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1.
In an effort to prime our mass spectrometry (MS)-based sulfoglycomic mapping platform technology for facile identification of sulfated lacdiNAc (GalNAcβ1-4GlcNAcβ1-), we have re-examined the N-glycans of bovine thyroid stimulating hormone. We showed that MALDI-MS mapping of permethylated glycans in negative ion mode can give an accurate representation of the sulfated glycans and, through MS/MS, diagnostic ions can be derived that we can collectively define the presence of a terminal sulfated lacdiNAc moiety at high sensitivity. Based on these ions, which can also be produced by nanoESI-MSn, we demonstrated that the glycome of an ovarian carcinoma cell line, RMG-1, comprises a high abundance of sulfated lacdiNAc epitopes carried on multiantennary complex type N-glycans alongside fucosylated, sialylated and/or sulfated lacNAc antennae. This represents the first report of a natural glycomic occurrence of sulfated lacdiNAc on a cell line, as opposed to other better-characterized presence on secreted glycoproteins from a handful of sources. It is anticipated that with improved methods of detection such as that developed in this work, we are likely to identify a wider occurrence of sulfated lacdiNAc and be able to more accurately delineate the regulatory mechanism dictating the choice of a cell type in synthesizing sulfated, sialylated, fucosylated and/or non-substituted lacdiNAc.  相似文献   

2.
Twenty-eight enzymes, encoded by different genes and secreted by different mutant strains of Chrysosporium lucknowense, were subjected to MALDI-TOF MS peptide fingerprinting followed by analysis of the MS data using the GlycoMod tool from the ExPASy proteomic site. Various N-linked glycan structures were discriminated in the C. lucknowense proteins as a result of the analysis. N-Glycosylated peptides with modifications matching the oligosaccharide compositions contained in the GlycoSuiteDB were found in 12 proteins. The most frequently encountered N-linked glycan, found in 9 peptides from 7 proteins, was (Man)(3)(GlcNAc)(2), that is, the core pentasaccharide structure forming mammalian-type high-mannose and hybrid/complex glycans in glycoproteins from different organisms. Nine out of 12 enzymes represented variably N-glycosylated proteins carrying common (Hex)(0-4)(HexNAc)(0-6)+(Man)(3)(GlcNAc)(2) structures, most of them being hybrid/complex glycans. Various glycan structures were likely formed as a result of the enzymatic trimming of a 'parent' oligosaccharide with different glycosidases. The N-glycosylation patterns found in C. lucknowense proteins differ from those reported for the extensively studied enzymes from Aspergilli and Trichoderma species, where high-mannose glycans of variable structure have been detected.  相似文献   

3.
Characterization of the N-glycans from human pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3) revealed completely different glycosylation patterns. RNase 1 from healthy cells contained neutral complex biantennary structures, with smaller amounts of tri- and tetraantennary compounds, and glycans with poly-N-acetyllactosamine extensions, all extensively fucosylated. In contrast, RNase 1 glycans from tumor cells (Capan-1) were fucosylated hybrid and complex biantennary glycans with GalNAc-GlcNAc antennae. RNase 1 glycans from Capan-1 and MDAPanc-3 cells also contained sialylated structures completely absent in the healthy pancreas. Some of these features provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Thus monoclonal antibodies to Lewis(y) reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis(x) and sialyl-Lewis(a) reacted only with RNase 1 secreted from the tumor cells. These glycosylation changes in a tumor-secreted protein, which reflect fundamental changes in the enzymes involved in the glycosylation pathway, open up the possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma.  相似文献   

