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Gain-of-function glycosylation mutants provide access to glycosylation pathways, glycosylation genes, and mechanisms that regulate expression of a glycotype. Previous studies have shown that the gain-of-function Chinese hamster ovary (CHO) mutants LEC12, LEC29, and LEC30 express an N-ethylmaleimide-resistant alpha(1, 3)fucosyltransferase (alpha(1,3)Fuc-T) activity that is not detected in CHO cells and that generates the Lewis(X) but not the sialyl-Lewis(X) determinant. The three mutants differ, however, in lectin resistance properties, expression of fucosylated antigens, and in vitro alpha(1,3)Fuc-T activities. In this paper we show that each mutant expresses Fuc-TIX, but only LEC30 cells express Fuc-TIV. Using genomic PCR and reverse-transcriptase (RT)-PCR strategies, we isolated coding portions of the CHO Fut4 and Fut9 genes. Each gene is present in a single copy in the CHO and mutant genomes. The Fut4 gene is expressed only in LEC30 cells, while all three mutants express the Fut9 gene. Interestingly, the fucosylation phenotypes of LEC12 and LEC29 cells do not correlate with the relative abundance of their Fut9 gene transcripts (LEC29 >> LEC12). Compared to LEC29 cells, LEC12 cells have an approximately 40-fold higher in vitro alpha(1,3)Fuc-T activity and bind the VIM-2 monoclonal antibody, whereas LEC29 cells do not bind VIM-2. Mixing experiments did not detect Fuc-TIX inhibitory activity in LEC29 cell extracts, and CHO cells expressing a transfected Fut9 gene behaved like LEC12 cells. Therefore, it seems that LEC29 cells may not translate their more abundant Fut9 gene transcripts efficiently or may not synthesize appropriate acceptors for internal alpha(1,3)fucosylation. Alternatively, LEC12 cells may possess, in addition to Fuc-TIX, a novel alpha(1,3)Fuc-T activity.

The carbohydrate portion of the G glycoprotein of vesicular stomatitis virus (VSV) grown in CHO cells (CHO/VSV) has been fractionated on BioGelP6, concanavalin A-Sepharose, and pea lectin-agarose. The results suggest that, in addition to sialic acid and fucose heterogeneity, the asparagine-linked complex carbohydrate moieties of CHO/VSV also display branching heterogeneity. Although the majority of the glycopeptides bind to concanavalin A-Sepharose in a manner typical of certain biantennary carbohydrate structures, a significant proportion do not bind to the lectin. The latter behavior is typical of tri- or tetraantennary (branched) carbohydrate structures. The CHO/VSV glycopeptides which do not bind to concanavalin A-Sepharose separate into bound and unbound fractions on pea lectin-agarose suggesting that they include at least two different types of (branched) carbohydrate structures. Glycopeptides from the G glycoprotein of VSV grown in two, independently derived CHO glycosylation mutants which belong to complementation group 4 (Lec4 mutants) were examined in the same manner. In contrast to glycopeptides from CHO/VSV, glycopeptides from Lec4/VSV which passed through concanavalin A-Sepharose did not contain a component which subsequently bound to pea lectin-agarose. A glycopeptide fraction with these lectin-binding properties was also missing from cell surface glycopeptides derived from Lec4 cells. The combined results are consistent with the hypothesis that Lec4 CHO glycosylation mutants lack a glycosyltransferase activity responsible for the addition of a (branch) N -acetylglucosamine residue linked β1,6 to mannose.

A biochemical basis for the pea and lentil lectin resistance of two Chinese hamster ovary (CHO) cell mutants, Lec13 and Lec13A, was investigated. Studies of the G glycopeptides of vesicular stomatitis virus grown in the mutants indicated that Lec13 cells essentially lack the ability to add fucose to complex carbohydrates while Lec13A cells synthesize significant proportions of fucosylated, complex moieties. However, both mutants were known to be reverted to lectin sensitivity by growth in l -fucose, making them similar to the mouse lymphoma mutant, PLR1.3, which is defective in the conversion of GDP-manose to GPD-fucose [ M. L. Reitman, I. S. Trowbridge, and S. Kornfeld (1980) J. Biol. Chem.255, 9900–9906 ]. Optimal conditions for the production of GDP-fucose from GDP-mannose by CHO cytosol were found to occur at pH 8 in the presence of 7.5 μ m GDP-mannose, 15 m m Mg2+, 0.2 m m NAD+, 0.2 m m NADPH, 10 m m niacinamide, 5 m m ATP, and 50 m m Tris-HCl. Under these conditions, Lec13 cytosol produced no detectable GDP-fucose nor GDP-sugar intermediates while Lec13A cytosol produced significant quantities of both. Mixing experiments with Lec13 cytosol identified the first enzyme of the conversion pathway (GDP-mannose 4,6-dehydratase, EC 4.2.1.47) as the site of the block. In addition to being markedly reduced, the Lec13A 4,6-dehydratase activity was relatively insensitive to changes in pH in comparison to the activity in parental cytosol, suggesting that Lec13A cells might possess a structurally altered GDP-mannose 4,6-dehydratase enzyme.

Carbohydrate moieties derived from the G glycoprotein of Vesicular Stomatitis Virus (VSV) grown in parental Chinese hamster ovary (CHO) cells and the glycosylation mutant Lec4 have been analyzed by high-field 1 H NMR spectroscopy. The major glycopeptides of CHO VSV and Lec4 VSV were purified by their ability to bind to concanavalin A-Sepharose. The carbohydrates in this fraction are of the biantennary, complex type with heterogeneity in the presence of α(2,3)-linked sialic acid and α(1,6)-linked fucose residues. A minor CHO VSV glycopeptide fraction, which does not bind to concanavalin A-Sepharose but which binds to pea lectin-agarose, was also investigated by 1 H NMR spectroscopy. These carbohydrates are complex moieties which appear to contain N -acetylglucosamine in β(1,6) linkage. Their spectral properties are most similar to those of a triantennary complex oligosaccharide containing a 2,6-disubstituted mannose α(1,6) residue. Carbohydrates of this type are not found among the glycopeptides of VSV grown in the Lec4 CHO glycosylation mutant.