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1.
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
2.
Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid 总被引:2,自引:1,他引:1
Duffin Kevin L; Lange Gary W.; Welply Joseph K.; Florman Richard; O'Brien Paul J.; Dell Anne; Reason Andrew J.; Morris Howard R.; Fliesler Steven J. 《Glycobiology》1993,3(4):365-380
The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn2 and Asn15. The predominant glycan (60% of total) had thestructure GlcNAcß12Man13(Man16)Manß14GlcNAcß14GlcNAc-(Asn),with the remaining structures containing 13 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn2 site. FAB-MS analysis of oligosaccharides releasedfrom the Asn2 site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc1Hex6HexNAc3(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin 相似文献
3.
Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry. 相似文献
4.
Joseph V. Lomino Andreas Naegeli Jared Orwenyo Mohammed N. Amin Markus Aebi Lai-Xi Wang 《Bioorganic & medicinal chemistry》2013,21(8):2262-2270
A chemoenyzmatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymatic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our experiments demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymatic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc–Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc–Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addition, the Glc–Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc–Asn linked glycopeptide. The chemoenzymatic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery. 相似文献
5.
von Witzendorff D Ekhlasi-Hundrieser M Dostalova Z Resch M Rath D Michelmann HW Töpfer-Petersen E 《Glycobiology》2005,15(5):475-488
The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides. 相似文献
6.
Characterization of the glycosylation of a human IgM produced by a human-mouse hybridoma 总被引:2,自引:2,他引:0
Monica Thomas J.; Williams Steven B.; Goochee Charles F.; Maiorella Brian L. 《Glycobiology》1995,5(2):175-185
We analysed the oligosaccharides of a human IgM produced bya human-human-mouse hybridoma at each of its five conservedheavy chain glycosylation sites. Consistent with previous reports,this IgM possesses sialylated oligosaccharides at Asn171, Asn332and Asn395, and high-mannose-type oligosaccharides at Asn402.In contrast to previous reports for human IgMs, we find thatAsn563 is not occupied by oligosaccharide on perhaps 25% ofIgM heavy chains, while occupied Asn563 sites contain both high-mannose-typeand sialylated oligosaccharides. These latter results are consistentwith the glycosylation at Asn563 previously reported for themouse MOPC 104E IgM. We demonstrate that both the human hybridomaIgM and the mouse MOPC 104E IgM are mixtures of pentamers andhexamers, raising the possibility that the unique findings concerningthe glycosylation at Asn563 in this study and the previous studyof the MOPC 104E IgM could be related, at least in part, tothe different packing requirements of the hexameric geometryand the accessibility of oligosaccharides in the hexameric geometryfor processing to complex type. In addition, we used high-pHanion-exchange (HPAE) chromatography, neutral anion-exchangechromatography, fluorophore-assisted carbohydrate electrophoresisand Western blots to compare the oligosaccharide compositionsof the human hybridoma IgM, pooled human serum IgM and two mousemonoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presenceof N-glycolylneuraminic acid (NeuGc) and N-acetymeuraminic acid(NeuAc) at a 2:1 ratio in the oligosaccharides of the humanhybridoma IgM. The presence of both NeuGc and NeuAc complicatesthe interpretation of HPAE chromato-graphs. glycosylation high-pH anion-exchange chromatography human IgM humanmouse hybridoma oligosaccharide 相似文献
7.
Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity. 总被引:17,自引:0,他引:17
T Tai K Yamashita M Ogata-Arakawa N Koide T Muramatsu S Iwashita Y Inoue A Kobata 《The Journal of biological chemistry》1975,250(21):8569-8575
Heterogeneities of the two ovalbumin glycopeptides, (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn, were revealed by borate paper electrophoresis of oligosaccharide alcohols obtained from the glycopeptides by endo-beta-N-acetylglucosaminidase H digestion and NaB3H4 reduction. The structures of the major components of the oligosaccharides were determined by the combination of methylation analysis, acetolysis, and alpha-mannosidase digestion. Based on the results, the whole structures of the major components of (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn were elucidated as Manalpha1 leads to 6[Manalpha1 leads to 3]-Manalpha1 leads to 6[Manalpha1 leads to 3[Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc leads to Asn and Manalpha1 leads to 6[Manalpha1 leads to 3]Manalpha1 leads to 6[Manalpha1 leads to 2Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to GlcNAc leads to Asn, respectively. Since endo-beta-N-acetylglucosamini dase D hydrolyzes (Man)5(GlcNAc)2Asn but not (Man)6(GlcNAc)2Asn, the presence of the unsubstituted alpha-mannosyl residue linked at the C-3 position of the terminal mannose of Manbeta1 leads to 4GlcNAcbeta1 leads to 4 GlcNAcAsn core must be essential for the action of the enzyme. 相似文献
8.
Joseph P. M. Hui Patricia Lanthier Theresa C. White Sylvia G. McHugh Makoto Yaguchi Ren Roy Pierre Thibault 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,752(2):1549
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
9.
Williams A.F.; Parekh R.B.; Wing D.R.; Willis A.C.; Barclay A.N.; Dalchau R.; Fabre J.W.; Dwek R.A.; Rademacher T.W. 《Glycobiology》1993,3(4):339-348
Protein structure and tissue type are known to influence glycosylationof proteins. We have previously investigated the N-glycans ateach of the three glycosylation sites of the cell surface glycoproteinThy-1 when isolated from rat brain and thymocytes. Here we reporta comparative analysis of the site-specific N-glycosylationpatterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) andhuman (Asn 23, 60, 100) neural Thy-1. Despite considerable differencesin amino acid sequence, the results show a remarkable conservationof the pattern of N-glycans at corresponding sites between thethree species, as judged by chromatographic comparisons andglycosidase susceptibility. This is particularly marked forsites at Asn 74/75 in rat/mouse and the equivalent site at 60in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively.The sites at Asn 23 in rat/mouse also contained almost identicalglycosylation paterns, but at this site human Thy-1 showed significantlydifferent glycosylation patterns. These site glycosylation patternsare discussed in relation to the likely accessibility of theoligosaccharides for processing. It is known that within a species,the glycosylation of Thy-1 is tissue specific; therefore, thisdegree of conservation of glycosylation of Thy-1 expressed inthe same tissue in different species is all the more striking,given the known variation between species in the amino acidsequence of Thy-1. It is therefore proposed that neural cellshave a particular requirement for specific surface carbohydratesand that the Thy-1 polypep-tide serves as an appropriate carrierfor these structures. glycosylation site-specific Thy-1 相似文献
10.
Three glycopeptides, obtained in quantity from ovalbumin by exhaustive digestion with Pronase and purified by ion-exchange chromatography and gel filtration, had mannose-2-acetamido-2-deoxyglucose-aspartic acid ratios of 5:4:1, 6:2:1, and 5:2:1. The structures of the glycopeptides have been investigated by sequential digestion with purified exo-glycosidases, Smith degradation, and selective acetolysis, and by methylation analysis of the glycopeptides and their degradation products. The resulting data indicated the structures to be α-d-Manp-(1→6)-[α-d- Manp-(1→3)]-α-d-Manp-(1→6)-[β-d-GlcNAcp-(1→4)]-[β-d-GlcNAcp-(1→2)-α-d- Manp-(1→3)]-β-d-Manp-(1→4)-β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn, α-d- Manp-(1→6)-[α-d-Manp-(1→3)]-α-d-Manp-(1→6)-[α-d-Manp-(1→2)-α-d-Manp- (1→3)]-β-d-Manp-(1→4)-β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn, and α-d-Manp- (1→6)-[α-d-Manp-(1→3)]-α-d-Manp-(1→6)-[α-d-Manp-(1→3)]-β-d-Manp-(1→4)- β-d-GlcNAcp-(1→4)-β-d-GlcNAcp→Asn. The glycopeptides had a common-core structure consisting of five mannose and two hexosamine residues, but the two larger glycopeptides were not homologous. 相似文献
11.
