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ABSTRACT: The transfection of glycoprotein glycosyltransferase genes into cells leads to modification of both the structure and function of the glycoproteins and as a result, changes in glycome patterns. N-glycan branching enzymes hold some promise as a model system for the identification of glycome patterns. Both N-acetylglucosaminyltransferase III and alpha 1-6 fucosyltransferase are typical glycosyltransferases, which are involved in the branching of N-glycans. The resulting enzymatic products, bisecting N-GlcNAc and alpha 1-6 fucose residues, are no longer modified by other glycosyltransferases and it is a relatively simple task to identify their modification by means of lectins. In this review, the glycome patterns of glycosyltransferase gene transfectants and the non-transfectants were compared by two-dimensional gel electrophoresis and lectin staining, and the biological significance of the two genes are described. Analyses of glycome patterns by transfecting glycosyltransferase genes will lead to new fields of study in the area of postgenome research.
PROTEOMICS 03/2001; 1(2):239-47. · 4.51 Impact Factor
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ABSTRACT: The mechanism for cell-cycle-dependent regulation of N-acetylglucosaminyltransferase III (GnT-III) activity was investigated using synchronized culture of Colo201, a human colon cancer cell line. In the synchronized culture, it was found that GnT-III activity rapidly increased in the M phase and the maximal activity was five times higher than the basal level found in the G1 phase. Northern blot and Western blot analyses revealed that the increase in the activity is due not to an increase in expression level of its mRNA but, rather, to the level of protein. Furthermore, it was shown by a pulse-chase experiment that the increased protein level of GnT-III is the result of its prolonged turnover rate. Lectin blotting with erythroagglutinating phytohemagglutinin showed that the content of bisecting N-acetylglucosamine structure in glycoproteins was transiently increased during the M phase in conjunction with the increased activity of GnT-III. These results suggest that GnT-III activity undergoes a cell-cycle-dependent regulation and thereby oligosaccharide structures of N-glycans vary specifically during the M phase of the cell cycle. Thus, it is possible that the cell-cycle-dependent alteration of N-glycans by GnT-III might play a role in biological events, such as the progression of cell cycle and cell division.
Archives of Biochemistry and Biophysics 03/2000; 374(1):52-8. · 2.93 Impact Factor
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ABSTRACT: N-acetylglucosaminyltransferases III (GnT-III) and V (GnT-V) play a pivotal role in the processing of N-linked glycoproteins, and are highly involved in cancer progression and metastasis. Expression of GnT-III and GnT-V in the liver is enhanced during hepatocarcinogenesis, although they are not expressed in the normal liver. Gene expression of GnT-V is regulated by a transcriptional factor, ets-1, which is involved in angiogenesis and invasion of tumor cells. When the formation of the product of GnT-V, GlcNAc-beta1-6 branches, is inhibited by overexpression of GnT-III, lung metastasis of melanoma cells is suppressed. Modification of glycoprotein receptors such as the receptors for epidermal growth factor and nerve growth factor by GnT-III sense transfection changes an intracellular signaling pathway, which may lead to a variety of biological alterations in tumor cells. In this review, we focus on cancer progression and metastasis in relation to GnT-III and GnT-V.
Biochimica et Biophysica Acta 11/1999; 1455(2-3):287-300. · 4.66 Impact Factor
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M Yoshimura, Y Ihara,
T Nishiura,
Y Okajima,
M Ogawa,
H Yoshida,
M Suzuki,
K Yamamura,
Y Kanakura,
Y Matsuzawa,
N Taniguchi
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ABSTRACT: Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing beta-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.
