The LEC11 Chinese hamster ovary mutant synthesizes N-linked carbohydrates containing sialylated, fucosylated lactosamine units. Analysis by one- and two-dimensional 1H NMR spectroscopy

Pamela Stanley, P. H. Atkinson

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Abstract

Glycoproteins synthesized by the Chinese hamster ovary cell mutants LEC11 and LEC12 carry the Le(x) determinant (Galβ1,4(Fucα1,3)GlcNAc), while those synthesized by LEC11 cells also carry the sialyl-Le(x) determinant (NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc), and both mutants have been shown to possess a distinct α(1,3)-fucosyltransferase of the appropriate specificity to synthesize these determinants (Campbell, C., and Stanley, P. (1983) Cell 35, 303-309; Campbell, C., and Stanley, P. (1984), J. Biol. Chem. 259, 11208-11214; Howard, D. R., Fukuda, M., Fukuda, M. N., and Stanley, P. (1987) J. Biol. Chem. 262, 16830-16837). The LEC11 cells therefore provide a source of carbohydrates terminating in sialylated, fucosylated lactosamine, a relatively rare structure not previously characterized by 1H NMR spectroscopy when in association with an N-linked carbohydrate. In this paper we use a monoclonal antibody specific for Le(x) to show that the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC11 and LEC12 cells possesses the Le(x) determinant and that G from LEC11/VSV also possesses sialylated Le(x). Biantennary carbohydrates purified from LEC11/VSV and LEC12/VSV were therefore used to examine the effects on the 1H NMR spectrum of the presence of α(1,3)-fucose residues on sialylated and unsialylated lactosamine units. Comparisons of one-dimensional spectra obtained at 500 MHz from LEC11/VSV and LEC12/VSV glycopeptides before and after neuraminidase treatment with spectra of biantennary carbohydrates lacking α(1,3)-fucose allowed the assignment of several new resonances. Resolution of certain signals and determinations of coupling constants were achieved by two-dimensional correlation spectroscopy (COSY) at 400 MHz and allowed the assignment of several more resonances in the one-dimensional spectrum.

Original languageEnglish (US)
Pages (from-to)11374-11381
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number23
StatePublished - 1988

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Vesicular Stomatitis
Cricetulus
Viruses
Nuclear magnetic resonance spectroscopy
Ovary
Magnetic Resonance Spectroscopy
Carbohydrates
Fucose
galactoside 3-fucosyltransferase
Glycoproteins
Glycopeptides
Neuraminidase
lactosamine
Proton Magnetic Resonance Spectroscopy
Spectrum Analysis
Monoclonal Antibodies
Cells
Nuclear magnetic resonance
Spectroscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{c0068a3b3b554b828a38e7d93121dfc1,
title = "The LEC11 Chinese hamster ovary mutant synthesizes N-linked carbohydrates containing sialylated, fucosylated lactosamine units. Analysis by one- and two-dimensional 1H NMR spectroscopy",
abstract = "Glycoproteins synthesized by the Chinese hamster ovary cell mutants LEC11 and LEC12 carry the Le(x) determinant (Galβ1,4(Fucα1,3)GlcNAc), while those synthesized by LEC11 cells also carry the sialyl-Le(x) determinant (NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc), and both mutants have been shown to possess a distinct α(1,3)-fucosyltransferase of the appropriate specificity to synthesize these determinants (Campbell, C., and Stanley, P. (1983) Cell 35, 303-309; Campbell, C., and Stanley, P. (1984), J. Biol. Chem. 259, 11208-11214; Howard, D. R., Fukuda, M., Fukuda, M. N., and Stanley, P. (1987) J. Biol. Chem. 262, 16830-16837). The LEC11 cells therefore provide a source of carbohydrates terminating in sialylated, fucosylated lactosamine, a relatively rare structure not previously characterized by 1H NMR spectroscopy when in association with an N-linked carbohydrate. In this paper we use a monoclonal antibody specific for Le(x) to show that the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC11 and LEC12 cells possesses the Le(x) determinant and that G from LEC11/VSV also possesses sialylated Le(x). Biantennary carbohydrates purified from LEC11/VSV and LEC12/VSV were therefore used to examine the effects on the 1H NMR spectrum of the presence of α(1,3)-fucose residues on sialylated and unsialylated lactosamine units. Comparisons of one-dimensional spectra obtained at 500 MHz from LEC11/VSV and LEC12/VSV glycopeptides before and after neuraminidase treatment with spectra of biantennary carbohydrates lacking α(1,3)-fucose allowed the assignment of several new resonances. Resolution of certain signals and determinations of coupling constants were achieved by two-dimensional correlation spectroscopy (COSY) at 400 MHz and allowed the assignment of several more resonances in the one-dimensional spectrum.",
author = "Pamela Stanley and Atkinson, {P. H.}",
year = "1988",
language = "English (US)",
volume = "263",
pages = "11374--11381",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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T1 - The LEC11 Chinese hamster ovary mutant synthesizes N-linked carbohydrates containing sialylated, fucosylated lactosamine units. Analysis by one- and two-dimensional 1H NMR spectroscopy

