tified in various fish lineages including cartilaginous fishes, ray-finned fishes, and lungfish. 20142041 However, a comparative study of the KKS components among fishes and other vertebrates has not been conducted. Instead, our knowledge of the fish systems has been largely extrapolated from mammalian studies. Moreover, since the discovery of KKS in fishes, the endogenous BK types and levels have not been determined due to the lack of a 1 KKS Cascade in Teleosts doi: 10.1371/journal.pone.0081057.g001 reliable immunoassay. Recently, evidence from genome data suggests that the components of KKS in fishes are different from mammals. These differences included the simple gene structure of kng1 in fish and lamprey, and the absence of an orthologous KLK group in the fish lineage. Doolittle suggested that the contact activation system for Hageman factors in blood coagulation as well as plasma kallikrein are absent in fishes, and the enzyme responsible for the formation of 22576162 BK is not known. Because of the putative difference among the composition of KKS in mammals and fishes, we aim to define systemically the components of KKS in teleosts in the present study. Through biochemical methods, molecular cloning, and genome data-mining, we demonstrated that some of the well-known mammalian KKS components are missing in the teleost lineages. Furthermore, the discovery of the coevolution of KLKB1 and HMW KNG in a lobe-finned fish has shed light on the evolutionary history and origin of plasma KKS in tetrapods. Materials and Methods Animal husbandry and sampling Juvenile, sexually immature Japanese eel, tilapia, rainbow trout, and adult medaka of both sex were kept 
in a freshwater recirculating aquarium system in the Atmosphere and Ocean Research Institute, the University of Tokyo. The water was maintained at 12 C for trout, 18 C for eel, 25 C for tilapia, and 27 C for medaka, and all AMI-1 cost animals were exposed to a 14/10 h light/dark cycle throughout the experiment. All animal experimental procedures were approved by the Animal Experiment Committee of the University of Tokyo. Fish were anesthetized by 0.1% ethyl 3-aminobenzoate methanesulfonate neutralized with sodium bicarbonate. Blood samples were obtained by caudal puncture into syringes containing an inhibitor cocktail to prevent clotting and peptide degradation. The v/v ratio of inhibitor cocktails to blood was kept at 0.03 to 1. Blood samples were centrifuged immediately after collection at 10,000 rpm for 5 min at 4 C and the plasma fraction was obtained and stored at -30 C until further use. A pooled plasma sample from 10 eel individuals was prepared for validating the immunoassay. Kininogen cloning in eel Initially, partial sequence of kng1 in eel was obtained from the draft genome of Anguilla japonica. The full- 2 KKS Cascade in Teleosts length cDNA of eel KNG was obtained using the SMART cDNA Library Construction kit according to the manufacturer’s protocol. All sequencing procedures were performed using a BigDye Terminator Cycle sequencing kit and an ABI 3130 DNA sequencer. Nglycosylation and O-glycosylation sites were predicted using the CBS prediction server. Distribution of kininogen mRNA in various tissue Eel was terminally anesthetized as mentioned previously and various tissues including brain, pituitary, gill, atrium, ventricle, liver, kidney, esophagus, stomach, anterior intestine, posterior intestine, spleen, rete mirabilis, and interrenal were dissected out, snap frozen in liquid nitrogen and sto
