Báo cáo khoa học: Purification and characterization of zebrafish hatching enzyme – an evolutionary aspect of the mechanism of egg envelope digestion pot

Purification and characterization of zebrafish hatching enzyme – an evolutionary aspect of the mechanism of egg envelope digestion Kaori Sano 1 , Keiji Inohaya 2 , Mari Kawaguchi 3 , Norio Yoshizaki 4 , Ichiro Iuchi 5 and Shigeki Yasumasu 5 1 Graduate Program of Biological Science, Graduate School of Science and Technology, Sophia University, Tokyo, Japan 2 Department of Biological Information, Tokyo Institute of Technology, Yokohama, Japan 3 Ocean Reseach Institute, The University of Tokyo, Japan 4 Department of Biological Diversity, Faculty of Agriculture, Gifu University, Japan 5 Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan Hatching enzyme is an enzyme that digests an egg envelope at the time of embryo hatching. Fish hatch- ing enzymes have been purified from several fish species [1–5]. Among them, the hatching enzyme of medaka Oryzias latipes has been extensively studied, and its study field was extended not only to character- ization of the enzyme itself, but also to the mechanism of its egg envelope digestion [6,7]. The hatching of Keywords astacin family; egg envelope; hatching enzyme; molecular evolution Correspondence S. Yasumasu, Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan Fax / Tel: +81 3 3238 3393 E-mail: s-yasuma@hoffman.cc.sophia.ac.jp (Received 17 June 2008, revised 22 September 2008, accepted 2 October 2008) 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 chorio- lytic 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 analo- gous 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. 5934 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS medaka embryos is performed by two enzymes, high choriolytic enzyme, choriolysin H (HCE; EC 3.4.24.67) and low choriolytic enzyme, choriolysin L (LCE; EC 3.4.24.66), which cooperatively digest egg envelope. Two enzymes have been separately purified from hatching liquid [4,5]. HCE swells the egg enve- lope by its limited proteolytic action, whereas LCE efficiently digests the HCE-swollen envelope and solu- bilizes it completely. We have named this digesting system the ‘HCE-LCE system’. cDNA cloning analysis revealed that both enzymes belong to the astacin fam- ily and comprise 200 amino acid residues in mature enzyme portions with 55% identity in amino acid sequence [8]. In addition, two hatching enzymes have been purified from killifish Fundulus heteroclitus embryos, and hatching has been demonstrated to be performed by the HCE-LCE system [9]. Two types of enzymes homologous to medaka HCE (MHCE) and medaka LCE (MLCE) were cloned from other euteleostean fishes, such as fugu Takifugu rubripes, spotted green pufferfish Tetraodon nigroviridis and ayu Plecoglossus altivelis altivelis [10]. Thus, the hatching of euteleostean fishes can be performed by the HCE- LCE system. According to the phylogenetic tree based on the mitochondorial DNA of Teleostei, Osteoglossomorpha first branched off from an ancestor, followed by Elopomorpha, and then branched paraphyletically to Otocephala and Euteleostei [11–14]. The cDNA cloning analysis using Japanese eel Anguilla japonica belonging to Elopomorpha revealed that several hatch- ing enzyme cDNAs were present, their nucleotide sequences were similar to each other, and all formed a monophyletic clade in the phylogenetic tree of fish hatching enzymes [15]. These results suggest that the hatching of eel embryos is performed by a single type of enzyme. Therefore, HCE and LCE are considered to have been produced by a gene duplication event after Elopomorpha had diverged [16]. At present, and in contrast with such genetic information, information at the protein level is restricted to euteleostean fishes and is not available for fishes belonging to Elopomorpha and Otocepha- la. In the present study, we purified hatching enzyme from embryos of zebrafish Danio rerio belonging to Cypriniformes in Otocephala, analyzed the mecha- nism of its egg envelope digestion and compared it with that of medaka hatching enzyme. Finally, the evolution of the mechanism of egg envelope diges- tion is discussed on the basis of the manner of the reciprocal or cross-species egg envelope digestion using enzymes and substrates of both species: zebra- fish and medaka. Results Expression of zebrafish hatching enzyme ZHE1 and ZHE2 genes It has been reported that two cDNAs, ZHE1 and ZHE2, are cloned from the RNA of prehatching embryos [17]. According to the zebrafish genome pro- ject, three orthologues, ZHE1a, ZHE1b and ZHE2, were clustered in the genome [16]. The amino acid sequence of ZHE1a is 99% identical to that of ZHE1b, and 60.8% identical to that of ZHE2. Therefore, we considered that two types of hatching enzyme genes are present in the zebrafish genome. First, we observed the expression of ZHE1 and ZHE2 genes by northern blot analysis (Fig. 1A). The expression of ZHE1 was detected in embryos at 11.5 h, and a strong signal was observed at 24 h. After hatching, no expression was observed. The size of the band ( 1 kbp) was in agreement with that deduced from ZHE1 cDNA (800 bp). By contrast, no signal for ZHE2 gene expression was detected at any of the developmental stages (Fig. 1A). Next, RT-PCR, a method more sensitive than nor- thern blot analysis, was used to detect expression