Purification and characterization of zebrafish hatching enzyme – an evolutionary aspect of the mechanism of egg envelope digestion

Abstract

Hatching enzyme is an enzyme that digests an egg envelope at the time of embryo hatching. Fish hatching enzymes have been purified from several fish species [1][2][3][4][5]. Among them, the hatching enzyme of medaka Oryzias latipes has been extensively studied, and its study field was extended not only to characterization of the enzyme itself, but also to the mechanism of its egg envelope digestion [6,7]. The hatching of 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.Abbreviations MCA, 7-amino-4-methylcoumarin; MHCE, medaka high choriolytic enzyme; MLCE, medaka low choriolytic enzyme; ZHE, zebrafish hatching enzyme; ZPD, ZP domain.

Crystal Structure of Zebrafish Hatching Enzyme 1 from the Zebrafish Danio rerio

“…3), consistent with the previously reported cleavage efficiency difference. 6 Alanine-scanning mutagenesis of the most preferable 10-residue substrate (TVQQS↓DYLIK, the major substrate peptide or WT-major) has shown that the Asp-to-Ala mutation at P1′ decreases the cleavage efficiency to 3.9% that of the original substrate. On the other hand, the mutation at P5′ increases the cleavage efficiency to 128%.…”

“…The major site is cleaved three times more efficiently than the minor site, and is also cleaved four times more efficiently than the most preferable MCA substrate reported, N-benzyloxycarbonyl-Phe-Arg-MCA. 6 These differences in cleavage efficiency suggest that ZHE1 recognizes both the amino acid sequence and the conformation of the substrate. Two other zinc metalloproteases that are related to ZHE1 in amino acid sequence-astacin and bone morphogenetic protein 1 (BMP1)-exhibit different substrate preferences from ZHE1.…”

“…4 The substrate specificity of ZHE1 for 7-amino-4-methylcoumarin (MCA) substrates has been reported to be somewhat broad. 6 However, the cleavage of the chorion protein by ZHE1 occurs only at two sites: TVQQS↓DYLIK (major cleavage site) and KLMLK↓APEPF (minor cleavage site). The major and minor cleavage sites of ZHE1 exist in the N-terminal regions of zona pellucida glycoproteins 2 and 3, respectively, and 866 Crystal Structure of Zebrafish Hatching Enzyme 1 Fig.…”

“…6 The zebrafish chorion is constructed mainly of two glycoproteins, zona pellucida glycoproteins 2 and 3, that are cross-linked by transglutaminase to form N ɛ -(γ-L-glutamyl)-Llysine isopeptides in the fertilized egg. ZHE1 swells and softens the zebrafish chorion by cleaving chorion proteins.…”

Aquatic toxicity of manufactured nanomaterials: challenges and recommendations for future toxicity testing

“…[47] The hatching process in zebrafish is initiated by the release of a protease, zebrafish hatching enzyme (ZHE1), into the embryonic fluid, and it culminates with zebrafish movements physically disrupting the ZHE1 weakened chorion. [171] Although Ong et al [47] observed no effect on the movement of hatched larvae, ZnO NMs inhibited protease activity in isolates of chorionic fluid extracted from zebrafish embryos in vitro. This specific inhibition of ZHE1 by ZnO NMs has also been further elucidated since by Lin et al [172] who reported the in vitro inhibition of the proteolytic activity of recombinant ZHE1 expressed in Escherichia coli.…”