Seya T, Hara T, Matsumoto M, Kiyohara H, Nakanishi I, Kinouchi T, Okabe M, Shimizu A, Akedo H
Department of Immunology, Center for Adult Diseases Osaka, Japan.
Eur J Immunol. 1993 Jun;23(6):1322-7. doi: 10.1002/eji.1830230620.
A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46). These proteins served as factor I cofactors for the cleavage of methylamine-treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor-activity-blocking mAb. Thus, these semen proteins are antigenic and functional homologous of MCP. On SDS-PAGE analysis these MCP migrated as single-band proteins which differed from the two-band forms of MCP expressed on other cells. smMCP was N-glycosylated but not O-glycosylated, while ssMCP was O-glycosylated: after deglycosylation of these proteins bands were detected at 38-40 kDa and 43 kDa on SDS-PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and "sterile" subject groups was determined by sandwich enzyme-linked immunosorbent assay. Seminal plasma in the two groups contained 250-700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from "sterile" subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS-PAGE/immunoblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra-lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP. The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome-reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.
通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE），随后用四种针对膜辅因子蛋白（MCP，CD46）的单克隆抗体（mAb）进行免疫印迹，鉴定出一种分子量为43 kDa的精子蛋白（精子细胞膜辅因子蛋白，smMCP）和一种60 kDa的精浆蛋白（ssMCP）。这些蛋白作为I因子辅因子参与甲胺处理的C3（C3ma）的裂解，其活性被MCP辅因子活性阻断单克隆抗体M75所阻断。因此，这些精液蛋白是MCP的抗原性和功能同源物。在SDS - PAGE分析中，这些MCP以单条带蛋白形式迁移，这与在其他细胞上表达的两条带形式的MCP不同。smMCP进行N - 糖基化但不进行O - 糖基化，而ssMCP进行O - 糖基化：这些蛋白去糖基化后，在SDS - PAGE上分别在38 - 40 kDa和43 kDa处检测到条带。因此，这些精液MCP在结构上与传统MCP不同。通过夹心酶联免疫吸附测定法测定了正常和“不育”受试者组中的ssMCP。两组精浆中ssMCP含量为250 - 700 ng/ml。两组之间的差异很小，尽管正常受试者的样本往往比“不育”受试者的样本显示出更高浓度的ssMCP。通过SDS - PAGE /免疫印迹分析，两组中的ssMCP和smMCP未观察到分子差异。免疫组织化学分析表明，MCP在前列腺的腺上皮细胞和管腔以及睾丸的大多数管腔内细胞中呈阳性。使用抗体M177，通过免疫印迹分析溶解的前列腺和睾丸，并与其他细胞的MCP进行比较。睾丸中MCP的主要条带为60 kDa，而前列腺中没有，这与ssMCP一致。然而，睾丸或前列腺MCP条带与smMCP不一致。ssMCP可能在睾丸中产生，而smMCP的来源仍然未知。我们假设ssMCP在精子存活中很重要，保护它们免受女性生殖道中免疫球蛋白和补体的局部分泌影响，并且在顶体反应的精子上表达的smMCP在精子与卵母细胞的相互作用中起重要作用。
