共查询到20条相似文献,搜索用时 421 毫秒
1.
Yoon SJ Nakayama K Takahashi N Yagi H Utkina N Wang HY Kato K Sadilek M Hakomori SI 《Glycoconjugate journal》2006,23(9):639-649
GM3 ganglioside interacts specifically with complex-type N-linked glycans having multivalent GlcNAc termini, as shown for
(1) and (2) below. (1) Oligosaccharides (OS) isolated from ConA-non-binding N-linked glycans of ovalbumin, whose structures
were identified as penta-antennary complex-type with bisecting GlcNAc, having five or six GlcNAc termini (OS B1, B2), or bi-antennary
complex-type having two GlcNAc termini (OS I). OS I is a structure not previously described. (2) Multi-antennary complex-type
N-linked OS isolated from fetuin, treated by sialidase followed by β-galactosidase, having three or four GlcNAc termini exposed.
These OS, conjugated to phosphatidylethanolamine (PE), showed clear interaction with 3H-labeled liposomes containing GM3, when various doses of OS-PE conjugate were adhered by drying to multi-well polystyrene
plates. Interaction was clearly observed only with liposomes containing GM3, but not LacCer, Gb4, or GalNAcα1-3Gb4 (Forssman
antigen). GM3 interaction with PE conjugate of OS B1 or B2 was stronger than that with PE conjugate of OS I. GM3 interacted
clearly with PE conjugate of N-linked OS from desialylated and degalactosylated fetuin, but not native fetuin. No binding
was observed to cellobiose-PE conjugate, or to OS-PE conjugate lacking GlcNAc terminus. Thus, GM3, but not other GSL liposomes,
interacts with various N-linked OS having multiple GlcNAc termini, in general. These findings suggest that the concept of
carbohydrate-to-carbohydrate interaction can be extended to interaction of specific types of N-linked glycans with specific
GSLs. Natural occurrence of such interaction to define cell biological phenomena is under investigation.
All solvent ratios are by volume.
An erratum to this article can be found at 相似文献
2.
Self-recognition of N-linked glycans with multivalent GlcNAc, determined as ceramide mimetic conjugate 总被引:1,自引:0,他引:1
Aminoceramide mimetic was synthesized and conjugated to N-linked oligosaccharides having multivalent GlcNAc by reductive amination. Ceramide mimetic conjugates with "complex-type" glycan having five or six GlcNAc termini (termed Os Fr. B-Cer) were purified, analyzed by thin-layer chromatography (TLC), and finally characterized by MS/MS analysis through liquid chromatography/mass spectrometry. Binding of Os Fr. B-Cer placed on solid phase polystyrene surface with [3H]cholesterol-labeled liposomes containing Os Fr. B-Cer, or containing various glycosphingolipids (GSLs) was determined. The binding of Os Fr. B-Cer liposomes to Os Fr. B-Cer coated plate was significantly higher than binding of GM3 liposomes. Other GSL liposomes showed no binding. Thus, self-recognition of Os Fr. B-Cer was clearly demonstrated using ceramide mimetic conjugates. 相似文献
3.
We investigated interaction of GM3 with N-acetylglucosamine (GlcNAc) termini of N-linked glycans of epidermal growth factor receptor (EGFR), as the underlying mechanism for inhibitory effect of GM3 on EGFR activation, using ldlD cells transfected with EGFR gene. These cells, defective in UDP-Gal/UDP-GalNAc 4-epimerase, are incapable of synthesizing galactose (Gal)-containing glycans, unless Gal is provided in culture (+Gal). Key observations: (1) Expression of GlcNAc termini was high in -Gal cells, and strongly reduced in +Gal cells. (2) Comparative study of inhibitory effect of exogenously-added GM3 on EGFR activation in +Gal versus -Gal cells indicated that higher level of GlcNAc termini on EGFR is correlated with greater inhibitory effect of GM3. (3) GM3-, but not GM1-, coated beads bound to EGFR in lysate of -Gal cells, which have highly exposed GlcNAc termini. Such binding was inhibited in the presence of EDTA, similarly to other carbohydrate-carbohydrate interactions. 相似文献
4.
Seon-Joo Yoon Natalia Utkina Martin Sadilek Hirokazu Yagi Koichi Kato Sen-itiroh Hakomori 《Glycoconjugate journal》2013,30(5):485-496
High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans. 相似文献
5.
