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1.
Xiao-ling Jin Hou-bao Liu Ying Zhang Bing-sheng Wang Hui-Li Chen 《Glycoconjugate journal》2003,20(6):399-406
The activities of three N-acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins
in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from
18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using
a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively
of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation
and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins
in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E4-PHA. The increased GnT-V activity led to the elevation of the β1,6GlcNAc branch (or antennary number) on the N-glycans in
cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest
that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in
the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis
and prognosis evaluation in EBDC patients. Published in 2004.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
2.
3.
Fengchao Chen Ikuko Kakizaki Masanori Yamaguchi Kaoru Kojima Keiichi Takagaki Masahiko Endo 《Glycoconjugate journal》2009,26(5):559-566
When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid
chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each
minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based
on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer,
and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH. 相似文献
4.
Folkert Reck Ernst Meinjohanns Matthias Springer Roland Wilkens Johannes A. L. M. Van Dorst Hans Paulsen Gabriele Möller Inka Brockhausen Harry Schachter 《Glycoconjugate journal》1994,11(3):210-216
UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K
i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K
i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K
i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis.
Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate. 相似文献
5.
We showed previously that Chinese hamster ovary cells took up and utilized a variety of N-acetylglucosaminides as primers of oligosaccharide biosynthesis (Ding et al., 1999, J. Carbohydr. Chem., 18:471–475). In this study, a library of N-acetylglucosaminides was enzymatically galactosylated in vitro to yield type 2 chain N-acetyllactosaminides bearing a variety of aglycones. Those disaccharides are potential acceptors for fucosyltransferases. As an extension of the previous study, we tested the type 2 chain disaccharyl glycosides (Gal1,4-GlcNAc-R) for their aglycone-dependent acceptor specificity for -L-fucosyltransferase III (Fuc-TIII). The enzyme activity significantly depended on the aglycone structures, suggesting that, in addition to the polar groups on the sugar moiety, the hydrophobic aglycone can substantially contribute to recognition in this reaction. 相似文献
6.
7.
Alessandro Datti Aldo Orlaccio Katherine A. Siminovitch James W. Dennis 《Glycoconjugate journal》1984,1(2):127-135
Serine/threonine (O)-linked oligosaccharide on cell-surface CD43 has been reported to be abnormal in haemopoietic lineages of patients with the X-linked immunodeficiency, Wiskott-Aldrich syndrome (WAS). This defect largely appears to be the result of abnormal regulation of UDP-GlcNAc:Gal1-3GalNAc-R1-6-N-acetylglucosaminyltransferase (also known as core 2 GlcNAc-T), an enzyme in the Golgi apparatus that is subject to regulation during haemopoietic differentiation. To determine whether core 2 GlcNAc-T activity provides a reliable marker for WAS, we studied 12 unrelated WAS patients with respect to their expression of this enzyme activity. Compared with healthy subjects, the WAS patients showed levels of core 2 activity that were, on average, 2.5- and 3.9-fold higher in fresh lymphocytes and platelets respectively. These data suggest that altered core 2 GlcNAc-T activity is consistently found in lymphocytes and platelets of WAS patients and as such may provide a diagnostic marker for the disease. In view of the relatively limited amounts of blood sample generally available from infants and young children, we have also tested a more sensitive coupled assay that permits assessment of core 2 GlcNAc-T activity in very small samples of cells and which would therefore render this assay of wide clinical applicability. 相似文献
8.
Folkert Reck Matthias Springer Ernst Meinjohanns Hans Paulsen Inka Brockhausen Harry Schachter 《Glycoconjugate journal》1995,12(6):747-754
UDP-GlcNAc:Man1-3R 1-2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) catalyses the conversion of [Man1-6(Man1-3)Man1-6][Man1-3]Man-O-R to [Man1-6(Man1-3)Man1-6] [GlcNAc1-2Man1-3]Man-O-R (R=1-4GlcNAc1-4GlcNAc-Asn-X) and thereby controls the conversion of oligomannose to complex and hybrid asparagine-linked glycans (N-glycans). GlcNAc-T I also catalyses the conversion of Man1-6(Man1-3)Man-O-octyl to Man1-6(GlcNAc1-2Man1-3)Man-O-octyl. We have therefore tested a series of synthetic analogues of Man1-6(Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T I. The 2-deoxy and the 3-, 4- and 6-O-methyl derivatives are all good substrates confirming previous observations that the hydroxyl groups of the Man1-6 residue do not play major roles in the binding of substrate to enzyme. In contrast, all four hydroxyl groups on the Man1-3 residue are essential since the corresponding deoxy derivatives either do not bind (2- and 3-deoxy) or bind very poorly (4- and 6-deoxy) to the enzyme. The 2- and 3-O-methyl derivatives also do not bind to the enzyme. However, the 4-O-methyl derivative is a substrate (K
m
=2.6mm) and the 6-O-methyl compound is a competitive inhibitor (K
i=0.76mm). We have therefore synthesized various 4- and 6-O-alkyl derivatives, some with reactive groups attached to anO-pentyl spacer, and tested these compounds as reversible and irreversible inhibitors of GlcNAc-T I. The 6-O-(5-iodoacetamido-pentyl) compound is a specific time dependent inhibitor of the enzyme. Four other 6-O-alkyl compounds showed competitive inhibition while the remaining compounds showed little or no binding indicating that the electronic properties of the attachedO-pentyl groups influence binding.Abbreviations GlcNAc-T I
UDP-GlcNAc:Man1-3R 1-2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)
- GlcNAc-T II
UDP-GlcNAc:Man1-6R 1-2-N-acetylglucosaminyltransferase II (EC 2.4.1.143)
- MES
2-(N-morpholino)ethane sulfonic acid monohydrate 相似文献
9.
