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

P. Stanley, P. H. Atkinson

Research output: Contribution to journalArticle

17 Scopus citations

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 - Jan 1 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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