using RNA of 24 h embryos. An amplified band for ZHE1 transcript became visible at the 19th cycle of PCR, whereas only a faint band for the ZHE2 transcript was observed at the 28th cycle (Fig. 1B). The result suggests that the amount of ZHE1 transcript is quite different from that of ZHE2. The amount of ZHE2 transcript is considered to be much lower than that of ZHE1. Taken together with the results of the northern blot analysis, the ZHE2 gene in developing embryos is considered to be expressed to a very small extent. Finally, the expression of ZHE genes was observed by whole mount in situ hybridization. ZHE1 gene expression was observed in the hatching gland cells located on the yolk sac of 24 h embryos (Fig. 1C). However, no positive signal for ZHE2 was observed in the same staining condition. Several more days of incubation with a substrate solution showed only a weak ZHE2 signal in the cells (Fig. 1C). Thus, the ZHE1 gene, and not the ZHE2 gene, is predominantly expressed in zebrafish embryos. Purification of ZHE The hatching liquid (i.e. culture medium after embryos hatched out) was used to purify ZHE. First, we applied the concentrated hatching liquid onto a Super- dex 75 10 ⁄ 300 GL column in the HPLC system (Fig. 2A). Most of the protein was eluted just after the K. Sano et al. Hatching enzyme of zebrafish FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5935 void volume, and a single peak of caseinolytic activity was eluted near the bed volume. After dialysis against a25mm Tris–HCl buffer (pH 7.5), the fraction having caseinolytic activity was applied onto a Source 15S column in the HPLC system and eluted with a linear gradient of 0–1 m NaCl (Fig. 2B). Most of the activity was retained in the column and eluted at the concen- tration of approximately 0.35 m NaCl as a sharp single peak. SDS ⁄ PAGE of the active fraction gave a single band, with an estimated molecular mass of 23 kDa (Fig. 3). A partial amino acid sequence from the N-ter- minus of the 23 kDa protein was NALIXE, which matched with the sequence from the N-terminus of mature protein portion deduced from ZHE1 cDNA, but not from ZHE2. Thus, a single enzyme, ZHE1, was contained in the hatching liquid. This is consistent with the results of the gene expression analysis: ZHE1 10 20 300 0.01 0.2 0.1 Time (min) 0.02 0 1 NaCI ( M ) Caseinolytic activity (ΔA 280 ) Caseinolytic activity (ΔA 280 ) 0.5 10 20 30 40 500 Time (min) 0.1 0.2 0.3 0.2 0.4 0.6 0.8 B A A 280 A 280 Fig. 2. Purification of the hatching enzyme of zebrafish. (A) Super- dex 75 10 ⁄ 300 GL column chromatogram of hatching liquid. The solid line indicates A 280 and the dotted line shows caseinolytic activity. (B) Source-15S column chromatogram of the caseinolytic active fractions obtained from the Superdex 75 10 ⁄ 300 GL column chromatograqphy. The sample was eluted with a line gradient from 0–1 M NaCl (broken line). The solid line indicates the protein amount measured at A 280 . The dotted line indicates the caseinolytic activity. Fig. 3. SDS ⁄ PAGE patterns of rec. ZHE1 (lane 1) and purified ZHE1 (lane 2). The gel was stained with silver. Numbers on the left refer to the sizes of molecular markers. A B C Fig. 1. Expression analysis of ZHE1 and ZHE2 genes. (A) The result of the northern blot analysis probed with ZHE1 and ZHE2 cDNAs. Total RNAs were isolated from 11.5 h embryos (lane 1), 24 h embryos (lane 2) or embryos after hatching (lane 3). Arrowheads indicate the positions of 28S and 18S rRNA. (B) Expression of ZHE1 and ZHE2 was analyzed by RT-PCR using RNA isolated from 24 h embryos. b-actin was used as a control. (C) The result of whole mount in situ hybridization probed with ZHE1 and ZHE2 cDNAs. The color precipitation was developed for 2 h (ZHE1) or several days (ZHE2). Arrowheads in (C) indicate positive signals observed in hatching gland cells. Scale bars = 200 lm. Hatching enzyme of zebrafish K. Sano et al. 5936 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS is mainly expressed in the developing embryo, but very little ZHE2 is expressed. Generation of recombinant ZHE1 Recombinant ZHE1 (rec. ZHE1) was generated by an Escherichia coli expression system using pET3c as an expression vector, and the active enzyme was obtained by the astacin-refolding method with slight modifica- tions [18]. The specific caseinolytic activity of rec. ZHE1 (900 min )1 Æmg )1 protein) was higher than that of purified medaka hatching enzymes (800 min )1 Æmg )1 protein for MHCE, 540 min )1 Æmg )1 protein for MLCE) [4,5]. The result suggests that almost all rec. ZHE1 molecules were correctly refolded and had activity. By contrast, rec. ZHE2 failed in the refolding. rec. ZHE1 was completely inhibited by 1 mm EDTA, but not by 10 mm diisopropylfluorophosphate or 10 mm iodoacetic acid, consistent with the fact that fish hatching enzymes generally belong to the metallo- protease family. The substrate specificity of rec. ZHE1 was determined using various 7-amino-4-methylcou- marin (MCA) peptides (Table 1). ZHE1 cleaved the peptide bonds at the C-terminal side of Arg, Tyr, Asn, Trp, Ala, Asp, Phe and Gly, suggesting that ZHE1 has broad substrate specificity. One of the most suitable substrates was Z-Phe-Arg-MCA, and the specific activ- ity was 27.02 nmolÆ30 min )1 Æmg )1 protein. The sub- strate specificity of rec. ZHE1 was similar to that of the protease contained in hatching liquid. This result supported