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-mannose 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 microM
GDP-mannose, 15 mM Mg2+, 0.2 mM
NAD+, 0.2 mM
NADPH, 10 mM
niacinamide, 5 mM
ATP, and 50 mM 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.