Y111是维持血管内皮细胞生长抑制因子生物活性的关键氨基酸

The structure-function relationship analysis of VEGI: Y111 is an important residue in biological activity

目的血管内皮细胞生长抑制因子(Vascu lar endothe-lial cell growth inh ib itor,VEG I)是近年来发现的一类TNF家族新成员,具有抑制内皮细胞增殖与新生血管生成的作用。但目前其结构功能关系研究尚为空白,该文研究VEG I空间结构上重要残基的作用极其对生物活性的影响。方法采用定点突变技术突变了四个关键氨基酸E45R,G47A,Y111F,Y111T,构建了表达载体并在大肠肝菌中表达及纯化,采用人脐静脉内皮细胞增殖和鸡胚尿囊膜血管生成实验研究了四个突变体的活性。结果E45R突变体的活性显著下降,G47A突变体的活性略低于野生型VEG I,Y111F,Y111T突变体的活性比野生型VEGI下降10倍以上。结论VEGI的第111位氨基酸Y是发挥其功能的关键氨基酸,可能与受体形成直接结合,且氨基酸Y上的苯环与酚羟基均对其活性的发挥具重要作用;第45位氨基酸E对其活性的发挥也具重要作用,而第47氨基酸对其活性的发挥可能不具重要作用。

Aim Vascular endothelial cell growth inhibitor（VEGI） is a recently discovered novel member of the TNF superfamily, which is expressed predominantly in endothelial cells. As an endothelial cell-specific negative regulator of angiogenesis, the relationship between structure and function of VEGI is not understood at present. Methods In order to explore the functional key amino acids of VEGI, four mutants of VEGI（E45→R,G47→A,Y111→F, Y111→T） were construced by site-directed mutagenesis, and recombinant proteins were generated from E. coli. Four mutant proteins behaved similar to the wild type VEGI in various physico-chemical assays. The proliferation of HUVEC and chick choriallantic membrane assay were performed to study the activity of four mutants. Results The mutant E45→R significantly decreased the biological activity, and the mutant G47→ A caused a slight drop on activity, but the mutants Y111→F, Y111→T almost completely abolished biological activity. Conclusion It suggests that Y111 is an important residue in biological activity, which may play a direct role in receptor recognition. Moreover, the tyrosine ring and hydroxy group of the amino acid are important determinant of biological activity. Additionally, E45 also plays an important role in biological activity of VEGI.