Abstract: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 electro… Show more
“…The specific caseinolytic activities of the nickel column-purified rMfHE1 and rMfHE3 were 26.8 and 14.2 Δ 280 · min −1 · mg protein −1 , respectively. These values are similar to those of other fish hatching enzymes (30.7 Δ 280 · min −1 · mg protein −1 for ZHE1, 27.3 Δ 280 · min −1 · mg protein −1 for MHCE, and 18.4 Δ 280 · min −1 · mg protein −1 for MLCE) [19]. Thus, we concluded that recombinant enzymes were of sufficient quality to use in the egg envelope digestion experiments.Figure 2
Generation of recombinant milkfish hatching enzymes.…”
Section: Resultssupporting
confidence: 84%
“…Seven hatching enzyme cDNAs with 85–95% amino acid identity were cloned from the Japanese eel Anguilla japonica (Elopomorpha) [18]. However, these isozymes were functionally the same in terms of their role in egg envelope digestion [19]. Gene diversification has occurred in the common ancestor of Otocephala and Euteleostei, such that members of these subdivisions possess two types of genes, belonging to two clades, clade I and clade II.…”
Section: Introductionmentioning
confidence: 99%
“…A single type of hatching enzyme digests the N-terminal region of ZPB and ZPC into short peptide fragments. Its cleavage leads to cancelation of the inter-molecular Glu-Lys cross-links of ZP proteins and renders the egg envelope swollen and softened [19]. …”
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.
“…The specific caseinolytic activities of the nickel column-purified rMfHE1 and rMfHE3 were 26.8 and 14.2 Δ 280 · min −1 · mg protein −1 , respectively. These values are similar to those of other fish hatching enzymes (30.7 Δ 280 · min −1 · mg protein −1 for ZHE1, 27.3 Δ 280 · min −1 · mg protein −1 for MHCE, and 18.4 Δ 280 · min −1 · mg protein −1 for MLCE) [19]. Thus, we concluded that recombinant enzymes were of sufficient quality to use in the egg envelope digestion experiments.Figure 2
Generation of recombinant milkfish hatching enzymes.…”
Section: Resultssupporting
confidence: 84%
“…Seven hatching enzyme cDNAs with 85–95% amino acid identity were cloned from the Japanese eel Anguilla japonica (Elopomorpha) [18]. However, these isozymes were functionally the same in terms of their role in egg envelope digestion [19]. Gene diversification has occurred in the common ancestor of Otocephala and Euteleostei, such that members of these subdivisions possess two types of genes, belonging to two clades, clade I and clade II.…”
Section: Introductionmentioning
confidence: 99%
“…A single type of hatching enzyme digests the N-terminal region of ZPB and ZPC into short peptide fragments. Its cleavage leads to cancelation of the inter-molecular Glu-Lys cross-links of ZP proteins and renders the egg envelope swollen and softened [19]. …”
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.
“…As a result of hardening, the fertilized egg envelope becomes insoluble in denaturing reagents, such as urea and SDS. In contrast, the unfertilized egg envelope is generally solubilized by boiling in those reagents, and the component proteins (ZP proteins) dissociate into their monomeric forms (Sano et al., , , ; Yasumasu et al., ). However, the unfertilized Pacific herring egg envelopes did not dissolve completely by boiling in SDS, suggesting that they were partially hardened in the developing oocyte before fertilization.…”
Teleost egg envelope generally consists of a thin outer layer and a thick inner layer. The inner layer of the Pacific herring egg envelope is further divided into distinct inner layers I and II. In our previous study, we cloned four zona pellucida (ZP) proteins (HgZPBa, HgZPBb, HgZPCa, and HgZPCb) from Pacific herring, two of which (HgZPBa and HgZPCa) were synthesized in the liver and two (HgZPBb and HgZPCb) in the ovary. In this study, we raised antibodies against these four proteins to identify their locations using immunohistochemistry. Our results suggest that inner layer I is constructed primarily of HgZPBa and Ca, whereas inner layer II consists primarily of HgZPBa. HgZPBb and Cb were minor components of the envelope. Therefore, the egg envelope of Pacific herring is primarily composed of liver-synthesized ZP proteins. A comparison of the thickness of the fertilized egg envelopes of 55 species suggested that egg envelopes derived from liver-synthesized ZP proteins tended to be thicker in demersal eggs than those in pelagic eggs, whereas egg envelopes derived from ovarian-synthesized ZP proteins had no such tendency. Our comparison suggests that the prehatching period of an egg with a thick egg envelope is longer than that of an egg with a thin egg envelope. We hypothesized that acquisition of liver-synthesized ZP proteins during evolution conferred the ability to develop a thick egg envelope, which allowed species with demersal eggs to adapt to mechanical stress in the prehatching environment by thickening the egg envelope, while pelagic egg envelopes have remained thin.
“…The liver is one of the largest organs in the body; therefore, it is conceivable that the liver has an advantage in synthesizing a large amount of protein. In fact, the Euteleosts, such as salmon and medaka, also have a thick and tough egg envelope (approximately 30–40 and 20 µm, respectively, Yamagami et al, ), whereas, many elopomorph and otophysian fishes, who synthesize egg envelope proteins exclusively in the ovary, generally have a thin egg envelope (zebrafish, approximately 5 µm; Japanese eel, 1–2 µm; Sano et al, ). Conversely, a possible disadvantage of synthesizing egg envelope proteins in the liver is the transportation cost from the liver to the oocytes.…”
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.
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