Biochemical Journal 06/1998; 331 ( Pt 3):733-42. · 4.90 Impact Factor
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Y Ihara,
M Yoshimura,
E Miyoshi,
A Nishikawa,
A S Sultan,
S Toyosawa,
A Ohnishi,
M Suzuki,
K Yamamura,
N Ijuhin,
N Taniguchi
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ABSTRACT: N-Acetylglucosaminyltransferase III (GnT-III) produces "bisecting-GlcNAc" and regulates the branching of N-glycans. GnT-III activity is elevated during hepatocarcinogenesis, which is in contrast to the undetectable level found in normal hepatocytes. To determine the biological significance of GnT-III in hepatocytes, transgenic mice that specifically express GnT-III in the liver were established and characterized. The transgenic hepatocytes had a swollen oval-like morphology, with many lipid droplets. Apolipoprotein B, which contained increased level of bisecting-GlcNAc accumulated in the transgenic hepatocytes. In the transgenic serum, triglycerides, the beta- and pre-beta-lipoprotein fractions, and apolipoprotein B100 were significantly decreased, compared with levels in nontransgenic serum. These abnormal phenotypes were more prominent in the mice with more copies of the transgene and a resulting high GnT-III activity. We demonstrate that aberrant glycosylation, as the direct result of the formation of bisecting-GlcNAc, disrupts the function of apolipoprotein B, leading to the generation of fatty liver. This observation suggests a novel mechanism for the pathogenesis of fatty liver.
Proceedings of the National Academy of Sciences 04/1998; 95(5):2526-30. · 9.68 Impact Factor
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Advances in Enzyme Regulation 02/1998; 38:223-32.
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ABSTRACT: beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) is a pivotal glycosyltransferase which participates in branch formation by catalysis of the synthesis of a bisecting GlcNAc structure in N-glycans. These structures are thought to be one of the unique features of the N-glycans of neural tissues. To examine the intracellullar role of GnT-III expression and its product in neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells which normally express a low level of GnT-III. In the GnT-III gene-transfected cells, lectin blot analysis showed that some glycoproteins showed increased levels of bisecting GlcNAc structures. Following treatment with nerve growth factor (NGF) the control cells showed neurite outgrowth for differentiation whereas the transfectants showed no morphological response or change in the rate of cell growth. Transient tyrosine phosphorylation of the Trk/NGF receptor was detected at 5-15 min after NGF treatment in control cells, but not detected in the GnT-III gene-transfected cells despite the intact binding of NGF to the cells. Moreover the dimerization of Trk with NGF treatment was not induced in the GnT-III transfectant as compared with the dimerization seen in control cells. These results indicate that overexpression of GnT-III gene in PC12 cells affects some functions of glycoprotein receptors such as Trk by alteration of N-glycan structures, and results in changes in the intracellular signaling pathway of tyrosine phosphorylation modified by NGF.
Journal of Biological Chemistry 05/1997; 272(15):9629-34. · 4.77 Impact Factor
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ABSTRACT: GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a culture supernatant of human gastric cancer cell line MKN45. The purification procedures included chromatographies on Q-Sepharose Fast Flow, synthetic GDP-hexanolamine-Sepharose, and GnGn-bi-Asn-Sepharose columns. SDS-PAGE of the purified enzyme gave a major band corresponding to an apparent molecular mass of 60 kDa. The enzyme was recovered in a 12% final yield with an approximately 4,600-fold increase in specific activity. The pH optimum was 7.5, and the enzyme was fully active in the presence of 5 mM EDTA and did not require divalent cations, Mg2+ and Ca2+. Oligonucleotide primers designed from partial amino acid sequences were used to amplify and clone alpha1-6 FucT cDNA from a cDNA library of MKN45 cells. The cDNA encodes 575 amino acids in length, and contains the predicted N-terminal and internal amino acid sequences derived on lysyl endopeptidase digestion. The homology to porcine brain alpha1-6 FucT is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence of the human MKN45 cell enzyme or that of porcine brain. Thus, the enzyme is distinct from other fucosyltransferases which catalyze alpha1-2, alpha1-3, and alpha1-4 fucose addition.
