The Chemistry and Biology of Human Relaxin‐3
The Chemistry and Biology of Human Relaxin‐3
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DOI：
10.1196/annals.1282.008
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发表时间：
2005-05
影响因子：
5.2
通讯作者：
G. Tregear;R. Bathgate;S. Layfield;T. Ferraro;A. Gundlach;Sherie Ma;F. Lin;Nicola F Hanson;R. Summers;J. Rosengren;D. Craik;J. Wade
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文献类型：
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作者：
G. Tregear;R. Bathgate;S. Layfield;T. Ferraro;A. Gundlach;Sherie Ma;F. Lin;Nicola F Hanson;R. Summers;J. Rosengren;D. Craik;J. Wade
摘要：最近在基因组学数据库搜索期间发现了胰岛素超家族的人松弛素亚类的新成员，并命名为松弛素-3。与人松弛素-1和松弛素-2一样，松弛素-3预计由两条链结构和三个二硫键组成，其排列与胰岛素相同。为了对肽进行详细的生物物理和生物学表征，进行了其化学合成。然而，与人松弛素-1和松弛素-2相反，松弛素-3不能通过单个链的简单组合成功制备，因此需要求助于使用区域选择性二硫键形成策略。固相合成分离的、选择性S-保护的A和B链，然后对其进行纯化，随后逐步形成三个二硫化物中的每一个，从而成功获得人松弛素-3。全面的化学表征证实了合成产物的正确链取向和完整性。松弛素-3被发现在体外结合并激活天然松弛素受体，并在体内通过中枢松弛素受体刺激饮水。最近的研究表明，松弛素-3在体外会结合并激活人LGR 7，而不是LGR 8。二级结构分析表明，它采用了比松弛素-1或松弛素-2更少的有序确认，反映了前者存在更大比例的非螺旋形成氨基酸。NMR光谱和模拟退火计算用于确定松弛素-3的三维结构，并确定人类松弛素之间的关键结构差异。
Abstract: A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin‐3. Like human relaxin‐1 and relaxin‐2, relaxin‐3 is predicted to consist of a two‐chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin‐1 and relaxin‐2, however, relaxin‐3 could not be successfully prepared by simple combination of the individual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S‐protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin‐3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin‐3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin‐3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin‐1 or relaxin‐2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three‐dimensional structure of relaxin‐3 and to identify key structural differences between the human relaxins.