Identification and biochemical characterization of a second zebrafish autotaxin gene
Identification and biochemical characterization of a second zebrafish autotaxin gene
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DOI：
10.1093/jb/mvy114
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发表时间：
2019-03-01
影响因子：
2.7
通讯作者：
Aoki, Junken
中科院分区：
文献类型：
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作者：
Kise, Ryoji;Okasato, Ryohei;Aoki, Junken
自体趋化蛋白(ATX)是一种分泌酶，能产生一种具有生物活性的溶血磷脂酸(LPA)。ATX在胚胎的血管和神经发育中发挥作用，但其机制尚不清楚。在本研究开始时，只有一个斑马鱼ATX基因(Atxa)已知并已被研究。在本研究中，我们通过TALEN靶向Atxa产生了ATX基因敲除(KO)FISH。出乎意料的是，Atxa KO FISH既没有显示出血管缺陷，也没有显示ATX活性降低，这意味着基因组中存在一个或多个其他ATX。以Atxa蛋白片段为查询，通过BLAST搜索，我们发现了一个与Atxa外显子13、14和15非常相似的基因组序列。因此，我们克隆了一个编码第二斑马鱼自体分类蛋白(ATXb)的cDNA，并发现它在多种组织中转录。Atxb基因编码一个832个氨基酸的蛋白质(而atxa编码850个氨基酸)，与atxa有60%的氨基酸同源性，并与其他物种的atxs聚在一起。重组ATXb蛋白具有溶血磷脂酶D(LysoPLD)活性，底物特异性与Atxa和哺乳动物ATXs相似。这些结果表明，ATXb是第二个斑马鱼ATX，它可能与Atxa在胚胎发育中具有多余的作用。
Autotaxin (ATX) is a secreted enzyme that produces a bioactive lysophospholipid, lysophosphatidic acid (LPA). ATX plays a role in vascular and neural development in embryos but its mechanisms remain unclear. At the beginning of this study, only one zebrafish atx gene (atxa) was known and had been investigated. In this study, we generated ATX knockout (KO) fish by TALEN targeting atxa. Unexpectedly, atxa KO fish showed neither vascular defects nor reduction of ATX activity, implying the existence of one or more other ATXs in the genome. By a BLAST search using ATXa protein fragments as a query, we found a genomic sequence that closely resembled atxa exons 13, 14 and 15. Consequently, we cloned a cDNA encoding a second zebrafish autotaxin (ATXb), and found that it was transcribed in various tissues. The atxb gene encoded a protein of 832 amino acids (compared to 850 amino acids in ATXa) with 60% amino acid identity to ATXa and clustered with ATXs from other species. A recombinant ATXb protein showed lysophospholipase D (lysoPLD) activities with substrate specificities similar to those of ATXa and mammalian ATXs. These results indicate that ATXb is a second zebrafish ATX, which possibly shares redundant roles with ATXa in embryonic development.