3 years ago

Biochemical characterization of a medaka (Oryzias latipes) orthologue for mammalian Factor XIII and establishment of a gene-edited mutant

Biochemical characterization of a medaka (Oryzias latipes) orthologue for mammalian Factor XIII and establishment of a gene-edited mutant
Yuko Watanabe, Ryota Ogawa, Masato Kinoshita, Kiyotaka Hitomi, Rima Horimizu, Hisashi Hashimoto, Hideki Tatsukawa
In the final process of blood coagulation, fibrin molecules are stabilized via a catalytic reaction by Factor XIIIA (FXIIIA), a member of the transglutaminase (TGase) family that catalyzes protein cross-linking reactions. In this study, we characterized the orthologue of this enzyme in medaka (Oryzias latipes), an established model fish in which a coagulation system is also preserved. The recombinant protein of this orthologue enzyme was produced in baculovirus-infected insect cells and used for analysis of its biochemical properties including activation by thrombin proteolysis and calcium dependence of the TGase enzymatic activity. Immunostaining and immunoblotting revealed that medaka FXIIIA is expressed in the kidney, bone, and esophagus in addition to blood cells. Furthermore, a gene-mutant fish was established using the CRISPR/Cas9 system. The loss of FXIIIA expression was validated in the mutants, and phenotypes, such as absence of fibrin cross-linking, were investigated in the established mutant fish. Factor XIII is a transglutaminase that catalyzes fibrin cross-linking. The orthologue of this enzyme was characterized in medaka (Oryzias latipes), an established model fish. The recombinant protein of this orthologue enzyme was produced for analysis of its biochemical properties. Immunochemical analysis revealed its tissue distribution, which includes bone. Furthermore, a gene-mutant fish was established.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1111/febs.14153

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