3Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received on August 9, 2001; revised on November 9, 2001; accepted on November 12, 2001.
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Abstract |
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Key words: donor specificity/kinetic analysis/ N-acetylglucosaminyltransferase V/recombinant enzyme/UDP-GlcNAc
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Introduction |
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Some divalent metal-requiring GlcNAc transferases, such as GnTs-I (Nishikawa et al., 1988), -III (Nishikawa et al., 1992
; Ihara et al., 1993
), -IV (Oguri et al., 1997
; Yoshida et al., 1998
), and -VI (Taguchi et al., 2000
; Sakamoto et al., 2000
), have the so-called DxD motif, which is thought to be involved in Mn2+ ion binding and catalysis (Busch et al., 1998
; Wiggins and Munro, 1998
; Breton and Imberty, 1999
). On the other hand, GnT-V does not appear to require divalent cation for its transfer reaction, as is also the case for other ß1,6GlcNAc transferases. A common sequence motif involved in the catalytic mechanism has not been identified in this class of glycosyltransferase, because no significant sequence homology has not been found among the ß1,6GlcNAc transferases. Therefore considerably less is known about the mechanism of GnT-V compared to other GlcNAc transferases in terms of chemical and structural bases, in spite of the clinical importance of GnT-V.
It is generally believed that GnT-V is involved in cancer metastasis, and, as a result, the development of potent and specific inhibitors has been attempted (Kanie et al., 1993; Brockhausen et al., 1995
; Lu et al., 1996
). These inhibitors have been designed so as to mimic the acceptor oligosaccharide, probably because substrate specificity with respect to the acceptor has been intensively examined. However, the design of more potent inhibitors, such as transition analogs and slow-binding inhibitors, must await the elucidation of the catalytic mechanism. Because some major chemical reactions, specifically cleavage and reformation of bonds and inversion of configuration, during the transfer reaction occurs at a position C-1 of the transferred monosaccharide in the donor, it would be desirable to investigate the enzymatic properties of GnT-V in terms of its action with respect to the donor substrate, for example, structural requirements of the donor nucleotide-sugar for enzyme action, which would be expected to be involved in the catalytic mechanism.
In addition, though a detailed analysis of the properties of GnT-V would be useful for characterization, it may also provide valuable information as to whether GnT-V actually participates in the biosynthesis of the oligosaccharides in living cells. For example, it is possible that in some cells the production of ß1,6-branched oligosaccharides might be unexpectedly low, even though the expression and activity of GnT-V are significantly high. A more detailed analysis could explain this possible discrepancy and would provide a more clear explanation of the relation between tumor progressions, the content of ß1,6-branched oligosaccharides, and the expression or activity of the enzyme. Considering the importance of GnT-V and its product in tumor progression, such findings would be expected to be clinically useful.
In the present study, we report on the preparation of a polyhistidine-tagged soluble form of human GnT-V proteins using a baculovirusinsect cell expression system and kinetic analyses using various donor nucleotide-sugar derivatives to elucidate the structural requirements of the transferred monosaccharides. We show that not only UDP-GlcNAc but also UDP-Glc and TDP-Glc serve as donor substrates for GnT-V and that the specificity toward the nucleotide portion of the donor can be attributed to the catalytic step rather than the binding step. We also demonstrate that the in vivo level of UDP-GlcNAc is a critical factor in the production of ß1,6-branched N-glycans.
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Results |
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As shown in the kinetic analysis, the Km value of GnT-V for the donor is 4.0 mM (Table I) and seems to be relatively larger, compared to other GlcNAc transferases (Szumilo et al., 1987; Nishikawa et al., 1988
; Ikeda et al., 2000
). Furthermore, it would be more probable that this high Km value is sufficiently higher than the intracellular or intra-Golgi concentration of UDP-GlcNAc (Murphy et al., 1973
; Waldman and Rudnick, 1990
), therefore it would be expected that the enzyme responds to variations in the donor concentration in a linear manner. Under these conditions, the concentration is clearly a critical regulating factor in the biosynthesis of ß1,6-branched oligosaccharides.
