Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-<i>N</i>-acetylglucosaminyltransferase

Cheng, Pi-Wan; Wingert, William E.; Little, Mark R.; Wei, Ran

doi:10.1042/bj2270405

Cited by 15 publications

(4 citation statements)

References 37 publications

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“…The reaction mixtures were incubated at 37 • C for 2 h and terminated with 0.3 ml of 10 mM ZnCl 2 . The products were isolated by solid phase extraction on C18 cartridges with 4 × 0.5 ml of methanol as previously described [17]. The isolated products were dried by Automatic Environmental SpeedVac System (Savant, Holbrook, NY) and re-suspended in 500 µl of H 2 O.…”

Section: Methodsmentioning

confidence: 99%

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

et al. 2005

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Cytomegalovirus (CMV) immediate early promoter is a powerful promoter frequently used for driving the expression of transgenes in mammalian cells. However, this promoter gradually becomes silenced in stably transfected cells. We employed Chinese Hamster Ovary (CHO) and human pancreatic cancer (Panc 1) cells stably tansfected with three glycogenes driven by a CMV promoter to study the activation of silenced glycogenes. We found that butyrate, tricostatin A (TSA), and 5-aza-2 -deoxycytidine (5-Aza-dC) can activate these CMV-driven glycogenes. The increase in mRNA and protein of a glycogene occurred 8-10 h after butyrate treatment, suggesting an indirect effect of butyrate in the activation of the transgene.The enhanced expression of the trangenes by butyrate and TSA, known inhibitors of histone deacetylase, was independent of the transgene or cell type. However, the transgene can be activated by these two agents in only a fraction of the cells derived from a single clone, suggesting that inactivation of histone deacetylase can only partially explain silencing of the transgenes. Combination treatment of one or both agents with 5-Aza-dC, a known inhibitor of DNA methylase, resulted in a synergistic activation of the transgene, suggesting a cross-talk between histone acetylation and DNA demethylation. Understanding the mechanisms of the inactivation and reactivation of CMV promoter-controlled transgenes should help develop an effective strategy to fully activate the CMV promoter-controlled therapeutic genes silenced by the host cells. Published in 2005.

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Section: Methodsmentioning

confidence: 99%

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

et al. 2005

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“…Core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT, EC 2;4.1.102) is a glycosyltransferase that catalyses the transfer of GlcNAc from UDP-GlcNAc to the O-glycan core 1 structure, Galβ1-3GalNAc-R, to form the core 2 structure, GlcNAcβ1-6(Galβ1-3)GalNAc-R. Although various C2GnT activities have been found and characterized in a variety of tissues, it is not known how many different C2GnTs exist [1][2][3][4][5][6][7][8][9][10][11]. Substrate competition studies on C2GnT activity in mucin-secreting tissues revealed that a single enzyme (M type C2GnT) catalyses the addition of the β-6GlcNAc branch to form mucin core 2, core 4 [GlcNAcβ1-6(GlcNAcβ1-3)GalNAc-] and the branch of the blood group I antigenic determinant, GlcNAcβ1-6(GlcNAcβ1-3)Galβ- [4,5].…”

Section: Introductionmentioning

confidence: 99%

Expression of stable human O-glycan core 2 β-1,6-N-acetylglucosaminyltransferase in Sf9 insect cells

Toki

Sarkar

Yip

et al. 1997

Biochemical Journal

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UDP-GlcNAc:Galbeta1-3GalNAc-R (GlcNAc to GalNAc) beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) catalyses the formation of O-glycan core 2. Purification and characterization of C2GnT from natural sources has been hampered by the instability of this enzyme. We have been able to prepare a stable partly purified recombinant human C2GnT by expression of a truncated form of the enzyme in the baculovirus/Spodoptera frugiperda 9 (Sf9) insect cell system. C2GnT activity was secreted into the Sf9 culture medium (15 pmol/min per microl; approx. 0.2 mg/l) and was stable at 4 degrees C either in solution or after lyophilization. Endoglycosidase H and N-glycanase F treatment of the radiolabelled C2GnT indicated the presence of N-glycans at both potential N-glycosylation sites. The elimination of one or both of the two potential N-glycosylation sites or treatment of the virus-infected insect cells with tunicamycin resulted in loss of enzyme activity due in part to protein degradation.

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“…Analysis of the acceptor specificity showed that the recombinant bC2TF can transfer GlcNAc from UDP-GlcNAc only to core 1 disaccharide acceptor forming trisaccharide product, but not to core 3 and blood group i disaccharide acceptors. Analysis of the trisaccharide product by 1 H NMR showed that the NMR spectrum was identical to that of Gal␤3(GlcNAc␤6)Gal-NAc␣Bzl, core 2 structure (16,21).…”

Section: Construction Of a Recombinant Dna Containing The Complete Ormentioning

confidence: 94%

Mucin Biosynthesis: Molecular Cloning and Expression of Bovine Lung Mucin Core 2 N-Acetylglucosaminyltransferase cDNA

Adler²,

Cheng³

1998

Am J Respir Cell Mol Biol

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A cDNA clone containing a 2,150-bp insert was isolated from a bovine lung lambdagt10 cDNA library by cross-species hybridization using a DNA probe generated by polymerase chain reaction (PCR) employing a human cDNA that encodes mucin core 2 beta6-N-acetylglucosaminyltransferase (hC2TF) as the template. The bovine cDNA (bcDNA) insert was devoid of 220 bp of the 5' portion of the C2TF open reading frame (ORF), as predicted from the human counterpart. Southern blotting analysis suggested that the coding region of this C2TF gene is in one exon. To construct a full-length bovine C2TF (bC2TF) cDNA, a genomic DNA fragment containing the 5' portion of the ORF of the bC2TF gene was cloned from a lambdaEMBL bovine genomic DNA library and ligated to the 5' end of the cloned cDNA insert. DNA sequence analysis showed that the complete ORF of bC2TF gene was 1,281 bp in length, which corresponds to a polypeptide of 427 amino acids. Catalytically active bC2TF was expressed in sf21 insect cells infected with recombinant baculovirus containing the ORF of the bC2TF gene. The recombinant bC2TF catalyzed the synthesis of core 2, but not core 4 and blood group I structures. Western blotting analysis showed that the recombinant bC2TF migrated with the same mobility (approximately 55 kD) as the native bovine tracheal C2TF. Immunohistochemical analysis showed that in bovine trachea, the bC2TF was present at the surface epithelium and in the submucosal glands, with the latter being the major site of distribution.

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Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

Cited by 15 publications

References 37 publications

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Expression of stable human O-glycan core 2 β-1,6-N-acetylglucosaminyltransferase in Sf9 insect cells

Mucin Biosynthesis: Molecular Cloning and Expression of Bovine Lung Mucin Core 2 N-Acetylglucosaminyltransferase cDNA

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