Journal of Biochemistry 04/1997; 121(3):626-32. · 2.37 Impact Factor
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ABSTRACT: The bisecting N-acetylglucosamine residue is formed by UDP-N-acetylglucosamine:beta-D-mannoside-beta-1, 4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enzyme for N-glycans. We found that forskolin, an adenylyl cyclase activator, markedly enhanced GnT-III at the transcriptional level in various hepatoma cells and hepatocytes, resulting in an increase of bisecting GlcNAc residues in various glycoproteins, as judged from the lectin binding to erythroagglutinating phytohemagglutinin (E-PHA). In whole cell lysates, the E-PHA binding was increased, and leukoagglutinating phytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the maximum levels. In contrast, the binding capacity as to E-PHA, determined by fluorescence-activated cell sorting on the cell surface, was decreased, suggesting that bisecting GlcNAc structures in certain glycoproteins changed the expression levels of glycoproteins and decreased their sorting on the cell surface. Fractionated organelles of M31 cells showed that the binding capacity as to E-PHA was mainly localized in Golgi membranes and lysosomes. This was also supported by a fluorescence microscopy. In order to determine whether or not the bisecting GlcNAc residue acts as a sorting signal for glycoproteins, N-oligosaccharide structures of lysosomal-associated membrane glycoprotein 1 and beta-glucuronidase, gamma-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and alpha-fetoprotein were measured by E-PHA and L-PHA blotting after immunoprecipitation. The expression levels of lysosomal membrane glycoprotein 1 and gamma-glutamyltranspeptidase on the cell surface were decreased at 12 h after forskolin treatment, indicating that the bisecting GlcNAc structure may act as a negative sorting signal for the cell surface glycoproteins and may alter the characteristics of hepatoma cells. This is the first report on glycoprotein sorting related to a specific structure of oligosaccharides, bisecting GlcNAc.
Journal of Biological Chemistry 02/1997; 272(5):2866-72. · 4.77 Impact Factor
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ABSTRACT: GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-->6fucosyltransferase (alpha1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 2)Manbeta1-4GlcNAcbet a1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440, 000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The alpha1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. alpha1-6FucT activity was found to be high in the transfected cells, as compared with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that alpha1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.
Journal of Biological Chemistry 12/1996; 271(44):27810-7. · 4.77 Impact Factor
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Glycobiology 11/1996; 6(7):691-4. · 3.58 Impact Factor
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ABSTRACT: N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-6-D-mannoside to produce the beta1-6 linked branching of N-glycan oligosaccharides, which controls the polylactosamine content. The expression of N-acetylglucosaminyltransferase V, which contains 17 exons and spans 155 kilobase pairs, is expressed in a tissue- and cell type-specific manner and is regulated at the level of transcription by multiple promoters (Saito, H., Gu, J., Nishikawa, A., Ihara, Y., Fujii, J., Kohgo, Y., and Taniguchi, N. (1995) Eur. J. Biochem. 233, 18-26). To elucidate the mechanism by which the GnT-V gene is expressed in a cell- and tissue-specific manner, cell-restricted expression was analyzed using the 5'-upstream regions of the human GnT-V gene spanning base pairs -2760 to +23 in a human bile duct carcinoma cell line, HuCC-T1. We characterized two cis-acting elements that are potentially important in HuCC-T1 cell-specific expression. The two elements each contain an Ets-1 binding site, 5'-GGA-3'. Specific binding of Ets-1 to the respective elements was demonstrated by competition analysis as well as by antibody supershift experiments. Cotransfection of an Ets-1 expression plasmid along with a GnT-V promoter-luciferase reporter plasmid revealed the participation of Ets-1 in the regulation of the GnT-V gene transcription. These data indicated that the transcriptional regulation of the GnT-V gene was mediated by transcription factor Ets-1.
Journal of Biological Chemistry 11/1996; 271(43):26706-12. · 4.77 Impact Factor
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N Uozumi,
T Teshima,
T Yamamoto,
A Nishikawa,
Y E Gao,
E Miyoshi,
C X Gao,
K Noda,
K N Islam, Y Ihara,
S Fujii,
T Shiba,
N Taniguchi
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ABSTRACT: An assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-6FucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. [equation: see text] The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have alpha 1-6fucose at the reducing end GlcNAc. This method is highly specific for alpha 1-6FucT and is applicable for various experiments, including purification and cell culture ones.
