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Open Access Research article

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

Mari Kawaguchi124, Koji Inoue1, Ichiro Iuchi3, Mutsumi Nishida1 and Shigeki Yasumasu3*

Author Affiliations

1 Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan

2 Research Fellow of the Japan Society for the Promotion of Science (JSPS), Chiyoda-ku, Japan

3 Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan

4 Current address: Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan

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BMC Evolutionary Biology 2013, 13:231  doi:10.1186/1471-2148-13-231

Published: 25 October 2013



Hatching 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.


Hatching 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.


The 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.

Co-evolution; Hatching enzyme; Astacin family; Egg envelope; Chorion; ZP domain; Ancestral hatching enzyme