title: Acute intermittent porphyria: expression of mutant and wild-type porphobilinogen deaminase in COS-1 cells.
abstract: BACKGROUND
  Acute intermittent porphyria (AIP) is an autosomal dominant disorder that results from the partial deficiency of porphobilinogen deaminase (PBGD) in the heme biosynthetic pathway. Patients with AIP can experience acute attacks consisting of abdominal pain and various neuropsychiatric symptoms. Although molecular biological studies on the porphobilinogen deaminase (PBGD) gene have revealed several mutations responsible for AIP, the properties of mutant PBGD in eukaryotic expression systems have not been studied previously.
  
  
  METHODS
  Seven mutations were analyzed using transient expression of the mutated polypeptides in COS-1 cells. The properties of mutated polypeptides were studied by enzyme activity measurement, Western blot analysis, pulse-chase experiments, and immunofluorescence staining.
  
  
  RESULTS
  Of the mutants studied, R26C, R167W, R173W, R173Q, and R225X resulted in a decreased enzyme activity (0-5%), but R225G and 1073delA (elongated protein) displayed a significant residual activity of 16% and 50%, respectively. In Western blot analysis, the polyclonal PBGD antibody detected all mutant polypeptides except R225X, which was predicted to result in a truncated protein. In the pulse-chase experiment, the mutant polypeptides were as stable as the wild-type enzyme. In the immunofluorescence staining both wild-type and mutant polypeptides were diffusely dispersed in the cytoplasm and, thus, no accumulation of mutated proteins in the cellular compartments could be observed.
  
  
  CONCLUSIONS
  The results confirm the causality of mutations for the half normal enzyme activity measured in the patients' erythrocytes. In contrast to the decreased enzyme activity, the majority of the mutations produced a detectable polypeptide, and the stability and the intracellular processing of the mutated polypeptides were both comparable to that of the wild-type PBGD and independent of the cross-reacting immunological material (CRIM) class.
authors.affiliation: Department of Medicine, Helsinki University Central Hospital, Finland. sami.mustajoki@helsinki.fi
authors.forename: S
authors.initials: S
authors.lastname: Mustajoki
authors.forename: M
authors.initials: M
authors.lastname: Laine
authors.forename: M
authors.initials: M
authors.lastname: Lahtela
authors.forename: P
authors.initials: P
authors.lastname: Mustajoki
authors.forename: L
authors.initials: L
authors.lastname: Peltonen
authors.forename: R
authors.initials: R
authors.lastname: Kauppinen
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D018389:Codon, Nonsense / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004795:Enzyme Stability; D005091:Exons / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D017118:Porphyria, Acute Intermittent / Q000201:enzymology* / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D014162:Transfection
chemical_list: D018389:Codon, Nonsense; D011994:Recombinant Proteins; D011163:Hydroxymethylbilane Synthase