Benkirane M M, Bon D, Costagliola S, Paolucci F, Darbouret B, Princé P, Carayon P
Endocrinology. 1987 Sep;121(3):1171-7. doi: 10.1210/endo-121-3-1171.
Although the amino acid sequence of the alpha- and beta-subunits of glycoprotein hormones in various species has been deciphered, data on their tertiary structure are not abundant. This impedes correlation between structure and function. The availability of monoclonal antibodies to human TSH (hTSH) offers the opportunity to enumerate the antigenic determinants present on the surface of hTSH and its subunits and to examine their spatial relationships. Twenty-eight monoclonal antibodies to hTSH were obtained from several fusions, and screens carried out separately in the laboratories involved in this study. Affinities for hTSH ranged from 10(8)-10(11) M-1. Cross-reactivity with bovine TSH (bTSH), human gonadotropins (hLH, hFSH, and hCG), and the alpha- and beta-subunits of hTSH distinguished 10 groups of monoclonal antibodies (mAb) according to their main cross-reactions: 1) hTSH alpha, hLH, hFSH, and hCG; 2) hTSH alpha, bTSH, hLH, hFSH, and hCG; 3) hFSH; 4) bTSH and hFSH; 5) bTSH, hLH, and hFSH; 6) bTSH, hLH, hFSH, and hCG; 7) hTSH beta; 8) hTSH beta and bTSH; 9) hTSH beta and hFSH; and 10) hTSH beta, hLH, hFSH, and hCG. mAb were incorporated into 2-site binding assays to probe hTSH by a 28 X 28 matrix, the free alpha-subunit by a 4 X 4 matrix, and the free beta-subunit by a 18 X 18 matrix. Regarding intact hTSH, 12 different clusters of mAb were distinguished and interpreted as reflecting 12 distinct antigenic regions on the surface of the hTSH molecule. Two of them were localized on the alpha-subunit, and 6 on the beta-subunit; 4 were only expressed by the holo-hormone and, thus were designated conformational antigenic regions (alpha beta). Surface mapping of the free alpha- and beta-subunits was virtually identical to that observed with the holo-hormone. Modification of the operative conditions of mAb reacting only with holo-hTSH shows that they recognize the alpha-subunit, but not the beta-subunit of hTSH. These results indicate that 1) hTSH beta presents epitopes that are evolutionary conserved; 2) hTSH alpha presents several epitopes that are species specific and 2 that are not hormone specific; 3) dissociation of hTSH does not modify the antigenic surface expressed by both subunits when they are associated; and 4) some of the conformational determinants expressed only by holo-hTSH are more likely derived from the alpha-subunit than from the beta-subunit.
尽管已破解了不同物种中糖蛋白激素α亚基和β亚基的氨基酸序列，但关于它们三级结构的数据并不丰富。这妨碍了结构与功能之间的关联。针对人促甲状腺激素（hTSH）的单克隆抗体的可得性，为列举hTSH及其亚基表面存在的抗原决定簇以及研究它们的空间关系提供了契机。通过多次融合获得了28种针对hTSH的单克隆抗体，并在参与本研究的各个实验室分别进行筛选。这些抗体对hTSH的亲和力范围为10⁸ - 10¹¹ M⁻¹（此处指数形式表示亲和力，10⁸表示10的8次方，10¹¹表示10的11次方）。根据它们与牛促甲状腺激素（bTSH）、人促性腺激素（hLH、hFSH和hCG）以及hTSH的α亚基和β亚基之间的交叉反应，将这些单克隆抗体分为10组：1）hTSHα、hLH、hFSH和hCG；2）hTSHα、bTSH、hLH、hFSH和hCG；3）hFSH；4）bTSH和hFSH；5）bTSH、hLH和hFSH；6）bTSH、hLH、hFSH和hCG；7）hTSHβ；8）hTSHβ和bTSH；9）hTSHβ和hFSH；10）hTSHβ、hLH、hFSH和hCG。将单克隆抗体用于双位点结合测定，通过28×28矩阵检测hTSH，通过4×4矩阵检测游离α亚基，通过18×18矩阵检测游离β亚基。对于完整的hTSH，区分出12个不同的单克隆抗体簇，并解释为反映了hTSH分子表面12个不同的抗原区域。其中2个位于α亚基上，6个位于β亚基上；4个仅由全激素表达，因此被指定为构象抗原区域（αβ）。游离α亚基和β亚基的表面图谱与全激素观察到的图谱基本相同。仅与全hTSH反应的单克隆抗体操作条件的改变表明，它们识别hTSH的α亚基，但不识别β亚基。这些结果表明：1）hTSHβ呈现出进化保守的表位；2）hTSHα呈现出几个物种特异性的表位以及2个非激素特异性的表位；3）hTSH的解离不会改变两个亚基结合时所表达的抗原表面；4）一些仅由全hTSH表达的构象决定簇更可能源自α亚基而非β亚基。
