层黏连蛋白α4链中αvβ3整合素结合肽的精细鉴定与功能分析

Identification of interacting motif of alphavbeta3 integrin in LNα4 and its function analysis

目的利用点突变、缺失突变及功能实验确定αvβ3整合素在已鉴定人层黏连蛋白α4链(human laminin alpha4,LNα4)G19肽段中的最小结合位点及关键氨基酸残基。方法采用同源序列比较确定G19肽中的保守氨基酸残基,点突变确定其中的关键氨基酸残基,缺失突变确定αvβ3整合素结合的最小肽段,并通过人脐静脉内皮细胞(humanumbilical vein endothelial cells,HUVEC)管样结构形成实验及鸡胚尿囊膜(chick chorioallantoic membrane,CAM)实验验证鉴定肽的生物学功能。结果同源性比较分析发现,G19肽中共存在10个保守性氨基酸残基位点。点突变分析发现第4位的I、第10位的H及第16位的Y是αvβ3整合素结合的关键位点,缺失突变分析确定G1124-1137肽段(G14肽)是αvβ3整合素结合的最小肽段,合成的G14肽可以浓度依赖性地促进HUVEC管样结构的形成及CAM的血管生成。结论成功鉴定了αvβ3整合素在LNα4中的最小结合位点及关键氨基酸残基。所鉴定的肽段具有促HUVEC成管样结构及促CAM血管生成的生物学功能。

Objective To identify the binding motif of alphavbeta3 integrin in human laminin alpha 4 subunit （LNα4）. Methods The conserved amino acid residues were determined by blast analysis based on G19 peptide sequence （G1121-1139）. The key sites were identified by single-site mutant and the binding motif was identified by deletion mutant. The function of identified motif was analyzed by tube-like structure formation of human umbilical vein endothelial cells （HUVECs） in vitro and angiogenesis assay on chick chorioallantoic membrane in vivo. Results Total 10 conserved amino acid residues were found in G19 peptide through blast analysis, and 3 key sites binding with alphavbeta3 integrin were identified by single-site mutant. The shortest fragment between G1124-1137 was identified by deletion mutant and fragment promoted tube-like structure formation by hUVECs and angiogenesis on CAM in a dose-dependent manner. Conclusion The key sites and the shortest fragment binding with alphavbeta3 integrin in LNα4 are identified successfully, which provides the evidence for the function of LNα4 in illness.