Sano Kaori, Inohaya Keiji, Kawaguchi Mari, Yoshizaki Norio, Iuchi Ichiro, Yasumasu Shigeki
Graduate Program of Biological Science, Graduate School of Science and Technology, Sophia University, Tokyo, Japan.
FEBS J. 2008 Dec;275(23):5934-46. doi: 10.1111/j.1742-4658.2008.06722.x.
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.
斑马鱼孵化酶ZHE1和ZHE2。Northern印迹和RT-PCR分析表明，ZHE1主要在孵化前的胚胎中表达，而ZHE2很少表达。这与在蛋白质水平进行的实验结果一致，该实验表明能够从孵化液中纯化出一种孵化酶ZHE1。因此，斑马鱼胚胎的孵化由单一酶完成，这与青鳉孵化酶是由两种酶——青鳉高溶膜酶（MHCE）和青鳉低溶膜酶（MLCE）组成的酶系统协同消化卵膜的发现不同。六个ZHE1切割位点位于卵膜亚基蛋白ZP2和ZP3的N端区域，而不在内部区域，如ZP结构域。ZHE1的消化方式似乎与MHCE非常相似，后者部分消化卵膜并使卵膜膨胀。使用斑马鱼和青鳉的酶及底物进行的种间消化表明，ZHE1和MHCE都切割两种物种卵膜蛋白的相同位点，这表明ZHE1的底物特异性与MHCE非常相似。然而，MLCE没有显示出这种相似性。由于HCE和LCE是硬骨鱼进化途径中基因复制的结果，本研究表明ZHE1和MHCE保留了祖先孵化酶的特征，而MLCE获得了新功能，如促进被MHCE膨胀的卵膜的完全消化。
