Bidart J M, Troalen F, Bousfield G R, Bohuon C, Bellet D
Unité de Biochimie Clinique, Institut Gustave-Roussy, Villejuif, France.
Endocrinology. 1989 Feb;124(2):923-9. doi: 10.1210/endo-124-2-923.
To improve our knowledge of the structural features of the alpha-subunit of hCG we have studied the antigenic site recognized by monoclonal antibody (MAb) ECG01 raised against equine CG (eCG) which binds to hormones and alpha-subunits from human and equine species. We have also delineated regions of hCG alpha comprising the epitope recognized by HT13 which was raised against hCG and binds to hCG and hCG alpha. To define the residues involved in the antigenic sites recognized by ECG01 and HT13, we have studied the reactivities of these two MAbs with native or chemically modified LH and CG with subunits from equine, human, or ovine (o) species or with synthetic peptides analogous to various portions of hCG alpha. We have also compared these reactivities with those displayed by MAbs AHT20 and FA 36, whose epitopes have been previously described; anti-hCG alpha MAb AHT20 is specific for the free alpha-subunits of various species and recognizes residues localized to the 36-41 region of hCG alpha, whereas antipeptide MAb FA36 binds to the 87-92 carboxyl-terminal part of hCG alpha. Our results show that the epitopes of HT13 and ECG01 are 1) probably discontinuous, as these MAbs did not bind to the reduced and S-carboxymethylated hCG alpha; and 2) constituted by residues borne on the 1-35 and 52-86 sequences, as they do recognize the hCG alpha core missing the 36-51 portion, yet do not recognize hCG alpha-(87-92) region recognized by FA36. The comparative studies performed with specific two-site immunoradiometric assays to determine the interspecies cross-reactivities of the MAbs allow us hypothetical assignment of residues on the primary structure of hCG alpha. The antigenic site recognized by ECG01 might include two to six amino acids, four of these residues being located at inverted places compared to those of oLH alpha (Asp6/Gly22 and Arg67/Lys75). These residues present important charged functional groups highly conserved among evolutionarily related variants, and it is likely that they are located on the surface of both the intact hormone and its alpha-subunit. Three peptidic portions of hCG alpha, 16-17, 64-66, and 73-76, respectively, might be involved in the epitope recognized by HT13, although we could not rule out the possibility that other residues were also involved in the antigenic site. These observations allow us to identify several residues as potentially constituting the epitopes recognized by two MAbs on both hCG and hCG alpha.
为了增进我们对人绒毛膜促性腺激素（hCG）α亚基结构特征的了解，我们研究了单克隆抗体（MAb）ECG01识别的抗原位点，该抗体是针对马绒毛膜促性腺激素（eCG）产生的，能与来自人和马物种的激素及α亚基结合。我们还划定了hCGα中包含HT13识别的表位的区域，HT13是针对hCG产生的，能与hCG和hCGα结合。为了确定参与ECG01和HT13识别的抗原位点的残基，我们研究了这两种单克隆抗体与天然或化学修饰的促黄体生成素（LH）和绒毛膜促性腺激素（CG）、来自马、人或绵羊（o）物种的亚基或与类似于hCGα不同部分的合成肽的反应性。我们还将这些反应性与先前已描述其表位的单克隆抗体AHT20和FA 36所显示的反应性进行了比较；抗hCGα单克隆抗体AHT20对各种物种的游离α亚基具有特异性，识别位于hCGα 36 - 41区域的残基，而抗肽单克隆抗体FA36与hCGα的87 - 92羧基末端部分结合。我们的结果表明，HT13和ECG01的表位：1）可能是不连续的，因为这些单克隆抗体不与还原型和S - 羧甲基化的hCGα结合；2）由1 - 35和52 - 86序列上的残基组成，因为它们确实能识别缺失36 - 51部分的hCGα核心，但不能识别FA36识别的hCGα -（87 - 92）区域。用特异性双位点免疫放射分析进行的比较研究以确定单克隆抗体的种间交叉反应性，使我们能够对hCGα一级结构上的残基进行假设性定位。ECG01识别的抗原位点可能包括两到六个氨基酸，其中四个残基与oLHα的残基位置相反（Asp6/Gly22和Arg67/Lys75）。这些残基具有重要的带电官能团，在进化相关变体中高度保守，并且很可能位于完整激素及其α亚基的表面。hCGα的三个肽段，分别为16 - 17、64 - 66和73 - 76，可能参与了HT13识别的表位，尽管我们不能排除其他残基也参与抗原位点的可能性。这些观察结果使我们能够确定几个残基可能构成两种单克隆抗体在hCG和hCGα上识别的表位。
