Powell P P, Kyle J W, Miller R D, Pantano J, Grubb J H, Sly W S
E.A. Doisy Department of Biochemistry, St. Louis University Medical Center, MO 63104.
Biochem J. 1988 Mar 1;250(2):547-55. doi: 10.1042/bj2500547.
A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.
分离出大鼠肝脏β-葡萄糖醛酸酶的互补DNA（cDNA），测定其序列，并研究其转染到COS细胞后的表达情况。大鼠肝脏克隆推导的氨基酸序列与人类胎盘β-葡萄糖醛酸酶cDNA推导的序列有77%的同源性，与大肠杆菌β-葡萄糖醛酸酶cDNA推导的序列有47%的同源性。发现大鼠肝脏的cDNA与先前报道的大鼠包皮腺的cDNA之间存在一些差异。只有一处变化导致成熟酶中的一个氨基酸差异。通过使用编码来自人类胎盘cDNA克隆信号序列前18个氨基酸残基的片段和来自大鼠克隆编码信号序列四个氨基酸残基的片段、成熟大鼠酶的所有626个氨基酸残基以及所有3'非翻译区，构建了一个嵌合克隆。转染到COS细胞后，嵌合克隆表达的β-葡萄糖醛酸酶活性可被大鼠β-葡萄糖醛酸酶抗体特异性免疫沉淀。表达的基因产物76,000的相对分子质量是糖基化大鼠酶的特征。在代谢标记6小时后，它在COS细胞中被蛋白酶加工成75,000的相对分子质量。转染后60小时，至少50%的表达酶被分泌，但分泌的酶未经历蛋白酶加工。这些结果提供了证据，表明从大鼠肝脏文库中分离的部分cDNA包含成熟大鼠肝脏酶的完整编码序列，并且嵌合信号序列允许转染的大鼠肝脏酶在COS细胞中正常生物合成和加工。
