Prentice L, Sanders J F, Perez M, Kato R, Sawicka J, Oda Y, Jaskolski D, Furmaniak J, Smith B R
FIRS Laboratories, RSR Ltd, Llanishen, Cardiff, United Kingdom.
J Clin Endocrinol Metab. 1997 Apr;82(4):1288-92. doi: 10.1210/jcem.82.4.3895.
An in vitro transcription/translation (TnT) system was used to produce 35S-labeled full-length TSH receptor (TSHR) and TSHR extracellular domain (TSHRex). The interaction of the labeled proteins with TSHR autoantibodies in Graves' sera was then studied using an immunoprecipitation assay. In the assay, 35S-labeled TSHR or TSHRex were incubated with test sera, and any immune complexes formed were precipitated with protein A-Sepharose (in the case of mouse monoclonal antibodies, antimouse IgG-agarose was used). Rabbit antibodies to the TSHR and a mouse monoclonal antibody precipitated as much as 50% of the 35S-labeled TSHR preparations compared with about 2% for normal rabbit serum and 4% for a control monoclonal antibody. However, none of 34 Graves' sera (TSHR autoantibody levels ranging from 14-95% inhibition of [125I]TSH binding) were able specifically to immunoprecipitate 35S-labeled TSHR or TSHRex. These negative findings were confirmed by analysis of the immunoprecipitates on SDS-PAGE followed by autoradiography. Our results indicate that the TnT system is not useful for producing labeled TSHR preparations that can bind TSHR autoantibodies well. This is in contrast to TnT produced 35S-labeled glutamic acid decarboxylase, thyroid peroxidase, and 21-hydroxylase, which react well with their respective autoantibodies. One main difference between these 3 autoantigens and the TSHR is that the receptor is highly glycosylated, and this extensive glycosylation may be of critical importance for correct folding of the receptor. Consequently, the inability of the TnT system to glycosylate proteins could explain in part why TnT-produced 35S-labeled TSHR and TSHRex do not bind TSHR autoantibodies.
利用体外转录/翻译（TnT）系统生成35S标记的全长促甲状腺激素受体（TSHR）和TSHR胞外结构域（TSHRex）。然后使用免疫沉淀试验研究标记蛋白与格雷夫斯病血清中TSHR自身抗体的相互作用。在该试验中，将35S标记的TSHR或TSHRex与测试血清一起孵育，形成的任何免疫复合物用蛋白A-琼脂糖沉淀（对于小鼠单克隆抗体，使用抗小鼠IgG-琼脂糖）。与正常兔血清的约2%和对照单克隆抗体的4%相比，兔抗TSHR抗体和一种小鼠单克隆抗体沉淀了多达50%的35S标记的TSHR制剂。然而，34份格雷夫斯病血清（TSHR自身抗体水平对[125I]TSH结合的抑制范围为14%-95%）均不能特异性免疫沉淀35S标记的TSHR或TSHRex。通过SDS-PAGE分析免疫沉淀物并随后进行放射自显影证实了这些阴性结果。我们的结果表明，TnT系统对于生成能与TSHR自身抗体良好结合 的标记TSHR制剂无用。这与TnT产生的35S标记的谷氨酸脱羧酶、甲状腺过氧化物酶和21-羟化酶形成对比，后者能与其各自的自身抗体良好反应。这三种自身抗原与TSHR之间的一个主要区别在于该受体高度糖基化，这种广泛的糖基化可能对受体的正确折叠至关重要。因此，TnT系统无法对蛋白质进行糖基化可能部分解释了为什么TnT产生 的35S标记的TSHR和TSHRex不与TSHR自身抗体结合。