4.
Analysis of protein glycosylation within the nematode Caenorhabditis elegans has revealed an abundant and unreported set of core chitobiose modifications (CCMs) to N-linked glycans. With hydrazine release, an array of glycomers and isobars were detected with hexose extensions on the 3- and 3,6-positions of the penultimate and reducing terminus, respectively. A full complement of structures includes a range of glycomers possessing a Galbeta(1-4)Fuc disaccharide at the 3- and 6-positions of the protein-linked GlcNAc. Importantly, enzymatic (PNGase F/A) release failed to liberate many of these extended structures from reduced and alkylated peptides and, as a consequence, such profiles were markedly deficient in a representation of the worm glycome. Moreover, the 3-linked Galbeta(1-4)Fuc moiety was notably resistant to a range of commercial galactosidases. For identification, the fragments were spectrum-matched with synthetic products and library standards using sequential mass spectrometry (MS(n)). A disaccharide observed at the 3-position of penultimate GlcNAc, indicating a Hex-Fuc branch on some structures, was not further characterized because of low ion abundance in MS(n). Additionally, a Hex-Hex-Fuc trisaccharide on the 6-position of proximal GlcNAc was also distinguished on select glycomers. Similar branch extensions on 6-linked core fucosyl residues have recently been reported among other invertebrates. Natural methylation and numerous isobars complement the glycome, which totals well over 100 individual structures. Complex glycans were detected at lower abundance, indicating glucosaminyltransferase-I (GnT-I) and GnT-II activity. A range of phosphorylcholine (PC)-substituted complex glycans were also confirmed following a signature two-stage loss of PC during MS(n) analysis, although the precursor ion was not observed in the mass profiles. In a similar manner, numerous other minor glycans may be present but unobserved in hydrazine-release profiles dominated by fucosylated structures. All CCM structures, including multiple isomers, were determined without chromatography by gas-phase disassembly (MS(n)) in Paul and linear ion trap (IT) instruments.  相似文献   

5.
蛋白的糖基化对蛋白的活性、高级结构及功能都有重要的影响。酵母表达的糖蛋白不同于哺乳动物表达的杂合型或复杂型糖蛋白,而是高甘露糖型或过度甘露糖化糖蛋白。在前期成功敲除毕赤酵母α-1,6-甘露糖转移酶(Och1p)基因、阻断毕赤酵母过度糖基化,获得毕赤酵母过度糖基化缺陷菌株GJK01 (ura3、och1) 的基础上,通过表达不同物种来源的α-1,2-甘露糖苷酶I (MDSI) 的活性区与酵母自身定位信号的融合蛋白,并通过DSA-FACE (基于DNA测序仪的荧光辅助糖电泳) 分析筛选报告蛋白HSA/GM-CSF (人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白) 的糖基结构,发现当编码酿酒酵母α-1,2-甘露糖苷酶 (MnsI) 基因的内质网定位信号与带有完整C-端催化区的拟南芥MDSI基因融合表达时,毕赤酵母工程菌株能够合成Man5GlcNAc2哺乳动物甘露糖型糖蛋白。这为在酵母体内合成类似于哺乳动物杂合型或复杂型糖基化修饰的糖蛋白奠定了基础。  相似文献   

6.
The carbohydrate moieties of equine chorionic gonadotropin alpha and beta subunits were released from the protein backbones by successive treatments with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the beta subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-, di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly alpha 1-6 fucosylated at the Asn-bound GlcNAc residue and having alpha 2-6 and alpha 2-3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the alpha subunit. The major part of the O-linked carbohydrate chains, occurring solely in the beta subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the beta subunit.  相似文献   

7.
Ovarian carcinoma is the leading cause of death from gynecological cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation and consequently in ovarian cancer. In this study, a detailed structure analysis of the N-linked glycans from total glycoproteins from the SKOV3 ovarian carcinoma cell line and from a recombinantly expressed secretory glycoprotein, erythropoietin (EPO), produced from the same cells has been performed using high-performance anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total cellular N-glycans contained high-mannose type and proximally fucosylated complex type partially agalactosylated structures. On the other hand, the recombinant human EPO secreted from SKOV3 cells contained predominantly core-fucosylated tetraantennary structures, which were partially lacking one or two galactose residues, and partially contained the LacdiNAc motif. Only minor amounts of di- and triantennary complex-type glycans were found, and high-mannose-type glycans were not present in the secreted EPO protein. A large amount of N-acetylneuraminic acid in α2,3-linkage was detected as well. Endogenous glycoproteins were also found to contain the LacdiNAc motif in N-linked glycans. This work contributes to the knowledge of the glycosylation of a human ovarian cancer cell line. It also establishes the basis to further explore high-mannose-type glycans, and the LacdiNAc motif as possible markers of ovarian carcinoma.  相似文献   

