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Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein
D J FitzGerald1, C M Fryling, A Zdanovsky
1Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.
Insights
This study identifies the alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) as crucial for Pseudomonas exotoxin (PE) entry into cells. Developing resistant cell lines confirmed LRP
Area of Science:
- Molecular and Cellular Biology
- Biochemistry
- Toxicology
Background:
- The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) mediates cellular uptake of various ligands, including proteases and lipid-related molecules.
- The 39-kD receptor-associated protein (RAP) is known to inhibit LRP-mediated ligand binding and uptake.
- Previous research indicated that Pseudomonas exotoxin (PE) binds to LRP, and RAP inhibits PE-mediated cell killing, suggesting LRP's role in PE entry.
Purpose of the Study:
- To investigate the role of LRP in Pseudomonas exotoxin (PE) internalization and cell entry.
- To generate and characterize cell lines with altered LRP expression to understand its function in PE toxicity.
Main Methods:
- Selection of PE-resistant Chinese hamster ovary (CHO) cell lines with modified LRP expression.
- Screening of resistant clones for sensitivity to diphtheria toxin and a chimeric anthrax toxin.
- Detailed characterization of LRP expression (mRNA, protein, cell surface localization) and ligand uptake (alpha 2M-chymotrypsin) in selected cell lines.
- Assessment of PE resistance in relation to LRP function, using a transferrin receptor-mediated PE chimeric toxin as a control.
Main Results:
- Two PE-resistant cell lines, 14-2-1 and 13-5-1, exhibited distinct alterations in LRP expression and localization.
- Line 14-2-1 showed intracellular LRP with reduced cell surface expression, leading to 10-fold PE resistance and impaired alpha 2M-chymotrypsin uptake.
- Line 13-5-1 lacked detectable LRP mRNA and protein, demonstrating 100-fold PE resistance and no alpha 2M-chymotrypsin internalization.
- Resistance to PE in these lines was specific to LRP-mediated entry, as they remained sensitive to a PE chimeric toxin internalized via the transferrin receptor.
Conclusions:
- The results confirm that LRP is the primary receptor mediating the internalization of Pseudomonas exotoxin (PE).
- Defects in LRP expression or cell surface display confer significant resistance to PE toxicity.
- This study validates LRP as a key mediator for PE entry into sensitive cells.