Molecular Expression and Characterization of Erythroid-Specific 5-Aminolevulinate Synthase Gain-of-Function Mutations Causing X-Linked Protoporphyria

Abstract

X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ΔAT and ΔAGTG ALAS2 enzymes, two novel mutations, c.1734ΔG (ΔG) and c.1642C>T (p.Q548X), and an engineered deletion c.1670–1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ΔAGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ΔAT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ΔAT, ΔAGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ΔG enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (ΔAT, ΔAGTG and p.Q548X) or stability (ΔG) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.

Porphyrias: A 2015 update

“…6) [60]. This first description of gain-of-function mutations in genes of the heme biosynthetic pathway strongly suggested that PPIX and its zinc chelate accumulate in erythrocytes because the rate of ALA formation is increased to such an extent that insertion of Fe 2+ into PPIX by FECH becomes rate limiting for heme synthesis in erythroid tissues [61]. The ALAS2 gain-of-function produces more PPIX than is required for hemoglobinization, and it accumulates in quantities sufficient to cause photosensitivity and liver damage, despite the normal FECH activity.…”

Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release

“…26 Subsequent analyses of the increased catalytic activities of hALAS2 variants with XLPP mutations in relation to those of engineered hALAS2 variants and wild-type enzyme led to the definition of the hALAS2 gain-of-function domain as an approximately 45 C-terminal amino acid region. 16,17 Significantly, an XLPP variant with the gain-of-function domain truncated could not bind either SUCLA2 or SCS. 16 …”

“…10,14 The molecular basis for XLSA is thus a partial loss of hALAS2 activity, due to reduced catalytic function and/or protein stability. 10,14,21 Unlike the XLSA mutations, those causing XLPP enhance ALAS2 activity, 16,17,20 hence their designation as ‘gain-of-function’ mutations, 16,20 and lead to protoporphyrin IX and zinc-protoporphyrin IX accumulation. 19,20 In fact, patients suffering from XLPP had traditionally been diagnosed as having erythropoietic protoporphyria (EPP; MIM 177000), which is symptomatically similar but results from a deficiency of ferrochelatase (EC 4.99.1.1), the enzyme responsible for converting protoporphyrin IX and ferrous iron into heme.…”

“…22 Approximately 5-10% of patients presenting EPP symptoms do not have a ferrochelatase deficiency. 15,20 Most of these patients instead have XLPP, with defective and more active forms of hALAS2, 16,17,20,23 and while they share the phenotypic hallmark of elevated protoporphyrin IX levels with EPP patients, they differ in that they also accumulate zinc protoporphyrin in their erythrocytes. 19,23,24 …”

“…In contrast to XLSA, 10,14,16,25 all known XLPP mutations reside in a single exon, exon 11, encoding the far-C-terminus of hALAS2, and give rise to truncated or extended variants of the enzyme. 10,14,15,17,20 The 33 C-terminal amino acids are highly conserved and yet have diverged from the ALAS1 isoform, suggesting that these residues endow the enzyme with an erythroid-specific function and/or regulation.…”