8.
A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.  相似文献   

9.
Isolation of MUC5AC mucins from the gastric mucosa from two secretor individuals (one from normal mucosa from a patient with gastric cancer and one from a control) showed different abilities to bind and induce the proliferation of the Helicobacter pylori strain J99. Analysis of the released O-linked oligosaccharides by LC-MS from these individuals showed a very heterogeneous mixture of species from the cancer patient containing both neutral and sialylated structures, whereas the normal sample showed dominating neutral blood group H terminating structures as well as neutral structures containing the di-N-acetyllactosamine (lacdiNAc) unit GalNAcβ1-4GlcNAcβ1- on the C-6 branch of the reducing end GalNAc. The linkage configuration of these epitopes were determined using C-4-specific fragmentation for the GalNAcβ1-4GlcNAcβ1- glycosidic linkage, comparison of the MS(3) fragmentation with standards for linkage configuration and N-acetylhexosamine type as well as exoglycosidase treatment. It was also shown that the lacdiNAc epitope is present in both human and porcine gastric mucins, indicating that this is an epitope preserved between species. We hypothesize that the termination on gastric MUC5AC with lacdiNAc is in competition with complex glycosylation such as the Le(b) and H type 1 as well as complex sialylated structures. These are epitopes known to bind the H. pylori BabA and SabA adhesins.  相似文献   

10.
Using the avidin-biotinyl glycan system reported previously (Shao, M.-C., and Wold, F. (1987) J. Biol. Chem. 267, 2968-2972), we have compared the processing efficiency of oviduct enzymes acting on different glycan-(biotinyl)Asn and glycan-(6-biotinamidohexanoyl)Asn derivatives when they are free and bound to avidin. The glycans were selected to permit exploration of the individual processing steps, and the two different groups of derivatives were used to assess both the close (biotinyl) and more distal (biotinamidohexanoyl) display of the glycan relative to the avidin surface. The direct comparison of the free and avidin-bound glycans demonstrated that mannosidase I is strongly inhibited by avidin in both the close and distal complexes, whereas GlcNAc transferase I and mannosidase II are strongly inhibited only in the close complex. GlcNAc transferases III, IV, and V, which could only be assessed individually by indirect means using different substrates, did not appear to be affected in any major way by the protein matrix; the data suggest that transferase III is inhibited only to a minor extent in the close complex. Gal transferase activity showed a minor effect of the avidin matrix for both complexes in the hybrid processing pathways. The most significant consequence of the avidin effect on Gal transferase was the apparent abolishment of the incorporation of a 2nd Gal residue in the two avidin complexes. This survey of the protein matrix effects on glycan processing by oviduct enzymes appears to provide reasonable clues to the origin of the very different glycan structures observed in oviduct-processed glycoproteins. Thus, ovalbumin and avidin itself, containing a mixture of oligomannose and hybrid glycans at their single glycosylation sites, may well present they glycans to the processing enzymes in a display very similar to that of the avidin close complex observed here. The inhibition of mannosidase I and GlcNAc transferase I lead to preservation of oligomannose structures, whereas the strong inhibition of mannosidase II favors the incorporation of the bisecting GlcNAc by GlcNAc transferase III to yield hybrid structures as the most processed products. Ovomucoid, which contains multiantennary complex structures at all glycosylation sites, may on the other hand display its glycans, unencumbered by the protein surface, in conformations similar to either the free glycans or the distal complexes observed in this work.  相似文献   

11.
N-Glycans linked to the human secreted form of epidermal growth factor receptor were isolated from A431 cells after swainsonine treatment. Analysis of the oligosaccharides by (1)H NMR spectroscopy and mass spectrometry shows the presence of oligomannose- and (alpha2-3)-sialylated hybrid-type glycans. The major hybrid-type oligosaccharide chains are fucosylated at the Asn-bound GlcNAc residue. Smaller amounts of the hybrid-type structures are also fucosylated at peripheral GlcNAc residues, constituting the sialyl-Le(x) antigen. No complex-type glycans are found, suggesting the absence of alpha-mannosidase III. An assay for alpha-mannosidase III on the A431 cells in the absence and presence of 6 microM swainsonine shows that Man(5)GlcNAc(2) is not converted into Man(3)GlcNAc(2), thereby confirming that these cells do not contain alpha-mannosidase III activity.  相似文献   