the findings of the purification indicating that only a single enzyme, ZHE1, was contained in hatching liquid. Changes of fertilized egg envelopes treated with recombinant ZHE1 Figure 4B shows an egg envelope after hatching. At the natural hatching of zebrafish embryo, the egg enve- lope was not completely solubilized, but was softened and ruptured by the contractile movement of the embryo. When isolated egg envelopes were incubated with rec. ZHE1, no marked structural changes could be observed under a binocular microscope (Fig. 4C). Using electron microscopy, we observed changes of the fine structure of envelope. Figure 4D shows the structure of an intact egg envelope, which was composed of a thick inner layer and a thin outer layer. The inner layer comprised a lamellar structure with microvillous Table 1. The specific activity of rec. ZHE1 examined by various MCA substrates. The activity of hatching liquid was normalized by caseinolytic activity per 1 lg of rec. ZHE1. ND, not detected. MCA substrate Specific activity (nmolÆ30 min )1 Ælg )1 enzyme) rec. ZHE1 Hatching liquid Z-Phe-Arg-MCA 27.02 32.26 Suc-Leu-Leu-Val-Tyr-MCA 11.09 8.36 Boc-Phe-Ser-Arg-MCA 2.57 3.91 Z-Ala-Ala-Asn-MCA 2.33 1.26 Suc-Ile-Ile-Trp-MCA 1.55 2.01 Boc-Val-Pro-Arg-MCA 1.00 2.34 Suc-Ala-Pro-Ala-MCA 0.48 0.14 Ac-Asp-Glu-Val-Asp-MCA 0.40 0.28 Bz-Arg-MCA 0.34 0.47 Suc-Ala-Ala-Pro-Phe-MCA 0.22 0.32 Z-Leu-Arg-Gly-Gly-MCA 0.19 0.23 Suc-Ala-Glu-MCA ND ND Z-Val-Lys-Met-MCA ND ND Suc-GLy-Pro-Leu-Gly-Pro-MCA ND 0.39 A D B E C F Fig. 4. Morphological changes of egg envelope. The isolated egg envelope of zebrafish before hatching (A), after hatching (B) and digested by rec. ZHE1 (C) were observed using a binocular micro- scope. Scale bars = 200 lm. (D–F) Electron microscopic observa- tion of zebrafish egg envelopes. Sections of the egg envelope isolated from pre-hatching embryo (D), the egg envelope after hatching (E) and the egg envelope digested by rec. ZHE1 (F). Arrowheads indicate outer layers. Scale bars = 1 lm. K. Sano et al. Hatching enzyme of zebrafish FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5937 channels. After incubation with rec. ZHE1, the fibrous structure of the inner layer became evident, and its thickness was increased two-fold more than that of the intact envelope. Figure 4E shows an egg envelope after natural hatching. Its fine structure was similar to that of the egg envelope incubated with rec. ZHE1 (Fig. 4F). Taken together with the result of the purification, the single enzyme, ZHE1, is suggested to act on the egg envelope at the time of natural hatching. Digestion of unfertilized egg envelope by ZHE1 It is well known that the egg envelope becomes hard- ened after fertilization. The hardening of the envelope is considered to be achieved by the polymerization of egg envelope subunits. The polymerization is due to the formation of e-(c-glutamyl) lysine isopeptide cross- links by transglutaminase [19–21]. Such cross-links make it difficult to clearly determine the sites of egg envelope cleaved by ZHE1. Therefore, initially, an unfertilized egg envelope was used as a substrate. The zebrafish egg envelope is known to be mainly constructed by two glycoproteins, ZP2 (44 kDa) and ZP3 (49 kDa), which were visualized by the SDS ⁄ PAGE analysis of unfertilized egg envelopes (Fig. 5, lane 1). The isolated unfertilized egg envelopes were digested by rec. ZHE1 and analyzed by SDS ⁄ PAGE. After incubation for 2 min, bands with molecular masses of 43 and 39 kDa were observed in addition to undigested bands of ZP2 and ZP3. After incubation for 10 min, three major bands with molecu- lar masses of 43, 39 and 36.5 kDa were observed (Fig. 5, lane 2). After further incubation (60 min), the 39 kDa band disappeared, and only two bands with the same mobility as the 43 and 36.5 kDa products were detected (Fig. 5, lane 3). These results indicate that the 43 and 39 kDa products appear first, and the 39 kDa product is then further digested and shifted to the 36.5 kDa product. To determine rec. ZHE1-cleaving sites in egg enve- lope subunits, we analyzed the sequence of each prod- uct from its N-terminus. The sequences were compared with those deduced from ZP2 and ZP3 cDNA [22,23]. It is well known that ZP2 is composed of an N-terminal region (95 amino acids), an internal trefoil domain and a C-terminal ZP domain ( 260 amino acids) including eight consensus Cys residues (Fig. 6A). ZP3 is composed of an N-terminal region (45 amino acids) and a C-terminal ZP domain (Fig. 6B). The detected N-terminal amino acid sequence of the 43 kDa product was APEPFT, which matched with the sequence from Ala80 of ZP3, and we therefore deduced that the cleaving site is Gln79 ⁄ Ala80 (Fig. 6, Fig. 6. Amino acid sequences of ZP2 and ZP3 deduced from their cDNAs. The arrows and capital letters (sites A to F) indicate the cleaving sites of ZHE1 determined from the N-terminal amino acid sequences of the ZHE1 digests. Circled Q indicates a glutamine residue that is presumed to form a e-(c-glutamyl) lysine cross-link by the sequence analysis. ZP domains and trifoiled domain are indi- cated in light gray and dark gray boxes, respectively. Predicted N-glycosylation site is underlined. Black and white triangles indicate putative signal sequence cleaving sites and predicted C-terminal processing sites, respectively. Fig. 5. SDS ⁄ PAGE patterns of unfertilized egg envelopes digested by