Wang Zengjun, Widgren Esther E, Richardson Richard T, O'Rand Michael G
Laboratories for Reproductive Biology and Department of Cell & Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Biol Reprod. 2007 Sep;77(3):476-84. doi: 10.1095/biolreprod.107.060194. Epub 2007 Jun 13.
Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.
附睾蛋白酶抑制因子（Eppin，即丝氨酸蛋白酶抑制剂样蛋白1，含Kunitz和WAP结构域1（Eppin）；附睾蛋白酶抑制剂）覆盖在人类射出精子的表面，其来源是支持细胞和附睾上皮细胞。在本研究中，我们使用柱色谱法和二维SDS-PAGE从射出精液上清液（精浆）和洗涤后的射出精子中分离出天然附睾蛋白酶抑制因子，并通过质谱分析和蛋白质免疫印迹法鉴定出一种包含乳铁蛋白（LTF，也称为乳铁传递蛋白）、簇集素（CLU）和精液凝固蛋白1（SEMG1）的附睾蛋白酶抑制因子蛋白复合物（EPC）。为了证实附睾蛋白酶抑制因子与乳铁蛋白、簇集素的关联，我们使用针对簇集素和乳铁蛋白的抗体从人类精子裂解物中免疫沉淀簇集素和乳铁蛋白。在这两种情况下都得到了相同的结果，即EPC的免疫沉淀物。此外，我们在人类支持细胞以及人类睾丸和射出精子上定位了附睾蛋白酶抑制因子、乳铁蛋白和簇集素，这意味着从精子离开生精小管时起EPC就存在于精子上。在射出精子上，附睾蛋白酶抑制因子、乳铁蛋白和簇集素共定位于尾部。EPC成分的鉴定表明，乳铁蛋白、簇集素和/或附睾蛋白酶抑制因子受体可能作为EPC的精子质膜受体发挥作用，这表明该复合物是人类精子表面蛋白质-蛋白质相互作用网络中的核心参与者。EPC可能提供一个具有杀菌特性的表面网络，既能保护精子，又能调节精子向有运动能力、获能精子的转变。
