Pactacin is a novel digestive enzyme in teleosts

Abstract

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.

Molecular evolution of hatching enzymes and their paralogous genes in vertebrates

“…In bony fishes, therefore, the characteristics of the novel C6ast genes—conservation of consensus sequences, the molecular phylogenetic tree, and the domain structure were consistent with the characteristics of previously identified C6ast genes [ 3 , 9 ]. Cartilaginous fishes C6asts were found to retain the CUB domain structure, unlike the other C6ast found in other vertebrates.…”

“…The gene family with this characteristic is called C6astacin (C6ast) [ 8 ]. During the evolution of teleosts, the C6ast genes have been duplicated, and they are classified into the subfamily, which contains the C6astacin4/5 (C6ast4/5), nephrosin (npsn), patristacin (pastn), pactacin (pac), and HEs [ 9 ].

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“…The functions and expression patterns of these subfamily genes have diverged. It has been suggested that in zebrafish, npsn plays a role in biodefense, in neutrophils [ 10 ], while pac is expressed in the pancreas of seahorse and medaka and secreted into the intestine via the pancreatic duct, acting as a digestive enzyme [ 9 ]. It has been suggested that pastn (also called cimp1 in cichlid fish) may have contributed to the extension of the mandibular bones [ 11 ] and, further, to the parental internal brooding of eggs in Syngnathiformes and Cyprinodontiformes because they have divergent pastn genes [ 12 – 14 ].…”

Molecular evolution of patristacin genes in teleosts based on the genome survey

“…The three major clades are well supported by high bootstrap values. The pactacin clade was further divided into two subclades, which correspond to subclade 1 and subclade2/3 in a previous study (Kawaguchi et al, 2021). The bootstrap values within the patristacin clade were low, however, and we further performed ML analysis using nucleotide sequences corresponding to mature patristacin enzymes together with selected nephrosin and pactacin genes (Figure 2).…”

Survival Rates of Oryzias Celebensis Embryo Reared in Different Media in an Attempt to Provide Embryos for Ecotoxicological Studies

“…The hatching enzymes in medaka only digest the inner layer of the chorion in which the process consists of two steps, namely choriolytic swelling caused by HCE (High choriolytic enzyme) and dissolution of the swollen structure by LCE (low choriolytic enzyme) [45], [48], [49]. Besides these two enzymes, one more enzyme that works in the hatching process has been found, namely the pactacin enzyme [50].…”