The sugar chain structures of the cell surface change dramatically during cellular differentiation. A human neuroblastoma cell line, GOTO, is known to differentiate into neuronal cells and Schwannian cell-like cells on treatments with dibutyryl cAMP and bromodeoxyuridine, respectively. We have examined the expression of UDP-N-acetylglucosamine: -d-mannoside -1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) and UDP-N-acetylglucosamine: -6-d-mannoside -1,6N-acetylglucosaminyltransferase V (GnT-V: EC 2.4.1.155), two major branch forming enzymes inN-glycan synthesis, in GOTO cells on two distinct directions of differentiation.In neuronal cell differentiation, GnT-III activity showed a slight increase during initial treatment with Bt2cAMP for 4 days and decreased drastically after the fourth day, but the mRNA level of GnT-III did not show a decrease but in fact a slight increase. GnT-V activity increased to approximately two- to three-fold the initial level with increasing mRNA level after 8 days, and lectin blot analysis showed an increase in reactivity toDatsura stramonium (DSA) of the immunoprecipitated neural cell adhesion molecule (NCAM). In Schwannian cell differentiation, the activity and mRNA level of GnT-III showed no significant change on treatment with BrdU. GnT-V activity also showed no change in spite of the gradual increase in the mRNA level. These results suggest that the activation of GnT-V during neuronal cell differentiation of GOTO cells might be a specific change for branch formation in N-glycans, and this affects the sugar chain structures of some glycoproteins such as NCAM.Abbreviations and trivial names GnT
N-acetylglucosaminyltransferase
- Bt2cAMP
N
6,O
6-dibutyryl cAMP
- BrdU
bromodeoxyuridine
- DSA
Datsura stramonium
- NCAM
neural cell adhesion molecule
- PAGE
polyacrylamide gel electrophoresis 相似文献
10.
11.
A comprehensive analysis of low molecular weight compounds in biological samples by the hyphenated method of GC/MS in general detects a large number of peaks which can not be identified by searching of commercially available mass spectral libraries. Therefore, more information is required for a successful identification of these compounds. Some structural features like molecular weight and number of derivatization groups present in the molecule can be determined by variation of the derivatization prior to GC/MS. The use of deuterated derivatizing reagents and the newly developed N-methyl-N-ethyldimethylsilyl-trifluoracetamide for this purpose is described. The knowledge of these structural properties alone undoubtedly will not lead to the structure elucidation of novel metabolites. However, it may be helpful in identifying derivatives of known metabolites or artifacts. Thus, by use of the molecular weight as search criterion it is possible to find plausible candidates in metabolic pathway databases. It is shown that the application of this method to a hydrophilic extract of C. glutamicum lead to the identification of 31 in previous analyses unidentified peaks, in their majority representing minor derivatives of common metabolites. 相似文献
12.
13.
A series of glycosyl haloacetamides were synthesized as potential inhibitors of cytoplasmic peptide:N-glycanase (PNGase), an enzyme that removes N-glycans from misfolded glycoproteins. Chloro-, bromo-, and iodoacetamidyl chitobiose and chitotetraose derivatives exhibited
a significant inhibitory activity. No inhibitory activity was observed with of fluoroacetamididyl derivatives. Moreover, N-acetylglucosamine derivatives, β-chloropropionamidyl chitobiose, and chloroacetamidyl cellooligosaccharide derivatives did
not show any activity. These results underscore the importance of the N-acetyl groups of chitobiose for PNGase recognition. In addition, reactivity and position of the leaving group at the reducing
end are also important factors. 相似文献
14.
Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome ofAspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in theAspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.Abbreviations CM
complete medium
- Gal-T
UDP-Gal:GlcNAc -1,4-galactosyltransferase (EC 2.4.1.38/90)
- GnT I
UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)
- HPLC
high performance liquid chromatography
- M3-octyl
Man1-6[Man1-3]Man-octyl
- PAGE
polyacrylamide gel electrophoresis
- MES
2-(N-morpholino)ethane sulfonate
- PCR
polymerase chain reaction
- PEG
polyethylene glycol
- PMSF
phenyl methyl sulfonyl fluoride
- SDS
sodium dodecyl sulfate
- SSC (1×)
0.15m NaCl/0.015m sodium citrate (pH 7.0)
- STC
1.2m sorbitol, 100mm Tris-HCl, pH 7.4, and 10mm CaCl2
- STET
0.1m NaCl, 10mm Tris-HCl, pH 8.0, 1mm EDTA, pH 8.0, 5% Triton-X-100
Deceased. This paper is dedicated to the memory of Lorne S. Reid. 相似文献
15.
Haruko Ogawa Ako Hijikata Maho Amano Kyoko Kojima Hisako Fukushima Ineo Ishizuka Yoshie Kurihara Isamu Matsumoto 《Glycoconjugate journal》1996,13(4):555-566
The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing 1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.Abbreviations
Cry j I
a major allergenic glycoprotein ofCryptomeria japonica
- B-SJA-II
Sophora japonica bark lectin II
- CTA
Clerodendron trichotomum lectin
- TFMS
trifluoromethanesulfonic acid
- HRP
horseradish peroxidase 相似文献
16.
A family of dispersed repetitive sequences (Hch1) which is present in the genome of the wild barley Hordeum chilense was studied in detail. Hch1 sequences are found both as part of short tandem arrays and dispersed throughout the H. chilense chromosomes. Subcloning of sections of the sequence reveals that it is composed of unrelated classes of sequences which can also be found separately in other genomic locations. Analysis of these sequences in the genomes of wheat and two other wild barley species strongly suggests that specific amplifications and arrangements of the repeated sequences have taken place during speciation. Nucleotide sequence analysis fails to detect, in their entirity, the features shown by plant transposons. 相似文献
17.
L. M. V. Jonsson M. E. G. Aarsman J. van Diepen P. de Vlaming N. Smit A. W. Schram 《Planta》1984,160(4):341-347
An anthocyanin 5-O-glucosyltransferase from flowers of Petunia hybrida was purified about 30-fold. Using uridine 5-diphosphoglucose as glucose donor (Km 0.22 mM), the enzyme glucosylated the 3-(p-coumaroyl)-rutinoside derivatives of delphinidin and petunidin (Km 3 M), isolated from pollen of Petunia. Delphinidin 3-rutinoside, cyanidin 3-rutinoside and delphinidin 3-glucoside did not serve as substrates. The glucosylation of petunidin 3-(p-coumaroyl)-rutinoside showed a pH-activity optimum at pH 8.3 and was neither stimulated by Mg2+ or Ca2+, nor inhibited by ethylenediaminetetraacetic acid. After separating the 5-O-glucosyltransferase from the anthocyanidin 3-O-glucosyltransferase by means of chromatofocusing, it was shown that both enzymes exhibit a high degree of positional specificity. The 5-O-glucosyltransferase activity was correlated with the gene An1, but not with the gene Gf.Abbreviations HPLC
high performance liquid chromatography
- 3GT
3-O-glucosyltransferase
- 5GT
5-O-glucosyltransferase
- 3RGac
3-(p-coumaroyl)-rutinoside
- 3RGac5G
3-(p-coumaroyl)-rutinoside-5-glucoside
- UDPGlc
uridine 5-diphosphoglucose 相似文献
18.
Díaz-Quintana A Navarro JA Hervás M Molina-Heredia FP De la Cerda B De la Rosa MA 《Photosynthesis research》2003,75(2):97-110
Plastocyanin and cytochrome c
6 are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes
b
6
f and Photosystem I. Despite plastocyanin and cytochrome c
6 differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern
(one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the
two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar
way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c
6 reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria,
in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed
using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins
have been shown to be critical for the electron transfer reaction. Cytochrome c
6 does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic
patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial
protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction
with Photosystem I.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
19.
20.
Genetic divergence was evaluated in 31 breeding lines from fourBrassica species using Mahalanobis'D
2
. A new method of grouping usingD
2
values was used to group the 31 lines, based on diagnostic morphological traits (called morphoqts). Isozyme variation of
the individual enzymes esterase and glutamate oxaloacetate was quantified by five parameters (called isoqts) developed earlier.
Grouping by the same method was also done based on the isoqts, and the grouping by isozymes was compared with that by morphoqts.
Overall, there was an agreement of 73% suggesting that isoqts can be used in the choice of parents and also first stage selection
of segregants in the laboratory. It was suggested that such an exercise would help to take care of season-bound and field-related
problems of breeding. The new isozyme QTs, within lane variance of relative mobility and relative absorption, accounted for
about 50% of the total divergence. The utility of the new method and isoqts in cost-effective breeding were highlighted. 相似文献