抗血管生成活性的重组人proEMAPⅡ/p43蛋白缺失突变体的构建和筛选(英文)  

Construction and Screening of Truncated Mutantsof Recombinant Human Anti-angiogenicProtein proEMAPⅡ/p43

作　　者：邢玉华[1,2] 刘大涛 谭俊杰[1] 胡立德 刘刚[1] 付学奇[2] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]吉林大学生命科学学院,长春130012 [3]上药集团信谊药厂,上海201206

出　　处：《生物化学与生物物理进展》2014年第6期567-574,共8页Progress In Biochemistry and Biophysics

基　　金：supported by a grant from Shanghai Sine Pharmaceutical Laboratories Co.,Ltd,China~~

摘　　要：proEMAPⅡ（又称p43蛋白）是哺乳动物氨酰tRNA合成酶的辅助因子，近年来发现，p43具有细胞因子活性以及抗新生血管生成的作用，具有潜在的抗肿瘤疗效．p43的C端结构域即为EMAPⅡ，其结构和功能都已知，具有细胞因子和抗血管生成的双重功能．但是N端的结构还不清楚，研究表明全长的p43比EMAPⅡ具有更高的生物学活性，但是p43的结构和作用机制尚不明确．此外，作为蛋白质药物，更小的分子能够减少免疫原性和副作用，从而发挥更好的活性．本文利用生物信息学方法，对p43 N端的二级结构进行预测，并且在不破坏二级结构的前提下，构建了10个p43的缺失突变体，在体外对10个缺失突变体与全长的p43蛋白进行抗新生血管生成活性验证比较，最终我们获得了3个活性高的p43缺失突变体，并且发现N端的1～16，1～79位氨基酸和C端的264～312位氨基酸不是p43发挥抗血管生成功能所必需的，而且它们的删除使得活性位点更好地呈现并发挥活性．通过我们的研究，有助于揭示p43蛋白结构和功能的关系以及其作用机制，同时为临床筛选肿瘤治疗候选药物奠定基础．proEMAPⅡ/p43 was originally described as a scaffolding protein that is a component of the multi-aminoacyl-tRNA synthetase complex. Recently, proEMAPⅡ/p43 was found to be a cytokine as well as an endogenic anti-angiogenic protein. The p43 protein is thought to be a precursor of endothelial monocyte-activating polypeptideⅡ （EMAPⅡ）. p43 showed higher biological activity than EMAPⅡ, making it a promising anti-angiogenesis inhibitors for cancer therapy. However, the structure of p43 and its function in angiogenesis remain unknown. Here, we constructed p43-like proteins with low molecular mass and high activity. We also determined the functional domains for the anti-angiogenic activity of p43. First, we predicted the secondary structure of p43 using a bioinformatics method, and then constructed 10 p43 truncated mutants. We compared the anti-angiogenic activity of the full-length p43 with the activities of the truncated proteins. We found that all the truncated proteins inhibited the migration of endothelial cells and prevent tubule formation. The deletion of up to 79 amino acids at the N-terminus or 47 amino acids at the C-terminus of p43 increased the activity to 2～3 times that of full-length p43. We identified three p43 truncations with lower molecular mass and higher activity than the full-length p43. These findings will help improve our understanding of the structure and function of p43, which, in turn, will be helpful to the further studies on the possible clinical applications of p43-like drugs.

关 键 词：proEMAPⅡ P43 缺失突变体 抗血管生成剂 内皮细胞迁移 管腔形成 

分 类 号：Q71[生物学—分子生物学] Q936