Two types of carbohydrate to carbohydrate interaction (CCI) have been known to be involved in biological processes. One is the CCI between molecules expressed on interfacing cell membranes of different cells to mediate cell to cell adhesion, and subsequently induce cell signaling, and is termed trans-CCI. It has been indicated that the Lex to Lex interaction at the morula stage in mouse embryos plays an important role in the compaction process in embryonic development. GM3 to Gg3 or GM3 to LacCer interaction has been suggested to be involved in adhesion of tumor cells to endothelial cells, which is considered a crucial step in tumor metastasis. The other is the CCI between molecules expressed within the same microdomain of the cell surface membrane, and is termed cis-CCI. The interaction between ganglioside GM3, and multi (>3) GlcNAc termini of N-linked glycans of epidermal growth factor receptor (EGFR), has been indicated as the molecular mechanism for the inhibitory effect of GM3 on EGFR activation. Also, the complex with GM3 and GM2 has been shown to inhibit the activation of hepatocyte growth factor (HGF) receptor, cMet, through its association with tetraspanin CD82, and results in the inhibition of cell motility. Since CCI research is still limited, more examples of CCI in biological processes in development, and cancer progression will be revealed in the future. 相似文献
6.
Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While
structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding
N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis
but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass
spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high
energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives
of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect
of terminal sialylation on the tight binding of rat sublingual mucin to Galβ1→4GlcNAc specific lectins and three oligomannose
specific lectins were clearly demonstrated in this study. 相似文献
7.
North SJ Koles K Hembd C Morris HR Dell A Panin VM Haslam SM 《Glycoconjugate journal》2006,23(5-6):345-354
With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment
of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles
of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic
genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex
glycans were observed with GlcNAc and Galβ1–4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type
core 1 Galβ1-3GalNAc sequence. 相似文献
8.
Velkova L Dolashka P Lieb B Dolashki A Voelter W Van Beeumen J Devreese B 《Glycoconjugate journal》2011,28(6):385-395
The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans. The proposed structures are based on MALDI-TOF-MS
data before and after treatment with the specific exoglycosidases β1-3,4,6-galactosidase and α1-6(>2,3,4) fucosidase followed by sequence analysis via electrospray ionization MS/MS-spectra. In total, 15 glycans were
identified as a highly heterogeneous group of structures. As in most molluscan hemocyanins, the glycans of HtH1 contain a
terminal MeHex, but more interestingly, a novel structural motif was observed: MeHex[Fuc(α1-3)-]GlcNAc, including thus MeHex and (α1-3)-Fuc residues being linked to an internal GlcNAc residue. While the functional unit (FU) c (HtH1-c) is completely lacking
any potential glycosylation site, FU-h possesses a second exposed sugar attachment site between beta-strands 8 and 9 within
the beta sandwich domain compared to the other FUs. The glycosylation pattern/sites show a high degree of conservation. In
FU-h two prominent potential glycosylation sites can be detected. The finding that HtH1 is not able to form multidecameric
structures in vivo could be explained by the presence of the exposed glycan on the surface of FU-h. 相似文献
9.
The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure
of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc
glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples
from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different
IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi),
AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase
of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting
GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated
with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival
of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation
is related to pathogenesis of cancer and progression of the disease. 相似文献
10.
Previous studies on the carbohydrate specificities of Erythrina cristagalli lectin (ECL) were mainly limited to analyzing the binding of oligo-antennary Galβ1→4GlcNAc (II). In this report, a wider range of recognition factors of ECL toward known mammalian ligands and glycans were examined by
enzyme-linked lectinosorbent and inhibition assays, using natural polyvalent glycotopes, and a glycan array assay. From the
results, it is shown that GalNAc was an active ligand, but its polyvalent structural units, in contrast to those of Gal, were
poor inhibitors. Among soluble natural glycans tested for 50% molecular mass inhibition, Streptococcus pneumoniae type 14 capsular polysaccharide of polyvalent II was the most potent inhibitor; it was 2.1 × 104, 3.9 × 103 and 2.4 × 103 more active than Gal, tri-antennary II and monomeric II, respectively. Most type II-containing glycoproteins were also potent inhibitors, indicating that special polyvalent II and Galβ1-related structures play critically important roles in lectin binding. Mapping all information available, it can
be concluded that: [a] Galβ1→4GlcNAc (II) and some Galβ1-related oligosaccharides, rather than GalNAc-related oligosaccharides, are the core structures for lectin
binding; [b] their polyvalent II forms within macromolecules are a potent recognition force for ECL, while II monomer and oligo-antennary II forms play only a limited role in binding; [c] the shape of the lectin binding domains may correspond to a cavity type with
Galβ1→4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear
trisaccharide, Galβ1→4GlcNAcβ1→6Gal. These analyses should facilitate the understanding of the binding function of ECL.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal
surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4)
n
oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal,
and (GlcNAc β1,4)
n
oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3
GalNAc, (GlcNAc β1,4)
n
oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system
contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4)
n
oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most
of these glycoproteins probably corresponds to the H+K+-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose,
(GlcNAc β1,4)
n
oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive
tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly
in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier. 相似文献
12.
