X-linked Sideroblastic Anemia Due to Carboxyl-terminal ALAS2 Mutations That Cause Loss of Binding to the β-Subunit of Succinyl-CoA Synthetase (SUCLA2)

Bishop, David F.; Tchaikovskii, Vassili; Hoffbrand, A. V.; Fraser, M.E.; Margolis, S.

doi:10.1074/jbc.m111.306423

Cited by 41 publications

(65 citation statements)

References 37 publications

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“…The ALAS2 C365 mutant had enzyme kinetics similar to that of the WT enzyme (Table 1). However, the mutant enzyme was unstable, consistent with the notion that PLP binding plays a key role in stabilizing ALAS2 (17,18). When the enzymes were expressed as maltose-binding protein (MBP) fusions, the yield of the mutant enzyme in the initial crude extract was only 3.3% of the WT enzyme yield ( Figure 2B).…”

Section: Resultssupporting

confidence: 65%

X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

Sankaran¹,

Ulirsch²,

Tchaikovskii³

et al. 2015

J. Clin. Invest.

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Section: Resultssupporting

confidence: 65%

X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

Sankaran¹,

Ulirsch²,

Tchaikovskii³

et al. 2015

J. Clin. Invest.

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“…The wild-type ALAS2 enzyme always had two forms of approximately equal intensity with molecular masses of 54 and 52 kDa, irrespective of the purification method and inclusion of protease inhibitors (11). The human ALAS2 54-and 52-kDa subunits were also seen by immunodetection in human erythroid cell extracts (12).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the deletion or altered elongated sequence may render the active site more open, thereby increasing access to its substrates, resulting in increased enzyme activity. In addition, it has been shown recently that mutations in the carboxy-terminal region of ALAS2 can cause loss of binding to the ATP-using β subunit (SUCLA2) of succinyl-CoA synthetase as well as to the succinyl-CoA synthetase heterodimeric holoenzyme (11). Further, regulation in this region is complicated, since it was shown that two different exon 11 loss-offunction mutations that cause X-linked sideroblastic anemia (M567V and S568G) resulted in elimination of the binding of ALAS2 to SUCLA2.…”

Section: Discussionmentioning

confidence: 99%

“…Recombinant ALAS2 enzymatic activity was determined colorimetrically by using succinyl-CoA and glycine as substrates, as previously described (11). Briefly, the 0.5 mL reaction mixture contained 100 mmol/L glycine, 50 mmol/L potassium HEPES, pH 7.4, 10 mmol/L MgC1 2 , 100 μmol/L succinyl-CoA, 10 μmol/L PLP and 10-200 units ALAS2 activity.…”

Section: Enzyme and Protein Assaysmentioning

confidence: 99%

“…Recombinant wild-type and mutant ALAS2 proteins were purified as previously described (11). Briefly, an overnight culture of the expression vector was used to seed 1 liter of Luria broth (LB) media containing 0.2% glucose, 100 μg/mL ampicillin and 10 μmol/L pyridoxal 5′-phosphate (PLP), grown at 37ºC with shaking to a density of 0.6-0.…”

Section: Expression and Purification Of Human Recombinant Alas2 Enzymesmentioning

confidence: 99%

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

Bishop

Tchaikovskii

Nazarenko

et al. 2013

Mol Med

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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.

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Sideroblastic anemia: functional study of two novel missense mutations in ALAS2

Méndez

Moreno-Carralero

Morado‐Arias

et al. 2016

Molec Gen & Gen Med

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BackgroundX‐linked sideroblastic anemia (XLSA) is a disorder characterized by decreased heme synthesis and mitochondrial iron overload with ringed sideroblasts in bone marrow. XLSA is caused by mutations in the erythroid‐specific gene coding 5‐aminolevulinate synthase (ALAS2). Anemia in XLSA is extremely variable, characteristically microcytic and hypochromic with poikilocytosis, and the red blood cell distribution width is increased and prominent dimorphism of the red cell population. Anemia in XLSA patients responds variably to supplementation with pyridoxine.Methods and ResultsWe report four patients with XLSA and three mutations in ALAS2: c.611G>A (p.Arg204Gln), c.1218G>T (p.Leu406Phe) and c.1499A>G (p.Tyr500Cys). The in silico predictions of three ALAS2 mutations and the functional consequences of two ALAS2 mutations were assessed. We performed in silico analysis of these mutations using ten different softwares, and all of them predicted that the p.Tyr500Cys mutation was deleterious. The in vitro prokaryotic expression showed that the p.Leu406Phe and p.Tyr500Cys mutations reduced the ALAS2 specific activity (SA) to 14% and 7% of the control value, respectively.ConclusionIn view of the results obtained in this study, a clear relationship between genotype and phenotype cannot be established; clinical variability or severity of anemia may be influenced by allelic variants in other genes or transcription factors and environmental conditions.

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X-linked Sideroblastic Anemia Due to Carboxyl-terminal ALAS2 Mutations That Cause Loss of Binding to the β-Subunit of Succinyl-CoA Synthetase (SUCLA2)

Cited by 41 publications

References 37 publications

X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

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

Sideroblastic anemia: functional study of two novel missense mutations in ALAS2

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