rec. ZHE1. The envelopes isolated from unfertilized egg of zebrafish (lane 1) were incubated with rec. ZHE1 for 2 min (lane 2), 10 min (lane 3) and 40 min (lane 4). Numbers on the right show the molecular masses of the major bands. Hatching enzyme of zebrafish K. Sano et al. 5938 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS site E). The molecular mass of the 43 kDa product was somewhat larger than the molecular mass pre- dicted from ZP3 cDNA (39 070.60; from Ala80 to lle431; Fig. 6). Because the amino acid sequence of ZP3 contains one of the consensus sequences for N-glycosylation site, such a difference is considered to be due to the existence of a sugar chain. The N-termi- nal amino acid sequences of the 39 and 36.5 kDa prod- ucts were DYLIKEIVQP and VEEVVVK, respectively, and these matched with the sequences from Asp48 and Val67 deduced from ZP2 cDNA, respectively. There- fore, the cleaving sites are Ser47 ⁄ Asp48 and Arg66 ⁄ Val67 (Fig. 6, sites A and B). The molecular masses of the 39 and 36.5 kDa products of ZP2 were consistent with those calculated from ZP2 cDNA (39 107.06 from Asp48 to Arg405; 36 902.50 from Val67 to Arg405). Digestion of fertilized egg envelope by ZHE1 Next, a fertilized egg envelope was digested by rec. ZHE1. As a control, the SDS extract of intact egg envelopes was analyzed by SDS ⁄ PAGE. Several bands with a mobility that did not correspond to that of ZP2 and ZP3 were observed (Fig. 7, lane 1). These bands are considered to be proteins that are secreted from cortical alveoli and adhere to the envelope at fertiliza- tion. The hardened, fertilized egg envelopes are not considered to be solubilized by SDS. The egg envelope is not soluble but became swollen by rec. ZHE1. This swollen envelope was dissolved into SDS and analyzed by SDS ⁄ PAGE. SDS ⁄ PAGE of the fertilized egg envelope digested by rec. ZHE1 for 50 min gave three bands (150, 43 and 36.5 kDa), which were not found in the control (Fig. 7, lane 2). The N-terminal amino acid sequence of the 43 kDa product (APEPFT) was identical to that of the 43 kDa product obtained in the unfertilized egg envelope digestion, suggesting that rec. ZHE1 cleaves the com- mon sites of unfertilized and fertilized egg envelope (Fig. 6, site E). Amino acid sequence analysis of the 36.5 kDa product revealed that this sequence was a mixture of two peptide sequences. One of them was VEEV- VVKAGPVDK and matched that of the 36.5 kDa product from ZP2 in unfertilized egg envelope digests (Fig. 6, site B). The other sequence, APLDLXE, did not correspond to any cleaving site obtained in the unfertilized egg envelope digestion. However, this sequence was found in the sequence from Ala68 of ZP3 (Gln67 ⁄ Ala68; Fig. 6, site D). This cleaving site was located 12 amino acid residues upstream from the cleaving site obtained from the 43 kDa product of ZP3 (site E). Therefore, the finding that the 36.5 kDa prod- uct is a mixture of two peptides from ZP2 and ZP3 suggested that the 36.5 kDa product of ZP2 obtained from unfertilized digest binds the 12 amino acid resi- dues fragment (from site D to site E) of ZP3 via an e-(c-glutamyl) lysine cross-link. Further analysis revealed that 150 kDa product also contained two amino acid sequences identical to those of the 36.5 kDa product, VEEVVVKAGPVDK and APLDLXE. The sequence APLDLXE is quite similar to the sequence APLDLQE of ZP3 deduced from cDNA. However, the sixth glutamine residue (Q of APLDLQE; Fig. 6, circle) was not detected in sequenc- ing of the 36.5 and 150 kDa product by Edman degra- dation. There is evidence that Edman degradation did not release amino acid residues at the e-(c-glutamyl) lysine cross-linked position [24]. Although further investigation is necessary, we conclude that Gln73 in ZP3 is one of the glutamine acceptor sites for e-(c-glut- amyl) lysine cross-link formation. The lysine donor site presumed to exist in the ZP2 sequence of the 36.5 and 150 kDa product was not determined in the present study. The 150 kDa proteins disappeared after further digestion (90 min; Fig. 7, lane 3), and this analysis identified a new cleaving site, Gln79 ⁄Ala80 peptide bond (Fig. 6, site E) in ZP3, which is identical to the site found in the 43 kDa product. Therefore, this digestion probably resulted in further digestion of the 150 kDa products into the 36.5 and 43 kDa product. All the results suggest that rec. ZHE1 cleaves the N-terminal portions of ZP2 and ZP3 and eliminates Fig. 7. SDS ⁄ PAGE patterns of fertilized egg envelopes. Lane 1, SDS extract of intact fertilized egg envelopes; lanes 2 and 3, SDS extract of egg envelopes digested by rec. ZHE1 for 50 and 90 min, respectively; lane 4, SDS extract of egg envelopes after hatching. Numbers on the left show the molecular masses of the major bands. K. Sano et al. Hatching enzyme of zebrafish FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5939 the tight bindings between subunits by cleaving out the small portion of peptides including e-(c-glutamyl) lysine isopeptide cross-links. We compared the cleaving sites determined from egg envelopes after natural hatching (post-hatching) with those artificially digested by rec. ZHE1. As shown in Fig. 7 (lane 4), the SDS ⁄ PAGE pattern of the proteins of the post-hatching egg envelopes was similar to that digested by rec. ZHE1 for 90 min. The 43 kDa band became weaker than that of the 50 min incubation, suggesting further