Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai.
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.
['Heo|Yunwi|Y|', 'Kwon|Young Chul|YC|', 'Bae|Seong Kyeong|SK|', 'Hwang|Duhyeon|D|', 'Yang|Hye Ryeon|HR|', 'Choudhary|Indu|I|', 'Lee|Hyunkyoung|H|', 'Yum|Seungshic|S|', 'Shin|Kyoungsoon|K|', 'Yoon|Won Duk|WD|', 'Kang|Changkeun|C|', 'Kim|Euikyung|E|']
D000595:Amino Acid Sequence D000818:Animals D001483:Base Sequence D001665:Binding Sites D020134:Catalytic Domain D053818:Chymases D002918:Chymotrypsin D003001:Cloning, Molecular D018076:DNA, Complementary D019143:Evolution, Molecular D007700:Kinetics D010664:Phenylmethylsulfonyl Fluoride D010802:Phylogeny D011485:Protein Binding D037401:Scyphozoa D015842:Serine Proteinase Inhibitors D013329:Structure-Activity Relationship D013379:Substrate Specificity
cloning a chymotrypsin-like 1 (CTRL-1) protease Nemopilema nomurai amidolytic kinetic assay full-length cDNA sequence genomic DNA sequence