To investigate the effect of the UDP-GlcNAc concentration in vivo on the production of this type of sugar chain, GlcNAc was added to the culture of mouse B16 melanoma cells, which express a high level of GnT-V (Yoshimura et al., 1995; Taniguchi et al., 2000
), to increase the intracellular UDP-GlcNAc level. Because nucleotide-sugar transporters are known to be a class of antiporter, transport of cytoplasmic UDP-GlcNAc into the lumen of the Golgi depends on the electrochemical potential differences of both UDP-GlcNAc and UMP across the membrane. Therefore, although the intra-Golgi level cannot be determined, it is conceivable that the intra-Golgi level of UDP-GlcNAc reflects the increase in the cytoplasmic level. The cells treated with sugars were analyzed by an UDP-GlcNAc determination and a lectin blot (Figure 7). As a result of the addition of 20 mM GlcNAc into the culture medium, the amount of UDP-GlcNAc was increased four times larger (Figure 7A). When the cells were analyzed by SDSPAGE followed by Coomassie brilliant blue staining and carbohydrate staining, essentially no differences were found in the staining patterns (Figure 7C). In addition, the addition of GlcNAc also had no effect on the activity of GnT-V (Figure 7B). However, in the lectin blot analysis with leukoagglutinating phytohemagglutinin (L-PHA), which recognizes sugar chains with a core ß1,6-branch, an enhancement of signals was observed (Figure 7C). It may be possible that the L-PHA staining is sometimes affected by the substitution and different linkages of ß1,6-branch. However, because the addition of GlcNAc to the transfected GnT-V-overexpressing cells led to no alteration in the lectin binding, this treatment does not appear to cause such a structural alteration (data not shown). Therefore, it is suggested that the increase in the L-PHA binding results from the increased levels of ß1,6-branch rather than other structural changes. On the other hand, the addition of 20 mM glucosamine did not affect the amount of UDP-GlcNAc and the reactivity toward L-PHA (Figure 7A, C). The same treatment of the GnT-III-transfected B16 cells did not increase the levels of bisected sugar chains, the products of GnT-III, as probed by E-PHA, a lectin that is specific for bisected oligosaccharides (data not shown). The treatment with 20 mM of the monosaccharides did not affect the viability of the cells. Thus these results suggest that the increase in intracellular UDP-GlcNAc level facilitates the in vivo production of ß1,6-branched sugar chains without an increase in GnT-V activity.
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Discussion |
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It has been generally thought that the core ß1,6-branched oligosaccharides are participating in cancer metastasis and malignancy, and, in this context, the potential importance of GnT-V has been emphasized (Dennis and Laferte, 1989; Demetriou et al., 1995
, 2001; Granovsky et al., 2000
). Hence the development of a potent and specific inhibitor of GnT-V has been attempted on the basis of specificity toward the acceptor substrates. This strategy was based on the definition of the minimal structural requirement for an oligosaccharide acceptor for GnT-V because the minimal requirements are different among the glycosyltransferases even though they share a common substrate. The results of investigation of donor substrate specificity suggest that the configuration at the C-4 position of the transferred monosaccharide is an essential factor in the action of GnT-V, as revealed by the inability of UDP-Gal and UDP-GalNAc to act as donor substrates. On the other hand, it was found that the catalytic efficiency toward UDP-Glc is about 6% of the values for UDP-GlcNAc, suggesting that the presence of a 2-N-acetyl group is not necessarily essential. In addition, it was shown that the thymidine nucleotide sugar also appears to be tolerated by GnT-V. These properties of GnT-V are different from those of another GlcNAc transferase, which is involved in core structure formation, such as GnT-III (Ikeda et al., 2000
), and the different characteristics indicate that the mechanism underlying the donor specificity of GnT-V is unique.
