Chugunov Anton O, Dvoryakova Elena A, Dyuzheva Maria A, Simonyan Tatyana R, Tereshchenkova Valeria F, Filippova Irina Yu, Efremov Roman G, Elpidina Elena N
M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.
L.D. Landau School of Physics, Moscow Institute of Physics and Technology (State University), 141701 Dolgoprudny, Russia.
Int J Mol Sci. 2023 Aug 2;24(15):12369. doi: 10.3390/ijms241512369.
Roughly 1% of the global population is susceptible to celiac disease (CD)-inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect -cathepsin L-hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2-4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L-loss of the catalytically competent rotameric state of one of the active site residues, His 275. To "fix" the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people.
全球约1%的人口易患乳糜泻（CD），这是一种遗传性自身免疫性小肠炎症，由对小麦、黑麦和大麦籽粒中存在的醇溶蛋白不耐受引起，小麦中的这种蛋白称为麸质。传统治疗方法是终身无麸质饮食，这种方法受限且成本高昂。另一种方法是基于开发并口服有效的肽酶，这些肽酶可在胃中降解具有免疫原性的富含脯氨酸和谷氨酰胺的醇溶蛋白肽，而这些肽是肠道严重反应的原因。在先前的研究中，我们已经确定一种昆虫的主要消化肽酶——组织蛋白酶L——在谷氨酰胺残基后水解免疫原性醇溶蛋白，但在人胃的极端酸性环境（pH 2 - 4）中不稳定，不能用作消化辅助剂。在这项工作中，我们使用分子动力学模拟，发现了组织蛋白酶L在pH值下不稳定的可能原因——活性位点残基之一的组氨酸275失去了具有催化活性的旋转异构体状态。为了“固定”该残基的正确取向，我们设计了一个V277A突变变体，它扩展了肽酶在酸性环境中的稳定性范围，同时保留了其大部分活性。我们建议将这种蛋白质作为开发口服药物制剂的主要麸质酶，用于对抗易感人群的乳糜泻和麸质不耐受。
