Suda S A, Gettins P G, Patston P A
Department of Oral Medicine and Diagnostic Sciences, Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, 60612, USA.
Arch Biochem Biophys. 2000 Dec 1;384(1):31-6. doi: 10.1006/abbi.2000.2110.
Thyroxine binding globulin (TBG) is the major carrier of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in plasma. TBG is member of the serpin family of proteins although it has no proteinase inhibitory activity. In this study we show that TBG has properties typical of a metastable serpin and provide evidence that occupancy of the hormone binding site alters the conformation of the reactive center loop. After reactive center loop cleavage by endoproteinase Asp-N or neutrophil elastase the protein became more stable to guanidine hydrochloride denaturation compared to the native protein, as a result of loop insertion. In addition, incubation of the native protein with a reactive center loop peptide, caused a change in mobility on a native gel. This is consistent with the idea that thyroxine binding globulin is able to form a binary complex with the peptide as a result of beta-sheet A expansion. To assess the effect of cleavage and loop insertion on the hormone binding site we used the specific binding of a fluorophore, 1,8-anilinonaphthalene sulfonic acid (ANS). Loop insertion itself had no effect on ANS affinity, but cleavage with elastase at the P4'-P5' bond caused a reduction in affinity, presumably because this cleavage site is located within the hormone binding site. These data support the concept that cleavage of TBG by proteinases released in inflammation is a mechanism to deliver thyroid hormones to target tissues. A linkage between the occupancy state of the hormone binding site and the conformation of the reactive center loop was indicated by the observation that binding of T3 to native TBG reduced proteolytic susceptibility by both endoproteinase Asp-N and elastase.
甲状腺素结合球蛋白（TBG）是血浆中甲状腺激素三碘甲状腺原氨酸（T3）和甲状腺素（T4）的主要载体。TBG是丝氨酸蛋白酶抑制剂（serpin）家族的成员，尽管它没有蛋白酶抑制活性。在本研究中，我们表明TBG具有亚稳态serpin的典型特性，并提供证据表明激素结合位点的占据会改变反应中心环的构象。在用内肽酶Asp - N或中性粒细胞弹性蛋白酶切割反应中心环后，与天然蛋白相比，由于环插入，该蛋白对盐酸胍变性更稳定。此外，天然蛋白与反应中心环肽一起孵育会导致在天然凝胶上的迁移率发生变化。这与甲状腺素结合球蛋白能够由于β - 折叠A的扩展而与该肽形成二元复合物的观点一致。为了评估切割和环插入对激素结合位点的影响，我们使用了荧光团1,8 - 苯胺基萘磺酸（ANS）的特异性结合。环插入本身对ANS亲和力没有影响，但在P4' - P5'键处用弹性蛋白酶切割会导致亲和力降低，推测是因为该切割位点位于激素结合位点内。这些数据支持了这样的概念，即炎症中释放的蛋白酶对TBG的切割是将甲状腺激素递送至靶组织的一种机制。T3与天然TBG的结合降低了内肽酶Asp - N和弹性蛋白酶的蛋白水解敏感性，这一观察结果表明激素结合位点的占据状态与反应中心环的构象之间存在联系。