Because of the clinical importance of GnT-V and the ß1,6-branched oligosaccharide, numerous efforts have been made to investigate correlations between GnT-V expression, to examine the content of the ß1,6-branched sugar chain and malignant potential, and to develop specific inhibitors on the basis of acceptor specificity. However, as suggested by the present study involving kinetic analysis, the UDP-GlcNAc level also appears to be a particularly important factor in the regulation of ß1,6-branched oligosaccharide formation, due to the unique kinetic properties of GnT-V. In some malignant cells in which UDP-GlcNAc levels are low, the levels of the ß1,6-branched structure may be lower than expected, compared to GnT-V expression levels. Furthermore, a definition of the difference in the mechanism of the substrate specificity among glycosyltransferases with respect to donor as well as acceptor would be useful in designing more specific inhibitors.
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Materials and methods |
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Site-directed mutagenesis
Site-directed mutagenesis was carried out according to the method of Kunkel (1985), for the creation of a poly-histidine tag (His x 6) at the C-terminus of the GnT-V. Briefly, the 3' Hind IIIXba I 0.7-kb fragment of human GnT-V cDNA was subcloned into a pBluescript SK+ (Stratagene). The uracil-substituted single-stranded template was prepared from Escherichia coli CJ236, which had been transformed by the plasmid. The uracil template was used with a synthetic oligonucleotide primer to replace the STOP codon by Gly and to extend the C-terminal sequence by Gly-Gly-His-His-His-His-His-His-(Stop). The oligonucleotide primer used in this study was 5'-AAA GAC TGC CTA GGG GGG GGA CAT CAT CAT CAC CAC CAC TAG CGG CCG GGG AAG ACA GTG-3'. The resulting mutation was verified by dideoxy sequencing using a DNA sequencer (DSQ-1000, Shimazu), as were the entire sequences that had been subjected to mutagenesis.
Transfer plasmids for baculovirus system
In this study, two forms of the recombinant proteins, namely, BVV73 and BVV
188, were used. These represent C-terminus polyhistidine-tagged proteins that are fused to a cleavable signal peptide derived from baculoviral protein gp67. To introduce a polyhistidine tag at the C-terminus of the GnT-V, Hind IIIXba I fragments of expression plasmid for GnVd73 and GnV187, which are chimeric proteins with GnT-III (Sasai et al., 2001
), were replaced by the 3' Hind IIIBam HI fragment in which the tag sequence had been created as described (designed for GnVd73HIS and GnVd187HIS, respectively). For the preparation of BVV
73 protein, Bsp EIEag I fragment of GnVd73HIS was ligated to Xma IEag I site of a transfervector, pAcGP67-A vector (PharMingen). For the preparation of BVV
188 protein, expression plasmid for GnVd187HIS was digested by Kpn I and Eag I. The resulted fragment was treated with T4 polymerase and ligated in to Sma I site of a pAcGP67-A vector.
Expression of soluble recombinant hGnT-V in insect cells
Spodoptera frugiperda (Sf) 21 cells were maintained at 27°C in Graces insect media (Gibco-BRL) supplemented with 10% fetal bovine serum, 3.33 g/L of yeastolate, 3.33 g/L of lactalbumin hydrolysate, and 100 mg/L of kanamycine. Recombinant viruses were manipulated as described previously (Murphy et al., 1997). The purified transfer plasmid (1 µg) was cotransfected into 5 x 105 Sf21 cells with 10 ng of Baculo Gold DNA (PharMingen), which was used as Autographa californica nuclear polyhedrosis viral genome. The transfection experiments were carried out by the Lipofectin (Gibco-BRL) method (Felgner et al., 1987
), as described previously (Ikeda et al., 2000
). The recombinant virus, the titer of which was 510 multiples of infection, was infected to the cells (5 x 107, 80% confluent state) in a 175-cm2 flask for 1 h. The medium was collected about 5 days postinfection for purification of the secreted GnT-V.
