Identification of cDNA clones encoding a precursor of rat liver cathepsin B.
Identification of cDNA clones encoding a precursor of rat liver cathepsin B.
复制标题
编码大鼠肝脏组织蛋白酶 B 前体的 cDNA 克隆的鉴定。
DOI：
10.1073/pnas.82.8.2320
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发表时间：
1985
影响因子：
11.1
通讯作者：
Steiner,DF
中科院分区：
文献类型：
--
作者：
SanSegundo,B;Chan,SJ;Steiner,DF
最近的研究表明，许多溶酶体酶，包括组织蛋白酶B（EC 3.4.22.1），可以合成为较大的前体，并通过蛋白水解加工成其成熟形式。为了确定组织蛋白酶B的初级翻译产物的结构，我们筛选了编码大鼠肝组织蛋白酶B的噬菌体cDNA文库。我们合成了两个延伸的DNA寡核苷酸用作杂交探针：一个50-mer对应于成熟组织蛋白酶B残基215-231的编码区段，一个54-mer对应于残基117-134。在筛选600，000个噬斑后，获得了与32 P标记的50-mer杂交的5个克隆;其中，2个（λ rCB 3和λ rCB 5）也与54-mer反应。DNA序列分析证实λ rCB 3和λ rCB 5都编码大鼠肝组织蛋白酶B，并且翻译的序列与测定的序列一致[Takio，K.，Towatari，T.，Katunuma，N.，Teller，D. C. & Titani，K.等人（1983）Proc. Acad. Sci. USA 80，3666-3670]，除了在残基78处色氨酸取代甘氨酸以及在轻链和重链的连接位点处存在两个氨基酸。此外，该DNA序列揭示了延伸超过5'（NH 2末端）的开放阅读框，并且成熟蛋白质的编码序列的预测COOH末端延伸了6个氨基酸。这些结果证实了组织蛋白酶B的生物合成涉及较大的前体形式，并证明了长寡核苷酸探针用于筛选以检测稀有克隆mRNA的有效性。
Recent studies have suggested that many lysosomal enzymes, including cathepsin B (EC 3.4.22.1), may be synthesized as larger precursors and proteolytically processed to their mature forms. To determine the structure of the primary translation product of cathepsin B, we have screened a phage cDNA library for clones encoding rat liver cathepsin B. We synthesized two extended DNA oligonucleotides to use as hybridization probes: a 50-mer corresponding to the coding segment for residues 215-231 of mature cathepsin B and a 54-mer corresponding to residues 117-134. After screening 600,000 plaques, five clones were obtained that hybridized to the 32P-labeled 50-mer; of these, two (lambda rCB3 and lambda rCB5) also reacted with the 54-mer. DNA sequence analysis confirmed that lambda rCB3 and lambda rCB5 both encoded rat liver cathepsin B, and the translated sequence is in agreement with the sequence determined [Takio, K., Towatari, T., Katunuma, N., Teller, D. C. & Titani, K. (1983) Proc. Natl. Acad. Sci. USA 80, 3666-3670], except for a tryptophan for glycine substitution at residue 78 and the presence of two amino acids at the junction site of the light and heavy chains. Moreover, the DNA sequence reveals an open reading frame extending beyond the 5' (NH2 terminus), and the predicted COOH terminus of the coding sequence for the mature protein is extended by six amino acids. These results confirm that the biosynthesis of cathepsin B involves a larger precursor form and demonstrate the effectiveness of long oligonucleotide probes for screening to detect rare cloned mRNAs.