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We purified eel hatching enzyme (EHE) from the hatching liquid of Japanese eel Anguilla japonica belonging to Elopomorpha to a single band on SDS/PAGE. TOF-MS analysis revealed that the purified EHE contained several isozymes with similar molecular masses. Comparison of the egg envelope digestion specificities of the purified EHE and of recombinant EHE4, one of the EHE isozymes, suggested that the isozymes contained in the purified EHE were functionally the same in terms of egg envelope digestion. By electron microscopy, the egg envelope became swollen after treatment with the purified EHE. The EHE cleavage sites on the zona pellucida (ZP) protein of the egg envelope were located in the N-terminal repeat regions. In previous phylogenetic analysis, we suggested that fishes included in Elopomorpha, as basal teleosts, possess a single type of hatching enzyme genes, and that fishes in Otocephala and Euteleostei gain two types of hatching enzyme genes called clade I and II genes by duplication. Further, the clade I enzymes, zebrafish hatching enzyme (ZHE1) and medaka high choriolytic enzyme (HCE), swell the egg envelope by cleaving the N-terminal regions of ZP proteins, while the clade II enzyme, medaka low choriolytic enzyme (LCE), solubilizes the swollen envelope by cleaving the site at the middle region on the ZP domain. In this evolutionary scenario, our findings support that hatching of Japanese eel conserves the ancestral mechanism of fish egg envelope digestion. The clade I enzymes inherit the ancestral enzyme function, and the clade II enzymes gain a new function during evolution to Otocephala and Euteleostei. |