Purification of the recombinant enzyme
Cell debris in the culture medium was precipitated by saturated ammonium sulfate and pelleted by centrifugation. The pellet was dissolved in and dialyzed against 50 mM TrisHCl (pH 8.0) and 200 mM NaCl. The dialyzed materials were applied to a Ni2+-chelating Sepharose fast flow column (Amersham Pharmacia) equilibrated with 50 mM TrisHCl (pH 8.0) containing 200 mM NaCl and 40 mM imidazole. The column was then washed thoroughly with 50 mM TrisHCl (pH 8.0) containing 200 mM NaCl and 80 mM imidazole. The soluble GnT-V proteins were eluted from the column with 50 mM TrisHCl (pH 8.0) containing 200 mM NaCl and 160 mM imidazole.
SDSPAGE and immunoblotting
SDSPAGE was carried out according to Laemmli (1970), and the protein bands were visualized using a silver-staining kit (Daiichi Pure Chemicals) or Coomassie brilliant blue. The separated proteins were electrophoretically transferred onto a PROTORAN (Schleicher and Schuell), followed by blocking with 5% skim milk. The resulting membrane was incubated with the first antibody. After washing, the membrane was then reacted with the anti-mouse antibody. The reactive protein bands were visualized by a chemiluminescence using an electrochemiluminescence system (Amersham-Pharmacia).
GnT-V activity assay and kinetic analysis
The GnT-V activity was assayed using a fluorescence-labeled oligosaccharide acceptor, as described previously (Taniguchi et al., 1989; Sasai et al., 2001
). Cell homogenates (20 µg proteins) and purified enzyme (50 ng) were incubated at 37°C for 15 min with 10 µM GnGn-bi-PA as an acceptor and 2 mM UDP-GlcNAc as the donor in 125 mM MES-NaOH (pH 6.25) containing 200 mM GlcNAc, 0.5% Triton X-100 and 10 mM ethylenediamine tetra-acetic acid. The reaction was terminated by heating the mixture at 100°C for 2 min, and the sample was then centrifuged at 15,000 rpm for 5 min in a microcentrifuge. The resulting supernatant was analyzed by reversed-phase HPLC (Shimazu) using a TSKgel ODS-80TM (4.6 x 150, Tosoh). The solvent used was a 20 mM ammonium acetate buffer (pH 4.0), and the substrate and the product were isocratically separated. Fluorescence was detected with a fluorescence detector (RF-10AXL, Shimazu) at excitation and emission wavelengths of 320 nm and 400 nm, respectively. For kinetic analyses, the purified recombinant GnT-V proteins were incubated with various concentrations of GnGn-bi-PA and various nucleotide sugar derivatives.
ESI-MS
ESI-MS was carried out as described previously (Ikeda et al., 2000) using an LCQ (Finnigan) quadrupole mass spectrometer. The PA-labeled oligosaccharide was dissolved in a 50% aqueous methanol and introduced into the ion source by direct infusion at a flow rate of 3 µl/min using a syringe pump integrated into system. ESI-MS spectra were obtained using the positive ion mode. The ion spray voltage and capillary voltage were 4.5 kV and 10 V, respectively, and capillary temperature was 200°C. Full scan spectra were obtained in the range of 10001800.
Cell culture
B16-F10 melanoma cells were routinely maintained at 37°C in Dulbeccos modified Eagle medium (Nikken) supplemented with 10% fetal calf serum (Gibco-BRL), 50 U/ml penicillin G, and 50 µg/ml streptomycin under a humidified atmosphere of 95% air and 5% CO2.
Monosaccharide treatment
When B16-F10 cells reached at approximately 50% confluence, monosaccharides, GlcNAc, or glucosamine were supplemented with normal culture media at the concentration of 20 mM. After 72 h, the extracted cellular proteins were subjected to SDSPAGE, and the separated proteins were electrophoretically transferred onto a PROTORAN. The transferred membranes were blocked with 2% bovine serum albumin and incubated with biotin-labeled lectin. After washing, the membrane was reacted with an avidin-biotin complex with the peroxidase. The reactive glycoprotein bands were visualized by chemiluminescence using an ECL system (Amersham-Pharmacia). For the detection of sugars in glycoconjugates, the transferred membrane was treated with a DIG Glycan Detection Kit (Boehringer Mannheim) according to the manufactures protocol.
