Fares F A, Levi F, Reznick A Z, Kraiem Z
Department of Biochemistry, Carmel Medical Center and the Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 34362 Israel.
J Biol Chem. 2001 Feb 16;276(7):4543-8. doi: 10.1074/jbc.M008093200. Epub 2000 Nov 16.
Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, alpha and beta. We have recently converted the hTSH heterodimer to a biologically active single chain (hTSHbeta.CTPalpha) by fusing the common alpha-subunit to the C-terminal end of the hTSH beta-subunit in the presence of a approximately 30-amino acid peptide from hCGbeta (CTP) as a linker. The hTSHbeta.CTPalpha single chain was used to investigate the role of the N-linked oligosaccharides of alpha- and beta-subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the alpha-subunit (hTSHbeta.CTPalpha(1+2)) and the other lacking the oligosaccharide chain on the beta-subunit (hTSHbeta.CTPalpha(deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSHbeta.CTPalpha wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSHbeta.CTPalpha(1+2) variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroid-stimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma.
促甲状腺激素（TSH）和促性腺激素（FSH、LH、hCG）是一类异源二聚体糖蛋白激素家族，由两个非共价连接的亚基α和β组成。我们最近通过在人绒毛膜促性腺激素β亚基（CTP）的约30个氨基酸的肽作为连接子的情况下，将常见的α亚基融合到人促甲状腺激素β亚基的C末端，将人促甲状腺激素异源二聚体转化为具有生物活性的单链（hTSHβ.CTPα）。hTSHβ.CTPα单链用于研究α和β亚基的N-连接寡糖在人促甲状腺激素分泌和功能中的作用。使用重叠PCR诱变制备了两种去糖基化变体：一种在α亚基上缺乏两条寡糖链（hTSHβ.CTPα(1+2)），另一种在β亚基上缺乏寡糖链（hTSHβ.CTPα(deg)）。单链变体在CHO细胞中表达并分泌到培养基中。在培养的人甲状腺滤泡中，缺乏寡糖链的人促甲状腺激素变体在cAMP形成和甲状腺激素分泌方面比hTSHβ.CTPα野生型效力更低。两种去糖基化变体均以剂量依赖性方式与人促甲状腺激素竞争。hTSHβ.CTPα(1+2)变体阻断了由人促甲状腺激素以及由抗体、促甲状腺免疫球蛋白刺激的cAMP形成和甲状腺激素分泌，促甲状腺免疫球蛋白是导致甲状腺功能亢进最常见原因——格雷夫斯病的病因。因此，这种变体表现为一种潜在的拮抗剂，为治疗由格雷夫斯病和分泌促甲状腺激素的垂体腺瘤引起的甲状腺毒症提供了一种新的治疗策略。
