title: Disease-causing missense mutations affect enzymatic activity, stability and oligomerization of glutaryl-CoA dehydrogenase (GCDH).
abstract: 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 wild-type enzyme. Western blot and pulse-chase analyses demonstrated that the amount of expressed p.Arg402Trp protein was significantly reduced compared with cells expressing wild-type 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 underlie GA1.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Keyser
authors.forename: Chris
authors.initials: C
authors.lastname: Mühlhausen
authors.forename: Achim
authors.initials: A
authors.lastname: Dickmanns
authors.forename: Ernst
authors.initials: E
authors.lastname: Christensen
authors.forename: Nicole
authors.initials: N
authors.lastname: Muschol
authors.forename: Kurt
authors.initials: K
authors.lastname: Ullrich
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D005808:Genes, Recessive; D005977:Glutarates / Q000378:metabolism*; D050770:Glutaryl-CoA Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense / Q000235:genetics*; D020836:Protein Structure, Quaternary / Q000235:genetics*
chemical_list: D005977:Glutarates; D024101:Mitochondrial Proteins; D050770:Glutaryl-CoA Dehydrogenase; C035736:glutaric acid