Kaori Sano

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

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.

Mechanism of egg envelope digestion by hatching enzymes, HCE and LCE in medaka, Oryzias latipes

Hatching of medaka embryos from the fertilized egg envelope involves two enzymes, HCE and LCE. HCE swells the envelope and then LCE completely dissolves it. We determined HCE and LCE cleavage sites on the egg envelope that are primarily constructed of two groups of subunit proteins, ZI-1,2 and ZI-3. HCE and LCE cleaved different target sequences on the egg envelope proteins but shared one common cleavage site. HCE cleaved the N-terminal region of ZI-1,2 and ZI-3, mainly the Pro-Xaa-Yaa repeat sequence of ZI-1,2 into hexapeptides, but not the site within a zona pellucida (ZP) domain that is considered to be the core structure of the egg envelope. The cleavage of these N-terminal regions results in swelling and softening of the envelope. LCE cleaved the middle of the ZP domain of ZI-1,2, in addition to the upstream of the trefoil domain of ZI-1,2 and the ZP domain of ZI-3. This middle site is in the intervening sequence connecting two subdomains of the ZP domain. Cleaving this site would result in the solubilization of the swollen egg envelope by the disruption of the filamentous structure that is thought to be formed by the non-covalent polymerization of ZP domains.

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE‐swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N‐terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro‐X‐Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE‐swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three‐spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved.

Evolution of the teleostean zona pellucida gene inferred from the egg envelope protein genes of the Japanese eel, Anguilla japonica

A fish egg envelope is composed of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among vertebrate species. Euteleost fishes synthesize ZP proteins in the liver, while otocephalans synthesize them in the growing oocyte. We investigated ZP proteins of the Japanese eel, Anguilla japonica, belonging to Elopomorpha, which diverged earlier than Euteleostei and Otocephala. Five major components of the egg envelope were purified and their partial amino acid sequences were determined by sequencing. cDNA cloning revealed that the eel egg envelope was composed of four ZPC homologues and one ZPB homologue. Four of the five eel ZP (eZP) proteins possessed a transmembrane domain, which is not found in the ZP proteins of Euteleostei and Otocephala that diverged later, but is found in most other vertebrate ZP proteins. This result suggests that fish ZP proteins originally possessed a transmembrane domain and lost it during evolution. Northern blotting and RT‐PCR revealed that all of the eZP transcripts were present in the ovary, but not in the liver. Phylogenetic analyses of fish zp genes showed that ezps formed a group with other fish zp genes that are expressed in the ovary, and which are distinct from the group of genes expressed in the liver. Our results support the hypothesis that fish ZP proteins were originally synthesized in the ovary, and then the site of synthesis was switched to the liver during the evolutionary pathway to Euteleostei.

Inferring the Evolution of Teleostean zp Genes Based on Their Sites of Expression

Fish egg envelopes consist of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among chordates. Euteleosts synthesize ZP proteins in the liver, while elopomorphs synthesize them in the ovaries. In Cypriniformes, zp genes are expressed in the ovaries. We investigated the zp genes of two Otocephalan orders: Clupeiformes (Pacific herring and Japanese anchovy) and Gonorynchiformes (milkfish), which diverged earlier than Cypriniformes. cDNA cloning of zp gene homologs revealed that Pacific herring and Japanese anchovy possessed both ovary- and liver-expressed zp genes; however, the zp genes of milkfish were only expressed in the ovaries. Molecular phylogenetic analysis showed that ovary- and liver-expressed zpc genes of two the Clupeiformes formed independent clades. Based on this, we hypothesized the evolution of teleostean zp genes, focusing on the organ expressing zp gene. As in other chordates, the original site of expression of zp genes was likely the ovary. In the early stage of teleostean evolution, the ancestral zp genes acquired the ability to express in the liver. Later, one of the two expression sites became dominant. The liver-expressed zp genes are component proteins of the egg envelope in the Euteleostei. In Otocephala, Clupeiformes possess both ovary- and liver-expressed genes that presumably participate in egg envelope formation, whereas the Gonorynchiformes and Cypriniformes have primarily preserved ovary expressed zp genes.

