X连锁原卟啉症(XLP)(MIM300752)是由于最后一个外显子的功能获得突变而导致的红细胞生成卟啉症(Ducamp等人。,红系特异性氨基乙酰丙酸合酶基因(ALAS2)的HumMolGenet22:1280-88,2013)。通过NHBLI外显子组测序项目鉴定的五个ALAS2外显子11变体(p。R559H,p.E565D,p.R572C,p.S573F和p.Y586F)表示,纯化和表征,以评估它们对XLP的可能贡献。为了进一步表征XLP功能增益区域,五个新的ALAS2截短突变(p。P561X,p.V562X,p.H563X,p.E569X和p.F575X)也被表达和研究。
定点诱变用于产生ALAS2突变体克隆,并且全部原核表达,纯化至接近均一,并通过蛋白质和酶动力学测定进行表征。计算3次或更多次测定重复的标准偏差。
五个ALAS2单核苷酸变体的琥珀酰-CoAVmax增加1.3至1.9倍,热稳定性增加2至3倍,表明大多数可能是卟啉症的功能获得修饰剂而不是原因。一个SNP(p。R559H)的纯化率明显较低,表明酶不稳定是X连锁铁粒母细胞性贫血的老年患者XLSA的可能原因。五个新的ALAS2截短突变增加了琥珀酰辅酶A和甘氨酸底物的Vmax值(比野生型高1.4至5.6倍),而两种底物的Kms仅略有变化。感兴趣的,截短的ALAS2突变体的热稳定性明显低于野生型,与Vmax倍数增加成反比。
应始终评估患有卟啉症的患者是否存在此处确定的ALAS2功能获得修饰变体。通过此处研究的截短突变鉴定了ALAS2羧基末端区域的关键区域,并且增加的可热性与活性的相关性表明,分子灵活性/活性位点开放性的增加是该区域突变功能增强的机制,从而进一步了解ALAS2的羧基末端区域在调节类红细胞血红素合成中的作用。
X-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.
Site-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.
The 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.
Patients 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.