title: Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Münster, Albert-Schweitzer-Str. 33, 48149 Muenster, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Engel
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Vuissoz
authors.forename: Sandra
authors.initials: S
authors.lastname: Eggimann
authors.forename: Murielle
authors.initials: M
authors.lastname: Groux
authors.forename: Christoph
authors.initials: C
authors.lastname: Berning
authors.forename: Liyan
authors.initials: L
authors.lastname: Hu
authors.forename: Vera
authors.initials: V
authors.lastname: Klaus
authors.forename: Dorothea
authors.initials: D
authors.lastname: Moeslinger
authors.forename: Saadet
authors.initials: S
authors.lastname: Mercimek-Mahmutoglu
authors.forename: Sylvia
authors.initials: S
authors.lastname: Stöckler
authors.forename: Bendicht
authors.initials: B
authors.lastname: Wermuth
authors.forename: Johannes
authors.initials: J
authors.lastname: Häberle
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nuoffer
mesh_terms: D001123:Argininosuccinate Lyase / Q000096:biosynthesis / Q000235:genetics*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology; D006720:Homozygote; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D010641:Phenotype; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001123:Argininosuccinate Lyase