Capony F, Morisset M, Barrett A J, Capony J P, Broquet P, Vignon F, Chambon M, Louisot P, Rochefort H
J Cell Biol. 1987 Feb;104(2):253-62. doi: 10.1083/jcb.104.2.253.
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.
我们研究了52-kD蛋白的翻译后修饰，该蛋白是一种雌激素调节的自分泌促有丝分裂原，由培养的几种人乳腺癌细胞分泌（韦斯特利，B.，和H. 罗什福尔，1980年，《细胞》，20:353 - 362）。发现分泌的52-kD蛋白大部分（94%）在高甘露糖型N-连接寡糖链上发生磷酸化，并鉴定出甘露糖-6-磷酸信号。磷酸信号通过碱性磷酸酶水解被完全去除。分泌的52-kD蛋白部分通过甘露糖-6-磷酸受体被MCF7细胞摄取，并像溶酶体水解酶一样被加工成48-kD和34-kD的蛋白部分。通过电子显微镜观察，免疫过氧化物酶染色显示溶酶体中有大部分反应性蛋白。通过免疫亲和层析完全纯化后，我们鉴定出分泌的52-kD蛋白及其加工后的细胞形式为天冬氨酸蛋白酶和酸性蛋白酶，它们被胃蛋白酶抑制剂特异性抑制。52-kD蛋白酶以无活性的酶原形式分泌到乳腺癌细胞中，在酸性pH下可自动激活，分子量略有降低。与正常组织的组织蛋白酶D相比，发现乳腺癌细胞的这种酶在分子量、酶活性（抑制剂、底物、比活性）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-乙酰葡糖胺糖苷酶H（Endo H）完全敏感，而正常组织的几种细胞组织蛋白酶D对Endo H有部分抗性。除了其他关于组织分布、促有丝分裂活性和激素调节的差异外，这一差异强烈表明乳腺癌细胞的52-kD组织蛋白酶D样酶与先前描述的组织蛋白酶D不同。52-kD雌激素诱导的溶酶体蛋白酶可能在促进乳腺癌细胞增殖、迁移和转移方面具有重要功能。
