Acute Intermittent Porphyria: Expression of Mutant and Wild-Type Porphobilinogen Deaminase in COS-1 Cells

Molecular Medicine 6(8): 670–679, 2000 Molecular Medicine © 2000 The Picower Institute Press Acute Intermittent Porphyria: Expression of Mutant and Wild-Type Porphobilinogen Deaminase in COS-1 Cells Sami Mustajoki,1 Minna Laine,2 Maija Lahtela,3 Pertti Mustajoki,1 Leena Peltonen,2 and Raili Kauppinen1 1 Department of Medicine, Division of Endocrinology, Helsinki University Central Hospital, Helsinki, Finland 2 Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland 3 Center for Scientific Computing, Espoo, Finland Accepted April 23, 2000 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. Materials and 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. Introduction pattern of inheritance (1). The disease results from the partial deficiency of porphobilinogen deaminase (PBGD; also known as hydroxymethylbilane synthase [EC 4.3.1.8]), which is the third enzyme in the heme biosynthetic pathway. PBGD activity is usually decreased to about 50% of normal when determined in patients’ erythrocytes (2). The clinical manifestations of AIP vary even within families. Only 10–20% of Acute intermittent porphyria (AIP) is a metabolic disorder with an autosomal dominant Address correspondence to: Sami Mustajoki, Department of Medicine, Division of Endocrinology, Helsinki University Central Hospital, P.O. Box 340, 00029 HYKS, Finland. Phone: +358-9-4717-2324; Fax: +358-9-4717-4012; E-mail: S. Mustajoki et al.: Expression of Mutant Porphobilinogen Deaminase patients experience occasional acute attacks, which consist of abdominal pain and various neuropsychiatric symptoms; however, milder symptoms typical for AIP are more common (3). Biochemical and immunological studies have shown that AIP is attributable to a heterogenous group of biochemical defects, and the disease has previously been classified into four subtypes according to ratio of PBGD polypeptide concentration (cross-reacting immunological material [CRIM] negative and positive subtypes) to the enzyme activity in erythrocytes (4). The crystal structure of E. coli PBGD indicates that the polypeptide chain is folded into three domains of similar sizes (5). Each of them is comprised of a -sheet, an -helical secondary structure, and a hydrophobic core. PBGD catalyzes the polymerization of porphobilinogen yielding the linear tetrapyrrole, hydroxymethylbilane (preuroporphyrinogen). The reaction requires the presence of the dipyrromethane cofactor, which arises from the autocatalytic coupling of two porphobilinogen rings and is attached to the invariant cysteine-242 (human C261) in domain 3 (6). The cofactor stabilizes the enzyme (7), and it is not incorporated in the product but functions as a primer to which the four substrate molecules are sequentially attached (6,8). The large volume of the active site cavity and the multipoint interaction between the enzyme and the growing polypyrrole chain imply that mutations altering multiple amino acids can result in deficient PBGD activity. The PBGD gene has been sequenced and thoroughly characterized (9–14). The gene is assigned to chromosome 11q24.1-24.2 and it contains 15 exons. The size of the gene is approximately 10 kb of which 1.3 kb represent coding sequence. Two tissue-specific isoforms have been characterized. Both transcripts arise from two separate promoters via alternative splicing of exons 1 and 2. The mRNA of the housekeeping (nonerythropoietic) isoform contains exons 1 and 3–15, coding for an enzyme of 361 amino acids (M  42,000), whereas the erythroid isoform (M  40,000) is encoded by exons 2–15 lacking the first 17 amino acids of the amino terminus. Recent molecular biological studies on the PBGD gene have revealed approximately 160 mutations responsible for AIP, limiting the potential of DNA diagnostics in this disease. We have previously characterized 26 mutations 671 from 38 AIP families, which cover 95% of the 40 families known to have AIP in the Finnish population of 5 million (15–17). In addition, we have studied the steady-state mRNA levels of the mutant alleles in lymphocytes and shown large variations in the transcript levels among different mutants (18). These variations do not correlate with the CRIM class, the localization of the mutation in the PBGD gene, or the clinical phenotype of AIP. To further characterize the functional consequences of the mutations responsible for AIP, we have performed in vitro site-directed mutagenesis of seven different mutations in the PBGD gene and studied the transient expression of each mutated polypeptide in an eukaryotic cell line. Material and Methods Seven different mutations representing different CRIM classes, amino acid substitutions in the same codon, or a deletion removing normally utilized termination codon in the PBGD gene were chosen for this study (Table 1). All of them have previously been identified among Finnish AIP patients (15) and the steady-state transcript levels of the mutant alleles have previously been determined in the case of five mutations (18) (Table 1). Mutagenesis Mutagenesis was performed using the Chameleon Double- (...truncated)