Rationally engineered prolyl endopeptidases from Sphingomonas capsulata with improved hydrolytic activity towards pathogenic peptides of celiac diseases
Rationally engineered prolyl endopeptidases from Sphingomonas capsulata with improved hydrolytic activity towards pathogenic peptides of celiac diseases
复制标题
来自荚膜鞘氨醇单胞菌的合理工程脯氨酰内肽酶，对乳糜泻致病肽具有改善的水解活性
DOI：
10.1016/j.ejmech.2020.112499
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发表时间：
2020
影响因子：
6.7
通讯作者：
Zheng Yongxiang
中科院分区：
文献类型：
--
作者：
Xiao Bin;Zhang Chun;Song Xiaotong;Wu Miao;Mao Jianping;Yu Rong;Zheng Yongxiang
乳糜泻影响约1%的人口，是全球主要的公共卫生问题。它被谷蛋白衍生肽所取代，谷蛋白衍生肽具有异常高的脯氨酸-谷氨酰胺基序含量，并且对胃肠道消化酶的蛋白水解具有高度抗性。乳糜泻的唯一治疗方法是严格的，终身坚持无麸质饮食，这是有效的，但昂贵，难以维持。因此，迫切需要用于乳糜泻的新型非饮食疗法。谷蛋白降解酶是有前途的非膳食治疗，并且一些酶已经在临床前或临床研究中进行了研究。来自鞘氨醇单胞菌的脯氨酰内肽酶（SC PEP）和一种谷氨酰胺特异性内切蛋白酶（来自大麦的EP-B2）的组合被称为宽谷蛋白酶，在II期临床试验中显示出不足的益处，可能是因为其在胃环境中的酶活性低。因此，提高酶活性对SC PEP的临床应用至关重要。酶活性可以使用计算机辅助的合理蛋白质设计工具来增强。在本研究中，我们结合分子对接和分子动力学模拟来合理设计SC PEP突变体，并对其活性进行实验评价。我们鉴定了比活性增加高达90-103%和催化速率增加高达80-202%的突变体。我们研究了这些突变体增强酶活性的机制，发现SC PEP的β-螺旋桨结构域和催化结构域的构象转变对酶活性至关重要，这种转变受到催化结构域和结构域界面残基的影响;底物Pro与含氧阴离子空穴之间较短的距离也是提高SC PEP催化活性的关键。我们的研究结果提供了有用的信息，合理设计的高活性SC PEP，以加速开发酶治疗候选人的腹腔疾病。
Celiac disease affects approximately 1% of the population and is a major public health problem worldwide. It is trigged by gluten-derived peptides, which have unusually high proline-glutamine motif content and are highly resistant to proteolysis by digestive enzymes of the gastrointestinal tract. The only treatment for celiac disease is strict, lifelong adherence to a gluten-free diet, which is effective but costly and difficult to maintain. Therefore, novel non-dietary therapies for celiac disease are urgently needed. Gluten-degrading enzymes are promising non-dietary treatments, and some enzymes have been investigated in preclinical or clinical studies. A combination of prolyl endopeptidase from Sphingomonascapsulata(SC PEP) and a glutamine-specific endoprotease (EP-B2 from barley) known as latiglutenase showed insufficient benefits in phase II clinical trials, likely because of its low enzyme activity in the gastric environment. Therefore, improving enzyme activity is essential for the clinical application of SC PEP. Enzyme activity can be enhanced using computer-aided rational protein design tools. In this study, we combined molecular docking and molecular dynamics simulation to rationally design SC PEP mutants and experimentally evaluated their activities. We identified mutants with up to 90–103% increases in specific activity and up to 80–202% increases in the catalytic rate. We have investigated the mechanism underlying the enhanced activity of these mutants, and found that a conformational transition of the β-propeller domain and catalytic domain of SC PEP was important for enzyme activity, and this transition was affected by residues in the catalytic domain and at the domain interface; a shorter distance between the substrate Pro and the oxyanion holes was also crucial for improving SC PEP catalytic activity. Our results provide useful information for the rational design of highly active SC PEPs to accelerate the development of enzyme therapeutics candidates for Celiac disease.