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Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin
Abstract:
Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance.
Insights
Two mouse cell lines resistant to ricin toxin were developed. CL 3 cells showed increased sialic acid, masking ricin binding sites, while CL 6 cells had altered sugar content and glycosyltransferase activity, revealing multiple resistance mechanisms.
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- Ricin is a potent galactose-binding lectin with significant cytotoxic activity.
- Understanding cellular resistance mechanisms to toxins like ricin is crucial for developing countermeasures.
Purpose of the Study:
- To investigate the molecular basis of ricin resistance in selected mouse L cell lines (CL 3 and CL 6).
- To characterize the changes in cell surface carbohydrates and glycosyltransferase activities associated with ricin resistance.
Main Methods:
- Selection of ricin-resistant mouse L cell lines (CL 3 and CL 6).
- Analysis of cell membrane composition, including sialic acid, galactose, mannose, and hexosamine content.
- Measurement of specific glycosyltransferase activities (CMP-sialic acid:glycoprotein sialyltransferase, GM3 synthetase, UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase, DPU-galactose:glycoprotein galactosyltransferase).
- Neuraminidase treatment to assess the role of sialic acid in ricin binding and resistance.
Main Results:
- CL 3 cells exhibited a 50-70% decrease in ricin binding and 300-500 fold resistance, with a 200% increase in sialic acid content and altered glycoprotein/glycolipid composition.
- CL 6 cells showed decreased sialic acid, galactose, and hexosamine, with normal mannose content, and altered glycosyltransferase activities.
- Neuraminidase treatment of CL 3 cells restored ricin binding but only partially reduced resistance, suggesting additional resistance mechanisms.
Conclusions:
- Increased sialic acid content in CL 3 cells contributes to ricin resistance by masking binding sites.
- Alterations in glycosyltransferase activities and cell surface carbohydrate composition are key to ricin resistance in both CL 3 and CL 6 cells.
- CL 3 cells possess a secondary ricin resistance mechanism beyond sialic acid masking, potentially involving altered ricin binding site accessibility or intracellular pathways.