Characterization of cysteine proteases from the carcinogenic liver fluke, Opisthorchis viverrini
Characterization of cysteine proteases from the carcinogenic liver fluke, Opisthorchis viverrini
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DOI：
10.1007/s00436-007-0831-1
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发表时间：
2008-03-01
影响因子：
2
通讯作者：
Sripa, Banchob
中科院分区：
文献类型：
--
作者：
Kaewpitoon, Natthawut;Laha, Thewarach;Sripa, Banchob
采用明胶酶谱法和荧光肽底物研究了蛇胸草提取物的蛋白酶活性。利用明胶浸渍x线片，对2 μ g猪瘟弧菌的排泄产物（Ov-ES）和成虫体提取物（Ov-SE）进行了蛋白水解实验。两种菌株的酶谱图均显示了30 kDa的谱带。利用荧光肽底物，确定了O. viverrini的大部分活性是组织蛋白酶l样半胱氨酸蛋白酶（裂解的z - ph -精氨酸氨基甲基香豆素（AMC）），而其他类别的蛋白酶很少或没有活性。pH值为6.0时，瘤胃O. viverrini半胱氨酸蛋白酶活性最高，且活性被类特异性抑制剂E-64和Z-Ala-CHN2抑制。从Ov-ES和Ov-SE中富集了30 kDa左右的巯基磷酸酯，对O. viverrini半胱氨酸蛋白酶进行了层析纯化。纯化酶的活性谱与Ov-SE和Ov-ES中组织蛋白酶l样活性相似。此外，对不同发育阶段的半胱氨酸蛋白酶活性测定显示，囊蚴可溶性提取物的蛋白酶活性最高，其次是Ov-ES、卵溶提取物和Ov-SE。这些发现表明，O. viverrini具有组织蛋白酶l样半胱氨酸蛋白酶(s)，并提示在囊蚴中存在丰富的半胱氨酸蛋白酶活性，其水解酶可能参与哺乳动物感染时的囊肿脱落。
Protease activities in extracts of Opisthorchis viverrini were investigated using gelatin zymography and fluorogenic peptide substrates. Using gelatin-impregnated X-ray film, 2 mu g of O. viverrini excretory-secretory products (Ov-ES) and adult somatic extract (Ov-SE) showed proteolytic activity. Zymography of both O. viverrini extracts revealed bands at similar to 30 kDa. Using fluorogenic peptide substrates, the majority of O. viverrini activity was determined to be cathepsin L-like cysteine protease (cleaved Z-Phe-Arg-aminomethylcoumarin (AMC)) whereas little or no activity was ascribable to other classes of proteases. The O. viverrini cysteine protease activity was greatest at pH 6.0 and the activity was inhibited by the class-specific inhibitors, E-64 and Z-Ala-CHN2. Chromatographic purification of O. viverrini cysteine proteases on thiol-sepharose enriched for protein(s) of similar to 30 kDa from Ov-ES and Ov-SE. The activity profile of the purified enzyme was similar to that of the cathepsin L-like activity characterized in Ov-SE and Ov-ES. Furthermore, determination of cysteine protease activity in several developmental stages of the parasite revealed the highest protease activity in metacercariae soluble extract, followed by Ov-ES, egg soluble extract, and Ov-SE. These findings demonstrated that O. viverrini has a cathepsin L-like cysteine protease(s) and suggested that abundant cysteine protease activity was present in metacercariae where the hydrolase might be involved in cyst excystation during mammalian infection.