A. Reeber J.P. Zanetta G. Vincendon 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,801(3):444-455
Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed. 相似文献
12.
Arkwright Peter D.; Rademacher Thomas W.; Boutignon Francois; Dwek Raymond A.; Redman Christopher W.G. 《Glycobiology》1994,4(1):39-47
Immunosuppressive factors isolated from trophoblast are knownto block both innate and major histocompatability complex (MHC)-dependentcell-mediated immune responses in vitro and, in some cases,in vivo. We investigated the biochemical nature of these factors,which is presently unknown. Immunosuppressive activity, assessedby inhibition of two-way MLR, was extracted from term syncytiotrophoblastmicrovilli using 3 M KCl. The activity resisted both extensivepronase digestion and heating to 90°C for 1 h, demonstratingthat intact membrane proteins were not required. Although purifiedprotein-linked oligosaccharides released by hydrazinolysis fromthe syncytiotrophoblast membrane were themselves inactive, theyblocked the immunosuppressive activity of the KCl extract. Afterpronase digestion, the activity could be fractionated by TSK55S gel filtration, followed by C18 reverse-phase chromatography.Sequential exoglycosidase digestion of hydrazine-released sugarsof the active fraction demonstrated that it contained neutralN-linked oligomannose and hybrid oligosaccharides, which normallymake up <3% of the total syncytiotrophoblast-derived proteinglycan Library. These glycopeptides of the active fraction wereassociated with membrane phospholipid micelles. The possiblemechanism by which incompletely processed N-linked oligosaccharidesexpressed by a variety of syncytiotrophoblast membrane glycoproteinsmay block allogeneic reactivity when presented as polyvalentsugar groups is discussed. glycoprotein immunosuppression oligosaccharides pregnancy trophoblast 相似文献
13.
Kamar M Alvarez-Manilla G Abney T Azadi P Kumar Kolli VS Orlando R Pierce M 《Glycobiology》2004,14(7):583-592
N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V. 相似文献
14.
Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, is a N-linked glycoprotein with one high-mannose-type and one xylose-containing oligosaccharide side chain per polypeptide. The high-mannose-type glycan is attached to Asn12 and the complex-type glycan to Asn60 [Sturm, A. & Chrispeels, M. J. (1986) Plant Physiol. 81, 320-322]. The structures of the oligosaccharides were elucidated from two glycopeptides obtained from the lectin by Pronase digestion, affinity chromatography on concanavalin-A--Sepharose and gel-filtration chromatography on a column of BioGel P-4. The N-linked glycan structures were investigated by 500-MHz 1H-NMR spectroscopy and were established to be: [formula; see text] 相似文献
15.
Glycan decorations dictate protein functions and thus have crucialimportance in life sciences. Previously glycoprotein analysiswas mainly focused on the analysis of the liberated glycansallowing detailed structural, but lacking positional information.Analysis of intact glycopeptides required purified glycoproteinsand manual interpretation of spectra. We developed an approachwhere mixtures of native glycopeptides were analyzed with tandemmass spectrometry and the spectra were analyzed with automatedin silico workflows. The latter included combination of theoriginal spectra, generation of a human N-glycopeptide library,matching the glycopeptide spectra to the theoretical peptidefragments, scoring the observations, predicting the glycan composition,which were then matched against the observed spectra, statisticalvalidation of the results with target–decoy filtering,and finally the calculation of glycan structures. We verifiedthis approach with the 150 serotransferrin glycopeptide spectra,where we automatically generated 105 putative interpretationsfrom >109 theoretical glycopeptides. After scoring 62 glycopeptidespectra obtained validated interpretation with concomitant aminoacid sequences, glycan compositions, and structures. When applyingthis method to an unknown mixture of human plasma glycoproteinswe identified 80 glycopeptides with their glycan compositionsor structures. Instead of weeks and months of interpretationwork of mass spectrometry files our automated workflow can beexecuted in few hours and provide information concomitantlyfrom both the amino acid and glycan moieties of intact glycopeptidesin mixtures. No advanced computational skills were needed touse these preformed and tested workflows. In case users wantto add complexity to the analysis they are allowed to alterall parameters and rebuild the workflows. 相似文献
16.
