Human Molecular Genetics Advance Access published online on September 5, 2008
Human Molecular Genetics, doi:10.1093/hmg/ddn284
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Disease-causing missense mutations affect enzymatic activity, stability, and oligomerization of glutaryl-CoA dehydrogenase (GCDH)
1 Department of Biochemistry, Childre
s Hospital, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
2 Department of Molecular Structural Biology, Göttingen Center for Molecular Biosciences, Georg-August-University, 37075 Göttingen, Germany
3 Department of Clinical Genetics, Juliane Marie Center, Rigshospitalet, DK-2100 Copenhagen, Denmark
* Correspondence to: Dr. Thomas Braulke Department of Biochemistry, Childres Hospital University Medical Center Hamburg-Eppendorf Martinistrasse 52, 20246 Hamburg, Germany Phone: ++49-40-42803-4493, Fax: ++49-40-42803-8504 Email: braulke{at}uke.uni-hamburg.de
Received June 30, 2008; Revised August 21, 2008; Accepted September 4, 2008
Glutaric aciduria type 1 (GA1) is an autosomal recessive neurometabolic disorder caused by mutations in the glutaryl-CoA dehydrogenase gene (GCDH), leading to an accumulation and high excretion of glutaric acid and 3-hydroxyglutaric acid. Considerable variation in severity of the clinical phenotype is observed with no correlation to the genotype. We report here for the first time on expression studies of four missense mutations c.412A>G (p.Arg138Gly), c.787A>G (p.Met263Val), c.1204C>T (p.Arg402Trp), and c.1240G>A (p.Glu414Lys) identified in GA1 patients in mammalian cells. Biochemical analyses revealed that all mutants were enzymatically inactive with the exception of p.Met263Val which showed 10 % activity of the expressed wildtype enzyme. Western blot and pulse-chase analyses demonstrated that the amount of expressed p.Arg402Trp protein was significantly reduced compared with cells expressing wildtype protein which was due to rapid intramitochondrial degradation. Upon cross linkage the formation of homotetrameric GCDH was strongly impaired in p.Met263Val and p.Arg402Trp mutants. In addition, GCDH appears to interact with distinct heterologous polypeptides to form novel 97, 130 and 200 kDa GCDH complexes. Molecular modeling of mutant GCDH suggests that Met263 at the surface of the GCDH protein might be part of the contact interface to interacting proteins. These results indicate that reduced intramitochondrial stability as well as the impaired formation of homo- and heteromeric GCDH complexes can underly GA1.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.