Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A
Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A
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DOI：
10.1074/jbc.m113.539262
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发表时间：
2014-03-07
影响因子：
4.8
通讯作者：
Craik, David J.
中科院分区：
文献类型：
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作者：
Jia, Xinying;Kwon, Soohyun;Craik, David J.
背景：索尔特酶A(Sortase A，SrtA)是一种能够催化酰胺键形成的转肽酶。结果：SrtA被用来主链环化富含二硫键的多肽，包括Kalata B1、-Conooxin Vc1.1和SFTI-1。结论：srtA介导的环化反应适用于二硫键含量较高的小分子多肽。意义：srtA介导的环化反应是一种替代天然化学连接的方法，用于环化具有治疗意义的小肽。富含二硫键的环肽因其对化学、酶或热攻击的特殊稳定性而引起了人们对基于多肽的治疗药物开发的极大兴趣。特别是，它们已经被用作支架，可以在其上嫁接生物活性表位，以利用富含二硫键的环肽的良好生物物理性质。到目前为止，最常用的多肽从头到尾环化的方法是天然的化学连接。然而，近年来，酶介导的环化反应因其高效、安全和成本效益高而成为一种很有前途的新技术。山梨酸酶A(SrtA)是一种具有转肽酶活性的细菌酶。它识别C-末端的五个氨基酸基序LPXTG，并切割Thr和Gly之间的酰胺键形成硫代酰基连接的中间体。该中间体受到N端多聚甘氨酸序列的亲核攻击，在Thr和N端Gly之间形成酰胺键。在这里，我们证明了索尔特酶A可以成功地用于环化各种富含二硫键的小肽，包括环肽Kalata B1、-ConotoxVc1.1和向日葵胰蛋白酶抑制剂1。这些多肽的大小从14到29个氨基酸不等，在它们头尾的环状骨架中分别含有三个、两个或一个二硫键。我们的发现为具有治疗潜力的富含二硫键的多肽的酶环化潜在的广泛适用性提供了概念证据。
Background: Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds. Results: SrtA was used to backbone-cyclize disulfide-rich peptides, including kalata B1, -conotoxin Vc1.1, and SFTI-1. Conclusion: SrtA-mediated cyclization is applicable to small disulfide-rich peptides. Significance: SrtA-mediated cyclization is an alternative to native chemical ligation for the cyclization of small peptides of therapeutic interest.Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, -conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.