与SW结合的山羊溶酶体α-甘露糖苷酶结构域重置及功能确证

Locoweeds are main poisonous plants on the western lands in China. The indolizidine alkaloid swainsonine (SW), as a toxic component in the locoweeds, inhibits the function of α-mannosidase (α-AMA) resulting in animal poisoning and death. Which has produced huge economic losses every year. However, there is lack research work about the mechanism of SW inhibition α-AMA, and has not find out the miracle drug as yet. The site-directed mutagenesis to reduce the sensitivity of Capra hircus lysosomal α-mannosidase (chLAM) to SW has been successfully completed in the early stage of the project. The active site of chLAM was determined. But chLAM expression level is low. In this work, truncation expressing will be performed to high conservative level subunit fragments chA、chC、chD、chT、chD222 and chD220. Selection subunit fragments with high expressive activity to decrease the susceptibility of α-AMA to SW by performing the point mutation and replacement of domain binding SW. The replaced of domain on truncated chLAM is completed. Obtaining the mutant chLAM with high expression and low susceptibility to SW. Confirming active center of wild type chLAM and function of domain. Clarifying structure-activity relationship of chLAM. Finally, the molecular mechanism about SW inhibits α-mannosidase and the structure activity relationship will be elucidated. The preventive experimentations of the mutant chLAM to the cell and toxic animal will be done to ensure the function of detoxification. Which will provide the foundation for development of special effect medicine, and provide scientific foundation to solve the problem of locoweed poisoning in animal.

疯草是我国西部草场的主要有毒植物，其毒性成分苦马豆素（SW）通过抑制α-甘露糖苷酶（α-AMA）的活性引起动物中毒死亡，每年造成数以亿计的经济损失。由于缺乏疯草中毒的分子机制研究，无法研制出特效治疗药物。项目组成功完成了降低山羊α-甘露糖苷酶（chLAM）对SW敏感性的定点突变，确定了chLAM的活性位点，但存在表达量低的不足；本项目对chLAM含有活性位点的亚基片段chA、chC、chD、chT、chD222和chD220进行截短表达，选择高表达活性的亚基片段，进行降低对SW敏感性的定点突变，完成截短型chLAM的结构域重置，获得高表达量、对SW的敏感性降低的chLAM，确证野生型chLAM活性中心和结构域功能，阐明chLAM构效关系，揭示SW抑制α-AMA的分子机制；通过突变型chLAM对细胞和动物的保护性试验，检测其解毒功能，为研制特效药，防治动物疯草中毒病提供科学依据。

[背景]疯草是我国西部草场的主要有毒植物，其毒性成分苦马豆素（SW）通过抑制α-甘露糖苷酶的活性引起动物中毒死亡，每年造成数以亿计的经济损失。由于缺乏疯草中毒的分子机制研究，无法研制出特效治疗药物。[研究内容]项目在前期完成山羊α-甘露糖苷酶（chLAM）的表达，确定其的活性位点的基础上，完成chLAM的截短表达，降低截短型chLAM对SW敏感性的定点突变，检测突变型chLAM解毒的功能。[重要结果]本项目对chLAM含有活性位点的亚基片段chA、chC、chD、chT、chD222和chD220进行截短表达，chT、chD222和chD220截短表达产物都具有酶活性。对野生型chLAM和截断型chT片段进行了降低对SW敏感性的定点突变，完成截短型chLAM的结构域重置，获得对SW的敏感性降低的chLAM；细胞保护性试验发现，突变后的山羊乳腺上皮细胞产生对SW敏感性低的突变型chLAM，SW染毒对突变后的山羊乳腺上皮细胞损伤明显减小。[关键数据] 确证野生型chLAM活性中心由A、C、D三个链上大约9个氨基酸构成，TrpA28和TyrD58突变为结构简单的Gly，可以使SW与chLAM的作用减弱，阐明了chLAM构效关系。[科学意义]这些结果为深入研究SW抑制α-AMA的分子机制，研制特效药，防治动物疯草中毒病提供科学依据。