Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP)

Abstract

Frameshift mutations in the last coding exon of the 5-aminolevulinate synthase (ALAS) 2 gene were described to activate the enzyme causing increased levels of zinc- and metal-free protoporphyrin in patients with X-linked dominant protoporphyria (XLDPP). Only two such so-called gain-of-function mutations have been reported since the description of XLDPP in 2008. In this study of four newly identified XLDPP families, we identified two novel ALAS2 gene mutations, a nonsense p.Q548X and a frameshift c.1651-1677del26bp, along with a known mutation (delAGTG) found in two unrelated families. Of relevance, a de novo somatic and germinal mosaicism was present in a delAGTG family. Such a phenomenon may explain the high proportion of this mutation in XLDPP worldwide. Enhancements of over 3- and 14-fold in the catalytic rate and specificity constant of purified recombinant XLDPP variants in relation to those of wild-type ALAS2 confirmed the gain of function ascribed to these enzymes. The fact that both p.Q548X and c.1651-1677del26bp are located in close proximity and upstream from the two previously described mutations led us to propose the presence of a large gain-of-function domain within the C-terminus of ALAS2. To test this hypothesis, we generated four additional nonsense mutants (p.A539X, p.G544X, p.G576X and p.V583X) surrounding the human XLDPP mutations and defined an ALAS2 gain-of-function domain with a minimal size of 33 amino acids. The identification of this gain-of-function domain provides important information on the enzymatic activity of ALAS2, which was proposed to be constitutively inhibited, either directly or indirectly, through its own C-terminus.

Red cells from ferrochelatase-deficient erythropoietic protoporphyria patients are resistant to growth of malarial parasites

“…The mutant enzyme upregulates the entry of succinyl-CoA and glycine precursors into the heme biosynthesis pathway, 29 resulting in substrate build up at the next ratelimiting step, which is ferrochelatase. 30,31 Individuals with XLDPP are clinically identical to EPP patients and their red cells contain similarly elevated levels of PPIX and are morphologically and physiologically very similar; ferrochelatase activity is, however, normal (supplemental Table 1). The growth of P falciparum in red cells from 3 XLDPP patients was virtually identical to rates of parasite growth in normal red cells (Figure 1), nor were there differences in the ratios of immature or mature parasite growth stages (supplemental Figure 1B).…”

Porphyrias: A 2015 update

“…This new syndrome, X-linked protoporphyria (XLPP) (MIM 300752), results from an increased activity of ALAS2 (EC 2.3.1.3.7), the rate-controlling enzyme of erythroid heme synthesis, which is essential for hemoglobin formation [59]. All other previously described mutations in ALAS2 are loss-of-function mutations that cause X-linked recessive sideroblastic anemia (XLSA) (Fig.…”

Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia

“…Different forms of Congenital sideroblastic anemia have been defined at the molecular level, each of which has provided some insight into cellular pathways associated with dysfunctional mitochondrial iron metabolism (7). X-linked sideroblastic anemia is a form of non-syndromic congenital sideroblastic anemia caused by a defect of the ␦-aminolevulinate synthase 2 (ALAS2) 3 gene, which encodes the first mitochondrial enzyme of heme biosynthesis in erythroid cells (8,9). The ALAS2 catalyzes the condensation into mitochondria of glycine with succinyl-coenzyme A to yield ␦-aminolevulinic acid (ALA) and requires pyridoxal 5Ј-phosphate (PLP; vitamin B6) as the cofactor (10,11).…”