Geadkaew-Krenc Amornrat, Grams Rudi, Siricoon Sinee, Kosa Nanthawat, Krenc Dawid, Phadungsil Wansika, Martviset Pongsakorn
Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Khlong Luang, Pathum Thani 12120, Thailand.
Thailand Institute of Scientific and Technological Research, Khlong Luang, Pathum Thani 12120, Thailand.
Pathogens. 2023 Jul 18;12(7):949. doi: 10.3390/pathogens12070949.
A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, . Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of . Six cDNA coding sequences of cystatins, Cys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, Cys1 and Cys2 are classified as type 1 cystatins, while Cys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins Cys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, Cys1, 3 and 4 were selected for inhibition analysis and immune reactivity with -infected hamster sera. Cys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that cystatins are not major antigenic components in the ES product of this parasite and that other effects of cystatins should be investigated.
在泰国已观察到胆管癌（胆管癌）的高发病率。这种通常罕见的癌症与人类肝吸虫感染有关。寄生虫与宿主相互作用的分泌物被认为是其致病性的主要组成部分，而蛋白水解是分泌分子的关键生物学活性。在本研究中，我们对肝吸虫的半胱氨酸蛋白酶抑制剂（胱抑素）进行了分子分析。使用RT-PCR从寄生虫的成虫阶段克隆了6个胱抑素的cDNA编码序列，即Cys1-6。根据它们的序列，Cys1和Cys2被归类为1型胱抑素，而Cys3-6被归类为2型胱抑素，每个都含有一个信号肽且仅含有一个C端二硫键。与其他胱抑素成员相比，它们的C端区域序列各不相同。使用蛋白质印迹检测在成虫寄生虫的粗虫提取物和排泄分泌（ES）产物中发现了胱抑素Cys1、3和4，而其他同工型未被发现。因此，选择Cys1、3和4进行抑制分析以及与感染肝吸虫的仓鼠血清的免疫反应性分析。Cys1、3和4对哺乳动物组织蛋白酶L的抑制作用比对组织蛋白酶B更有效。它们完全活性的pH范围非常宽（pH 3-9），并且在至少3小时内对热稳定。与肝吸虫胱抑素不同，基于间接ELISA，它们与感染仓鼠血清没有免疫反应性。我们的研究结果表明，肝吸虫胱抑素不是该寄生虫ES产物中的主要抗原成分，应研究肝吸虫胱抑素的其他作用。