Journal of Biochemistry 09/1996; 120(2):385-92. · 2.37 Impact Factor
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ABSTRACT: We have isolated cDNA clones for the human N-acetylglucosaminyltransferase III (GlcNAc-transferase III) gene. Two of them, H15 and H20, contain 5' non-coding regions that are totally different from each other except for 8 bp adjacent to the putative initiation codon. Analysis of one of the genomic cosmid clones containing the GlcNAc-transferase III coding region, Hug3, revealed the 5' non-coding regions of H15 and H20 contain two and one exons, respectively, in addition to the exon containing the coding region (exon 1). These have arisen as the result of alternative splicing. The transcription-initiation sites were determined by primer-extension analysis and 5'-rapid amplification of cDNA ends (RACe). Both H15-specific and H20-specific primers gave cDNAs longer than those expected from the lengths of H15 and H20, and a primer complementary to the region around the intron/exon junction near the putative initiation codon also gave distinct signals. Promoter activities of the 5'-flanking regions of H15, H20 and exon 1 were measured in a human hepatoblastoma cell line, HuH-6 cells by luciferase assays. The 5'-flanking region of exon 1 was the most active, whilst that of H15 was several times less active, and that of H20 was inactive. Our study suggests that multiple promoters of the GlcNAc-transferase III gene contribute to the complex regulation of this gene.
European Journal of Biochemistry 07/1996; 238(3):853-61. · 3.58 Impact Factor
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ABSTRACT: Introduction of the beta1-4 N-acetylglucosaminyltransferase (GnT-III) gene was reported to suppress metastasis in highly metastatic B16-hm murine melanoma cells (Yoshimura, M., Nishikawa, A. , Ihara, Y., Taniguchi, S., and Taniguchi, N.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8754-8758). In this study, the effect of GnT-III gene transfer on E-cadherin was studied, since E-cadherin acts as a suppressor of metastasis. E-cadherin expression at cell-cell contacts of B16-hm cells expressing high GnT-III activity was greater than controls without affecting transcription. Lectin blotting showed that E-cadherin from GnT-III transfectants was glycosylated by ectopically expressed GnT-III. The glycosylated E-cadherin exhibited the delayed turnover and the decreased release from cell surface, as compared with the native E-cadherin, resulting in the elevated expression at the cell-cell border of GnT-III transfectants. Furthermore, cell-cell aggregation was enhanced in GnT-III transfectants, indicating that the glycosylated E-cadherin is biologically functional. These results suggest that the glycosylated E-cadherin contributes to the suppression of metastasis by the introduction of GnT-III gene into melanoma cells.
Journal of Biological Chemistry 07/1996; 271(23):13811-5. · 4.77 Impact Factor
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ABSTRACT: beta 1-4 N-acetylglucosaminyltransferase (GnT-III) catalyzes the formation of bisecting N-acetylglucosamine (GlcNAc) in the biosynthesis of N-linked oligosaccharides. To examine the effect of bisecting GlcNAc on the natural killer (NK) cytotoxicity, the GnT-111 gene was introduced into NK-sensitive K562 cells that have no detectable GnT-III activity. We obtained three clones stably expressing high GnT-III (positive transfectants). Introduction of the GnT-III gene resulted in an increase of bisecting GlcNAc and a decrease of external sialic acid as well as tri- and tetraantennary sugars, as judged by flow cytometry. Compared to controls, the NK cytotoxicity was completely blocked against positive transfectants. The binding of effector cells to positive transfectants was also decreased. After s.c. injection into nude mice, positive transfectants produced spleen colonization, although no spleen lesions were formed by control cells. In nude mice depleted of NK cells by anti-asialo GM1 antibody, both positive transfectants and controls produced spleen colonization equally. These results indicate that K562 cells expressing GnT-III are resistant to NK cytotoxicity, resulting in spleen colonization in nude mice.
Cancer Research 02/1996; 56(2):412-8. · 7.86 Impact Factor
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ABSTRACT: 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: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) and UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase V (GnT-V: EC 2.4.1.155), two major branch forming enzymes in N-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 to Datsura 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.
