Fazleabas A T, Verhage H G, Waites G, Bell S C
Department of Obstetrics and Gynecology, University of Illinois College of Medicine, Chicago 60680.
Biol Reprod. 1989 Apr;40(4):873-85. doi: 10.1095/biolreprod40.4.873.
The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin [alpha 1-PEG]), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta. Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues. Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections. Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000). Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP. Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta. Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D. The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM. The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP. These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans. The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy.
人蜕膜的主要分泌蛋白（妊娠相关子宫内膜α1球蛋白[α1-PEG]）是一种胰岛素样生长因子结合蛋白（IGF-BP），在免疫学和生物化学方面与从足月人胎盘中提取的胎盘蛋白12（PP12）相似。由于先前的研究表明，狒狒和人的子宫内膜在培养中能合成并释放多种生物化学和免疫学相关的多肽，因此进行本研究以进一步鉴定狒狒胎盘组织中的一种相关IGF-BP。通过剖宫产从妊娠134至160天的怀孕狒狒获取蜕膜、带有附着蜕膜的绒毛膜羊膜（CAM-D）和胎盘绒毛。部分组织要么在35S-甲硫氨酸存在下培养，固定用于免疫细胞化学，要么在液氮中冷冻用于胞质溶胶提取。组织培养基（TCM）的二维聚丙烯酰胺凝胶电泳（2D-PAGE）显示，蜕膜和CAM-D的主要分泌产物是一种酸性多肽（分子量33,000）。用针对人α1-PEG的鼠单克隆抗体（B2H10）对TCM进行蛋白质印迹分析和免疫沉淀表明，这种由狒狒蜕膜和CAM-D而非胎盘绒毛分泌的分子在免疫学上与人IGF-BP相同。IGF-BP的免疫细胞化学定位在蜕膜和CAM-D的基质细胞胞质中强烈，而在胎盘中不存在。对TCM和胞质溶胶进行凝胶过滤，随后筛选洗脱液与125I-IGF-I的结合，在蜕膜和CAM-D中解析出两个特异性IGF-BP峰（分子量大于100,000和35,000）。分子量35,000的峰的结合能力是分子量大于100,000峰的100 - 200倍，解离常数为1.14 - 1.83 nM。通过多克隆放射免疫测定法检测，分子量35,000峰中的洗脱液对α1-PEG也具有免疫反应性。用125I-IGF-I进行亲和交联，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，显示出一个分子量为36,000的免疫反应性复合物，证实狒狒蛋白代表一种高亲和力IGF-BP。这些研究表明，狒狒蜕膜和CAM-D的肥大基质细胞合成并释放一种IGF-BP作为其主要分泌产物，类似于人类的情况。本研究结果表明，这种蛋白可能在妊娠期间IGF作用的调节中发挥作用。
