing neutral and acidic glycoforms of both N- and O-linked sugars were obtained in good yield by this method. Upon isolation, glycopeptides may be subjected to further glycoproteomic and glycomic workflows for the purpose of identifying glycoproteins present in the sample and characterizing their glycosylation sites, as well as their global and site-specific glycosylation profiles at the glycopeptide level. Detailed structural analysis of glycoforms may then be performed at the glycan level upon chemical or enzymatic release of the oligosaccharides. Aiming at complementing other purification methods, this technique is extremely simple, cost-effective, and efficient. Glycopeptide enrichment was verified and validated by nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining electron-transfer dissociation (ETD) and collision-activated dissociation (CAD) fragmentation techniques.  相似文献   

16.
Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.  相似文献   

17.
Some aberrant N‐glycosylations are being used as tumor markers, and glycoproteomics is expected to provide novel diagnosis markers and targets of drug developments. However, one has trouble in mass spectrometric glycoproteomics of membrane fraction because of lower intensity of glycopeptides in the existence of surfactants. Previously, we developed a glycopeptide enrichment method by acetone precipitation, and it was successfully applied to human serum glycoproteomics. In this study, we confirmed that this method is useful to remove the surfactants and applicable to membrane glycoproteomics. The glycoproteomic approach to the human fetal lung fibroblasts membrane fraction resulted in the identification of over 272 glycoforms on 63 sites of the 44 glycoproteins. According to the existing databases, the structural features on 41 sites are previously unreported. The most frequently occurring forms at N‐glycosylation site were high‐mannose type containing nine mannose residues (M9) and monosialo‐fucosylated biantennary oligosaccharides. Several unexpected N‐glycans, such as fucosylated complex‐type and fucosylated high‐mannose and/or fucosylated pauci‐mannose types were found in ER and lysosome proteins. Our method provides new insights into transport, biosynthesis, and degradation of glycoproteins.  相似文献   

18.
The technology of glycopeptide synthesis has recently developed into a fully mature science capable of creating diverse glycopeptides of biological interest, even in combinatorial displays. This has allowed biochemists to investigate substrate specificity in the biosynthetic processing and immunology of various protein glycoforms. The construction of all the mucin core structures and a varietyof cancer-related glycopeptides has facilitated detailed analysis of the interaction between MHC-bound glycopeptides and T cell receptors. Novel dendritic neoglycopeptide ligands have been shown to demonstrate high affinity for carbohydrate receptors and these interactions are highly dendrimer specific. Large complex N-linked oligosaccharides have been introduced into glycopeptides using synthetic or chemoenzymatic procedures, both methods affording pure glycopeptides corresponding to a single glycoform in preparative quantities. The improved availability of glycosyl transferases has led to increased use of chemoenzymatic synthesis. Chemical ligation has been introduced as a method of attaching glycans to peptide templates. Combinatorial synthesis and the analysis of resin-bound glycopeptide libraries have been successfully carried out by applying the ladder synthesis principle. Direct quantitative glycosylation of peptide templates on solid phase has paved the way for the synthesis of templated glycopeptide mixtures as libraries of libraries.  相似文献   

19.
Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), and capillary liquid chromatography–electrospray mass spectrometry (cLC–ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC–ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man8GlcNAc2) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC–PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.  相似文献   

20.
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   

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