Abstract

The dominant Chinese hamster ovary cell glycosylation mutant, LEC18, was selected for resistance to pea lectin (Pisum sativum agglutinin (PSA)). Lectin binding studies show that LEC18 cells express altered cell surface carbohydrates with markedly reduced binding to 125I-PSA and increased binding to 125I-labeled Datura stramonium agglutinin (DSA) compared with parental cells. Desialylated [3H]Glc-labeled LEC18 cellular glycopeptides that did not bind to concanavalin A-Sepharose exhibited an increased proportion of species that were bound to DSA-agarose. Most of these glycopeptides bound to ricin-agarose and were unique to LEC18 cells. This fraction was purified from approximately 10(10) cells and shown by 1H NMR spectroscopy and methylation linkage analysis to contain novel N-linked structures. Digestion of these glycopeptides with mixtures of beta-D-galactosidases and N-acetyl-beta-D-glucosaminidases gave core glycopeptides that, in contrast to cores from parental cells, were mainly not bound to concanavalin A-Sepharose or to PSA-agarose. 1H NMR spectroscopy, matrix-assisted laser desorption ionization/time of flight mass spectrometry, electrospray mass spectrometry, and collision-activated dissociation mass spectrometry showed that the LEC18 core glycopeptides contained a new GlcNAc residue that substitutes the core GlcNAc residues. Methylation linkage analysis of the parent compound provided evidence that the GlcNAc is linked at O-6 to give the following novel, N-linked core structure. [formula: see text]

Highlights

  • LEC18 Chinese hamster ovary (CHO) cells are rare mutants that were isolated following a selection for resistance to PSA [8]

  • Datura stramonium agglutinin (DSA) binds to poly-N-acetyllactosamine chains [22, 23], and it can be seen that LEC18 cells bound significantly more 125I-DSA than parental cells

  • The novel core structure was found on branched, polylactosamine-containing, N-glycans from LEC18 cells that were not found in parent CHO cells and that bound to both DSA and RCAII (Fig. 2)

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Summary

The abbreviations used are

Fucose; Xyl, xylose; PSA, P. sativum (pea) agglutinin; CHO, Chinese hamster ovary; LCA, L. culinaris agglutinin; ConA, concanavalin A; L-PHA, Phaseolus vulgaris leukoagglutinin; RCAI, ricin agglutinin I; RCAII, ricin agglutinin II; MS/MS, collision-activated dissociation mass spectrometry; PBS, phosphatebuffered saline; BSA, bovine serum albumin; GLC-MS, gas-liquid chromatography-mass spectrometry; ES-MS, electrospray mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption ionization/time of flight mass spectrometry; DSA, D. stramonium agglutinin.

EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION

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