Determination of the amount of cellular UDP-GlcNAc
B16-F10 cells were scraped and homogenized in 0.7 M perchloric acid, followed by microcentrifugation at 15,000 rpm. The resulting supernatants were neutralized with 5 M potassium carbonate and microcentrifuged at 15,000 rpm. The resulting supernatants, containing the extracted nucleotide-sugars, were separated on a Partisil SAX anion exchange HPLC column (4.6 x 250 mm) and quantified by UV absorption at 254 nm as described previously (Robinson et al., 1995).
Protein determination
Protein concentrations were determined using the method described by Bradford (1976) using bovine serum albumin as a standard.
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Acknowledgment |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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Breton, C. and Imberty, A. (1999) Structure/function studies of glycosyltransferases. Curr. Opin. Struct. Biol., 9, 563571.[CrossRef][ISI][Medline]
Brockhausen, I., Reck, F., Kuhns, W., Khan, S., Matta, K.L., Meinjohanns, E., Paulsen, H., Shah, R.N., Baker, M.A., and Schachter, H. (1995) Substrate specificity and inhibition of UDP-GlcNAc:GlcNAc ß 1-2Man alpha 1-6R ß 1, 6-N-acetylglucosaminyltransferase V using synthetic substrate analogues. Glycoconj. J., 12, 371379.[ISI][Medline]
Brockhausen, I., Carver, J.P., and Schachter, H. (1988) Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V, and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes. Biochem. Cell Biol., 66, 11341151.[ISI][Medline]
Busch, C., Hofmann, F., Selzer, J., Munro, S., Jeckel, D., and Aktories, K. (1998) A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J. Biol. Chem., 273, 1956619572.
Cummings, R.D., Trowbridge, I.S., and Kornfeld, S. (1982) A mouse lymphoma cell line resistant to the leukoagglutinating lectin from Phaselusvulgaris is deficient in UDP-GlcNAc: -D-mannoside ß 1, 6 N-acetylglucosaminyltransferase. J. Biol. Chem., 257, 1342113427.
Demetriou, M., Granovsky, M., Quaggin, S., and Dennis, J.W. (2001) Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation. Nature, 409, 733739.[CrossRef][ISI][Medline]
Demetriou, M., Nabi, I.R., Coppolino, M., Dedhar, S., and Dennis, J.W. (1995) Reduced contact-inhibition and substratum adhesion in epithelial cells expressing GlcNAc-transferase V. J. Cell Biol., 130, 383392.[Abstract]
Dennis, J.W. and Laferte, S. (1989) Oncodevelopmental expression of GlcNAc ß 1-6Man 1-6Man ß 1branched asparagine-linked oligosaccharides in murine tissues and human breast carcinomas. Cancer Res., 49, 945950.[Abstract]
Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl Acad. Sci. USA, 84, 74137417.[Abstract]
Granovsky, M., Fata, J., Pawling, J., Muller, W.J., Khokha, R., and Dennis, J.W. (2000) Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nat. Med., 6, 306312.[CrossRef][ISI][Medline]
Gu, J., Nishikawa, A., Tsuruoka, N., Ohno, M., Yamaguchi, N., Kangawa, K., and Taniguchi, N. (1993) Purification and characterization of UDP-N-acetylglucosamine: -6-D-mannoside ß 1-6N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line. J. Biochem. (Tokyo), 113, 614619.[Abstract]
Ihara, Y., Nishikawa, A., Tohma, T., Soejima, H., Niikawa, N., and Taniguchi, N. (1993) cDNA cloning, expression, and chromosomal localization of human N-acetylglucosaminyltransferase III (GnT-III). J. Biochem. (Tokyo), 113, 692698.[Abstract]
Ikeda, Y., Koyota, S., Ihara, H., Yamaguchi, Y., Korekane, H., Tsuda, T., Sasai, K., and Taniguchi, N. (2000) Kinetic basis for the donor nucleotide-sugar specificity of ß1, 4-N-Acetylglucosaminyltransferase III. J. Biochem. (Tokyo), 128, 609619.[Abstract]
Kanie, O., Crawley, S.C., Palcic, M.M., and Hindsgaul, O. (1993) Acceptor-substrate recognition by N-acetylglucosaminyltransferase-V: critical role of the 4-hydroxyl group in ß-D-GlcpNAc-(1-2)--D-Manp(1-6)-ß-D-Glcp-OR. Carbohydr. Res., 243, 139164.[CrossRef][ISI][Medline]
Korczak, B., Le, T., Elowe, S., Datti, A., and Dennis, J.W. (2000) Minimal catalytic domain of N-acetylglucosaminyltransferase V. Glycobiology, 10, 595599.
