In vivo expression of an alternatively spliced human tumor message that encodes a truncated form of cathepsin B -: Subcellular distribution of the truncated enzyme in COS cells
In vivo expression of an alternatively spliced human tumor message that encodes a truncated form of cathepsin B -: Subcellular distribution of the truncated enzyme in COS cells
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DOI：
10.1074/jbc.273.21.13236
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发表时间：
1998-05-22
影响因子：
4.8
通讯作者：
Frankfater, A
中科院分区：
文献类型：
--
作者：
Mehtani, S;Gong, QM;Frankfater, A
组织蛋白酶B是一种溶酶体半胱氨酸蛋白酶，其表达增加被认为与肿瘤的恶性进展有关。选择性剪接和在人类中使用选择性转录起始位点产生在其5 '-和3'-非翻译末端不同的组织蛋白酶B mRNA。一些人类肿瘤还含有组织蛋白酶B相关的转录物，其缺乏编码N-末端信号肽的外显子3和62个氨基酸的抑制性前肽中的34个。在这项研究中，我们发现一个这样的转录本，CB（-2，3），这是缺失外显子2和3，很可能是一个功能性的信息在肿瘤中。因此，发现CB（-2，3）在其他方面是完整的，含有组织蛋白酶B编码序列的剩余部分和3 '非翻译区的一部分，该部分是所有先前表征的人类组织蛋白酶B mRNA所共有的。其体外翻译产物可被折叠以产生针对组织蛋白酶B特异性底物N-α-苄氧基羰基-L-Arg-L-Arg-4-甲基香豆酰-7-酰胺的酶活性，来自转移性人黑素瘤细胞系A375 M的内源性CB（-2，3）与多聚核糖体共沉积，表明其参与这些细胞中的真核翻译机制。在瞬时转染入COS细胞后，产生了与正常蛋白质相当的量的来自CB（-2，3）的截短组织蛋白酶B的表位标记形式。免疫荧光显微镜和亚细胞分级显示，这种新的肿瘤形式的组织蛋白酶B与细胞核和其他膜细胞器，在那里它很可能被绑定到细胞膜的细胞质面。这种亚细胞分布不同于COS细胞中表位标记的全长组织蛋白酶B所示的溶酶体模式。这些结果表明，缺失外显子2和3的信息可能被翻译成催化活性酶，并且选择性剪接（外显子跳跃）可能有助于在一些人类癌症中观察到的组织蛋白酶B的异常细胞内运输。
Cathepsin B is a lysosomal cysteine protease whose increased expression is believed to be linked to the malignant progression of tumors. Alternative splicing and the use of alternative transcription initiation sites in humans produce cathepsin B mRNAs that differ in their 5'- and 3'-untranslated ends. Some human tumors also contain cathepsin B-related transcripts that lack exon 3 which encodes the N-terminal signal peptide and 34 of the 62-amino acid inhibitory propeptide. In this study we show that one such transcript, CB(-2,3), which is missing exons 2 and 3, is likely to be a functional message in tumors. Thus, CB(-2,3) was found to be otherwise complete, containing the remainder of the cathepsin B coding sequence and the part of the 3'-untranslated region that is common to all previously characterized cathepsin B mRNAs in humans. Its in vitro translation product can be folded to produce enzymatic activity against the cathepsin B-specific substrate, N-alpha-benzyloxycarbonyl-L-Arg-L-Arg-4-methycoumaryl-7- amide, Endogenous CB(-2,3) from the metastatic human melanoma cell line, A375M, co-sediments with polysomes, indicating that it engages the eukaryotic translation machinery in these cells. Epitope-tagged forms of the truncated cathepsin B from CB(-2,3) are produced in amounts comparable to the normal protein after transient transfection into COS cells. Immunofluorescence microscopy and subcellular fractionation show this novel tumor form of cathepsin B to be associated with nuclei and other membranous organelles, where it is likely to be bound to the cytoplasmic face of the membranes. This subcellular distribution was different from the lysosomal pattern shown by the epitope-tagged, full-length cathepsin B in COS cells. These results indicate that the message missing exons 2 and 3 is likely to be translated into a catalytically active enzyme, and that alternative splicing (exon skipping) could contribute to the aberrant intracellular trafficking of cathepsin B that is observed in some human cancers.