E21R突变增强血清环境中人α防御素5抗病原微生物活性的分子机制与生物学意义

防御素是一种内源性阳离子多肽，因抗病原微生物作用突出和不易产生耐药而有望成为新一代抗生素，但在血清等环境中防御素的抗微生物活性显著受阻，限制了其开发应用。在前一个基金资助下，我们通过体外进化与筛选获得一株在较高浓度血清中仍具有显著抗菌和抗病毒活性的人α防御素5突变体HD5-E21R（第21位谷氨酸E突变为精氨酸R）。根据HD5的空间结构和电荷分布特点，推测血清中盐离子和相关蛋白对HD5活性影响存在"位点+电荷"效应，而E21可能是外界因素干扰HD5的一个敏感位点。为此，本项目拟采用SPR、HPLC等方法，结合位点突变和空间结构分析，从氨基酸位置、电荷数量与分布、血清蛋白封阻等方面，探讨E21R突变提高HD5抗微生物活性和逃避血清干扰的分子机制，并增加相关位点突变以进一步提高HD5抗血清干扰能力和体内应用效果。旨在深入解读防御素结构-效应-环境因素影响之间的关系，为加速其开发和应用奠定基础。

Human defensins,a kind of small cationic peptides produced endogenously, are of significant interest to be developed into a new generation of antibiotics due to their specific antimicrobial properties. However, the antimicrobial activities of defensins were dramatically decreased in serum and other complicated conditions, which encumbers the exploitation and clinical application of defensins. Recently, through in vitro evolution and selection in a study supported by NSFC,we obtained a mutant of human αdefensin 5(HD5)termed HD5-E21R, with glutamic acid residue(E) substituted by arginine residue (R)at position 21. Compared with natural HD5, HD5-E21R displays much higher antibacterial and antiviral activities even in the conditions with high concentration of serum.According to the crystal structure and distribution of charged residues, we presume that E21 is a sensitive region for HD5 to be influenced by salt and serum protein during antimicrobial actions. Therefore,in this study by application of HPLC, surface plasmon resonance (SPR) assay, site mutation and crystal structure analyse, we plan to investigate the effects of E21R mutation on the changes of electric charge and distribution of HD5, as well as serum protein binding and blocking to HD5. Based on the discoveries, subsequent experiments will be carried out to search for more effective HD5 mutant by adding new potential point mutation to E21R. The aim of this study is to uncover the molecular mechanism of E21R mutation in enhancing the antimicrobial activities of HD5 in serum, so as to provide deep insights into the relationship among the space structure,antimicrobial activity and the environmental influence of defensins, and lay a foundation for the exploitation of human defensins.