LEC14, a dominant Chinese hamster ovary glycosylation mutant expresses complex N-glycans with a new N-acetylglucosamine residue in the core region

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.