A Cysteine Protease Inhibitor Stored in the Large Granules of Horseshoe Crab Hemocytes: Purification, Characterization, cDNA Cloning and Tissue Localization.
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概要
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A cysteine protease inhibitor with an apparent Mr=12, 600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08nM. It also inhibits cathepsin L (Ki=0.17nM) and ficin (Ki=0.52nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.
- 社団法人 日本生化学会の論文
著者
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Agarwala Kishan
Department Of Molecular Biology Graduate School Of Medical Science Kyushu University
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Kawabata Shun-ichiro
Department Of Bilolgy Faculty Of Science Kyushu University
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HIRATA Michimasa
Department of bacteriology, School of Medicine, Iwate Medical University
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Iwanaga Sadaaki
Department Of Biology Faculty Of Scienc Kyushu University
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Miyagi Masaru
Biotechnology Research Laboratories Takara Shuzo Co.
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Tsunasawa Susumu
Biotechnology Research Laboratories Takara Shuzo Co. Ltd.
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