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Published on: November 23, 2013
MDR1 gene-specific monoclonal antibody C494 cross-reacts with pyruvate carboxylase
V V Rao1, D C Anthony, D Piwnica-Worms
1Department of Radiology, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Abstract:
Overexpression of P-glycoprotein, the plasma membrane protein product of the MDR1 gene, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents. A battery of antibodies, including the MDR1 gene-specific monoclonal antibody (mAb) C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials. In rat liver fractions, we report that mAb C494 strongly cross-reacted with a nonmembranous M(r) approximately 130,000 protein, comigrating with core-glycosylated human MDR1 on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By immunoblotting and microsequence analysis, this protein was identified as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. A search of the National Center for Biotechnology Information data base, using the epitope-specific sequence of mAb C494, revealed that PC (mouse) contains four of the five most reactive amino acids (TLEG), located near the COOH-terminal end of PC at positions 1167-1170. mAb C494 specifically reacted with PC purified from bovine liver; immunoreactivity was completely abolished by preincubating mAb C494 in the presence of excess synthetic C494 epitope-specific peptide. Furthermore, in cryosections of human skeletal muscle, a tissue known not to express P-glycoprotein, peptide-displaceable immunohistochemical staining with mAb C494 showed a distinct mitochondrial pattern specific to type 1 fibers. Variable immunostaining results were obtained with formaldehyde-fixed, paraffin-embedded muscle and isolated liver mitochondrial preparations. In summary, mAb C494 cross-reacted strongly with rat, bovine, and human PC. Caution is warranted in interpretation of immunoblots and immunohistochemical sections with this putative MDR1 gene-specific mAb.
Insights
Monoclonal antibody C494, intended to detect P-glycoprotein, unexpectedly cross-reacts with pyruvate carboxylase, a mitochondrial enzyme. This cross-reactivity necessitates caution when interpreting results from MDR1 gene expression studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- P-glycoprotein, encoded by the MDR1 gene, is crucial in multidrug resistance in cancer chemotherapy.
- Monoclonal antibody (mAb) C494 is utilized in clinical trials for MDR1 surveillance.
- Previous studies assumed mAb C494 specificity for P-glycoprotein.
Purpose of the Study:
- To investigate the specificity of monoclonal antibody (mAb) C494.
- To identify proteins cross-reactive with mAb C494.
- To assess the implications of cross-reactivity for MDR1 gene expression studies.
Main Methods:
- Immunoblotting and microsequence analysis of rat liver fractions.
- Epitope mapping using the National Center for Biotechnology Information database.
- Immunohistochemical staining of human skeletal muscle cryosections.
- Peptide competition assays with purified bovine liver pyruvate carboxylase.
Main Results:
- mAb C494 strongly cross-reacted with pyruvate carboxylase (PC), an M(r) approximately 130,000 nonmembranous protein.
- PC shares sequence homology with the C494 epitope.
- Immunohistochemistry revealed mitochondrial staining patterns for PC in human skeletal muscle, independent of P-glycoprotein expression.
- Peptide preincubation abolished mAb C494 immunoreactivity with PC.
Conclusions:
- Monoclonal antibody C494 exhibits significant cross-reactivity with pyruvate carboxylase across species (rat, bovine, human).
- The findings challenge the exclusive specificity of mAb C494 for P-glycoprotein.
- Caution is advised when interpreting immunoblots and immunohistochemical data using mAb C494 for MDR1 gene expression analysis.
