Purification and characterization of zebrafish hatching enzyme - an evolutionary aspect of the mechanism of egg envelope digestion
Purification and characterization of zebrafish hatching enzyme - an evolutionary aspect of the mechanism of egg envelope digestion
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DOI：
10.1111/j.1742-4658.2008.06722.x
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发表时间：
2008-12-01
期刊：
影响因子：
5.4
通讯作者：
Yasumasu, Shigeki
中科院分区：
文献类型：
--
作者：
Sano, Kaori;Inohaya, Keiji;Yasumasu, Shigeki
斑马鱼基因组中有两个孵化酶同源物：斑马鱼孵化酶ZHE1和ZHE2。Northern blot和RT-PCR分析显示，ZHE1主要在未孵化的胚胎中表达，而ZHE2很少表达。这与在蛋白水平上的实验结果一致，表明可以从孵化液中纯化出一种孵化酶ZHE1。因此，斑马鱼胚胎的孵化是由单一的酶完成的，而不是由发现的medaka孵化酶是由medaka高绒毛膜溶解酶（MHCE）和medaka低绒毛膜溶解酶（MLCE）两种酶组成的酶系统，它们共同消化卵包膜。6个zhe1切割位点均位于卵包膜亚基蛋白ZP2和ZP3的n端区域，而不在ZP结构域等内部区域。ZHE1的消化方式似乎与MHCE的消化方式高度相似，部分消化卵包膜并使卵包膜膨胀。利用斑马鱼和medaka的酶和底物进行跨种酶切实验发现，ZHE1和MHCE切割了两种鱼卵包膜蛋白的相同位点，表明ZHE1的底物特异性与MHCE非常相似。然而，MLCE没有表现出这种相似性。由于HCE和LCE是Teleostei进化途径中基因复制的结果，本研究认为ZHE1和MHCE保持了祖先孵化酶的特征，而MLCE获得了新的功能，如促进MHCE肿胀的卵包膜的完全消化。
There are two hatching enzyme homologues in the zebrafish genome: zebrafish hatching enzyme ZHE1 and ZHE2. Northern blot and RT-PCR analysis revealed that ZHE1 was mainly expressed in pre-hatching embryos, whereas ZHE2 was rarely expressed. This was consistent with the results obtained in an experiment conducted at the protein level, which demonstrated that one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid. Therefore, the hatching of zebrafish embryo is performed by a single enzyme, different from the finding that the medaka hatching enzyme is an enzyme system composed of two enzymes, medaka high choriolytic enzyme (MHCE) and medaka low choriolytic enzyme (MLCE), which cooperatively digest the egg envelope. The six ZHE1-cleaving sites were located in the N-terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. The digestion manner of ZHE1 appears to be highly analogous to that of MHCE, which partially digests the egg envelope and swells the envelope. The cross-species digestion using enzymes and substrates of zebrafish and medaka revealed that both ZHE1 and MHCE cleaved the same sites of the egg envelope proteins of two species, suggesting that the substrate specificity of ZHE1 is quite similar to that of MHCE. However, MLCE did not show such similarity. Because HCE and LCE are the result of gene duplication in the evolutionary pathway of Teleostei, the present study suggests that ZHE1 and MHCE maintain the character of an ancestral hatching enzyme, and that MLCE acquires a new function, such as promoting the complete digestion of the egg envelope swollen by MHCE.