Acute intermittent porphyria: Expression of mutant and wild-type porphobilinogen deaminase in COS-1 cells

Background: Acute intermittent porphyria (AIP) is an autosomal dominant dis order 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 sym ptoms. Although molecular biological studies on the porphobilinogen deamina se (PBGD) gene have revealed several mutations responsible for AIP, the pro perties of mutant PBGD in eukaryotic expression systems have not been studi ed previously. Materials and Methods: Seven mutations were analyzed using transient expres sion of the mutated polypeptides in COS-I cells. The properties of mutated polypeptides were studied by enzyme activity measurement, Western blot anal ysis, pulse-chase experiments, and immunofluorescence staining. Results: Of the mutants studied, R26C, R167W, R173W, R173Q, and R225X resul ted in a decreased enzyme activity (0-5%), but R225G and 1073delA (elongate d protein) displayed a significant residual activity of 16% and 50%, respec tively. In Western blot analysis, the polyclonal PBGD antibody detected all mutant polypeptides except R225X, which was predicted to result in a trunc ated protein. In the pulse-chase experiment, the mutant polypeptides were a s stable as the wild-type enzyme. In the immunofluorescence staining both w ild-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 no rmal enzyme activity measured in the patients' erythrocytes. In contrast to the decreased enzyme activity, the majority of the mutations produced a de tectable polypeptide, and the stability and the intracellular processing of the mutated polypeptides were both comparable to that of the wild-type PBG D and independent of the cross-reacting immunological material (CRIM) class .