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

Kawaguchi, Mari; Yasumasu, Shigeki; Shimizu, Akio; Kudo, Norio; Sano, Kôkichi; Iuchi, Ichiro; Nishida, Mutsumi

doi:10.1242/jeb.069716

Cited by 17 publications

(25 citation statements)

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“…This suggests that even though there is homology between the hatching enzymes in different aquatic embryos, differences in media (i.e. freshwater versus seawater) play an important role in metal ion toxicity (Kawaguchi et al, 2013;Nomura et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Developmental effects of two different copper oxide nanomaterials in sea urchin (Lytechinus pictus) embryos

et al. 2015

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Copper oxide nanomaterials (nano-CuOs) are widely used and can be inadvertently introduced into estuarine and marine environments. We analyzed the effects of different nano-CuOs (a synthesized and a less-pure commercial form), as well as ionic copper (CuSO 4 ) on embryo development in the white sea urchin, a well-known marine model. After 96 h of development with both nano-CuO exposures, we did not detect significant oxidative damage to proteins but did detect decreases in total antioxidant capacity. We show that the physicochemical characteristics of the two nano-CuOs play an essential role in their toxicities. Both nano-CuOs were internalized by embryos and their differential dissolution was the most important toxicological parameter. The synthesized nano-CuO showed greater toxicity (EC 50 ¼ 450 ppb of copper) and had increased dissolution (2.5% by weight over 96 h) as compared with the lesspure commercial nano-CuO (EC 50 ¼ 5395 ppb of copper, 0.73% dissolution by weight over 96 h). Copper caused specific developmental abnormalities in sea urchin embryos including disruption of the aboral-oral axis as a result in changes to the redox environment caused by dissolution of internalized nano-CuO. Abnormal skeleton formation also occurred.

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Section: Discussionmentioning

confidence: 99%

Developmental effects of two different copper oxide nanomaterials in sea urchin (Lytechinus pictus) embryos

et al. 2015

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“…The egg envelope digestion activity of HCE was determined by turbidimetry, using fragments of fertilized egg envelope as substrate [27]. The specific activity was expressed as ΔT 610 · μg protein -1 .…”

Section: Methodsmentioning

confidence: 99%

“…The specific activity was expressed as ΔT 610 · μg protein -1 . For LCE activity, the amounts of peptides liberated from the swollen envelope were measured [27], and the specific activity was expressed as ΔA 595 · μg protein -1 .…”

Section: Methodsmentioning

confidence: 99%

“…Mutant FLCE or MLCE plasmids were constructed by PCR using mutagenic primers and codon-optimized FLCE or MLCE plasmids as a template. The recombinant proteins were synthesized using a protocol described previously [27]. Because the refolding efficiencies of mutant recombinant proteins differed from sample to sample, the amount of active enzyme in the sample was estimated by CA.…”

Section: Methodsmentioning

confidence: 99%

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Molecular co-evolution of a protease and its substrate elucidated by analysis of the activity of predicted ancestral hatching enzyme

et al. 2013

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BackgroundHatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo. We have comprehensively studied the molecular mechanisms of the enzyme action to its substrate egg envelope, and determined the gene/protein structure and phylogenetic relationships. Because the hatching enzyme must have evolved while maintaining its ability to digest the egg envelope, the hatching enzyme-egg envelope protein pair is a good model for studying molecular co-evolution of a protease and its substrate.ResultsHatching enzymes from medaka (Oryzias latipes) and killifish (Fundulus heteroclitus) showed species-specific egg envelope digestion. We found that by introducing four medaka-type residue amino acid substitutions into recombinant killifish hatching enzyme, the mutant killifish hatching enzyme could digest medaka egg envelope. Further, we studied the participation of the cleavage site of the substrate in the species-specificity of hatching enzyme. A P2-site single amino acid substitution was responsible for the species-specificity. Estimation of the activity of the predicted ancestral enzymes towards various types of cleavage sites along with prediction of the evolutionary timing of substitutions allowed prediction of a possible evolutionary pathway, as follows: ancestral hatching enzyme, which had relatively strict substrate specificity, developed broader specificity as a result of four amino acid substitutions in the active site cleft of the enzyme. Subsequently, a single substitution occurred within the cleavage site of the substrate, and the recent feature of species-specificity was established in the hatching enzyme-egg envelope system.ConclusionsThe present study clearly provides an ideal model for protease-substrate co-evolution. The evolutionary process giving rise to species-specific egg envelope digestion of hatching enzyme was initiated by amino acid substitutions in the enzyme, resulting in altered substrate specificity, which later allowed an amino acid substitution in the substrate.

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“…Moreover, the egg envelope (chorion o r zona radiata) proteins are members of a unique group of structural proteins (Hyllner, Westerlund, Olsson, & Schopen, 2001;Arukwe & Goksøyr, 2003), which can be decomposed during the last stage of hatching by the hatching protease (chorionase) (Yamagami, 1973;Hagenmaier, 1974a,b;Luczynski, Strzczek, & Brzu zan, 1987) o r chorio lysins (Kawaguchi et al, 2013). Therefore, the phenomenon that egg chorion remains on the head of larvae can probably be related to the impaired act ivity of chorionase on egg envelope proteins and the above mentioned embryotoxicity.…”

Section: +mentioning

confidence: 99%

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Ljubobratović¹,

Csengeri²,

Kucska³

et al. 2017

Turk. J. Fish. Aquat. Sci.

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The methods for removal of adhesiveness in pikeperch eggs were compared in this study . Following the hormonal induction with carp pituitary, eggs of three females were used in each of two trials. Firstly, three common methods were tested: tannic acid, enzyme and milk. Tannic acid treatment led to poor hatching (2.5±0.5%), significantly lower than the other treatments (75.5±19.3% and 72.3±28.8%, enzyme and milk, respectively). In order to find the source of negative outcome, in the second trial the influence of water hardness on the tannic acid bath was tested through three treatments with varying concentrations of disodium ethylenediaminetetraacetic acid salt (NA 2 EDTA). Softening of the tannic acid bath did not lead to improved hatching rates (5.5±1.3%, 3.5±1.5% and 2.7±1.3% for 0, 400 and 800 ppm of EDTA, respectively). Evaluation of the effects of tannin and EDTA on water chemistry revealed that the use of tannin can alter the embryonic development and hatching process. While de-adhesion procedure with milk appeared to be impractical, the application of tannic acid bath leads to variable and lower hatching outcomes. Thus, usage of enzymes was found to be the most promising method which should be further described for straightforward implementation in practice.

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

Abstract: SUMMARY Embryos of medakaSupplementary material available online at

Cited by 17 publications

References 23 publications

Developmental effects of two different copper oxide nanomaterials in sea urchin (Lytechinus pictus) embryos

Developmental effects of two different copper oxide nanomaterials in sea urchin (Lytechinus pictus) embryos

Molecular co-evolution of a protease and its substrate elucidated by analysis of the activity of predicted ancestral hatching enzyme

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