Biochemical Characterization of Medaka (Oryzias latipes) Transglutaminases, OlTGK1 and OlTGK2, as Orthologues of Human Keratinocyte-Type Transglutaminase

RESEARCH ARTICLE Biochemical Characterization of Medaka (Oryzias latipes) Transglutaminases, OlTGK1 and OlTGK2, as Orthologues of Human Keratinocyte-Type Transglutaminase Ayaka Kikuta1☯, Eri Furukawa1☯, Ryota Ogawa1☯, Natsuki Suganuma1, Mai Saitoh1, Toshiyuki Nishimaki2, Takafumi Katsumura2, Hiroki Oota2, Tadafumi Kawamoto3, Hideki Tatsukawa1, Hisashi Hashimoto4, Kiyotaka Hitomi1* 1 Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, 464–8601 Japan, 2 Kitasato University School of Medicine, Sagamihara, 252–0734 Japan, 3 Radioisotope Research Institute, School of Dental Medicine, Tsurumi University, Tsurumi, Yokohama, 230–8501 Japan, 4 Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464–8601 Japan ☯ These authors contributed equally to this work. * OPEN ACCESS Citation: Kikuta A, Furukawa E, Ogawa R, Suganuma N, Saitoh M, Nishimaki T, et al. (2015) Biochemical Characterization of Medaka (Oryzias latipes) Transglutaminases, OlTGK1 and OlTGK2, as Orthologues of Human Keratinocyte-Type Transglutaminase. PLoS ONE 10(12): e0144194. doi:10.1371/journal.pone.0144194 Editor: Richard L. Eckert, University of Maryland School of Medicine, UNITED STATES Received: August 5, 2015 Accepted: November 13, 2015 Published: December 29, 2015 Copyright: © 2015 Kikuta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data except for DNA sequences are within the paper and its Supporting Information files. Regarding the Medaka gene sequences, we deposited (DNA Data Bank Japan) as following accession numbers: OlTGB (LC068825), OlTGT (LC068826), OlTGK1 (LC068829), OlTGK2 (LC068830), OlTGK3 (LC068831), OlTGF (LC068827), and OlTGO (LC068828). DDBJ search site: http://getentry.ddbj. nig.ac.jp/top-e.html Abstract Calcium-dependent transglutaminases (TGs) are a family of enzymes that catalyze protein cross-linking and/or attachment of primary amines in a variety of organisms. Mammalian TGs are implicated in multiple biological events such as skin formation, blood coagulation, and extracellular matrix stabilization. Medaka (Oryzias latipes) has been used as a model fish to investigate the physiological functions of mammalian proteins. By analysis of the medaka genome, we found seven TGs orthologues, some of which apparently corresponded to the mammalian TG isozymes, TG1, TG2, and Factor XIII. All orthologues had preserved amino acid residues essential for enzymatic activity in their deduced primary structures. In this study, we analyzed biochemical properties of two orthologues (OlTGK1 and OlTGK2) of mammalian epithelium-specific TG (TG1) that are significantly expressed at the transcriptional level. Using purified recombinant proteins for OlTGK1 and OlTGK2, we characterized their catalytic reactions. Furthermore, immunohistochemical analyses of fish sections revealed higher expression in the pancreas (OTGK1), intervertebral disk (OlTGK2) and pharyngeal teeth (OlTGK2) as well as in the skin epidermis. Introduction Transglutaminases (TGs) are the enzymes that catalyze formation of isopeptide-bonds between glutamine and lysine residues of their substrate proteins in a calcium-dependent manner [1– 2]. In addition to lysine residues, primary amines (e.g., polyamine) and water molecules can also react with glutamine residues, resulting in the attachment of the amine and conversion of the glutamine to a glutamic acid residue, respectively. In mammals, these enzymatic post- PLOS ONE | DOI:10.1371/journal.pone.0144194 December 29, 2015 1 / 13 Medaka Skin-Type Transglutaminases Funding: This work was supported by Grant-in-Aid for Scientific Research (B) (No. 23380200 and No. 26292912 to K.H.) from the Ministry of Education, Sports, Science and Technology (JSPS, KAKENHI, Japan), from the Japanese Science and Technology agency (to K.H.). Competing Interests: The authors have declared that no competing interests exist. Abbreviations: bio-Cd, biotin-labeled cadaverine; PBS, phosphate-buffered saline; TBS, Tris-based buffered saline. translational modifications are observed in multiple biological processes such as blood coagulation, skin formation, extracellular matrix stabilization, apoptosis, and also with non-catalytic functions [3]. In humans, these catalytic reactions are conducted in several tissues and cells by TGs family members comprising eight isozymes; Factor XIII and TG1-TG7. Because the physiological roles of TGs are diverse, their complete characterization remains incomplete. Among the tissues, skin formation is a prominent target of TG studies because the crosslinking reaction products formed in epidermal keratinocytes clearly contribute to their integrity and barrier function [4–6]. Skin-type TGs (TG1, TG3, and TG5) have been reported to be responsible for the cooperative formation of these cross-links. In our recent studies, TG6, mainly expressed in neuronal cells, also appeared to have enzymatic activity in the epidermis [7]. Furthermore, TG1 is also expressed in other epithelial tissues during development and in the matured various tissues in mice [8, 9]. Thus, studies on the physiological significance of skin-type TGs during the epidermis formation have advanced through several aspects of biochemical characterization of these TGs and through use of knockout mouse [10, 11]. However, cooperative cross-linking of their epidermal substrates and functional expression in tissues other than the skin remain still unclear for these isozymes. In recent years, small fish such as zebrafish (Danio rerio) and medaka (Oryzias latipes) have been used for several studies, including investigation of the mechanisms of basic biological phenomena, drug screening, and phenotype analysis of diseases [12–15]. These species have advantages, such as short generation time, high fertility, and low maintenance cost. In particular, the reverse-genetic approach to knock down a specific gene is more feasible in these organisms than in mammals. Characterization of the TG orthologues of zebrafish has recently been reported: Twelve TG orthologues exist in this organism and some appear to be responsible for bone formation and signal transduction [16, 17]. In this study, we targeted the medaka, as alterative model fish, to discern the expression patterns and physiological functions of skin-type TGs, particularly TG1. Because TG1 is a major skin enzyme, knockout mice die after birth due to aberrant skin formation and damages in other tissues. Although it is possible to establish a mouse with tissue-specific ablation of TG1 expression, we considered that medaka might be suitable for studies on loss of function. However, information about biochemical properties of medaka TGs and their functional roles in skin formation and/or other tissues has not been repor (...truncated)