Publikation: Characterization of Wild-Type Human Medium-Chain Acyl-CoA Dehydrogenase (MCAD) and Mutant Enzymes Present in MCAD-Deficient Patients by Two-Dimensional Gel Electrophoresis: Evidence for Posttranslational Modification of the Enzyme
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Two-dimensional gel electrophoresis was used to study and compare wild type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and missense mutant enzyme found in patients with MCAD deficiency. By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wildtype protein. After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation. Detection of both wild-type and K304E mutant MCAD was achieved in lymphoblastoid cells from patients and carriers of the mutation. Both spots for the wild-type but only the nonmodified spot for the K304E mutant could be detected. In lymphoblastoid cells from carriers, the intensity of the spot representing the mutant protein is much weaker than the two spots representing wild-type MCAD, emphasizing that the K304E mutant protein is more susceptible to degradation than wild-type MCAD. The absence of detectable amounts of modified K304E mutant MCAD protein in these cells suggest that the conclusion drawn from COS-7 cell expression is also valid in patient cells.
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BROSS, Peter, Thomas G. JENSEN, Brage S. ANDRESEN, Margrethe KJELDSEN, Andreas NANDY, Steen KØLVRAA, Sandro GHISLA, Ihab RASCHED, Lars BOLUND, Niels GREGERSEN, 1994. Characterization of Wild-Type Human Medium-Chain Acyl-CoA Dehydrogenase (MCAD) and Mutant Enzymes Present in MCAD-Deficient Patients by Two-Dimensional Gel Electrophoresis: Evidence for Posttranslational Modification of the Enzyme. In: Biochemical medicine and metabolic biology. 1994, 52(1), pp. 36-44. ISSN 0885-4505. Available under: doi: 10.1006/bmmb.1994.1031BibTex
@article{Bross1994Chara-8842, year={1994}, doi={10.1006/bmmb.1994.1031}, title={Characterization of Wild-Type Human Medium-Chain Acyl-CoA Dehydrogenase (MCAD) and Mutant Enzymes Present in MCAD-Deficient Patients by Two-Dimensional Gel Electrophoresis: Evidence for Posttranslational Modification of the Enzyme}, number={1}, volume={52}, issn={0885-4505}, journal={Biochemical medicine and metabolic biology}, pages={36--44}, author={Bross, Peter and Jensen, Thomas G. and Andresen, Brage S. and Kjeldsen, Margrethe and Nandy, Andreas and Kølvraa, Steen and Ghisla, Sandro and Rasched, Ihab and Bolund, Lars and Gregersen, Niels} }
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By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wildtype protein. After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation. Detection of both wild-type and K304E mutant MCAD was achieved in lymphoblastoid cells from patients and carriers of the mutation. Both spots for the wild-type but only the nonmodified spot for the K304E mutant could be detected. In lymphoblastoid cells from carriers, the intensity of the spot representing the mutant protein is much weaker than the two spots representing wild-type MCAD, emphasizing that the K304E mutant protein is more susceptible to degradation than wild-type MCAD. The absence of detectable amounts of modified K304E mutant MCAD protein in these cells suggest that the conclusion drawn from COS-7 cell expression is also valid in patient cells.</dcterms:abstract> <dc:language>eng</dc:language> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/8842"/> <dcterms:rights rdf:resource="http://creativecommons.org/licenses/by-nc-nd/2.0/"/> <dc:creator>Bolund, Lars</dc:creator> <dc:contributor>Nandy, Andreas</dc:contributor> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dc:creator>Ghisla, Sandro</dc:creator> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/8842/1/ghislascan90.pdf"/> <dc:contributor>Bross, Peter</dc:contributor> <dc:creator>Nandy, Andreas</dc:creator> <dc:creator>Gregersen, Niels</dc:creator> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/8842/1/ghislascan90.pdf"/> <dc:format>application/pdf</dc:format> <dc:rights>Attribution-NonCommercial-NoDerivs 2.0 Generic</dc:rights> <dc:contributor>Kjeldsen, Margrethe</dc:contributor> <dcterms:title>Characterization of Wild-Type Human Medium-Chain Acyl-CoA Dehydrogenase (MCAD) and Mutant Enzymes Present in MCAD-Deficient Patients by Two-Dimensional Gel Electrophoresis: Evidence for Posttranslational Modification of the Enzyme</dcterms:title> <dcterms:bibliographicCitation>First publ. in: Biochemical medicine and metabolic biology 52 (1994), 1, pp. 36-44</dcterms:bibliographicCitation> <dc:creator>Jensen, Thomas G.</dc:creator> <dc:contributor>Bolund, Lars</dc:contributor> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dc:contributor>Jensen, Thomas G.</dc:contributor> <dc:contributor>Andresen, Brage S.</dc:contributor> <dc:contributor>Gregersen, Niels</dc:contributor> <dc:creator>Rasched, Ihab</dc:creator> <dc:contributor>Kølvraa, Steen</dc:contributor> <dc:creator>Kølvraa, Steen</dc:creator> <dc:creator>Bross, Peter</dc:creator> <dcterms:issued>1994</dcterms:issued> <foaf:homepage rdf:resource="http://localhost:8080/"/> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:47:16Z</dcterms:available> <dc:creator>Kjeldsen, Margrethe</dc:creator> <dc:contributor>Ghisla, Sandro</dc:contributor> </rdf:Description> </rdf:RDF>