A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia.
Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia.
Int J Mol Sci. 2022 Jun 23;23(13):7001. doi: 10.3390/ijms23137001.
Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest efficiently hydrolyzes gliadins. The main digestive peptidase of is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.
小麦醇溶蛋白含有大量富含谷氨酰胺和脯氨酸的肽，这些肽不能被人体消化肽酶水解，可在易感性人群中引起自身免疫性乳糜泻和其他形式的麸质不耐受。能有效切割此类免疫原性肽的肽酶可用于酶治疗。储存产品昆虫 能有效地水解醇溶蛋白。 的主要消化肽酶是组织蛋白酶 L，它属于木瓜 C1 家族，具有谷氨酰胺后切割活性。我们描述了 重组原酶（rpTcCathL1，NP_001164001）的分离和表征，该原酶在 细胞中表达。原酶的激活是通过自动催化处理进行的。研究了反应混合物中 pH 值和原酶浓度对处理的影响。成熟酶在 pH5.0 到 9.0 的范围内保持高活性，并在 20°C 时在 4.0 到 8.0 的范围内表现出高 pH 稳定性。根据天然条件下的电泳迁移率、在不同 pH 值下的活性和稳定性、对各种抑制剂的敏感性以及底物特异性对该酶进行了表征，并证明了其对 8-、10-、26-和 33-mer 免疫原性醇溶蛋白肽的水解作用。我们的结果表明，rTcCathL1 是一种有效的肽酶，可用于开发用于各种类型的麸质不耐受的酶治疗药物。
