LEC14, A DOMINANT CHINESE-HAMSTER OVARY GLYCOSYLATION MUTANT EXPRESSES COMPLEX N-GLYCANS WITH A NEW N-ACETYLGLUCOSAMINE RESIDUE IN THE COREREGION

The Chinese hamster ovary cell (CHO) glycosylation mutant, LEC14, was previously selected for resistance to pea lectin (Pisum sativum agglut inin) and shown to behave dominantly. The lectin resistance properties of LEC14 cells are related to, but distinct from, those of LEC18, a d ominant Chinese hamster ovary mutant that synthesizes complex N-glycan s 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, [H-3]Glc-labeled LEC14 cellular glycopeptides were desialylated, and the fraction that did not bind to concanavalin A-Sepharose was found t o have an increased proportion of species that bound to tomato-agarose , and to ricin-agarose, H-1 NMR spectroscopy and methylation linkage a nalysis of the tomato and ricin-bound fractions purified from similar to 10(10) 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 beta-D-galact osidases and N-acetyl-beta-D-glucosaminidases to obtain core glycopept ides. The latter were largely unbound by concanavalin A-Sepharose or p ea lectin-agarose. H-1 NMR spectroscopy and electrospray ionization-ma ss spectrometry showed that the LEC14 core glycopeptides contain a new GlcNAc residue that substitutes the core beta(1,4)-Man residue at O-2 to give the following novel, N-linked core structure.