Molecular cloning and characterization of chymopasin, a novel serine protease from rat pancreas
Molecular cloning and characterization of chymopasin, a novel serine protease from rat pancreas
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DOI：
10.1097/00006676-200211000-00010
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发表时间：
2002-11-01
期刊：
影响因子：
2.9
通讯作者：
Yamaguchi, N
中科院分区：
文献类型：
--
作者：
Sogame, Y;Kataoka, K;Yamaguchi, N
简介：胰腺分泌许多消化食物的酶进入胰液。我们克隆了一种新的丝氨酸蛋白酶，凝乳酶，大鼠胰腺前壁。目的：了解该酶在胰腺中的定位，并分析其酶学特性。方法：利用3‘和5’RACE克隆凝乳酶基因。用Northern印迹和原位杂交的方法研究了该酶的表达。利用特异性抗体对大肠杆菌产生的重组凝乳酶蛋白进行Western印迹分析，并用市售合成底物纤维蛋白和明胶对其酶学性质进行检测。结果：大鼠凝乳酶的开放阅读框由792bp组成，编码264个氨基酸残基。推导出的氨基酸序列包含丝氨酸蛋白酶家族的基本催化三联体特征。没有推测的N-糖基化位点。大鼠凝乳酶B的氨基酸序列与大鼠凝乳酶B的同源性为54.5%。Northern印迹分析表明，该转录本在胰腺中有较强的表达。地高辛标记的cRNA探针原位杂交显示，在腺泡细胞中可见阳性信号，而在胰岛或导管细胞中未见阳性信号。在胰腺匀浆和胆胰液中检测到凝乳酶蛋白。此外，雨蛙素还能刺激大鼠胰液中凝乳酶的分泌。结论：大鼠凝乳酶可能是腺泡细胞分泌的一种消化酶。通过人工合成底物的酶活性测定，纯化的重组凝乳酶在大肠杆菌中表达，具有类凝乳酶活性。此外，大鼠重组凝乳酶具有纤溶和明胶溶解活性。这些结果提示在胰腺损伤的发病机制中有一定的作用。
Introduction: Pancreas secretes many enzymes for food digestion into the pancreatic juice. We cloned a novel serine protease, chymopasin, front rat pancreas. Aims: To know the localization of this enzyme in the pancreas and to analyze the enzymatic characteristics. Methodology: We cloned chymopasin cDNA using 3' and 5' RACEs. Northern blot and in situ hybridization were used to study the expression of this enzyme. Recombinant chymopasin protein produced by E. coli was analyzed by Western blot using specific antibody, and its enzymatic characteristics were examined using commercially available synthetic substrates, fibrin and gelatin. Results: The open reading frame of rat chymopasin consisted of 792 bp encoding 264 amino acid residues. The deduced amino acid sequence contained the essential catalytic triad characteristic of the serine protease family. There was no putative N-glycosylation site. The amino acid sequence of rat chymopasin showed 54.5% identity to rat chymotrypsin B. Northern blot analysis showed that the transcript was strongly expressed in the pancreas. In situ hybridization with digoxigenin-labeled cRNA probe showed that the positive signals were observed in the acinar cells, but not in the islet or duct cells. Chymopasin protein was detected in the pancreas homogenate and bile-pancreatic juice. Further, cerulein stimulated the secretion of rat chymopasin into bilepancreatic juice. Conclusion: These results suggested that rat chymopasin might be a digestive enzyme secreted from the acinar cells. From the enzyme assay using synthetic substrates, the purified recombinant chymopasin expressed in Escherichia coli showed chymotrypsin-like activity. In addition, rat recombinant chymopasin showed fibrinolytic and gelatinolytic activities. These results suggested a role in the pathogenesis of pancreatic damage.