Molecular expression, characterization and mechanism of ALAS2 gain-of-function mutants

Abstract

BackgroundX-linked protoporphyria (XLP) (MIM 300752) is an erythropoietic porphyria due to gain-of-function mutations in the last exon (Ducamp et al., Hum Mol Genet 22:1280-88, 2013) of the erythroid-specific aminolevulinate synthase gene (ALAS2). Five ALAS2 exon 11 variants identified by the NHBLI Exome sequencing project (p.R559H, p.E565D, p.R572C, p.S573F and p.Y586F) were expressed, purified and characterized in order to assess their possible contribution to XLP. To further characterize the XLP gain-of-function region, five novel ALAS2 truncation mutations (p.P561X, p.V562X, p.H563X, p.E569X and p.F575X) were also expressed and studied.MethodsSite-directed mutagenesis was used to generate ALAS2 mutant clones and all were prokaryotically expressed, purified to near homogeneity and characterized by protein and enzyme kinetic assays. Standard deviations were calculated for 3 or more assay replicates.ResultsThe five ALAS2 single nucleotide variants had from 1.3- to 1.9-fold increases in succinyl-CoA Vmax and 2- to 3-fold increases in thermostability suggesting that most could be gain-of-function modifiers of porphyria instead of causes. One SNP (p.R559H) had markedly low purification yield indicating enzyme instability as the likely cause for XLSA in an elderly patient with x-linked sideroblastic anemia. The five novel ALAS2 truncation mutations had increased Vmax values for both succinyl-CoA and glycine substrates (1.4 to 5.6-fold over wild-type), while the Kms for both substrates were only modestly changed. Of interest, the thermostabilities of the truncated ALAS2 mutants were significantly lower than wild-type, with an inverse relationship to Vmax fold-increase.ConclusionsPatients with porphyrias should always be assessed for the presence of the ALAS2 gain-of-function modifier variants identified here. A key region of the ALAS2 carboxyterminal region is identified by the truncation mutations studied here and the correlation of increased thermolability with activity suggests that increased molecular flexibility/active site openness is the mechanism of enhanced function of mutations in this region providing further insights into the role of the carboxyl-terminal region of ALAS2 in the regulation of erythroid heme synthesis.Electronic supplementary materialThe online version of this article (10.1186/s10020-019-0070-9) contains supplementary material, which is available to authorized users.

Application of Weighted Gene Coexpression Network Analysis to Identify Key Modules and Hub Genes in Systemic Juvenile Idiopathic Arthritis

“…ALAS2 (erythroid-specific 5-aminolevulinate synthase) is the first and rate-limiting enzyme in the erythroid heme biosynthetic pathway [ 26 ]. Mutations in ALAS2 may be related to porphyria and X-linked sideroblastic anemia [ 27 ]. AHSP (alpha hemoglobin–stabilizing protein) is also necessary for the proper assembly of nascent alpha-globin into hemoglobin-A [ 28 ].…”

Application of Weighted Gene Co-expression Network Analysis to Identify Key Modules and Hub Genes in Systemic Juvenile Idiopathic Arthritis

“…ALAS2 (Erythroid-speci c 5-aminolevulinate synthase) is the rst and rate-limiting enzyme in the erythroid heme biosynthetic pathway [26]. Mutations in ALAS2 may be related to Porphyria and X-linked sideroblastic anemia [27]. AHSP (Alpha hemoglobin-stabilizing protein) is also necessary for the proper assembly of nascent alpha-globin into hemoglobin-A [28].…”

Porphyrias in the Age of Targeted Therapies

“…XLP is caused by gain-of-function alleles in exon 11 of the ALAS2 gene and is X-linked in inheritance. These ALAS2 variants lead to activation of the erythrocyte-specific ALAS enzyme and result in the overproduction of protoporphyrin in excess of what is required for heme synthesis in bone marrow [63]. Since there is normal function of the FECH enzyme, a larger proportion of the accumulating protoporphyrin is zinc bound, and the rest is metal free (~50-85%) [12].…”