Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.
Amino Acids. 2019 Apr;51(4):619-626. doi: 10.1007/s00726-018-02694-y. Epub 2019 Jan 2.
The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural L-amino acids or the corresponding unnatural D-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, L-Ala or L-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, D-Ala or D-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, D-Ala or D-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family's activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.
胰岛素超家族是一组同源蛋白，根据其不同的受体进一步分为胰岛素家族和松弛素家族。所有胰岛素超家族成员都含有三个绝对保守的二硫键和紧随第一个 B 链半胱氨酸的非手性 Gly 残基。通过用天然 L-氨基酸或相应的非天然 D-氨基酸取代该胰岛素家族中保守 Gly 残基的功能已被研究。然而，对于松弛素家族成员尚未进行此类分析。在本研究中，我们对嵌合松弛素家族肽 R3/I5 的保守 B11Gly 进行了手性诱变，该肽是受体 RXFP3 和 RXFP4 的有效激动剂。与对胰岛素家族折叠能力的影响相似，L-Ala 或 L-Ser 取代完全消除了重组 R3/I5 前体的体外重折叠；然而，D-Ala 或 D-Ser 取代对半合成 R3/I5 前体的重折叠没有不利影响，表明保守 Gly 残基控制着松弛素家族成员的折叠能力。与对胰岛素家族活性的影响相反，D-Ala 或 D-Ser 取代对成熟 R3/I5 对 RXFP3 和 RXFP4 的结合和激活效力没有不利影响，表明保守 Gly 残基与松弛素家族的活性无关。本研究首次揭示了保守 B 链 Gly 残基对松弛素家族肽的功能，为其对胰岛素超家族折叠能力和活性的贡献提供了全面的认识。
