Ahn Sang Jung, Kim Na Young, Seo Jung Soo, Je Ju Eun, Sung Ji Hea, Lee Sang Hwan, Kim Moo-Sang, Kim Joong Kyun, Chung Joon Ki, Lee Hyung Ho
Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
Comp Biochem Physiol B Biochem Mol Biol. 2009 Oct;154(2):211-20. doi: 10.1016/j.cbpb.2009.06.005. Epub 2009 Jun 21.
Cathepsin F is a recently described papain-like cysteine protease of unknown function, and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. In the present study, the cDNA of olive flounder (Paralichthys olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The PoCtF gene was determined to consist of the 1844 bp nucleotide sequence which encodes for a 475-amino acid polypeptide. The results of RT-PCR analysis revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however the PoCtF expressions increased significantly in gill at 3h post-injection with lipopolysaccharide (LPS). Also, immunostaining using anti-PoCtF antibody was strongest on the epidermal mucus in the fin. The cDNA encoding mature enzyme of PoCtF was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of cysteine proteinases, and the optimal pH for the protease activity was 7.5. The findings of the present study suggest that PoCtF has a higher optimum pH than mammalian cathepsin F, and PoCtF is an interesting target for future investigations of the role of cathepsin F in the epidermal mucus and fish innate immune system.
组织蛋白酶F是一种最近被描述的功能未知的木瓜蛋白酶样半胱氨酸蛋白酶，由于其N端前肽区域延长且包含一个胱抑素结构域，在组织蛋白酶中独一无二。在本研究中，通过同源分子克隆和cDNA末端快速扩增（RACE）方法相结合，克隆了牙鲆（Paralichthys olivaceus）组织蛋白酶F（PoCtF）的cDNA。PoCtF基因由1844 bp的核苷酸序列组成，该序列编码一个475个氨基酸的多肽。RT-PCR分析结果显示，在整个健康牙鲆组织中均有普遍表达；然而，在注射脂多糖（LPS）后3小时，鳃中PoCtF的表达显著增加。此外，使用抗PoCtF抗体的免疫染色在鳍的表皮黏液上最强。利用pGEX-4T-1表达载体系统在大肠杆菌中表达了编码PoCtF成熟酶的cDNA。通过切割合成肽Z-Phe-Arg-AMC（一种常用于半胱氨酸蛋白酶功能表征的底物）来定量其活性，该蛋白酶活性的最适pH为7.5。本研究结果表明，PoCtF的最适pH比哺乳动物组织蛋白酶F高，并且PoCtF是未来研究组织蛋白酶F在表皮黏液和鱼类先天免疫系统中作用的一个有趣靶点。
