Generally, animals extract nutrients from food by degradation using digestive enzymes. Trypsin and chymotrypsin, one of the major digestive enzymes in vertebrates, are pancreatic proenzymes secreted into the intestines. In this investigation, we report the identification of a digestive teleost enzyme, a pancreatic astacin that we termed pactacin. Pactacin, which belongs to the astacin metalloprotease family, emerged during the evolution of teleosts through gene duplication of astacin family enzymes containing six cysteine residues (C6astacin, or C6AST). In this study, we first cloned C6AST genes from pot-bellied seahorse (Hippocampus abdominalis) and analyzed their phylogenetic relationships using over 100 C6AST genes. Nearly all these genes belong to one of three clades: pactacin, nephrosin, and patristacin. Genes of the pactacin clade were further divided into three subclades. To compare the localization and functions of the three pactacin subclades, we studied pactacin enzymes in pot-bellied seahorse and medaka (Oryzias latipes). In situ hybridization revealed that genes of all three subclades were commonly expressed in the pancreas. Western blot analysis indicated storage of pactacin pro-enzyme form in the pancreas, and conversion to the active forms in the intestine. Finally, we partially purified the pactacin from digestive fluid, and found that pactacin is novel digestive enzyme that is specific in teleosts.

During the evolution of astacin metalloprotease family genes, gene duplication occurred, especially in the lineage of teleosts, in which several types of astacins containing six conserved cysteines (c6ast) emerged. One of them is patristacin, originally found in syngnathid fishes, such as pipefishes and seahorses. Patristacin is expressed in the brood pouch and is present on the same chromosome as other c6ast (pactacin and nephrosin) genes. We first surveyed all the genes from 33 teleost species using a genome database, and characterized the genes by phylogenetic analysis. Pactacin and nephrosin gene homologs were found from all the examined species with only few exceptions, while patristacin gene homologs were found from only several lineages. The patristacin gene homologs were found as multicopy genes in most species of Percomorpha, one of the diverged groups in teleosts. Further diversification of the gene occurred during the evolution of Atherinomorphae, one of the groups in Percomorpha. Fishes of Atherinomorphae possess two types of patristacin, belonging to subclades 1 and 2. Among the Atherinomorpha, we chose the southern platyfish to examine the patristacin gene expression. Platyfish possess eight patristacin gene homologs, called XmPastn1, 2, 3, 4, 5, 7, 10, and 11. Of these genes, only XmPastn2 belongs to subclade 1, while the other seven belong to subclade 2. Only XmPastn2 showed strong expression in several organs of adult platyfish, as observed in reverse‐transcription polymerase chain reaction of RNA extracts. Cells expressing XmPastn2 were predominantly mucus‐secreting cells found in epidermis around the jaw, as revealed by in‐situ hybridization. This result suggests that XmPastn2 is secreted and may contribute to mucus formation or secretion.