Human serum contains a novel .beta.1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo(N-acetyllactosaminoglycans)

Leppänen, Anne; Penttilä, Leena; Niemelä, Ritva; Helin, Jari; Seppo, Antti; Lusa, Sari; Renkonen, Ossi

doi:10.1021/bi00102a022

Cited by 36 publications

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“…Glycans 1, 2, and 3 were synthesized essentially as described previously (15,17,19). Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc-PA (lacto-N-neotetraose-PA), Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc-PA (lacto-N-tetraose-PA), and Gal␤1-3GlcNAc␤1-3(Gal␤1-4GlcNAc␤1-6)Gal␤1-4Glc-PA (lacto-Nhexaose-PA) were prepared by pyridylamination of lacto-N-neotetraose, lacto-N-tetraose, and lacto-N-hexaose using the commercial pyridylamination apparatus Glyco TAG (Takara, Shiga, Japan), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…We refer to this enzyme activity as dIGnT6 to emphasize the distal site of its action (15,16). The second type of the enzyme activity acts at the midchain positions of the (completed or growing) poly-N-acetyllactosamine chains, converting Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-R to Gal␤1-4GlcNAc␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc␤1-R (13,(15)(16)(17). Accordingly, we refer to this enzyme activity as cIGnT6.…”

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confidence: 99%

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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

Sakamoto¹,

Taguchi²,

Tano³

et al. 1998

Journal of Biological Chemistry

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A ␤1,6N-acetylglucosaminyltransferase (␤1-6GnT) responsible for the formation of the ␤1,6-branched poly-Nacetyllactosamine structure has been purified 210,000-fold in 2.4% yield from a homogenate of hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni 2؉ -chelating Sepharose FF, and UDP-hexanolamine-agarose, using an assay wherein pyridylaminated lacto-N-neotetraose (Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc-PA) was used as an acceptor substrate, and the reaction product was Gal␤1-4Glc-NAc␤1-3(GlcNAc␤1-6)Gal␤1-4Glc-PA. The apparent molecular weight of the purified enzyme was 76,000 under nonreducing conditions. The enzyme has a pH optimum at 7.0 and has no requirement for any divalent metal ions. The K m values for pyridylaminated lacto-Nneotetraose and UDP-GlcNAc were 0.96 and 2.59 mM, respectively. For its activity, this enzyme was shown to have an absolute requirement of at least a complete LacNAc (LacNAc ‫؍‬ Gal␤1-4GlcNAc) residue bound to position 3 of the acceptor Gal residues, i.e. it is capable of acting only on the Gal residues of internal LacNAc units. The data strongly suggest that this enzyme could be involved in generating branches to central positions of preformed as well as growing polylactosamine chains, but not in synthesizing the distal branches to growing polylactosamine chains.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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

Sakamoto¹,

Taguchi²,

Tano³

et al. 1998

Journal of Biological Chemistry

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“…Briefly, GlcNAcß 1-3'LacNAcß 1-3'LacNAc [8], was al,3-fucosylated partially by using al,3/4 fucosyltransferase(s) of human milk [9]. The fraction of monofucosylated products was isolated by paper chromatography and the hexasaccharide GlcNAcßl-3'(Fucal-3)LacNAcßl-3'LacNAc was isolated from this fraction by WGA-agarose chromatography (Niemelä et al, loe.…”

Section: Synthesis Of the Heptasaccharidementioning

confidence: 99%

Improved enzymatic synthesis of a highly potent oligosaccharide antagonist of L‐selectin

et al. 1997

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The polylactosamine sLexßl-3'(sLexßl-6')LacNAcßl-3'(sLexßl-6')LacNAcßl-3'(sLexßl-6')LacNAc (7) (where sLex is Neu5Aca2-3Galßl^t(Fucal-3)GlcNAc and LacNAc is Galßl^4GlcNAc) is a nanotnolar L-selectin antagonist and therefore a potential anti-inflammatory agent (Renkonen et al. (1997) Glycobiology, 7, 453). Here we describe an improved synthesis of 7. The octasaccharide LacNAcßl-3'LacNAcßl-3'LacNAcßl-3'LacNAc (4) was converted into the triply branched undecasaccharide LacNAcßl-3'(GlcNAcßl-6')LacNAcßl-3'(GlcNAcßl-6')LacNAcßl-3'(GlcNAcßl-6')-LacNAc (5) by incubation with UDP-GlcNAc and the midchain ßl,6-GlcNAc transferase activity of rat serum. Glycan 5 was enzymatically ßl,4-galactosylated to LacNAcßl-3'(LacNAcßl-6')LacNAcßl-3'(LacNAcßl-6')LacNAcßl-3'(LacNAcßl-6')-LacNAc (6). Combined with the enzymatic conversion of 6 to 7 (Renkonen et al., loe. cit.) and the available chemical synthesis of 4, our data improve the availability of 7 for full assessment of its anti-inflammatory properties.© 1997 Federation of European Biochemical Societies.

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“…Degradative Experiments-Digestions with endo-␤-galactosidase from Bacteroides fragilis (EC 3.2.1.103) (Boehringer Mannheim) were performed according to Leppä nen et al (9); parallel control reactions cleaved over 90% of radiolabeled GlcNAc␤1-3Gal␤1-4GlcNAc.…”

Section: Methodsmentioning

confidence: 99%

“…The "distally acting" dIGnT6 transfers a GlcNAc unit in the ␤1,6 linkage to the penultimate galactose residue at the growing end of the linear polylactosamine chain (Scheme 1) (2)(3)(4)(5)(6)(7)(8). By contrast, the "centrally acting" cIGnT6 transfers a GlcNAc residue in the ␤1,6 linkage to midchain galactose units of preformed as well as growing linear chains (3,9,10). The dIGnT6 reactions proceed only with acceptor chains bearing distal GlcNAc residues, whereas the cIGnT6 works with polylactosamine backbones carrying either a galactose or a GlcNAc residue at the distal position.…”

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Biosynthesis of Branched Polylactosaminoglycans

Leppänen¹,

Zhu²,

Maaheimo³

et al. 1998

Journal of Biological Chemistry

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Two types of ␤1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAc␤1-3Gal␤1-4GlcNAc␤1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAc␤1-3)Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Gal␤1-4GlcNAc␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc from the linear tetrasaccharide Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc-␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc and Gal␤1-4GlcNAc-␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc from the linear hexasaccharide Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAc␤1-3Gal␤1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.

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Human serum contains a novel .beta.1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo(N-acetyllactosaminoglycans)

Cited by 36 publications

References 35 publications

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

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

Improved enzymatic synthesis of a highly potent oligosaccharide antagonist of L‐selectin

Biosynthesis of Branched Polylactosaminoglycans

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Human serum contains a novel .beta.1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo(N-acetyllactosaminoglycans)

Cited by 36 publications

References 35 publications

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

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

Improved enzymatic synthesis of a highly potent oligosaccharide antagonist of L‐selectin

Biosynthesis of Branched Polylactosaminoglycans

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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

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