Silva Francine B, Batista João A N, Marra Brener M, Fragoso Rodrigo R, Monteiro Ana Carolina S, Figueira Edson L Z, Grossi-de-Sá Maria Fátima
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brasil.
Genet Mol Res. 2004 Sep 30;3(3):342-55.
Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.
半胱氨酸蛋白酶（CPs）以酶原形式合成，并通过蛋白水解切割和释放其前结构域肽转化为成熟的蛋白酶形式。从大豆孢囊线虫J2 cDNA文库中分离出一个编码木瓜蛋白酶样CP的cDNA，命名为hgcp-Iv，对其进行表达，并用于评估其前肽抑制活性形式蛋白酶的能力。hgcp-Iv cDNA序列编码一个374个氨基酸的多肽，其结构域组织与其他组织蛋白酶L样CP相同，包括一个疏水信号序列和一个前结构域区域。HGCP-Iv在大肠杆菌中作为与硫氧还蛋白的融合蛋白产生，可降解合成肽苄氧羰基-苯丙氨酸-精氨酸-7-氨基-4-甲基香豆素，并被E-64抑制，E-64是一种常用于CP功能表征的底物和抑制剂。在大肠杆菌中表达的HGCP-Iv重组前肽在体外对其同源酶和南方根结线虫雌虫的蛋白酶活性具有高抑制活性，表明其在生物系统中抑制线虫CP方面的有用性。来自其他物种的半胱氨酸蛋白酶没有产生明显的活性。
