caa a22 4500 397556039 CHVBK 20180308164802.0 cr unu---uuuuu 161202e199601 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021220 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021220 A Cysteine Protease Inhibitor Stored in the Large Granules of Horseshoe Crab Hemocytes: Purification, Characterization, cDNA Cloning and Tissue Localization [Elektronische Daten] [Kishan Lai Agarwala, Shun-ichiro Kawabata, Michimasa Hirata, Masaru Miyagi, Susumu Tsunasawa, Sadaaki Iwanaga] 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 (K1) of 0.08 nM. It also inhibits cathepsin L (K1 =0.17 nM) and ficin (K1, =0.52 nM), 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. © by the Journal of Biochemistry Regular Papers nationallicence antimicrobial activity nationallicence cystatin nationallicence cysteine protease nationallicence horseshoe crab nationallicence ion-spray mass spectrometry nationallicence Agarwala Kishan Lai Department of Molecular Biology, Graduate School of Medical Science Fukuoka 812-81 aut Kawabata Shun-ichiro Department of Molecular Biology, Graduate School of Medical Science Fukuoka 812-81 aut Hirata Michimasa Department of Bacteriology, School of Medicine, Iwate Medical University Morioka, Iwate 020 aut Miyagi Masaru Biotechnology Research Laboratories, Takara Shuzo Co., Ltd. Otsu, Shiga 520-21 aut Tsunasawa Susumu Biotechnology Research Laboratories, Takara Shuzo Co., Ltd. Otsu, Shiga 520-21 aut Iwanaga Sadaaki Department of Molecular Biology, Graduate School of Medical Science Fukuoka 812-81 aut The Journal of Biochemistry Oxford University Press 119/1(1996-01), 85-94 0021-924X 119:1<85 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021220 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021220 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Agarwala Kishan Lai Department of Molecular Biology, Graduate School of Medical Science Fukuoka 812-81 aut NATIONALLICENCE 700 1- Kawabata Shun-ichiro Department of Molecular Biology, Graduate School of Medical Science Fukuoka 812-81 aut NATIONALLICENCE 700 1- Hirata Michimasa Department of Bacteriology, School of Medicine, Iwate Medical University Morioka, Iwate 020 aut NATIONALLICENCE 700 1- Miyagi Masaru Biotechnology Research Laboratories, Takara Shuzo Co., Ltd. Otsu, Shiga 520-21 aut NATIONALLICENCE 700 1- Tsunasawa Susumu Biotechnology Research Laboratories, Takara Shuzo Co., Ltd. Otsu, Shiga 520-21 aut NATIONALLICENCE 700 1- Iwanaga Sadaaki Department of Molecular Biology, Graduate School of Medical Science Fukuoka 812-81 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/1(1996-01), 85-94 0021-924X 119:1<85 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford