Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells.
Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells.
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DOI：
10.1083/jcb.104.2.253
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发表时间：
1987-02
期刊：
影响因子：
--
通讯作者：
Rochefort H
中科院分区：
文献类型：
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作者：
Capony F;Morisset M;Barrett AJ;Capony JP;Broquet P;Vignon F;Chambon M;Louisot P;Rochefort H
我们已经研究了52-kD蛋白的翻译后修饰，所述52-kD蛋白是由培养物中的几种人乳腺癌细胞分泌的雌激素调节的自分泌有丝分裂原（Westley，B.，和H. Rochefort，1980，Cell，20：353-362）。分泌的52 kD蛋白被发现是磷酸化的大部分（94%）高甘露糖N-连接的寡糖链，甘露糖-6-磷酸信号被确定。磷酸信号被碱性磷酸酶水解完全去除。分泌的52-kD蛋白通过甘露糖-6-磷酸受体被MCF 7细胞部分摄取，并与溶酶体水解酶一样加工成48-和34-kD蛋白部分。电镜下，免疫过氧化物酶染色显示大部分反应蛋白在溶酶体中。完全纯化后，通过免疫亲和层析，我们确定了分泌的52 kD蛋白和其加工的细胞形式的天冬氨酸和酸性蛋白酶特异性抑制胃酶抑制剂。52 kD蛋白酶在乳腺癌细胞中以其失活的酶原形式分泌，其可以在酸性pH下自活化，分子量略有降低。与正常组织的组织蛋白酶D（s）相比，乳腺癌细胞的酶在分子量、酶活性（抑制剂、底物、特异性活性）和免疫反应性方面相似。然而，乳腺癌细胞的52- kD蛋白及其细胞加工形式对内切-β-N-乙酰氨基葡萄糖苷酶H（Endo H）完全敏感，而正常组织的几种细胞组织蛋白酶D（s）部分耐Endo H。这种差异，除了其他有关组织分布，促有丝分裂活性和激素调节，强烈表明，52-kD的组织蛋白酶D样酶的乳腺癌细胞是不同于以前描述的组织蛋白酶D（S）。雌激素诱导的52 kD溶酶体蛋白酶可能在促进乳腺癌细胞增殖、迁移和转移中起重要作用。
We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6- phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52- kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.