Prolyl endopeptidases to treat Celiac Sprue

描述（由申请人提供）：本提案的目标是设计适合治疗乳糜泻的候选酶。乳糜泻是一种在遗传易感个体中由膳食麸质引起的广泛流行的小肠免疫疾病。通过破坏肠绒毛，乳糜泻会引起一系列症状，包括腹泻、铁和维生素缺乏、骨密度降低、生长发育迟缓、疲劳、贫血和骨质疏松、肠癌和淋巴瘤风险增加。目前治疗这种疾病的唯一方法是完全不吃谷蛋白，这是非常困难的，因为几乎所有的谷物中都含有谷蛋白。

DESCRIPTION (provided by applicant): The goal of this proposal is to engineer enzyme candidates suitable for the treatment of Celiac Sprue. Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten in genetically susceptible individuals. By damaging the intestinal villi, Celiac Sprue causes a range of symptoms, including diarrhea, iron and vitamin deficiencies, reduced bone density, stunted growth, fatigue, anemia and osteoporosis, increased risks of intestinal cancer and lymphomas. The only treatment for the disease currently available is complete avoidance of gluten, which is very difficult because gluten proteins are present in almost all grains. Proteolytically resistant proline-rich peptides from gluten have been implicated as the immunotoxic agent for Celiac patients. Preliminary results have indicated that bacterial prolyl endopeptidases can degrade these peptides and may thus be an effective treatment for celiac patients. However the naturally occurring endopeptidases all have shortcomings that prevent their therapeutic application. The aim of this project is to use a sequence-activity based protein engineering technology to optimize the activities of prolyl endopeptidases for treatment of Celiac Sprue. Enzymes will be obtained with increased activity towards a full spectrum of gluten-derived proteolytically-resistant peptides, and optimal pH profiles for oral administration. Briefly the experimental methods are as follows. (1) Use phylogenetic and structural modeling to identify amino acid substitutions likely to improve these desired enzyme properties. (2) Design and synthesize specific enzyme variants. (3) Use HPLC to accurately measure the ability of each variant to degrade gluten and its immunotoxic peptides under appropriate conditions. (4) Use the results from activity testing to derive sequence-activity relationships and thus design further improved variants.