Nonspecific proteolytic digestion of glycoproteins is an established technique in glycomics and glycoproteomics. In the presence of pronase E, for example, glycoproteins are digested to small glycopeptides having one to six amino acids residues, which can be analyzed with excellent sensitivity using mass spectrometry. Unfortunately, the long digestion times (1-3 days) limit the analytical throughput. In this study, we used controlled microwave irradiation to accelerate the proteolytic cleavage of glycoproteins mediated by pronase E. We used ESI-MS and MALDI-MS analyses to evaluate the microwave-assisted enzymatic digestions at various digestion durations, temperatures, and enzyme-to-protein ratios. When digesting glycoproteins, pronase E produced glycopeptides within 5 min under microwave irradiation; glycopeptides having one or two amino acids were the major products. Although analysis of peptides containing multiple amino acid residues offers the opportunity for peptide sequencing and provides information regarding the sites of glycosylation, the signals of Asn-linked glycans were often suppressed by the glycopeptides containing basic amino acids (Lys or Arg) in MALDI-MS experiments. To minimize this signal-to-content dependence, we converted the glycopeptides into their sodiated forms and then methylated them using methyl iodide. This controlled methylation procedure resulted in quaternization of the amino group of the N-terminal amino acid residue. Using this approach, the mass spectrometric response of glyco-Asn was enhanced, compensating for the poorer ionization efficiency associated with the basic amino acids residues. The methylated products of glycopeptides containing two or more amino acid residues were more stable than those containing only a single Asn residue. This feature can be used to elucidate glycan structures and glycosylation sites without the need for MS/MS analysis. 相似文献
17.
Franc V Sebela M Rehulka P Končitíková R Lenobel R Madzak C Kopečný D 《Journal of Proteomics》2012,75(13):4027-4037
Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins. 相似文献
18.
We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-3H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A) Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[3H]fucitol quantitatively from [3H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [3H]fucose-labelledglycoproteins from Lec1 cells were separated by SDSPAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [3H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDSPAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[3H]fucitol. Trypsin treatment of [3H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked 相似文献
19.
The protein encoded by the envelope gene of Friend spleen focus-formingvirus is responsible for the acute leukaemogenicity of thisvirus. In order to correlate glycosylation and intracellularprocessing of this protein with viral pathogenicity, envelopegene products of pathogenic and apathogenic glycosylation mutantswere expressed in Rat-1 cells and metabolically labelled with[6-3H] glucosamine. Following immunoprecipitation, primary andsecondary gene products (gp55, gp65) were separated by preparativepolyacrylamide gel electrophoresis. Oligosaccharides were releasedfrom tryptic glycopeptides by treatment with endo-ß-N-acetylglucosaminidaseH (gp55), peptide-N4-(N-acetyl-ß-glucosaminyl)asparagineamidase F (gp65) or by reductive ß-elimination. Resultingglycans were characterized by cochromatography with authenticoligosaccharide standards using different HPLC systems and digestionwith exoglycosidases. The results revealed that the primaryenvelope gene products of pathogenic glycosylation mutants were,in part, further processed in Rat-1 cells similar to wild-typeglycoprotein, resulting in polypeptides carrying complex-typeN-glycans as well as partially sialylated O-linked oligosaccharides.In contrast, corresponding glycoproteins encoded by apathogenicmutants were found to remain at the level of the primary translationproduct exclusively comprising high-mannose-type N-glycans.Hence, intracellular maturation of the envelope gene productsin this model cell line seems to correlate with the in vivopathogenicityof the glycosylation mutants studied. carbohydrate structure glycoprotein murine leukaemia virus oligosaccharide processing SFFV 相似文献
20.
León IR da Costa Neves-Ferreira AG da Rocha SL de Oliveira Trugilho MR Perales J Valente RH 《Proteomics》2012,12(17):2753-2765
The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked. 相似文献