Molecular Expression and Characterization of Erythroid-Specific 5-Aminolevulinate Synthase Gain-of-Function Mutations Causing X-Linked Protoporphyria
Molecular Expression and Characterization of Erythroid-Specific 5-Aminolevulinate Synthase Gain-of-Function Mutations Causing X-Linked Protoporphyria
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DOI：
10.2119/molmed.2013.00003
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发表时间：
2013-01-01
影响因子：
5.7
通讯作者：
Desnick, Robert J.
中科院分区：
文献类型：
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作者：
Bishop, David F.;Tchaikovskii, Vassili;Desnick, Robert J.
X-连锁原卟啉症（XLP）（MIM 300752）是最近认识到的红细胞生成性卟啉症，由于红细胞特异性氨基乙酰丙酸合酶基因（ALAS 2）的功能获得性突变。先前，两个外显子11小缺失，c.1699_1670 Δ AT（Δ AT）和c.1706_1709 Δ AGTG（Δ AGTG），其过早地截短或延长ALAS 2多肽，据报道增加酶活性20- 40倍，引起原卟啉的红系蓄积、皮肤光敏性和肝脏疾病。突变Δ AT和Δ AGTG ALAS 2酶，两个新突变，c.1734 Δ G（Δ G）和c.1642 C>T（p.Q548X），以及工程化缺失c. 1670- 1671 TC>GA p.F557X的表达，并对其纯化的酶进行了表征。野生型和Δ AGTG酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上显示出相似量的54-和52-kDa多肽，而Δ AT和p.F557X仅具有52-kDa多肽。与纯化的野生型酶相比，Δ AT、Δ AGTG和Q548 X酶的比活性分别仅增加了1.8倍、3.1倍和1.6倍。有趣的是，结合研究表明，活性增加的Q548 X酶不结合琥珀酰辅酶A合成酶。伸长的Delta G酶具有野生型比活性、动力学和热稳定性;两倍于野生型纯化产率（56对25%）;并且主要是54-kDa形式，表明在体内更大的稳定性。突变酶的研究的基础上，最大的增益功能区跨越533和580之间的57个氨基酸。因此，这些ALAS 2功能获得性突变增加了酶的比活性（Δ AT、Δ AGTG和p.Q548X）或稳定性（Δ G），从而导致红系原卟啉积累增加，引起XLP。
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_1670 Delta AT (Delta AT) and c.1706_1709 Delta AGTG (Delta AGTG), 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 Delta AT and Delta AGTG ALAS2 enzymes, two novel mutations, c.1734 Delta G (Delta G) 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 Delta AGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the Delta AT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, Delta AT, Delta AGTG 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 Delta G 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 (Delta AT, Delta AGTG and p.Q548X) or stability (Delta G) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.