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Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in ‐ vitro enzyme activity with clinical phenotype in argininosuccinic aciduria
Author(s) -
Engel Katharina,
Vuissoz JeanMarc,
Eggimann Sandra,
Groux Murielle,
Berning Christoph,
Hu Liyan,
Klaus Vera,
Moeslinger Dorothea,
MercimekMahmutoglu Saadet,
Stöckler Sylvia,
Wermuth Bendicht,
Häberle Johannes,
Nuoffer JeanMarc
Publication year - 2012
Publication title -
journal of inherited metabolic disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 102
eISSN - 1573-2665
pISSN - 0141-8955
DOI - 10.1007/s10545-011-9357-x
Subject(s) - argininosuccinate lyase , mutant , microbiology and biotechnology , enzyme , biology , phenotype , mutation , wild type , in vitro , biochemistry , urea cycle , gene , amino acid , arginine
Background The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme. Methods Bacterial in ‐ vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299 T > C), p.V178M (c.532 G > A), p.E189G (c.566A > G), p.Q286R (c.857A > G), p.K315E (c.943A > G), p.R379C (c.1135 C > T) and p.R385C (c.1153 C > T) in comparison to the wildtype protein. Results In the bacterial in ‐ vitro expression system, ASL wild‐type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild‐type) and p.R379C (10 % of wild‐type) mutations in which K m values for argininosuccinic acid differed significantly from the wild‐type ASL protein. Conclusion The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.