Abstract
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.
Highlights
Hatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo
The inner layers are composed of two groups of subunit proteins called ZI-1,2 and ZI-3 [5], which are classified into vertebrate-common egg envelope protein groups ZPB and ZPC, respectively [6,7]
Medaka low choriolytic enzyme (LCE) (MLCE) sharply increased solubilization of the swollen medaka egg envelope with incubation time, whereas MLCE only slightly solubilized that of Fundulus (Figure 1C)
Summary
Hatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo. 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. The hatching enzyme of teleost species is secreted by the embryo to digest the egg envelope that surrounds. Both egg envelope protein and hatching enzyme have been studied in the model fish medaka (Oryzias latipes). ZI-3 is a homogenous glycoprotein derived from the precursor choriogenin L [8,9,10] Both ZI-1,2 and ZI-3 have a zona pellucida (ZP) domain consisting of about 260 amino acids, with eight conserved cysteine residues [11]. Additional characteristics of ZI-1,2 include a Pro-Xaa-Yaa repeat region and a trefoil domain upstream of the ZP domain
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