Weiner R S, Dias J A
Department of Biochemistry and Molecular Biology, Albany Medical College, New York 12208.
Endocrinology. 1992 Sep;131(3):1026-36. doi: 10.1210/endo.131.3.1380433.
Conformational features of two epitopes on the glycoprotein hormone alpha-subunit were investigated using two antihuman FSH (anti-hFSH) monoclonal antibodies (mAbs) 3A and 5F that recognize different epitopes and are specific for alpha-subunit. These mAbs were used to investigate whether the conformation of these epitopes was different in heterodimeric hFSH, hTSH, hLH, or hCG. Any differences in the mass of hormone in each preparation were accounted for by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis of all hormone preparations used in this study. Rabbit anti-hFSH alpha-(11-27) antipeptide antisera and [125I]protein-G were used in the Western blot analysis. Radioactivity associated with each band was determined and used to normalize the mass of alpha-subunit in each reference preparation used in the displacement assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also performed in order to examine the integrity of each of the hormone reference preparations. hTSH alpha and, to a lesser extent, hLH alpha preparations contained an internal nick in the polypeptide chain. RIA analysis performed using heterodimeric glycoprotein hormones as competitors revealed that an average 100-fold difference in the ED50 values for hFSH compared to the other glycoprotein hormones was seen with mAb 3A. Therefore, the conformation of 3A epitope appeared to be different in hFSH than in hTSH, hLH, or hCG. In comparison, the epitope recognized by mAb 5F only had an average 7-fold difference in reactivity (ED50 values) for hFSH compared to hTSH, hLH, and hCG. Likewise, competition assays using the respective alpha-subunits and mAb 5F revealed a pattern of competition similar to that observed with heterodimers, with an average 4-fold difference in the ED50 values for hFSH alpha compared to those for hTSH alpha, hLH alpha, and hCG alpha. Therefore, the conformation of the 5F epitope appears unaffected by association of alpha-subunit with beta-subunit. Accordingly, any differences in the conformation of the four alpha-subunits, as demonstrated by these small differences in ED50 values, appear to be inherent to each alpha-subunit. In fact, the 5F epitope appears to be quite rigid, since nicked alpha-subunit preparations could compete with [125I]hFSH for binding to 5F with comparable potency to non-nicked alpha-subunits. These findings support the concept that epitopes on heterodimeric hFSH alpha may have different conformational features. Some are specific for heterodimeric hFSH alpha, and we refer to these as conformationally active (flexible). Others are common to the four human glycoprotein hormone alpha-subunits, suggesting that they are conformationally constrained (rigid).
使用两种抗人促卵泡激素（anti-hFSH）单克隆抗体（mAb）3A和5F研究了糖蛋白激素α亚基上两个表位的构象特征，这两种抗体识别不同表位且对α亚基具有特异性。这些单克隆抗体用于研究这些表位的构象在异源二聚体hFSH、hTSH、hLH或hCG中是否不同。本研究中使用的所有激素制剂通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳/蛋白质印迹分析来解释每种制剂中激素质量的任何差异。蛋白质印迹分析中使用了兔抗-hFSHα-（11 - 27）抗肽抗血清和[125I]蛋白G。测定与每条带相关的放射性，并用于标准化置换试验中使用的每种参比制剂中α亚基的质量。还进行了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以检查每种激素参比制剂的完整性。hTSHα制剂以及程度稍轻的hLHα制剂在多肽链中存在内部切口。使用异源二聚体糖蛋白激素作为竞争者进行的放射免疫分析（RIA）显示，与其他糖蛋白激素相比，mAb 3A检测到hFSH的半数有效剂量（ED50）值平均相差100倍。因此，hFSH中3A表位的构象似乎与hTSH、hLH或hCG中的不同。相比之下，mAb 5F识别的表位与hTSH、hLH和hCG相比，hFSH的反应性（ED50值）平均仅相差7倍。同样，使用各自的α亚基和mAb 5F进行的竞争试验显示出与异源二聚体观察到的竞争模式相似，hFSHα的ED50值与hTSHα、hLHα和hCGα的相比平均相差4倍。因此，5F表位的构象似乎不受α亚基与β亚基缔合的影响。相应地，如ED50值的这些微小差异所示，四种α亚基构象的任何差异似乎是每个α亚基所固有的。事实上，5F表位似乎相当刚性，因为切口的α亚基制剂能够与[125I]hFSH竞争结合5F，其效力与未切口的α亚基相当。这些发现支持了异源二聚体hFSHα上的表位可能具有不同构象特征的概念。一些表位对异源二聚体hFSHα具有特异性，我们将其称为构象活性（灵活）表位。其他表位是四种人糖蛋白激素α亚基共有的，这表明它们是构象受限（刚性）的。