Adaptive evolution of fish hatching enzyme: one amino acid substitution results in differential salt dependency of the enzyme

SUMMARY Embryos of medaka Oryzias latipes hatch in freshwater, while those of killifish Fundulus heteroclitus hatch in brackish water. Medaka and Fundulus possess two kinds of hatching enzymes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), which cooperatively digest their egg envelope at the time of hatching. Optimal salinity of medaka HCE was found in 0 mol l−1 NaCl, and activity decreased with increasing salt concentrations. One of the two Fundulus HCEs, FHCE1, showed the highest activity in 0 mol l−1 NaCl, and the other, FHCE2, showed the highest activity in 0.125 mol l−1 NaCl. The results suggest that the salt dependencies of HCEs are well adapted to each salinity at the time of hatching. Different from HCE, LCEs of both species maintained the activity sufficient for egg envelope digestion in various salinities. The difference in amino acid sequence between FHCE1 and FHCE2 was found at only a single site at position 36 (Gly/Arg), suggesting that this single substitution causes the different salt dependency between the two enzymes. Superimposition of FHCE1 and FHCE2 with the 3-D structure model of medaka HCE revealed that position 36 was located on the surface of HCE molecule, far from its active site cleft. The results suggest a hypothesis that position 36 influences salt-dependent activity of HCE, not with recognition of primary structure around the cleavage site, but with recognition of higher ordered structure of egg envelope protein.

Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism.

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.

Evolutionary Changes in the Developmental Origin of Hatching Gland Cells in Basal Ray-Finned Fishes

Hatching gland cells (HGCs) originate from different germ layers between frogs and teleosts, although the hatching enzyme genes are orthologous. Teleostei HGCs differentiate in the mesoendodermal cells at the anterior end of the involved hypoblast layer (known as the polster) in late gastrula embryos. Conversely, frog HGCs differentiate in the epidermal cells at the neural plate border in early neurula embryos. To infer the transition in the developmental origin of HGCs, we studied two basal ray-finned fishes, bichir (Polypterus) and sturgeon. We observed expression patterns of their hatching enzyme (HE) and that of three transcription factors that are critical for HGC differentiation: KLF17 is common to both teleosts and frogs; whereas FoxA3 and Pax3 are specific to teleosts and frogs, respectively. We then inferred the transition in the developmental origin of HGCs. In sturgeon, the KLF17, FoxA3, and HE genes were expressed during the tailbud stage in the cell mass at the anterior region of the body axis, a region corresponding to the polster in teleost embryos. In contrast, the bichir was suggested to possess both teleost- and amphibian-type HGCs, i.e. the KLF17 and FoxA3 genes were expressed in the anterior cell mass corresponding to the polster, and the KLF17, Pax3 and HE genes were expressed in dorsal epidermal layer of the head. The change in developmental origin is thought to have occurred during the evolution of basal ray-finned fish, because bichir has two HGCs, while sturgeon only has the teleost-type.

Neofunctionalization of a duplicate hatching enzyme gene during the evolution of teleost fishes

BackgroundDuplication and subsequent neofunctionalization of the teleostean hatching enzyme gene occurred in the common ancestor of Euteleostei and Otocephala, producing two genes belonging to different phylogenetic clades (clade I and II). In euteleosts, the clade I enzyme inherited the activity of the ancestral enzyme of swelling the egg envelope by cleavage of the N-terminal region of egg envelope proteins. The clade II enzyme gained two specific cleavage sites, N-ZPd and mid-ZPd but lost the ancestral activity. Thus, euteleostean clade II enzymes assumed a new function; solubilization of the egg envelope by the cooperative action with clade I enzyme. However, in Otocephala, the clade II gene was lost during evolution. Consequently, in a late group of Otocephala, only the clade I enzyme is present to swell the egg envelope. We evaluated the egg envelope digestion properties of clade I and II enzymes in Gonorynchiformes, an early diverging group of Otocephala, using milkfish, and compared their digestion with those of other fishes. Finally, we propose a hypothesis of the neofunctionalization process.ResultsThe milkfish clade II enzyme cleaved N-ZPd but not mid-ZPd, and did not cause solubilization of the egg envelope. We conclude that neofunctionalization is incomplete in the otocephalan clade II enzymes. Comparison of clade I and clade II enzyme characteristics implies that the specificity of the clade II enzymes gradually changed during evolution after the duplication event, and that a change in substrate was required for the addition of the mid-ZPd site and loss of activity at the N-terminal region.ConclusionsWe infer the process of neofunctionalization of the clade II enzyme after duplication of the gene. The ancestral clade II gene gained N-ZPd cleavage activity in the common ancestral lineage of the Euteleostei and Otocephala. Subsequently, acquisition of cleavage activity at the mid-ZPd site and loss of cleavage activity in the N-terminal region occurred during the evolution of Euteleostei, but not of Otocephala. The clade II enzyme provides an example of the development of a neofunctional gene for which the substrate, the egg envelope protein, has adapted to a gradual change in the specificity of the corresponding enzyme.

Crystallization and preliminary X-ray analysis of ZHE1, a hatching enzyme from the zebrafish Danio rerio.

The hatching enzyme of the zebrafish, ZHE1 (29.3 kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0-1.80 and 50.0-1.14 A were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0-1.14 A. The space group was assigned as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 32.9, b = 62.5, c = 87.4 A. The crystal contained one ZHE1 molecule in the asymmetric unit.