Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

Tokugawa, Kazuhisa; Oguri, Suguru; Takeuchi, Makoto

doi:10.1007/bf01049679

Cited by 52 publications

(28 citation statements)

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“…Determination of Activities of GnT-IV-GnT-IV activity was assayed using fluorescence-labeled substrate according to the method of Nishikawa et al (19) with some modifications (26). Preparation of the substrate Gn 2 (2Ј,2)core-PA ( Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of the substrate Gn 2 (2Ј,2)core-PA ( Fig. 1) was done as reported (26). Enzyme solution (15 l) was incubated at 37°C for 4 h with 125 mM MOPS buffer, pH 7.3, containing 0.8 mM substrate, 20 mM UDP-GlcNAc, 7.5 mM MnCl 2 , 200 mM GlcNAc, 0.5% (w/v) Triton X-100, 10% glycerol, and 5 mg/ml bovine serum albumin in a total volume of 50 l. After the incubation, 50 l of water was added and the enzyme reaction was stopped by boiling for 2 min.…”

Section: Methodsmentioning

confidence: 99%

“…1. (GGn) 2 (2Ј,2)core-PA and Gn 2 (2Ј,2)core-PA was prepared from pyridylaminated biantennary sugar chains isolated from human apo-transferrin by glycosidase digestion (26). Core-PA was prepared from Gn 2 (2Ј,2)core-PA by digestion with ␤-N-acetylhexosaminidase in 150 mM sodium citrate phosphate buffer, pH 5.5, at 37°C for 20 h. GGn(2Ј),Gn(2)core-PA was prepared from (NeuAc␣2-6Gal␤1-4GlcNAc␤1-2Man␣1-6)(Gal␤1-4GlcNAc␤1-2Man␣1-3)Man␤1-4GlcNAc␤1-4GlcNAc-PA (Takara) and Gn(2Ј), GGn(2)core-PA was prepared from (Gal␤1-4GlcNAc␤1-2Man␣1-6)(NeuAc␣2-6Gal␤1-4GlcNAc␤1-2Man␣1-3)Man␤1-4GlcNAc␤1-4GlcNAc-PA (Takara) by sequential digestion with sialidase after ␤-galactosidase treatment.…”

Section: Methodsmentioning

confidence: 99%

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Purification and Characterization of UDP-N-Acetylglucosamine: α1,3-d-Mannoside β1,4-N-Acetylglucosaminyltransferase (N-Acetylglucosaminyltransferase-IV) from Bovine Small Intestine

Oguri¹,

Minowa²,

Ihara³

et al. 1997

Journal of Biological Chemistry

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A new ␤1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn 2؉, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the ␤1-4-linked GlcNAc residue on the Man␣1-3 arm. The purified enzyme shows a single band of M r 58,000 and behaves as a monomer. The substrate specificity demonstrated that the ␤1-2-linked GlcNAc residue on the Man␣1-3 arm (GnT-I product) is essential for the enzyme activity. ␤1-4-Galactosylaion to this essential ␤1-2-linked GlcNAc residue or N-acetylglucosaminylation to the ␤-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while ␤1-6-Nacetylglucosaminylation to the Man␣1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-Nacetylglucosamine:␣-3-D-mannoside ␤-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.The complex-type of oligosaccharides are synthesized through elongation by glycosyltransferases after trimming of the precursor oligosaccharides transferred to proteins in the endoplasmic reticulum. N-Acetylglucosaminyltransferases (GnTs) 1 take part in the formation of branches in the biosynthesis of complex-type sugar chains. In vertebrates, six GnTs, designated as GnT-I to -VI, which catalyze the transfer of GlcNAc to the core mannose residues of Asn-linked sugar chains, have been identified (1),

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Purification and Characterization of UDP-N-Acetylglucosamine: α1,3-d-Mannoside β1,4-N-Acetylglucosaminyltransferase (N-Acetylglucosaminyltransferase-IV) from Bovine Small Intestine

Oguri¹,

Minowa²,

Ihara³

et al. 1997

Journal of Biological Chemistry

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“…The substrate Gn 2 (2Ј,2)core-PA was prepared as reported previously (30). 10 g of each expression plasmid was incorporated into 5 ϫ 10 6 of COS-7 cells in 0.8 ml of phosphatebuffered saline by electroporation using a Gene Pulser (Bio-Rad) and conditions of 1,600 V, 25 F at room temperature.…”

Section: ј-Rapid Amplification Of Cdna Ends (Race)-5ј-race Was Carrimentioning

confidence: 99%

cDNA Cloning and Expression of Bovine UDP-N-Acetylglucosamine:α1,3-d-Mannoside β1,4-N-Acetylglucosaminyltransferase IV