digestion of the 43 kDa product. Furthermore, their 36.5 kDa bands became broader than that for the 50 min incubation. The sequence analyses of the 36.5 kDa bands revealed that two sequences of further digested products were detected, in addition to those of 36.5 kDa product obtained from the 50 min incubation. One was a minor sequence, QPASPG, which was found to locate at Q106 of ZP3; the cleaving site is K105 ⁄ Q106 (Fig. 6, site F), suggesting that the 43 kDa product of ZP3 is further digested and decreases its molecular mass to approximately 36.5 kDa. The other was AGPVDK (from Ala74 of ZP2; Fig. 6, site C), which was shifted seven amino acid residues to the C-terminal from the site B. Thus, the cleaving sites obtained from the 90 min incubation and the post-hatching egg envelopes are considered to contain the sites that can be cleaved, although inefficiently, by ZHE1. Considering that the perivitelline space where hatching enzyme is secreted is only a small area, a rather considerably high concen- tration of ZHE1 appears to act on egg envelope, and therefore the ZHE1-cleaving sites at natural hatching are suggested to include not only its preferred sites, but also inefficient cleaving sites for ZHE1. Specific activity of ZHE1 judged by synthetic peptide substrates The cleaving efficiency of ZHE1 was quantitatively estimated with synthetic peptide substrates that were designed from the determined ZHE1-cleaving sites. The specific activities of rec. ZHE1 toward five pep- tides (Fig. 6, sites A, B, C, D and E) were determined. The most efficient substrate was site A peptide and the second most efficient was site E peptide (Table 2). Sites A and E corresponded to the N-termini of the 39 kDa product of ZP2 and the 43 kDa product of ZP3 observed in the 2 min ZHE1 digestion of unfertilized egg envelopes, respectively. By contrast, the specific activities toward site B and D peptides were much lower than those toward the former two (5.86% and 2.47% of site A peptide, respectively). Therefore, these values well reflected the results of the egg envelope digestion experiment. According to the fertilized egg envelope digestion experiment, the e-(c-glutamyl) lysine isopeptide cross-links formed between ZP2 and ZP3 subunits are considered to be eliminated by cleaving of site E. Thus, site E is conjectured to be a key cleaving site leading to a conformational change that results in swelling of the egg envelope. Therefore, it is reasonable to consider that site E is one of the efficient cleaving sites for ZHE1. The cleaving activity at site C, which was detected in the 90 min digestion of the fertilized egg envelope and considered as an inefficient cleaving site, was not easily detected in this condition. Species specificity of digestion by hatching enzyme As described earlier, zebrafish hatching is performed by a single enzyme, ZHE1. Different from zebrafish, hatching of medaka is performed by a two enzyme sys- tem. To obtain an evolutionary aspect of the mecha- nism of egg envelope digestion by hatching enzyme, we changed the substrate–enzyme combination between zebrafish and medaka and performed the cross-species digestion experiment using unfertilized egg envelopes as substrate. First, the unfertilized egg envelopes of zebrafish were digested either by ZHE1, MHCE or MLCE, and their SDS ⁄ PAGE patterns were com- pared. SDS ⁄ PAGE of the MHCE digest after a 10 and 40 min incubation gave two bands (43 and 39 kDa), and an additional band (36.5 kDa) was observed after a 120 min incubation (Fig. 8A, lanes 3–5). These corre- sponded to three bands obtained from the ZHE1 digest after a 10 min incubation (Fig. 8A, lane 2). The N-terminal sequence analyses of three digests revealed that each of the MHCE-cleaving sites on zebrafish egg envelope was the same as the three ZHE1-cleaving Table 2. The specific activity of ZHE1, MHCE and MLCE examined by synthetic peptide substrates. The cleaving site of each peptide is indicated by an arrow. ND, not detected. Peptide name Peptide sequence Specific activity (nmolÆ30 min )1 Ælg )1 enzyme) ZHE1 MHCE MLCE Site A TVQQSflDYLIK 85.5 74.1 3.6 Site B PLPVRflVEEVV 6.2 5.4 ND Site C EVVVKflAGPVD ND – – Site D GKPVQflAPLDL 2.1 – – Site E KLMLKflAPEPF 32.4 39.0 2.4 Pro-X-Y-1 NPSYPQflNPSYPQ 27.3 41.7 0.12 Pro-X-Y-2 NPQVPQflYPSKPQ 14.4 32.1 1.5 ZPD-center EVQPPDflSPLSI 0.27 0.06 49.8 Hatching enzyme of zebrafish K. Sano et al. 5940 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS sites (Fig. 6, sites A, E and B). The results suggest that ZHE1 and MHCE have the same substrate specificity toward zebrafish unfertilized egg envelopes, although the digestion efficiency of MHCE appears to be some- what lower than that of ZHE1. By contrast, MLCE digested the zebrafish egg envelopes and produced two bands, the mobilities of which corresponded to 43 and 39 kDa ZHE1 digests (Fig. 8A, lanes 6–8). However, the cleaving efficiency of MLCE appears to be less than that of MHCE because a considerable amount of undigested bands remained in the digest after a 10 min incubation, and the 36.5 kDa band was not easily detected even after a 120 min incubation. The sequence analyses revealed that the N-terminal sequence of 43 kDa product digested by MLCE was identical to that of site E cleaved by ZHE1 (Fig. 6). However, the site in the 39 kDa product was not identical to the ZHE1-cleaving site of the 39 kDa product (site