AU - Stanley, Pamela

AU - Atkinson, P. H.

PY - 1988

Y1 - 1988

N2 - Glycoproteins synthesized by the Chinese hamster ovary cell mutants LEC11 and LEC12 carry the Le(x) determinant (Galβ1,4(Fucα1,3)GlcNAc), while those synthesized by LEC11 cells also carry the sialyl-Le(x) determinant (NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc), and both mutants have been shown to possess a distinct α(1,3)-fucosyltransferase of the appropriate specificity to synthesize these determinants (Campbell, C., and Stanley, P. (1983) Cell 35, 303-309; Campbell, C., and Stanley, P. (1984), J. Biol. Chem. 259, 11208-11214; Howard, D. R., Fukuda, M., Fukuda, M. N., and Stanley, P. (1987) J. Biol. Chem. 262, 16830-16837). The LEC11 cells therefore provide a source of carbohydrates terminating in sialylated, fucosylated lactosamine, a relatively rare structure not previously characterized by 1H NMR spectroscopy when in association with an N-linked carbohydrate. In this paper we use a monoclonal antibody specific for Le(x) to show that the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC11 and LEC12 cells possesses the Le(x) determinant and that G from LEC11/VSV also possesses sialylated Le(x). Biantennary carbohydrates purified from LEC11/VSV and LEC12/VSV were therefore used to examine the effects on the 1H NMR spectrum of the presence of α(1,3)-fucose residues on sialylated and unsialylated lactosamine units. Comparisons of one-dimensional spectra obtained at 500 MHz from LEC11/VSV and LEC12/VSV glycopeptides before and after neuraminidase treatment with spectra of biantennary carbohydrates lacking α(1,3)-fucose allowed the assignment of several new resonances. Resolution of certain signals and determinations of coupling constants were achieved by two-dimensional correlation spectroscopy (COSY) at 400 MHz and allowed the assignment of several more resonances in the one-dimensional spectrum.

AB - Glycoproteins synthesized by the Chinese hamster ovary cell mutants LEC11 and LEC12 carry the Le(x) determinant (Galβ1,4(Fucα1,3)GlcNAc), while those synthesized by LEC11 cells also carry the sialyl-Le(x) determinant (NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc), and both mutants have been shown to possess a distinct α(1,3)-fucosyltransferase of the appropriate specificity to synthesize these determinants (Campbell, C., and Stanley, P. (1983) Cell 35, 303-309; Campbell, C., and Stanley, P. (1984), J. Biol. Chem. 259, 11208-11214; Howard, D. R., Fukuda, M., Fukuda, M. N., and Stanley, P. (1987) J. Biol. Chem. 262, 16830-16837). The LEC11 cells therefore provide a source of carbohydrates terminating in sialylated, fucosylated lactosamine, a relatively rare structure not previously characterized by 1H NMR spectroscopy when in association with an N-linked carbohydrate. In this paper we use a monoclonal antibody specific for Le(x) to show that the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC11 and LEC12 cells possesses the Le(x) determinant and that G from LEC11/VSV also possesses sialylated Le(x). Biantennary carbohydrates purified from LEC11/VSV and LEC12/VSV were therefore used to examine the effects on the 1H NMR spectrum of the presence of α(1,3)-fucose residues on sialylated and unsialylated lactosamine units. Comparisons of one-dimensional spectra obtained at 500 MHz from LEC11/VSV and LEC12/VSV glycopeptides before and after neuraminidase treatment with spectra of biantennary carbohydrates lacking α(1,3)-fucose allowed the assignment of several new resonances. Resolution of certain signals and determinations of coupling constants were achieved by two-dimensional correlation spectroscopy (COSY) at 400 MHz and allowed the assignment of several more resonances in the one-dimensional spectrum.

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