Minowa¹,

Oguri²,

Yoshida

et al. 1998

Journal of Biological Chemistry

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UDP-N-acetylglucosamine:␣1,3-D-mannoside ␤1,4-Nacetylglucosaminyltransferase (GnT-IV) is one of the essential enzymes in the production of tri-and tetra-antennary Asn-linked sugar chains. Recently, we have successfully purified GnT-IV from bovine small intestine. Based on the partial amino acid sequence of the purified bovine GnT-IV enzyme, its cDNA has been cloned from bovine small intestine. The open reading frame is 1,605 base pairs long, and this sequence produced GnT-IV activity on transient expression in COS-7 cells. Although the deduced amino acid sequence does not have any significant homology with other known N-acetylglucosaminyltransferases (GnTs), the hydrophobicity profile showed a typical type II transmembrane protein structure, which is common to many glycosyltransferases. N-terminal amino acid sequencing of the purified GnT-IV revealed that 92 amino acids, including a transmembrane region, were truncated during purification. Of the three potential N-glycosylation sites Asn-458 was actually glycosylated in the purified enzyme, although this N-glycosylation site could be abolished without any reduction in GnT-IV activity. Serial deletions at both the N and C termini proved that the catalytic domain of GnT-IV is located in the central region of the enzyme. The GnT-IV mRNA level correlated with enzymatic activity in the various bovine tissues tested.UDP-N-acetylglucosamine:␣1,3-D-mannoside ␤1,4-N-acetylglucosaminyltransferase (GnT-IV) 1 (EC 2.4.1.145) is one of the essential enzymes in the synthesis of the multiantennary structure of Asn-linked sugar chains. It specifically generates the ␤1-4 linkage of GlcNAc to the ␣1-3 Man of the core structure of N-glycans (Man 3 GlcNAc 2 ). GnT-IV produces tetra-antennary structures in cooperation with GnT-V, which produces the ␤1-6 linkage of GlcNAc to the ␣1-6 Man of the core structure. This tetra-antennary structure is dominant in certain glycoproteins, such as erythropoietin (EPO) and ␣ 1 -acid glycoprotein, and very important for their bioactivity (1, 2). For instance, the in vivo biological activity of recombinant EPO has a positive correlation with the ratio of tetra-/biantennary sugar chains. Pharmacokinetics studies have shown that the bulky structure of the tetra-antennary branching prevents EPO from filtering out into the urine. The increase in the antenna number of ␣ 1 -acid glycoprotein observed under a chronic inflammatory state or liver damage suggested that some clinical situations and/or cellular disorders may also affect the branch formation of sugar chains (3). On the other hand, structural analyses of oligosaccharides in hepatoma cell lines and in choriocarcinoma have suggested that GnT-IV activity should be high in certain kinds of tumors (4 -7). Rat 3Y1 cells transformed with the adenovirus 12 gene also elevated the expression of the ␤1-4 GlcNAc on the ␣1-3 Man (8).To date, GnT-I, -II, -III, and -V have been purified and their cDNAs and/or genomic DNAs have been cloned (9 -23). Among the enzymes necessary to produce tetra-antennary sugar cha...

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“…They are not decisive although several puriˆcation methods have been devised. [1][2][3][4] The samples prepared by these puriˆca-tion steps still contain considerable amounts of impurities that had similar retention times as the PAoligosaccharides in the HPLC. Therefore we attempted to develop a new puriˆcation method for PA-oligosaccharides.…”

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confidence: 99%

Method for Purification of Fluorescence-Labeled Oligosaccharides by Pyridylamination

Natsuka

Adachi

Kawaguchi

et al. 2002

Bioscience, Biotechnology, and Biochemistry

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We developed a convenient method for puriˆcation of PA-oligosaccharides to remove contaminants originating from natural ‰uorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this puriˆcation method are described in this report.

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Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

Cited by 52 publications

References 4 publications

Purification and Characterization of UDP-N-Acetylglucosamine: α1,3-d-Mannoside β1,4-N-Acetylglucosaminyltransferase (N-Acetylglucosaminyltransferase-IV) from Bovine Small Intestine

Purification and Characterization of UDP-N-Acetylglucosamine: α1,3-d-Mannoside β1,4-N-Acetylglucosaminyltransferase (N-Acetylglucosaminyltransferase-IV) from Bovine Small Intestine

cDNA Cloning and Expression of Bovine UDP-N-Acetylglucosamine:α1,3-d-Mannoside β1,4-N-Acetylglucosaminyltransferase IV

Method for Purification of Fluorescence-Labeled Oligosaccharides by Pyridylamination

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