Kunkel, T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl Acad. Sci. USA, 82, 488492.[Abstract]
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Lu, P.P., Hindsgaul, O., Compston, C.A., and Palcic, M.M. (1996) New synthetic trisaccharide inhibitors for N-acetylglucosaminyltransferase-V. Bioorg. Med. Chem., 4, 20112022.[CrossRef][ISI][Medline]
Murphy, C.I., Piwnica-Worm, H., Grunwald, S., and Romanow, W.G. (1997) Maintenance of insect cell culture and generation of recombinant baculoviruses. In Ausubel, F.M., Brent, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) Current Protocol in Molecular Biology. John Wiley and Sons, Chichester, UK, 16.10.116.10.17.
Murphy, G., Ariyanayagam, A.D., and Kuhn, N.J. (1973) Progesterone and the metabolic control of the lactose biosynthetic pathway during lactogenesis in the rat. Biochem. J., 136, 11051116.[ISI][Medline]
Nishikawa, A., Ihara, Y., Hatakeyama, M., Kangawa, K., and Taniguchi, N. (1992) Purification, cDNA cloning, and expression of UDP-N-acetylglucosamine: ß-D-mannoside ß-1, 4N-acetylglucosaminyltransferase III from rat kidney. J. Biol. Chem., 267, 1819918204.
Nishikawa, Y., Pegg, W., Paulsen, H., and Schachter, H. (1988) Control of glycoprotein synthesis: Purification and characterization of rabbit liver UDP-N-acetylglucosamine:-3-D-mannoside ß-1, 2-N-acetylglucosaminyltransferase I. J. Biol. Chem., 263, 82708281.
Oguri, S., Minowa, M.T., Ihara, Y., Taniguchi, N., Ikenaga, H., Takeuchi, M. (1997) Purification and characterization of UDP-N-acetylglucosamine: 1, 3-D-mannoside ß1, 4-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase-IV) from bovine small intestine. J. Biol. Chem., 272, 2272122727.
Palcic, M.M., Ripka, J., Kaur, K.J., Shoreibah, M., Hindsgaul, O., and Pierce, M. (1990) Regulation of N-acetylglucosaminyltransferase V activity: kinetic comparisons of parental, Rous sarcoma virus transferomed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog. J. Biol. Chem., 265, 67596769.