Glycoconjugate Journal 01/1996; 12(6):787-94. · 2.12 Impact Factor
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ABSTRACT: N-acetylglucosaminyltransferases III and V (GnT-III and -V) are key enzymes in the synthesis of the branches of asparagine-linked oligosaccharides. Although their messenger RNAs (mRNAs) are expressed in various rat tissues, they are not detected in normal rat liver. Expression of the GnT-III and -V mRNAs, however, increased in regenerating liver after two-thirds partial hepatectomy compared with sham-operated rats. The enzymatic activities of GnT-III and -V increased in proportion to mRNA expression. To determine which type of cells in the liver have high activities of these glycosyltransferases, hepatocytes and nonparenchymal cells were separated by means of a two-step collagenase perfusion technique. GnT-III activity was only detected in nonparenchymal cells of normal rat liver. However, during liver regeneration, GnT-III activity increased and thus was also detectable in hepatocytes. GnT-V activity was detected in both types of cells. These data were supported by reverse transcription-polymerase chain reaction results. Although the mechanism underlying the induction of these glycosyltransferases is unknown, lectin blot analysis showed that oligosaccharides in many glycoproteins, including hepatocyte growth factor, a major growth factor associated with liver regeneration, were newly synthesized during liver regeneration. This is the first report on the expression of glycosyltransferases during liver regeneration and suggests that there are different mechanisms involved in regulation of the genes of GnT-III and -V during liver regeneration.
Hepatology 01/1996; 22(6):1847-55. · 11.66 Impact Factor
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ABSTRACT: beta-D-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of N-acetylglucosamine in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-linked oligosaccharides and forms a bisecting GlcNAc structure. Although the biological meaning of the bisecting GlcNAc structure remains unclear, it is known that the attachment of a bisecting GlcNAc inhibits further processing of oligosaccharides by other glycosyltransferases. To investigate whether or not structural changes of oligosaccharides affect secretion and gene expression of hepatitis B virus (HBV), we introduced the GnT-III gene into a human hepatoma cell line, HB611, which secreted HBV-related proteins into the medium. Positive transfectants were cloned by hygromycin resistant selection. Three clones have high activities of GnT-III and secreted lower levels of HBV-related proteins into the medium in comparison with other clones. These clones showed marked suppression of HBV-related mRNAs and an increased binding with E-PHA as judged by lectin blot. Expression of beta actin, alpha fetoprotein, albumin, and prealubmin was not correlated with GnT-III activity in all the seven clones. Treatment of these cells with tunicamycin or swainsonine resulted in enhanced expression of HBV-related mRNA. These results indicate that some glycoproteins whose oligosaccharide structures are changed by over-expression of GnT-III suppress HBV gene expression.
Journal of Biological Chemistry 12/1995; 270(47):28311-5. · 4.77 Impact Factor
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ABSTRACT: UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc transferase V), which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-6-D-mannoside, is an important enzyme regulating the branch formation in complex-type, N-linked oligosaccharides. It has been reported that the enzymic activity of GlcNAc transferase V increases after viral transformation and the enzymic product is closely related to the metastasis of tumors. We previously reported the purification, cDNA cloning and chromosomal mapping of human GlcNAc transferase V. In this study, we describe the isolation of genomic clones encoding human GlcNAc transferase V and the structure of the gene. The human GlcNAc transferase V gene is divided into 17 exons, and the open reading frame is encoded by exons 2-17, spanning 155 kb. Analysis of the 5'-untranslated regions of mRNAs from various cells showed multiple sequences depending on the cell types. The promoter region of the GlcNAc transferase V gene was characterized by searching for any consensus sequences matching those for transcription-factor binding. The consensus sequences for a TATA box, AP-1, AP-2, and some other transcription factors were found in the 5'-upstream region of exon 1, and consensus sequences for LF-A1, HNF1-HP1, liver-restricted transcription factors and other factors were also found in intron 1. Chloramphenicol acetyltransferase fusion plasmids with either the 5'-upstream region of exon 1 or intron 1 were constructed and transfected into COS-1 cells. Promoter activities of both DNA fragments were detected, indicating that transcription starts within this region. These data suggest that the human GlcNAc transferase V gene employs a multiple promoter system for its transcription, and gene expression may therefore be regulated in tissue-specific and cell-type-specific manners.
European Journal of Biochemistry 11/1995; 233(1):18-26. · 3.58 Impact Factor
