α-D-Galactosyltransferase activity in calf thymus

Halbeek, Herman van; Vliegenthart, Johannes F.G.; Winterwerp, H.H.K.; Blanken, Willem M.; Eijnden, Dirk H. van den

doi:10.1016/0006-291x(83)91269-x

Cited by 44 publications

(16 citation statements)

References 12 publications

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“…The position of this signal, together with its J 1 , 2 of 3.9 Hz, are characteristic of a galactose residue in an alinkage. The effects on the chemical shifts of the H-1 and the H-4 of the Galb4 are indicative of a substitution at its carbon-3 position (Van Halbeek et al 1983;Joziasse et al 1987). In summary, these data show that the recombinant enzyme forms a Galcxl + 3 linkage to the terminal Gal in both Galbl --t 4GlcNAc and Galfil + 4Glc.…”

Section: The Enzymatic Properties Of'the Recombinant Enzyme and The Cmentioning

confidence: 64%

α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates

Joziasse

Shaper²,

Salyer³

et al. 1990

European Journal of Biochemistry

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We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP-Gal:GalPI + 4GlcNAc a1 + 3-galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J . Biol. Chem. 264, 14290-142971. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera ,frugiperda (Sf9) insect cells with recombinant virus, resulted in high-level expression of enzymatically active a1 + 3-galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000-fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant a1 + 3-galactosyltransferase could be readily detergent-solubilized and subsequently purified by affinity chromatography on UDP-hexanolamineSepharose. The recombinant a1 + 3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (GalP1 + 4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus a1 Bid. Clzem. 260, 12927-129341. In pilot studies, the recombinant enzyme was examined for the ability to synthesize a1 + 3-galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 'H-NMR, methylation analysis, HPLC, and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Galal + 3GalP1+ 4-R. Our results demonstrate that catalysis by recombinant a1 -+ 3-galactosyltransferase can be used to obtain preparative quantities of various a1 + 3-galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.The enzyme UDP-Gal:GalPl + 4GlcNAc a1 + 3-galactosyltransferase (a1 + 3-galactosyltransferase) is a Golgimembrane-bound enzyme that catalyzes the following reaction :Gala1 +3Gal/31+4ClcNAc-R + UDP The preferred acceptor structure, GalPl + 4GlcNAc, may be present at the non-reducing terminus of either glycolipids, glycoproteins or oligosaccharides (Blanken and Van den Eijnden 1985). Consequently, the a1 -+ 3-linked galactose occupies the terminal non-reducing position of N-acetyllactosamine-type carbohydrate chains and of lactosaminoCorrespondr.nce to

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Section: The Enzymatic Properties Of'the Recombinant Enzyme and The Cmentioning

confidence: 64%

α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates

Joziasse

Shaper²,

Salyer³

et al. 1990

European Journal of Biochemistry

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“…The level of ␣-galactosylation observed in this study is unusually high, although the presence of these moieties could be anticipated, since the host in which the viruses were grown is a bovine cell line and this species is known to possess ␣-1,3-galactosyltransferase activity (24,25). Substrate specificity studies on ␣-1,3-galactosyltransferase purified from calf thymus demonstrated that the oligosaccharide acceptor for this enzyme is Gal␤4GlcNAc-R; the activity of this enzyme is mutually exclusive with ␣-2,6-sialyltransferase and is blocked by the presence of ␣-1,3-fucosylation on the GlcNAc residue (38).…”

Section: Discussionmentioning

confidence: 95%

“…gave no indication of the anomericity of the linkage between these Gal residues, they were likely to be ␣-linked, since previous studies have identified ␣-1,3-galactosyltransferase activity in bovine cells (24,25).…”

Section: Complex-type Oligosaccharidesmentioning

confidence: 99%

The Glycosylation of the Influenza A Virus Hemagglutinin by Mammalian Cells

Mir-Shekari

Ashford

Harvey

et al. 1997

Journal of Biological Chemistry

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We have characterized the glycans at individual sites on the hemagglutinin of three influenza A variants to obtain information on the role of cell-specific glycosylation in determining the receptor binding properties of this virus. The variants differ in whether they have a glycosylation site at residue 129 on the tip of the hemagglutinin and whether amino acid 184 (near to the receptor binding site) is His or Asn. We found that all sites on each variant are glycosylated in Madin-Darby bovine kidney cells, that the glycosylation is site-specific, and that the glycans at the same site in each variant are highly similar. One site that is buried in the hemagglutinin trimer contains only oligomannose glycans. The remaining sites carry complex glycans of increasing size as the distance of the site from the viral membrane decreases. Most of these complex glycans are terminated with ␣-galactose residues, a consequence in bovine cells of the removal of terminal sialic acids by the viral neuraminidase. Although the glycans at residue 129 are among the smallest on the molecule, they are large enough to reach the receptor binding pocket on their own and adjacent monomers. The results suggest that the reduction in receptor binding observed with MadinDarby bovine kidney cell-grown virus is due to the combined effect of large complex glycans at the tip of the hemagglutinin and a His to Asn substitution close to the receptor binding pocket.

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“…The probe temperature was 27°C. Solvent peak suppression for dilute samples was achieved by a water elimination Fourier transform (WEFT) pulse sequence (composite, non-selective 180" pulse-delay z -90 pulseacquisition) [21]. Chemical shifts (6) are expressed in pprn downfield from internal sodium 4,4-dimethyl-4-silapentane-1 -sulfonate but were actually measured by reference to internal acetone (6 2.225 ppm in 'H,O, 27 "C) with an accuracy of 0.002 ppm.…”

Section: Determination Of the Structure Of The Asialoglycopeptidesmentioning

confidence: 99%

“…This implies that it can only be deduced from the 'H-NMR spectrum of a given fraction, to which extent each of the elongations occurs, but not whether they occur together in one compound. Furthermore, it is worth mentioning that for reliable spectral integration only the nondisturbed spectra can be used (that is not the WEFT spectra [21] and, most importantly, not the spectra resolutionenhanced by computer techniques). A sufficient resolution in conjunction with comparable line-width (resulting in similar distortions by the WEFT technique and the Lorentzian-toGaussian transformation) made the following reporter groups most suited for determination of rough (f 10 %) centesimal proportions.…”

Section: -Mhz 'H-nmr Spectroscopymentioning

confidence: 99%

Primary structure of N-glycosidically linked asialoglycans of secretory immunoglobulins from human milk

et al. 1984

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The asialoglycopeptides obtained from secretory immunoglobulins A from human milk have been separated by gel filtration and affinity chromatography on Concanavalin A‐Sepharose and Lens culinaris agglutinin‐Sepharose columns. Their structures have been determined by sugar analysis, methylation studies including mass spectrometry and 500‐MHz 1H‐NMR spectroscopy. The glycans are of the biantennary N‐acetyllactosamine type differing in their degree of extension by fucosyl‐N‐acetyllactosamine residues. The overall structures of the glycopeptides are as follows: Most of the asialoglycospeptide structures possess an intersecting GlcNAc residue; they are suggested to be located on the α chain of the secretory immunoglobulins A of human milk. The non‐interesected structures probably occur on the secretory piece. The methodology applied to the structural analysis adequately coped with the exteremely high degree of heterogeneity shown by the structures.

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α-D-Galactosyltransferase activity in calf thymus

Cited by 44 publications

References 12 publications

α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates

α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates

The Glycosylation of the Influenza A Virus Hemagglutinin by Mammalian Cells

Primary structure of N-glycosidically linked asialoglycans of secretory immunoglobulins from human milk

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