Larsen M, Ressler S J, Lu B, Gerdes M J, McBride L, Dang T D, Rowley D R
Cell and Molecular Biology Program, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1998 Feb 20;273(8):4574-84. doi: 10.1074/jbc.273.8.4574.
We previously reported the purification of ps20 (Rowley, D. R., Dang, T. D., Larsen, M., Gerdes, M. J., McBride, L., and Lu, B. (1995) J. Biol. Chem. 270, 22058-22065), a urogenital sinus mesenchymal cell secreted protein having growth-inhibitory properties. We report here cloning of the 1.03-kilobase rat ps20 cDNA clone from the PS-1 (adult rat prostate smooth muscle) cDNA library. Partial clones were obtained by nested polymerase chain reaction with degenerate primers, and full-length ps20 cDNA clones were isolated by plaque hybridization. Sequence analysis revealed that ps20 protein contains a WAP-type "four-disulfide core" motif and is a novel member of the WAP signature protein family composed primarily of secreted serine protease inhibitors. Native ps20 immunoprecipitated from smooth muscle cells and recombinant ps20 both resolved on SDS-polyacrylamide gel electrophoresis with apparent molecular mass of 27-29 kDa under reducing conditions and 21-23 kDa under non-reducing conditions, respectively. Stable ps20-transfectant COS-7 cell lines secreted ps20 and were growth-inhibited relative to mock transfectants. In addition, COS-7 and prostate carcinoma PC-3 cells were growth-inhibited by bacterially expressed ps20. Northern analysis indicated differential expression by tissue with highest expression in the heart. Immunohistochemical localization of ps20 protein showed cell-specific expression by both visceral and vascular smooth muscle in all tissues, including the prostate gland. These results indicate ps20 is a novel growth-regulatory member of the WAP signature family expressed by smooth muscle cells.
我们之前报道过ps20的纯化（罗利，D.R.，当，T.D.，拉森，M.，格德斯，M.J.，麦克布赖德，L.，和卢，B.（1995年）《生物化学杂志》270，22058 - 22065），ps20是一种具有生长抑制特性的泌尿生殖窦间充质细胞分泌蛋白。我们在此报告从PS - 1（成年大鼠前列腺平滑肌）cDNA文库中克隆出1.03千碱基的大鼠ps20 cDNA克隆。通过用简并引物进行巢式聚合酶链反应获得部分克隆，并通过噬菌斑杂交分离出全长ps20 cDNA克隆。序列分析表明，ps20蛋白含有一个WAP型“四二硫键核心”基序，是主要由分泌型丝氨酸蛋白酶抑制剂组成的WAP特征蛋白家族的一个新成员。从平滑肌细胞免疫沉淀的天然ps20和重组ps20在SDS - 聚丙烯酰胺凝胶电泳上分别在还原条件下显示表观分子量为27 - 29 kDa，在非还原条件下为21 - 23 kDa。稳定转染ps20的COS - 7细胞系分泌ps20，相对于空载体转染细胞生长受到抑制。此外，COS - 7和前列腺癌PC - 3细胞受到细菌表达的ps20的生长抑制。Northern分析表明该基因在不同组织中差异表达，在心脏中表达最高。ps20蛋白的免疫组织化学定位显示，在包括前列腺在内的所有组织中，内脏和血管平滑肌均有细胞特异性表达。这些结果表明，ps20是平滑肌细胞表达的WAP特征家族的一个新的生长调节成员。