12.
H H Huang  P L Tsai  K H Khoo 《Glycobiology》2001,11(5):395-406
The glycobiology of Schistosoma mansoni is dominated by developmentally regulated expression of various fucosylated structures, most notably the Lewis X epitope and a multifucosylated sequence, Fuc alpha1-->2Fuc alpha1-->, in its various forms. For the infective cercarial stage, Lewis X has been structurally identified on glycosphingolipids and N-glycans of total glycoprotein extracts, and a population of multifucosylated glycoproteins were found to carry a unique terminal sequence, +/-Fuc alpha1-->2Fuc alpha1-->[3GalNAc beta1-->4(Fuc alpha1-->2Fuc alpha1--> 2Fuc alpha1-->3) GlcNAc beta1-->3Gal alpha1-->](n), on their O-glycans. Using a mass spectrometry approach coupled with chromatographic separation, sequential exoglycosidase digestion, periodate oxidation, and other chemical derivatization, we demonstrate that Lewis X could also be carried on the cercarial O-glycans, but the two distinctive sets of fucosylated epitopes were conjugated to two different core structures. Lewis X, lacNAc, or single GlcNAc was found to attach directly to the -->3Gal beta1-->3GalNAc core and indirectly via another beta-Gal residue branching off from C6 of the reducing end GalNAc to give a biantennary-like structure. The -->3(+/-Gal beta1-->6)Gal beta1-->3(-->3Gal beta1-->6)GalNAc core thus characterized represents a novel core type for O-glycans. In contrast, the previously characterized multifucosylated terminal sequences were carried on conventional type 1 and 2 cores. The smallest structures of the reductively released O-glycans were defined as GalNAc beta1-->4GlcNAc beta1-->3Gal beta1-->3GalNAcitol with a total of two to four fucoses attached to the terminal lacdiNAc. alpha-Galactosylation of the nonreducing terminal beta-GalNAc instead of fucose capping leads to further elongation with another lacdiNAc unit that could also extend directly from C6 of the reducing end GalNAc and similarly elongated or terminated.  相似文献   

13.
Site-specific N-glycan characterization of human complement factor H   总被引:1,自引:0,他引:1  
Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.  相似文献   

14.
Kim YG  Gil GC  Harvey DJ  Kim BG 《Proteomics》2008,8(13):2596-2610
The major barrier in transplantation of pig organs into humans is the presence of surface carbohydrate antigens (e.g., the Gal alpha 1-3 Gal beta 1-4GlcNAc-R (alpha-Gal) epitope) expressed on pig endothelial cells. In this study, total N-glycans from membrane glycoproteins derived from specific pathogen-free miniature pig kidney are identified by MALDI-TOF, negative ion ESI MS/MS and normal-phase HPLC (NP-HPLC) combined with exoglycosidase digestion. Over 100 N-glycans, including sialylated and neutral types, were identified. As well as the known alpha-Gal antigens, some of these glycans contained novel non-Gal carbohydrate antigens such as (Neu5Gc-Gal-GlcNAc) and Gal alpha 1-3 Lewis(x) (Gal-Gal-(Fuc)GlcNAc) which have not been reported before in N-glycans from pig organs. The ability of MALDI, ESI, and HPLC to measure the relative proportions of the glycans was evaluated. The HPLC resolution was insufficient for accurate work and some minor differences were noted in the ionization efficiencies of different glycan groups when measured by the two mass spectrometric techniques. However, the results indicated that the relative quantity of alpha-Gal epitope was in the region of 50% of the complex glycans. High-mannose type glycans were also abundant (35-43%) but appeared to be ionized more efficiently than the complex glycans by ESI than by MALDI.  相似文献   

15.
Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.  相似文献   

16.
Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions.  相似文献   

17.
To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the alpha-subunit of human chorionic gonadotropin have been studied by NMR spectroscopy. Significant differences in chemical shifts and NOE intensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straightforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be developed. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of extended glycans during the simulation. The values for phi and psi dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2(beta1-4)GlcNAc1 and Man3(beta1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycans. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures indicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generated conformers display especially for the beta-turn 20-23 a decreased structural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydrophilic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted beta-hairpins consisting of residues 10-28 and 59-84.  相似文献   

18.
The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.  相似文献   

19.
Kim YG  Kim SY  Hur YM  Joo HS  Chung J  Lee DS  Royle L  Rudd PM  Dwek RA  Harvey DJ  Kim BG 《Proteomics》2006,6(4):1133-1142
The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man(3-9)GlcNAc2) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with alpha-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the alpha-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.  相似文献   

20.
N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection. N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were Man1-3(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%). There was only approximately 6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue. However alpha(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.  相似文献   

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