TY - JOUR
T1 - LEC14, a dominant Chinese hamster ovary glycosylation mutant expresses complex N-glycans with a new N-acetylglucosamine residue in the core region
AU - Raju, T. Shantha
AU - Stanley, Pamela
PY - 1996/3/29
Y1 - 1996/3/29
N2 - The Chinese hamster ovary cell (CHO) glycosylation mutant, LEC14, was previously selected for resistance to pea lectin (Pisum sativum agglutinin) and shown to behave dominantly. The lectin resistance properties of LEC14 cells are related to, but distinct from, those of LEC18, a dominant Chinese hamster ovary mutant that synthesizes complex N-glycans with a novel O-6- linked GlcNAc residue in the core region (Raju, T. S., Ray. M., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302). Detailed structural studies of a complex N-glycan fraction from LEC14 cells have revealed yet another novel modification of the core region. [3H]Glc-labeled LEC14 cellular glycopeptides were desialylated, and the fraction that did not bind to concanavalin A-Sepharose was found to have an increased proportion of species that bound to tomato-agarose, and to ricin-agarose. 1H NMR spectroscopy and methylation linkage analysis of the tomato and ricin-bound fractions purified from ~1010 LEC14 cells showed they were complex N-glycans containing a 2,3,6-trisubstituted core Man residue. To examine the core region more closely, these N-glycans were digested with mixtures of β-D-galactosidases and N-acetyl-β-D-glucosaminidases to obtain core glycopeptides. The latter were largely unbound by concanavalin A-Sepharose or pea lectin-agarose. 1H NMR spectroscopy and electrospray ionization-mass spectrometry showed that the LEC14 core glycopeptides contain a new GlcNAc residue that substitutes the core β(1,4)-Man residue at O-2 to give the following novel, N-linked core structure.
AB - The Chinese hamster ovary cell (CHO) glycosylation mutant, LEC14, was previously selected for resistance to pea lectin (Pisum sativum agglutinin) and shown to behave dominantly. The lectin resistance properties of LEC14 cells are related to, but distinct from, those of LEC18, a dominant Chinese hamster ovary mutant that synthesizes complex N-glycans with a novel O-6- linked GlcNAc residue in the core region (Raju, T. S., Ray. M., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302). Detailed structural studies of a complex N-glycan fraction from LEC14 cells have revealed yet another novel modification of the core region. [3H]Glc-labeled LEC14 cellular glycopeptides were desialylated, and the fraction that did not bind to concanavalin A-Sepharose was found to have an increased proportion of species that bound to tomato-agarose, and to ricin-agarose. 1H NMR spectroscopy and methylation linkage analysis of the tomato and ricin-bound fractions purified from ~1010 LEC14 cells showed they were complex N-glycans containing a 2,3,6-trisubstituted core Man residue. To examine the core region more closely, these N-glycans were digested with mixtures of β-D-galactosidases and N-acetyl-β-D-glucosaminidases to obtain core glycopeptides. The latter were largely unbound by concanavalin A-Sepharose or pea lectin-agarose. 1H NMR spectroscopy and electrospray ionization-mass spectrometry showed that the LEC14 core glycopeptides contain a new GlcNAc residue that substitutes the core β(1,4)-Man residue at O-2 to give the following novel, N-linked core structure.
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U2 - 10.1074/jbc.271.13.7484
DO - 10.1074/jbc.271.13.7484
M3 - Article
C2 - 8631778
AN - SCOPUS:0029968244
SN - 0021-9258
VL - 271
SP - 7484
EP - 7493
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 13
ER -