Prolyl endopeptidases to treat Celiac Sprue

描述(由申请人提供)：这项提案的目标是设计出适合治疗腹腔炎的候选酶。乳糜泻是一种广泛流行的免疫性小肠疾病，由膳食面筋蛋白在遗传易感人群中诱发。通过破坏肠道绒毛，Celiac Sprue会导致一系列症状，包括腹泻、铁和维生素缺乏、骨密度降低、生长迟缓、疲劳、贫血和骨质疏松症，增加患肠癌和淋巴瘤的风险。目前唯一可用的治疗方法是完全避免面筋，这是非常困难的，因为面筋蛋白几乎存在于所有谷物中。 从面筋中提取的富含蛋白质的富含脯氨酸的多肽已被认为是腹腔病人的免疫毒剂。初步研究结果表明，细菌内多肽可降解这些多肽，因此可能是一种有效的治疗方法。然而，自然产生的内肽酶都有缺点，阻碍了它们的治疗应用。 本项目的目的是使用一种基于序列活性的蛋白质工程技术来优化治疗腹腔炎的Pro内肽酶的活性。酶的活性将增加，以获得全谱面筋衍生的蛋白水解性抗性多肽，以及口服给药的最佳pH曲线。简而言之，实验方法如下。(1)使用系统发育和结构建模来确定可能改善这些所需酶性质的氨基酸替换。(2)设计合成特异性酶变异体。(3)在适宜的条件下，用高效液相色谱法准确测定各变异体对面筋蛋白及其免疫毒素的降解能力。(4)使用活性测试的结果来推导序列-活性关系，从而设计进一步改进的变体。

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.