Purification and Characterization of UDP-GlcNAc:Galβ1–4GlcNAcβ1–3*Galβ1–4Glc(NAc)-R(GlcNAc to *Gal) β1,6N-Acetylglucosaminyltransferase from Hog Small Intestine

Sakamoto, Yoshihiro; Taguchi, Tomohiko; Tano, Yasuo; Ogawa, Tomoya; Leppänen, Anne; Kinnunen, Marjo; Aitio, Olli; Parmanne, Pinja; Renkonen, Ossi; Taniguchi, Naoyuki

doi:10.1074/jbc.273.42.27625

Cited by 18 publications

(16 citation statements)

References 35 publications

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“…These results suggest that human and rabbit erythroid precursor cells contain IGnT that is the same as or similar to the IGnT cloned from PA-1 cells. Hog small intestine may have similar poly-N-acetyllactosamine structures as seen in erythrocytes and PA-1 cells, since a cIGnT was purified from this tissue (46). The present study also demonstrated that intrinsic properties of ␤4Gal-TI and iGnT are critical in forming short I-branches.…”

Section: Addition Of Multiple Short I-branches 9301supporting

confidence: 51%

Regulation of I-Branched Poly-N-Acetyllactosamine Synthesis

Ujita

McAuliffe

Suzuki

et al. 1999

Journal of Biological Chemistry

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I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching ␤1,6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewis x , which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching ␤1,6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers ␤1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various ␤1,4-galactosyltransferases (␤4Gal-Ts), ␤4Gal-TI is most efficient in adding a galactose to linear and branched poly-Nacetyllactosamines. When a ␤1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of ␤4Gal-TI and i-extension ␤1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Gal␤134GlcNAc␤133 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that ␤4Gal-TI adds a galactose poorly to ␤1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while ␤1,3-N-acetylglucosaminyltransferase and ␤4Gal-TI efficiently add N-acetyllactosamine to linear poly-Nacetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.

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Section: Addition Of Multiple Short I-branches 9301supporting

confidence: 51%

Regulation of I-Branched Poly-N-Acetyllactosamine Synthesis

Ujita

McAuliffe

Suzuki

et al. 1999

Journal of Biological Chemistry

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“…A human GlcNAc-transferase, termed cIGnT6 (centrally acting), which catalyses the transfer of GlcNAc to the 6-position of internal galactose residues of poly-N-lactosamine chains of N-linked glycans has been characterized (Mattila et al, 1998). A similar enzyme has been characterized from hog kidney (Sakamoto et al, 1998) and a related enzyme from rat serum has been used to synthesize a poly-branched decasaccharide with poly-N-acetyllactosamine units . dIGnT6 (distally acting) transfers GlcNAc to the 6-position of b-GlcNAc-(1 !…”

Section: Glcnac-transferasesmentioning

confidence: 99%

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update covering the period 1999–2000

Harvey

2006

Mass Spectrometry Reviews

125

112

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This review describes the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of carbohydrates and glycoconjugates and continues coverage of the field from the previous review published in 1999 (D. J. Harvey, Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates, 1999, Mass Spectrom Rev, 18:349-451) for the period 1999-2000. As MALDI mass spectrometry is acquiring the status of a mature technique in this field, there has been a greater emphasis on applications rather than to method development as opposed to the previous review. The present review covers applications to plant-derived carbohydrates, N- and O-linked glycans from glycoproteins, glycated proteins, mucins, glycosaminoglycans, bacterial glycolipids, glycosphingolipids, glycoglycerolipids and related compounds, and glycosides. Applications of MALDI mass spectrometry to the study of enzymes acting on carbohydrates (glycosyltransferases and glycosidases) and to the synthesis of carbohydrates, are also covered.

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“…Hence, the elimination of the cytoplasmic and the membrane binding segment from the recombinant cIGnT6 of the present experiments was probably not associated with major changes in the substrate specificity. Consequently, it is worth noting that the recombinant cIGnT6 shares several features with the soluble cIGnT6 enzymes present in mammalian serum (18,31) and with the membrane-bound form of cIGnT6 recently isolated from hog small intestine by Sakamoto et al (46). The common features include (i) the ability to transfer one or several branches to midchain galactoses of long polylactosamine chains, (ii) the unability to react at peridistal galactose units in acceptors of the type GlcNAc␤1-3Gal␤1-4GlcNAc␤1-OR, and (iii) the inability to transfer at midchain galactoses that belong to Lewis x determinants.…”

Section: Discussionmentioning

confidence: 99%

The Centrally Acting β1,6N-Acetylglucosaminyltransferase (GlcNAc to Gal)

Mattila¹,

Salminen²,

Hirvas³

et al. 1998

Journal of Biological Chemistry

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In the present experiments the cDNA coding for a truncated form of the ␤1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 g of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Gal␤1-4GlcNAc␤1-3Gal␤1-4-GlcNAc, converting the acceptor to the branched pentasaccharide Gal␤1-4GlcNAc␤1-3(GlcNAc␤1-6)Gal␤1-4-GlcNAc as shown by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry, degradative experiments, and 1 H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAc␤1-3Gal␤1-4GlcNAc. Accordingly, we call the recombinant ␤1,6-GlcNAc transferase cIGnT6 to emphasize its action at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, ␤1,4galactosyltransferase and ␤1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

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Purification and Characterization of UDP-GlcNAc:Galβ1–4GlcNAcβ1–3Galβ1–4Glc(NAc)-R(GlcNAc to Gal) β1,6N-Acetylglucosaminyltransferase from Hog Small Intestine

Cited by 18 publications

References 35 publications

Regulation of I-Branched Poly-N-Acetyllactosamine Synthesis

Regulation of I-Branched Poly-N-Acetyllactosamine Synthesis

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update covering the period 1999–2000

The Centrally Acting β1,6N-Acetylglucosaminyltransferase (GlcNAc to Gal)

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