Takeda A, Takahashi N, Shimizu S
Aichi Cancer Center Research Institute, Nagoya, Japan.
Endocrinology. 1988 Jan;122(1):105-13. doi: 10.1210/endo-122-1-105.
An estrogen-inducible 97-kilodalton (kDa) secretory glycoprotein, designated as uterine secretory protein (USP)-1, synthesized by rat uterine epithelial cells was identified and characterized. Uterine luminal fluid (ULF) proteins were labeled by direct administration of [35S]methionine into uterine lumen of rats. Incorporation of [35S]methionine into ULF proteins was negligible in ovariectomized rats. However, when 17 beta-estradiol (E2) was administered in the ovariectomized rat as sc paraffin pellets, a marked increase of [35S]methionine incorporation was noted after 2 days of treatment, showing that the de novo synthesis and secretion of ULF proteins were induced by E2. Six estrogen-inducible polypeptides (130, 110, 97, 65, 42, and 39 kDa) were identified in the analysis of the labeled ULF proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Four of these polypeptides (110, 65, 42, and 39 kDa) were adsorbed by the immobilized antibody against rat serum proteins, indicating that these polypeptides are antigenically similar to serum proteins. Two polypeptides (130 and 97 kDa) not adsorbed by the antibody column were suggested to be uterine-specific secretory proteins. The 97-kDa protein (USP-1) was further purified by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis and electroelution. Reactivity of purified USP-1 to various lectins and carbohydrate composition analysis suggested that USP-1 possesses biantennary N-linked complex-type carbohydrate chain with fucose. Rabbit polyclonal antibody which can specifically immunoprecipitate [35S]methionine-labeled USP-1 was developed. Dot blot enzyme immunoassay showed that 4-day E2-treated ULF contains 78.3 +/- 24.1 (+/- SE) micrograms USP-1/mg protein. Immunohistochemical staining of rat uterine tissue showed that this protein localized only in the epithelial cells treated with estrogen (E2 and diethylstilbestrol). Testosterone, progesterone, and dexamethasone failed to induce synthesis and secretion of USP-1 as assessed by dot blot enzyme immunoassay, immunoprecipitation of [35S]methionine-labeled ULF proteins, and immunohistochemical staining of uterine tissue. The present result, for the first time, revealed that estrogen can induce synthesis and secretion of specific secretory protein which could be the useful marker to analyze the molecular mechanism of estrogen action in rat uterine epithelial cells.
一种雌激素诱导的97千道尔顿（kDa）分泌性糖蛋白，命名为子宫分泌蛋白（USP）-1，由大鼠子宫上皮细胞合成，已被鉴定和表征。通过将[35S]甲硫氨酸直接注入大鼠子宫腔来标记子宫腔液（ULF）蛋白。在去卵巢大鼠中，[35S]甲硫氨酸掺入ULF蛋白的量可忽略不计。然而，当以皮下石蜡丸剂形式给去卵巢大鼠注射17β-雌二醇（E2）时，在治疗2天后观察到[35S]甲硫氨酸掺入量显著增加，表明E2诱导了ULF蛋白的从头合成和分泌。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和荧光自显影分析标记的ULF蛋白，鉴定出六种雌激素诱导的多肽（130、110、97、65、42和39 kDa）。其中四种多肽（110、65、42和39 kDa）被固定化的抗大鼠血清蛋白抗体吸附，表明这些多肽在抗原性上与血清蛋白相似。未被抗体柱吸附的两种多肽（130和97 kDa）被认为是子宫特异性分泌蛋白。通过制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电洗脱进一步纯化了这种97 kDa的蛋白（USP-1）。纯化的USP-1与各种凝集素的反应性以及碳水化合物组成分析表明，USP-1具有带有岩藻糖的双天线N-连接复合型碳水化合物链。制备了能够特异性免疫沉淀[35S]甲硫氨酸标记的USP-1的兔多克隆抗体。斑点印迹酶免疫测定表明，经E2处理4天的ULF中含有78.3±24.1（±SE）微克USP-1/毫克蛋白。大鼠子宫组织的免疫组织化学染色表明，这种蛋白仅定位于用雌激素（E2和己烯雌酚）处理的上皮细胞中。通过斑点印迹酶免疫测定、[35S]甲硫氨酸标记的ULF蛋白的免疫沉淀以及子宫组织的免疫组织化学染色评估，睾酮、孕酮和地塞米松未能诱导USP-1的合成和分泌。本研究结果首次揭示，雌激素可诱导特定分泌蛋白的合成和分泌，这可能是分析雌激素在大鼠子宫上皮细胞中作用分子机制的有用标志物。