A), and the cleaving site was shifted one amino acid resi- due to the N-terminal side from site A. Therefore, the specificity of MLCE toward zebrafish egg envelope is similar to, but somewhat different from, that of ZHE1. By contrast, the medaka unfertilized egg envelope was digested by ZHE1. The SDS ⁄ PAGE pattern was similar to that by MHCE (Fig. 8B, lanes 10 and 11). The N-terminal sequences of the 47 and 35 kDa prod- ucts in ZHE1 digest matched with those of MHCE digests, suggesting that ZHE1-cleaving specificity toward medaka unfertilized egg envelope is similar to that of MHCE, and not MLCE (Fig. 8B, lane 12). Comparison of specific activities of ZHE1, MHCE and MLCE judged by synthetic peptide substrates Cleaving efficiencies of ZHE1, MHCE and MLCE were quantitatively estimated using synthetic peptide substrates (Fig. 9). For the zebrafish egg envelope, the peptides designed from sites A, B and E (Fig. 6) were employed. As mentioned earlier, the best substrate for ZHE1 was a site A peptide and the second best was a site E peptide, and the specific activity toward the site B peptide is lower than one tenth of that toward the site A peptide. In respective peptide substrates, the values of the specific activity of MHCE were similar to those of ZHE1. By contrast, the specific activity of MLCE was much lower than those of ZHE1 and MHCE. As was true in the egg envelope digestion experiment, the cleaving sites of site A peptides of MLCE did not coincide with those of ZHE1 and MHCE. However, the ratios of the specific activities of MLCE toward the three substrates were similar to that of ZHE1 and MHCE. In summary, the substrate specificity of MHCE toward peptides for zebrafish egg envelopes is quite similar to that of ZHE1, whereas that of MLCE is similar to a certain extent. The medaka egg envelope is known to consist of the subunits proteins having a ZP domain (i.e. ZI-1,2 and ZI-3) that are homologous to zebrafish ZP2 and ZP3, respectively [25,26]. One of the obvious differences of the subunit protein between medaka and zebrafish is AB Fig. 8. Cross-species digestion using hatching enzyme and the egg envelope of zebrafish and medaka. (A) Zebrafish unfertilized egg enve- lopes (lane 1) were incubated with either ZHE1 (lane 2), MHCE (lanes 3–5) or MLCE (lanes 6–8). After incubation for 10 min (lanes 2, 3 and 6), 40 min (lanes 4 and 7) and 120 min (lanes 5 and 8), each digest was separated by SDS ⁄ PAGE. (B) Medaka unfertilized egg envelopes (lane 9) were incubated with either ZHE1 (lane 10), MHCE (lane 11) or MLCE (lane 12) for 15 min. Each digest was separated by SDS ⁄ PAGE. Numbers on the left are the sizes (kDa) of molecular markers, and those on the right are the molecular masses for major bands. K. Sano et al. Hatching enzyme of zebrafish FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5941 that ZI1,2 possesses Pro-X-Y repeat sequences in its N-terminal region, which are not found in that of zebrafish ZP2. MHCE and MLCE are known to have different cleaving specificity toward the medaka egg envelope. MHCE mainly cleaves Pro-X-Y repeat sequences present in the N-terminal region of ZI-1,2 [7]. By contrast, the most efficient cleaving site of MLCE is in the center of the ZP domain of ZI-1,2. Therefore, two peptide substrates were designed from the amino acid sequences of Pro-X-Y repeat region, named Pro-X-Y-1 and Pro-X-Y-2, for MHCE, and another peptide substrate was designed from the amino acid sequence of ZP domain, named ZP domain (ZPD)-center for MLCE. As we expected, MHCE effi- ciently cleaved Pro-X-Y-1 and Pro-X-Y-2, whereas MLCE efficiently cleaved ZPD center (Fig. 9). In addi- tion, ZHE1 well cleaved Pro-X-Y-1 and Pro-X-Y-2, the sites for MHCE, and their specific activity values were approximately 80% of those of MHCE, whereas ZHE1 did not easily cleave the ZPD-center, the site for MLCE. The results suggest that ZHE1 has the MHCE-like activity toward medaka egg envelope; this was consistent with the results obtained in the diges- tion experiment using unfertilized egg envelopes. It is interesting to note that ZHE1 cleaves Pro-X-Y sequences that are not present in the subunit proteins of zebrafish egg envelope. Around the cleaving sites of ZHE1 and MHCE, we were unable to find a common or consensus amino acid sequence between zebrafish and medaka. This is sup- ported by the finding that ZHE1 has broad substrate specificity, as judged by the MCA substrate experiment. Discussion Gene expression analyses revealed that ZHE1, one of two zebrafish hatching enzyme genes, was mainly expressed, whereas ZHE2 was scarcely expressed. This was supported by the result that only a single enzyme, ZHE1, was purified from hatching liquid. In addition, the fine morphology of fertilized egg envelope digested by rec. ZHE1 was similar to that after natural hatch- ing. Thus, only one enzyme, ZHE1, is suggested to be essential for hatching of zebrafish embryo, and ZHE2 does not contribute to the hatching. We have suggested that the ZHE1 and ZHE2 genes were produced by gene duplication and subsequent diversification during the evolutionary process to zebrafish [16]. The whole mount in situ hybridization revealed that ZHE2 transcript was expressed specifi- cally, but weakly, in the hatching gland cells. At an earlier period of evolution, ZHE2 is inferred to have worked as a hatching enzyme and to have lost its abil- ity of egg envelope digestion during its further evolu- tionary process, namely a mutation(s) in the amino acid sequence of ZHE2 changed its substrate specificity as a proteolytic enzyme and, eventually, ZHE2 become uninvolved in the egg envelope digestion. Subse- quently, the amount of its expression would be decreased by accumulation of a mutation(s) in a regu- latory region(s) responsible for gene expression. 