Hua-Dong Liu Yu-Fen Zhao Yan-Mei Li 《International journal of peptide research and therapeutics》2006,12(3):237-241
The dynamic modification of many nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) on serine or threonine is catalyzed by O-GlcNAc transferase (OGT). The conserved GPGTF (glycogen phosphorylase/glycosyl transferase) motif, one of the α-helices of the second domain in OGT, was identified as a putative UDP-GlcNAc binding site. A miniature protein was designed which contains all of the conserved residues of GPGTF motif in the O-GlcNAc transferase, and was shown to adopt an alpha helix in 10% trifluoroethanol. It was anticipated that the miniature protein could shed light on the mechanism of dynamic O-GlcNAc modification and provide a potential drug for the diabetes and neurodegenerative diseases. 相似文献
13.
The rattlesnake (Crotalus atrox) venom lectin is a readily-prepared decameric C-type lectin, specific for Gal and GalNAc. Glycan microarray analysis showed
it reacted with a wide range of glycans, chiefly recognizing sets of compounds with Galβ1-4GlcNAc (LacNAc), α-Gal or α-GalNAc
non-reducing termini. Its array profile was therefore distinctly different from those of four previously studied mammalian
C-type lectins with the same Gal/GalNAc monosaccharide specificity, and it was more broadly reactive than several Gal- or
GalNAc-specific plant lectins commonly used for glycan blotting. Though a general reactivity towards glycoproteins might be
expected from the avidity conferred by its high valence, it showed a marked preference for glycoproteins with multiple glycans,
terminated by Gal or GalNAc. Thus its ten closely-spaced sites each with a KD for GalNAc of ~2 mM appeared to make RSVL more selective than the four more widely-spaced sites of soybean agglutinin, with
a ten-fold better KD for GalNAc. 相似文献
14.
Enzymatic glycosidation using sugar oxazolines 1-3 having a carboxylate group as glycosyl donors and compounds 4-6 as glycosyl acceptors was performed by employing a chitinase from Bacillus sp. as catalyst. All the glycosidations proceeded with full control in stereochemistry at the anomeric carbon of the donor and regio-selectivity of the acceptor. The N,N'-diacetyl-6'-O-carboxymethylchitobiose oxazoline derivative 1 was effectively glycosidated, under catalysis by the enzyme, with methyl N,N'-diacetyl-beta-chitobioside (4), pent-4-enyl N-acetyl-beta-D-glucosaminide (5), and methyl N-acetyl-beta-D-glucosaminide (6), affording in good yields the corresponding oligosaccharide derivatives having 6-O-carboxymethyl group at the nonreducing GlcNAc residue. The N,N'-diacetyl-6-O-carboxymethylchitobiose oxazoline derivative 2 was subjected to catalysis by the enzyme catalysis; however, no glycosidated products were produced through the reactions with 4, 5, and 6. Glycosidation reactions of the beta-d-glucosyluronic-(1-->4)-N-acetyl-D-glucosamine oxazoline derivative 3 proceeded with each of the glycosyl acceptors, giving rise to the corresponding oligosaccharide derivative having a GlcA residue at their nonreducing termini in good yields. 相似文献
15.
