title: Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai.
abstract: An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. yunwi0510@naver.com.
authors.forename: Yunwi
authors.initials: Y
authors.lastname: Heo
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. skyward2@hanmail.net.
authors.forename: Young Chul
authors.initials: YC
authors.lastname: Kwon
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. hotjjang002@naver.com.
authors.forename: Seong Kyeong
authors.initials: SK
authors.lastname: Bae
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. pooh9922@hanmail.net.
authors.forename: Duhyeon
authors.initials: D
authors.lastname: Hwang
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. tkwk565@naver.com.
authors.forename: Hye Ryeon
authors.initials: HR
authors.lastname: Yang
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. induchoudhary2u@gmail.com.
authors.forename: Indu
authors.initials: I
authors.lastname: Choudhary
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. leehy@gnu.ac.kr.
authors.forename: Hyunkyoung
authors.initials: H
authors.lastname: Lee
authors.affiliation: Marine Environment Research Center, Korea Institute of Ocean Science and Technology (KIOST), Geoje 656-834, Korea. syum@kiost.ac.kr.|Faculty of Marine Environmental Science, University of Science and Technology (UST), Geoje 656-834, Korea. syum@kiost.ac.kr.
authors.forename: Seungshic
authors.initials: S
authors.lastname: Yum
authors.affiliation: Ballast Water Research Center, Korea Institute of Ocean Science and Technology (KIOST), Geoje 656-834, Korea. ksshin@kiost.ac.kr.
authors.forename: Kyoungsoon
authors.initials: K
authors.lastname: Shin
authors.affiliation: Headquarters for Marine Environment, National Fisheries Research &amp; Development Institute, Shiran-ri, Gijang-eup, Gijang-gun, Busan 619-705, Korea. wdyoon@korea.kr.
authors.forename: Won Duk
authors.initials: WD
authors.lastname: Yoon
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. ckkang@gnu.ac.kr.|Institutes of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Korea. ckkang@gnu.ac.kr.
authors.forename: Changkeun
authors.initials: C
authors.lastname: Kang
authors.affiliation: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Korea. ekim@gnu.ac.kr.|Engineering Research Institute, Gyeongsang National University, Jinju-si 660-701, Korea. ekim@gnu.ac.kr.
authors.forename: Euikyung
authors.initials: E
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D053818:Chymases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019143:Evolution, Molecular; D007700:Kinetics; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010802:Phylogeny; D011485:Protein Binding; D037401:Scyphozoa / Q000201:enzymology* / Q000235:genetics*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D015842:Serine Proteinase Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D002918:Chymotrypsin; D053818:Chymases
keywords: Nemopilema nomurai; amidolytic kinetic assay; cloning a chymotrypsin-like 1 (CTRL-1) protease; full-length cDNA sequence; genomic DNA sequence