Keutmann H T, Charlesworth M C, Mason K A, Ostrea T, Johnson L, Ryan R J
Proc Natl Acad Sci U S A. 1987 Apr;84(7):2038-42. doi: 10.1073/pnas.84.7.2038.
Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. We prepared a series of peptides representing the intercysteine "loop" sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) beta subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG beta-(38-57) and hLH beta-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 X 10(-4) and 2.03 X 10(-5) M, respectively, while other peptide hormones and fragments from elsewhere in the beta subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 X 10(-5) M (hCG) and 2.18 X 10(-5) M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG beta-(38-57) peptide, native hCG and beta subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. Helical-wheel projection predicted an amphipathic region in the N-terminal portion of the 38-57 sequence, and circular dichroic measurements showed an increase in ordered structure, especially alpha-helix, when the 38-57 peptides were transferred from an aqueous to a more lipophilic (90% trifluoroethanol) environment. These results indicate that the 38-57 region of beta subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.
迄今为止，合成片段尚未广泛用于评估糖蛋白激素中的构效关系。我们制备了一系列代表人类绒毛膜促性腺激素（hCG）和促黄体生成素（hLH）β亚基中半胱氨酸间“环”序列（第38 - 57位氨基酸残基）的肽段，预期该序列可能朝向表面并易于与受体结合。对这些肽段进行了化学表征，并通过与大鼠卵巢膜受体结合以及刺激睾丸间质细胞睾酮生成来测试其生物活性。hCG β -（38 - 57）和hLH β -（38 - 57）肽段分别在1.51×10⁻⁴和2.03×10⁻⁵ M时，对¹²⁵I标记的hCG结合抑制作用达到半数最大效应，而β亚基其他部位的肽类激素和片段则无活性。两种肽段均刺激睾酮生成，hCG在3.55×10⁻⁵ M时、hLH在2.18×10⁻⁵ M时达到半数最大反应。用抗甲状腺球蛋白偶联的hCG β -（38 - 57）肽段的抗体进行放射免疫测定，天然hCG和β亚基具有高反应性，还原和羧甲基化的亚基及肽段也是如此。螺旋轮投影预测38 - 57序列N端部分存在一个两亲区域，圆二色性测量表明，当38 - 57肽段从水性环境转移到更具亲脂性的（90%三氟乙醇）环境中时，有序结构增加，尤其是α - 螺旋结构。这些结果表明，β亚基的38 - 57区域暴露于表面，是hCG和hLH受体结合域的一个组成部分。38 - 57序列中的两亲螺旋结构区域可能以与其他几种肽类激素类似的方式促进激素与受体的相互作用。