Structural diversity of N-glycans is essential for specific binding to their receptor proteins. To gain insights into structural and dynamic aspects in atomic detail not normally accessible by experiment, we here perform extensive molecular-dynamics simulations of N-glycans in solution using the replica-exchange method. The simulations show that five distinct conformers exist in solution for the N-glycans with and without bisecting GlcNAc. Importantly, the population sizes of three of the conformers are drastically reduced upon the introduction of bisecting GlcNAc. This is caused by a local hydrogen-bond rearrangement proximal to the bisecting GlcNAc. These simulations show that an N-glycan modification like the bisecting GlcNAc selects a certain “key” (or group of “keys”) within the framework of the “bunch of keys” mechanism. Hence, the range of specific glycan-protein interactions and affinity changes need to be understood in terms of the structural diversity of glycans and the alteration of conformational equilibria by core modification. 相似文献
16.
Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans. 总被引:8,自引:0,他引:8
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The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of beta 1-->2 xylose and alpha 1-->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan. 相似文献
17.
Mihoko Takahashi 《Genesis (New York, N.Y. : 2000)》1992,13(2):174-179
The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes. When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about l h after pair formation. The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2h, they were not distinguishable from the wild-type homotypic pairs. On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs. When tnrA was mated with tnrB more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect. Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca2+ channels. © 1992 Wiley-Liss, Inc. 相似文献
18.
Cell recognition and adhesion involving many kinds of cell surface molecules operate via homotypic and/or heterotypic protein-protein and protein-carbohydrate binding. Our investigations in marine sponges have provided direct evidence for a novel molecular mechanism of multivalent glycan-glycan binding related to cellular interactions. Biochemical characterization of purified proteoglycans revealed the presence of specific acidic glycans, different from classical glycosaminoglycans. Such acidic glycans of high molecular weight, containing fucose, glucuronic or galacturonic acids, and pyruvate and sulfate groups may represent a new class of primordial proteoglycans, named by us glyconectins. The thermodynamic and kinetic approaches of biological macromolecule interactions do not provide a direct measurement of the intermolecular binding forces that are fundamental for the function of the ligand-receptor association. Using the atomic force microscopy (AFM), we provided the first quantitative evaluation of the binding strength between cell adhesion proteoglycans. Measurement of binding forces intrinsic to cell adhesion glyconectin proteoglycans (AGPs) is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. (i) As a model, we selected the cell AGP isolated from the marine sponge Microciona prolifera; it mediates in vivo cell recognition and aggregation via homotypic, species-specific, multivalent, and calcium ion-dependent glycan-glycan interactions. (ii) Under physiological conditions, a large cohesive force theoretically able to hold the weight of approximately 1600 cells was measured. (iii) The C-2 autocomplementarity model for AGP-AGP interactions; and (iv) the requirement of the calcium ionic bridges suggest also that the self-recognition and multivalency of glycan-glycan interactions are essential for cell adhesion. (v) The evolution of glyconectin-like proteoglycan molecules may have been a fundamental prerequisite for the emergence of the first multicellular organisms. Glycan-glycan interactions may thus provide a new paradigm for molecular self-recognition. 相似文献
19.
Paul A. Verrell Stevan J. Arnold 《Evolution; international journal of organic evolution》1989,43(4):745-755
The behavioral basis of sexual isolation was investigated in 11 crosses staged between individuals from nine allopatric populations of the Appalachian mountain dusky salamander, Desmognathus ochrophaeus. Scan-sampled observations of within-population (homotypic) and between-population (heterotypic) heterosexual encounters yielded the following results. 1) Fewer heterotypic encounters than homotypic encounters resulted in insemination of females. Indices of the strength of sexual isolation based on insemination data ranged from 0.26 (lowest) to 0.82 (highest), indicating that mechanisms that confer sexual isolation may evolve during allopatric differentiation. 2) Heterotypic encounters that progressed as far as the stage of courtship during which the male stimulates the female to mate did so at the same time as homotypic encounters, although the number of the former reaching this stage was lower. 3) The time interval from courtship initiation to insemination was longer in heterotypic encounters than homotypic encounters. In some crosses, males did not attempt to initiate heterotypic encounters, indicating that this sex is responsible for sexual isolation in these instances. We use our results to formulate specific hypotheses concerning the behavioral basis of sexual isolation in these salamanders, and some possible experimental approaches are suggested. 相似文献
20.
Suzuki T Matsuo I Totani K Funayama S Seino J Taniguchi N Ito Y Hase S 《Analytical biochemistry》2008,381(2):224-232
It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells. 相似文献