Highly heterogeneous nature of δ-aminolevulinate dehydratase (ALAD) deficiencies in ALAD porphyria

Abstract

The properties of 9 ␦-aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and purified by glutathioneaffinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and delTC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions in the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified in ADP and indicates the highly heterogeneous nature of mutations in this disorder. IntroductionDelta-aminolevulinate dehydratase (ALAD) deficiency porphyria (ADP) is an autosomal recessive disorder caused by a homozygous ALAD deficiency. Patients with this condition have clinical symptoms of acute hepatic porphyria such as abdominal pain, vomiting, pain in the arms and legs, and neuropathy. ALAD is the second enzyme in the heme biosynthetic pathway. It catalyzes the Knorr-type condensation of 2 molecules of aminolevulinate acid (ALA) to form a monopyrrole, porphobilinogen (PBG). 1 This enzyme activity is present in large excess compared to other enzymes in the heme biosynthetic pathway, and its partial deficiency does not usually result in any clinical consequences. Seven patients with ADP have been reported to date, 2-6 though only 4 of them have been confirmed by immunochemical or molecular analysis. [2][3][4] Of these 4 cases, the first 2 were German patients reported by Doss and coworkers, 7 the third was a Swedish baby boy studied by Thunell and colleagues, 8 and the fourth was an elderly Belgian man reported by Hassoun and associates. 9 Recently, an asymptomatic Swedish baby girl has been identified to have a markedly decreased ALAD activity. 10 Thus far, a total of 9 point mutations of the alad gene have been identified in these 5 subjects, which resulted in different amino acid changes. However, the enzymatic activity of ALAD expressed by mutant ALAD was investigated only for 4 mutants, A274T and R240W, in German patient B, 3 and V153M and delTC in German patient H 11 (singleletter amino acid codes). This previous study with German patient B demonstrated that ALAD activity co...

The neurologic manifestations of the acute porphyrias

“…13 The majority of individuals with an inherited enzymatic defect of one of the autosomal dominant acute porphyrias do not go on to develop clinical manifestations. Penetrance has been most completely studied in AIP with estimated rates between 10% and 50%.…”

Single Amino Acid Mutations Alter the Distribution of Human Porphobilinogen Synthase Quaternary Structure Isoforms (Morpheeins)

“…In most cases, a single mutation to only one allele encoding PBGS does not result in a disease state, even though enzyme activity may be reduced to Ͻ50% of normal (18). For instance, individuals heterozygous for F12L are not porphyric.…”

Transposase-Derived Proteins FHY3/FAR1 Interact with PHYTOCHROME-INTERACTING FACTOR1 to Regulate Chlorophyll Biosynthesis by Modulating HEMB1 during Deetiolation in Arabidopsis

“…The deficiency of ALAD in humans results in increased excretion of ALA in the urine, leading to ALAD-deficient porphyria, an autosomal recessive disorder (Maruno et al, 2001). Thus, ALAD activity in neonatal blood is used as a screening tool for ADP (Schulze et al, 2001).…”