Robinson, K.A., Weinstein, M.L., Lindenmayer, G.E., and Buse, M.G. (1995) Effects of diabetes and hyperglycemia on the hexosamine synthesis pathway in rat muscle and liver. Diabetes, 44, 14381446.[Abstract]
Saito, H., Nishikawa, A., Gu, J., Ihara, Y., Soejima, H., Wada, Y., Sekiya, C., Niikawa, N., and Taniguchi, N. (1994) cDNA cloning and chromosomal mapping of human N-acetylglucosaminyltransferase V+. Biochem. Biophys. Res. Commun., 198, 318327.[CrossRef][ISI][Medline]
Sakamoto, Y., Taguchi, T., Honke, K., Korekane, H., Watanabe, H., Tano, Y., Dohmae, N., Takio, K., Horii, A., and Taniguchi, N. (2000) Molecular cloning and expression of cDNA encoding chicken UDP-N-acetyl-D-glucosamine (GlcNAc): GlcNAc ß1-6(GlcNAc ß1-2)-man 1-R[GlcNAc to man] ß1, 4 N-acetylglucosaminyltransferase-VI. J. Biol. Chem., 275, 3602936034.
Sasai, K., Ikeda, Y., Tsuda, T., Ihara, H., Korekane, H., Shiota, K., and Taniguchi, N. (2001) The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-Acetylglucosaminyltransferase V: the involvement of domain homophilic interaction. J. Biol. Chem., 276, 759765.
Shoreibah, M.G., Hindsgaul, O., and Pierce, M. (1992) Purification and characterization of rat kidney UDP-N-acetylglucosamine:-6-mannoside ß-1, 6-N-acetylglucosaminyltransferase. J. Biol. Chem., 267, 29202927.
Szumilo, T., Kaushal, G.P., and Elbein, A.D. (1987) Purification and properties of the glycoprotein processing N-Acetylglucosaminyltransferase II from plants. Biochemistry, 26, 54985505.[ISI][Medline]
Taguchi, T., Ogawa, T., Inoue, S., Inoue, Y., Sakamoto, Y., Korekane, H., and Taniguchi, N. (2000) Purification and characterization of UDP-GlcNAc: GlcNAcß 1-6(GlcNAcß 1-2)Man 1-R [GlcNAc to Man]-ß 1, 4-N-acetylglucosaminyltransferase VI from hen oviduct. J. Biol. Chem., 275, 3259832602.
Taniguchi, N., Jain, S.K., Takahashi, M., Ko, J.H., Sasai, K., Miyoshi, E., and Ikeda, Y. (2000) Glycosyltransferses: cell surface remodeling and regulation of receptor tyrosine kinases-induced signaling. Pure Appl. Chem., 71, 719728.[ISI]
Taniguchi, N., Nishikawa, A., Fujii, S., and Gu, J.G. (1989) Glycosyltransferase assays using pyridylaminated acceptors: N-acetylglucosaminyltransferase III, IV, and V. Meth. Enzymol., 179, 397408.[ISI][Medline]
Traynor, A.J., Hall, E.T., Walker, G., Miller, W.H., Melancon, P., and Kuchta, R.D. (1996) Inhibition of UDP-N-Acetylglucosamine import into Golgi membranes by nucleotide monophosphates. J. Med. Chem., 39, 28942899.[CrossRef][ISI][Medline]
Waldman B.C. and Rudnick, G. (1990) UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP. Biochemistry, 29, 4452.[ISI][Medline]
Wiggins, C.A. and Munro, S. (1998) Activity of the yeast MNN1 -1, 3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl Acad. Sci. USA, 95, 79457950.
Yoshida, A., Minowa, M.T., Takamatsu, S., Hara, T., Ikenaga, H., and Takeuchi, M. (1998) A novel second isoenzyme of the human UDP-N-acetylglucosamine:1, 3-D-mannoside ß1, 4-N-acetylglucosaminyltransferase family: cDNA cloning, expression, and chromosomal assignment. Glycoconj. J., 15, 11151123.[CrossRef][ISI][Medline]
Yoshimura, M., Nishikawa, A., Ihara, Y., Taniguchi, S., and Taniguchi, N. (1995) Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection. Proc. Natl Acad. Sci. USA, 92, 87548758.[Abstract]
Zhang, N., Peng, K.C., Chen, L., Puett, D., and Pierce, M. (1997) Circular dichroic spectroscopy of N-acetylglucosaminyltransferase V and its substrate interactions. J. Biol. Chem., 272, 42254229.