100 50 0 100 50 0 0 25 50 0 25 50 0 25 50 10 5 0 Site-A Site-B n mole –1 ·30 min –1 ·µg –1 enzymen mole –1 ·30 min –1 ·µg –1 enzyme n mole –1 ·30 min –1 ·µg –1 enzyme n mole –1 ·30 min –1 ·µg –1 enzymen mole –1 ·30 min –1 ·µg –1 enzyme n mole –1 ·30 min –1 ·µg –1 enzyme Site-E Pro XY-1 Pro XY-2 ZPD -Center Pro XY-1 Pro XY-2 ZPD -Center Pro XY-1 Pro XY-2 ZPD -Center Site-A Site-B Site-E Site-A Site-B Site-E ZHE 1 MHCE MLCE Fig. 9. Specific activity of ZHE1, MHCE and MLCE examined by synthetic peptide substrates. Names of the synthetic peptides are indicated at the bottom of the figures. Sites A, B and C indicate the ZHE1-cleaving sites on the zebrafish egg envelope. Pro XY-1 and Pro XY-2 indicate MHCE-cleaving sites, whereas ZPD-center is the MLCE-cleaving site on the medaka egg envelope. Hatching enzyme of zebrafish K. Sano et al. 5942 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS According to molecular phylogenetic analysis of fish hatching enzyme genes, hatching enzyme originally consisted of a single type of enzyme, and HCE and LCE were produced by duplication and diversification of the gene [16]. As comparing the egg envelope diges- tion mechanism between zebrafish and medaka, we will discuss the evolution of hatching enzyme function. In medaka egg envelope digestion, it has been reported that MHCE mainly cleaves Pro-X-Y repeat sequences located at the N-terminal region of ZI1,2 and releases small peptides containing most of the e-(c -glutamyl) lysine isopeptide cross-links [7]. The present study revealed that ZHE1 also cleaved the N-terminal regions of egg envelope subunits where most of cross-links are located, and swelled the egg envelope. Therefore, the manner of egg envelope diges- tion is analogous between ZHE1 and MHCE. The cross-species digestion experiments and the experiments using synthetic peptide substrates revealed that ZHE1 and MHCE cleaved the same sites on both zebrafish and medaka egg envelopes with a similar effi- ciency. ZHE1 swelled the medaka egg envelope but did not solublize its swollen envelope (data not shown). Such an agreement is surprising when it is considered that zebrafish and medaka diverged 140 million years ago [27]. From an evolutionary aspect on egg envelope digestion, ZHE1 and MHCE are presumed to maintain the substrate specificity of a common ancestral hatching enzyme. By contrast, the cleaving specificity of MLCE toward the zebrafish egg envelope was similar to those of ZHE1 and MHCE, but its cleaving efficiency was approximately ten-fold lower. Furthermore, MLCE had another efficient cleaving site, the center of ZP domain, where ZHE1 and MHCE hardly cleave. These results imply that MLCE changed its substrate specificity to one different from that of an ancestral enzyme, although its substrate specificity still remains to a certain extent. Therefore, we consider that the single enzyme-depen- dent swelling of the egg envelope in zebrafish is closely related to an original or ancestral form of egg envelope digestion, and the HCE-LCE system comprises a more developed form. HCE and ZHE1 would inherit the character of the ancestral enzyme with respect to the swelling of egg envelope. After gene duplication and diversification, LCE would be produced by changing its substrate specificity and would acquire a new function, the digestion of the swollen egg envelope. On comparing amino acid sequences between zebra- fish and medaka egg envelope subunits, we see that the identity of the ZP domains was approximately 60% (ZP3 ⁄ ZI3 = 55%; ZP2 ⁄ ZI1,2 = 65%); however, there was no similarity in their N-terminal regions in which the cleaving sites for ZHE1 or MHCE are located. Hatching enzyme recognition sites on the egg envelope are suggested to have changed with a relatively higher substitution rate during evolution. By contrast, one of the present studies using MCA substrates showed that ZHE1 had broad substrate specificity. MHCE also had broad substrate specificity [4]. In addition, some stud- ies report that astacin and meprin A, members of the same astacin family as hatching enzyme, have broad substrate specificity [28,29], suggesting that the sub- strate specificity of proteases belonging to this family is not so strict. Therefore, due to such a character common to the astacin family proteases, fish hatching enzymes could flexibly adapt the changes in amino acid residues around the cleaving sites on the N-termi- nal regions that had a relatively higher substitution rate, and the manner of egg envelope digestion was conserved between ZHE1 and MHCE. During evolution, mutations would be independently generated in the genes of egg envelope and hatching enzyme. Some mutations of the two genes would be selected and accumulated under a common pressure with respect to egg envelope digestion. Such evolution of an enzyme and substrate is one typical of the phe- nomena called ‘molecular co-evolution’. Therefore, the cleaving site recognition of both enzymes would be established under a rule that makes