The major splice variant of human 5-aminolevulinate synthase-2 contributes significantly to erythroid heme biosynthesis
The major splice variant of human 5-aminolevulinate synthase-2 contributes significantly to erythroid heme biosynthesis
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DOI：
10.1016/s1357-2725(03)00246-2
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发表时间：
2004-02-01
影响因子：
4
通讯作者：
May, BK
中科院分区：
文献类型：
--
作者：
Cox, TC;Sadlon, TJ;May, BK
红细胞血红素生物合成途径的初始步骤是由红细胞特异性的5-氨基乙酰酸合成酶-2 （ALAS2）异构体催化的。先前，虽然未知编码蛋白的功能意义，但已鉴定出ALAS2的选择性剪接mRNA异构体。我们试图描述这种ALAS2亚型对整个红系血红素生物合成的贡献。在这里，我们报告了三个新的ALAS2 mRNA剪接异构体的鉴定，除了先前描述的异构体，缺乏外显子4衍生序列。利用核糖核酸酶保护实验对这些mRNA进行定量分析，结果显示，不含外显子4衍生序列的同种异构体约占ALAS2 mRNA总量的35-45%，而新鉴定的转录本合计约占15%。尽管这三种新的转录本数量显著，但它们的特征表明它们不太可能对线粒体ALAS2的总体活性有实质性的贡献。相比之下，体外研究表明，主要剪接变体（缺乏外显子4编码序列）产生一种功能酶，尽管活性略有降低，并且与atp特异性琥珀酰辅酶a合成酶的β亚基具有亲和力，与成熟的ALAS2相当。研究还发现，ALAS2前蛋白的前49个氨基酸对于线粒体内膜的易位是必要和充分的，并且该过程不受缺乏外显子4编码序列的影响。我们得出结论，ALAS2的主要剪接异构体在体内具有功能，并可能显著促进红系血红素的生物合成和血红蛋白的形成。Elsevier Ltd.出版。
The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-aminolevulinate synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform, lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents similar to35-45% of total ALAS2 mRNA while the newly identified transcripts together represent similar to15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation. Published by Elsevier Ltd.