Two mutants of the BW5147 mouse
lymphoma cell line have been selected for their resistance to the toxic effects of
pea lectin. These cell lines, termed PLR1.3 and PHAR1.8 PLR7.2, have a decreased number of high affinity
pea lectin-binding sites (Trowbridge, I.S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Intact cell labeling experiments using [2-3H]
mannose indicated that PLR1.3 cells have a block in the conversion of
GDP-[3H]
mannose to
GDP-[3H]
fucose whereas PHAR1.8 PLR7.2 cells appear to be blocked in the transfer of
fucose from
GDP-[3H]
fucose to
glycoprotein acceptors. In vitro experiments with extracts of PLR1.3 cells confirmed the failure to convert
GDP-mannose to
GDP-fucose and indicated that the defect is in
GDP-mannose 4,6-dehydratase (EC 4.2.1.47), the first
enzyme in the conversion of
GDP-mannose to
GDP-fucose. The block in the PLR1.3 cells could be bypassed by growing the cells in the presence of
fucose, demonstrating that an alternate pathway for the production of
GDP-fucose presumably via
fucose 1-phosphate is functional in this line. PLR1.3 cells grown in 10 mM
fucose showed normal high affinity
pea lectin binding. PHRA1.8 PLR7.2 cells synthesize
GDP-fucose and have normal or increased levels of
GDP-fucose:
glycoprotein fucosyltransferase when assayed in vitro. The
fucosyltransferases of this clone can utilize its own
glycoproteins as
fucose acceptors in in vitro assays. These findings indicate that this cell line fails to carry out the
fucosyltransferase reaction in vivo despite the fact that it possesses the appropriate
nucleotide sugar,
glycoprotein acceptors, and
fucosyltransferase. The finding of decreased
glycoprotein fucose in two independent isolates of
pea lectin-resistant cell lines and the restoration of high affinity
pea lectin binding to PLR1.3 cells following
fucose feeding strongly implicates
fucose as a major determinant of
pea lectin binding.