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Mouse lymphoma cell lines resistant to pea lectin are defective in fucose metabolism
Abstract:
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.
Insights
Two pea lectin-resistant cell mutants were identified with defects in fucose metabolism. Restoring fucose levels in one mutant restored lectin binding, highlighting fucose
Area of Science:
- Cell Biology
- Biochemistry
- Glycobiology
Background:
- Pea lectin resistance in BW5147 mouse lymphoma cell lines (PLR1.3, PHAR1.8 PLR7.2) is linked to reduced high-affinity lectin binding sites.
- Investigating the biochemical basis of lectin resistance can elucidate cellular pathways involved in glycoprotein synthesis and cell surface interactions.
Purpose of the Study:
- To characterize the molecular defects in two pea lectin-resistant cell lines (PLR1.3 and PHAR1.8 PLR7.2).
- To determine the role of fucose metabolism and incorporation into glycoproteins in mediating pea lectin binding.
Main Methods:
- Cellular labeling with [2-3H]mannose to track GDP-fucose synthesis and transfer.
- In vitro enzymatic assays of GDP-mannose 4,6-dehydratase and GDP-fucose:glycoprotein fucosyltransferase activity.
- Supplementation of cell cultures with fucose to assess rescue of lectin binding.
Main Results:
- PLR1.3 cells exhibit a defect in GDP-mannose 4,6-dehydratase, blocking GDP-fucose synthesis; this defect is bypassed by exogenous fucose, restoring lectin binding.
- PHAR1.8 PLR7.2 cells possess functional GDP-fucose synthesis and transfer enzymes but fail to incorporate fucose into glycoproteins in vivo, despite in vitro enzyme activity.
- Reduced glycoprotein fucose content correlates with decreased pea lectin binding in both cell lines.
Conclusions:
- Fucose incorporation into glycoproteins is critical for high-affinity pea lectin binding.
- Defects in GDP-fucose biosynthesis (PLR1.3) or potentially in vivo fucosyltransferase activity (PHAR1.8 PLR7.2) lead to pea lectin resistance.
- These findings underscore the importance of specific glycosylation patterns in cell-surface recognition events.