it possible to co-evolve hatching enzyme and egg envelope subunit protein. To understand such a rule, it is necessary to obtain more information from other fish species, such as the Japanese eel belonging to Elopomorpha that is sister to the common ancestor of zebrafish and medaka. The present study is the first approach aiming to fully understand the molecular mechanism of co-evolution of hatching enzyme and egg envelope. A further study is now in progress. Experimental procedures Fish Wild-type embryos of the Ab strain of zebrafish were used. Embryos were obtained from natural mating and cultured in tap water at 30 °C. After 42 h of culture, the embryos were transferred into a beaker with a small amount of med- ium containing 10 mm NaCl and 2 mm NaHCO 3 and allowed to hatch. When 80–90% of the embryo hatched out, the culture medium, now termed hatching liquid, was filtered, frozen and stored at )20 °C. Northern blot analysis Ten microgram of total RNA extracted from embryos at 11.5 or 24 h, or after hatching, were electrophoresed on a K. Sano et al. Hatching enzyme of zebrafish FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5943 [...]... Structure and developmental expression of hatching enzyme genes of the Japanese eel Anguilla japonica: an aspect of the evolution of fish hatching enzyme gene Dev Genes Evol 214, 17 6–1 84 16 Kawaguchi M, Yasumasu S, Hiroi J, Naruse K, Suzuki T & Iuchi I (2007) Analysis of the exon-intron 5946 17 18 19 20 21 22 23 24 25 26 27 28 29 30 structures of fish, amphibian, bird and mammalian hatching enzyme genes,... Chang YS, Wang YW & Huang FL (2002) Cross-linking of ZP2 and ZP3 by transglutaminase is required for the formation of the outer layer of fertilization envelope of carp egg Mol Reprod Dev 63, 23 7–2 44 Wang H & Gong Z (1999) Characterization of two zebrafish cDNA clones encoding egg envelope proteins ZP2 and ZP3 Biochim Biophys Acta 1446, 15 6–1 60 Mold DE, Kim IF, Tsai CM, Lee D, Chang CY & Huang RC (2001)... partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes J Biochem 105, 20 4–2 11 FEBS Journal 275 (2008) 593 4–5 946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5945 Hatching enzyme of zebrafish K Sano et al 5 Yasumasu S, Iuchi I & Yamagami K (1989b) Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching. .. renaturation and affinity purification of the zinc endopeptidase astacin Biochem J 344, 85 1–8 57 Yamagami K, Hamazaki TS, Yasumasu S, Masuda K & Iuchi I (1992) Molecular and cellular basis of formation, hardening and breakdown of the egg envelope in fish Int Rev Cytol 136, 5 1–9 2 Sugiyama H & Iuchi I (2000) Molecular structure and hardening of egg envelope in fish Recent Res Dev Comp Biochem Physiol 1, 13 9–1 61 Chang... substrate, the hardened chorion FEBS Lett 39, 28 1–2 89 8 Yasumasu S, Iuchi I & Yamagami K (1994) cDNAs and the genes of HCE and LCE, two constituents of the medaka hatching enzyme Dev Growth Differ 36, 24 1–2 50 9 Kawaguchi M, Yasumasu S, Shimizu A, Hiroi J, Yoshizaki N, Nagata K, Tanokura M & Iuchi I (2005) Purification and gene cloning of Fundulus heteroclitus hatching enzyme: a hatching enzyme system... (hatching enzyme) of the teleost, Oryzias latipes Dev Biol 29, 34 3–3 48 2 Shoots AFM & Denuc_ JM (1981) Purification and e characterization of hatching enzyme of pike, Esox lucius Int J Biochem 13, 59 1–6 02 3 Yasumasu S, Iuchi I & Yamagami K (1988) Medaka hatching enzyme consists of two kinds of proteases which act cooperatively Zool Sci 5, 19 1–1 95 4 Yasumasu S, Iuchi I & Yamagami K (1989a) Purification and. .. Approximately 10 envelopes were incubated in 50 mm Tris–HCl (pH 7.5) containing 0.4 lg of rec ZHE1 at 30 °C and subjected to SDS ⁄ PAGE Hatching enzyme of zebrafish was incubated for 1 h at 30 °C The reaction was stopped by adding 250 lL of 20% perchloric acid, and the material was allowed to stand in ice for 10 min The mixture was centrifuged at 18 500 g for 5 min at 4 °C A280 of the supernatant was measured... residues were used in the analysis The synthetic sequences were designed from ZHE1-, MHCE- and MLCE-cleaving sites that were determined from the egg envelope digests A 100 lL reaction mixture was made comprising 100 nm of the peptide and an appropriate amount of enzyme in 50 mm Tris–HCl (pH 7.5) After incubation at 30 °C for 30 min, the reaction was stopped by addition of 10 lL of 0.1 m EDTA Such final... Japan) on the HPLC system equilibrated with 0.1% trifluoroacetic acid and eluted with a linear gradient of 0–3 6% MeCN in 0.1% trifluoroacetic acid The activity was calculated from the ratio of peaks areas of digested and undigested peptides The cleaving sites of peptides were determined by amino acid sequencing Determination of N-terminal amino acid sequences Egg envelopes were analyzed by SDS ⁄ PAGE and. .. comprising 3.3 mgÆmL)1 of casein and enzyme in a 50 mm Tris–HCl (pH 7.5) The mixture SDS ⁄ PAGE was performed by the method of Laemmli using a 12.5% gel [30] The gel was stained with Coomassie Brilliant Blue G or using a Silver Stain Kit (Wako, Osaka, Japan) Acknowledgements We express our cordial thanks to Professor F S Howell (Department of Materials and Life Sciences, Faculty of Science and Technology, . Purification and characterization of zebrafish hatching enzyme – an evolutionary aspect of the mechanism of egg envelope digestion Kaori Sano 1 ,. 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