日本七鳃鳗Arg-Gly-Asp毒素蛋白Lj-RGD3野生型与RGD全缺失突变体Lj-112的抗血管新生作用

Anti-angiogenic activities of Lj-RGD3 toxin protein from Lampetra japonica and its mutation protein Lj-112 without RGD motifs

七鳃鳗Arg-Gly-Asp(RGD)毒素肽Lj-RGD3与富含组氨酸糖蛋白HRG具有序列同源性,而RGD毒素蛋白及HRG都具有抑制血管新生的活性,但作用靶点不同。为研究Lj-RGD3结构与功能的关系,对野生型Lj-RGD3及其RGD全缺失突变体Lj-112进行了抗血管新生功能研究。将3个RGD模体的全缺失突变体基因Lj-112全序列合成后构建于pET-23b载体,对野生型Lj-RGD3及突变体Lj-112蛋白进行IPTG诱导表达,重组蛋白经组氨酸亲和层析纯化;采用MTT法测定野生型和突变体蛋白对人脐静脉内皮细胞ECV304细胞增殖的抑制作用;采用Transwell细胞培养板及人工基质膜Matrigel模仿体内环境研究rLj-RGD3与rLj-112对ECV304细胞迁移及浸润的抑制作用;运用鸡绒毛尿囊膜(CAM)模型检测rLj-RGD3与rLj-112对血管新生的抑制作用;ELISA法检测两种蛋白对整合素连接激酶ILK-1表达的影响。经组氨酸亲和层析获得了分子量约为15 kDa的rLj-RGD3及rLj-112纯化蛋白。MTT实验表明,rLj-RGD3与rLj-112均以剂量依赖方式抑制bFGF诱导的ECV304细胞增殖,rLj-RGD3的IC50为0.889μmol/L,rLj-112的IC50为0.160μmol/L。迁移及浸润结果表明,Lj-RGD3与Lj-112均能抑制ECV304细胞的迁移,抑制率分别为50%和63%;而以bFGF为趋化剂的ECV304细胞穿透Matrigel的浸润行为均明显受到抑制,且Lj-112的抑制功能强于Lj-RGD3。CAM血管新生实验结果表明,rLj-RGD3与rLj-112均能抑制CAM的血管新生,且同等剂量条件下rLj-112的作用强于rLj-RGD3。ILK-1的ELISA实验结果表明,rLj-RGD3与rLj-112均能下调ECV304细胞ILK-1的表达。野生型rLj-RGD3与其RGD全缺失突变体蛋白rLj-112均具有抑制血管新生功能,且突变体Lj-112的活性高于野生型Lj-RGD3,这说明Lj-112为HRG功能蛋白,而野生型Lj-RGD毒素蛋白与HRG序列的同源性并未使白其抗血管新生功能得到协同加强,二者的抗血管新生功能涉及了不同的信号通路。

Arg-Gly-Asp（RGD）-toxin protein Lj-RGD3 of Lampetra japonica shares homologous with a Histidine-rich glycoprotein（HRG）,and both RGD-toxin protein and HRG have antiangiogenic activities with different targets.To study the relationship between the function and the structure of Lj-RGD3,we studied the anti-angiogenic characteristics of both Lj-RGD3 and the mutation named Lj-112 of which three RGD motifs of Lj-RGD3 were deleted.We synthesized the gene of Lj-112,constructed it to the plasmid pET23b,and expressed the recombinant proteins in Escherichia coli BL21.Both recombinant proteins with the C-terminal his-tag were 15 kDa soluble proteins.Then we purified rLj-RGD3 and rLj-112 using the His-Bind affinity chromatography.To examine the effect of both proteins on bFGF-induced proliferation of ECV304 cell,we carried out the 3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide（MTT） assays.For cell migration and invasion assays,we used Transwell containing insert filter and Matrigel to imitate the in vivo environment.To examine whether both proteins were capable of interrupting the angiogenesis in vivo,we used the chick chicken embryonic chorioallantoic membrane（CAM） as an angiogenesis model.We used Integrin-linked kinase1（ILK1） ELISA method to study functionary mechanisms of rLj-RGD3 and rLj-112.Both rLj-RGD3 and rLj-112 inhibited bFGF-induced proliferation of ECV304 cells in a dose-dependent manner with IC50 at 0.889 μmol/L and 0.160 μmol/L,respectively.The results of migration and invasion assays revealed that both rLj-RGD3 and rLj-112 showed significant inhibition on bFGF induced migration and invasion of ECV304;and rLj-112 was more active than rLj-RGD3.The result of CAM angiogenesis assay demonstrated that both proteins inhibited the angiogenesis in chick CAM,and rLj-112 was more active than rLj-RGD3.ELISA assay of ILK1 showed that both rLj-RGD3 and rLj-112 down-regulated ILK1 expression of ECV304 cell.The fact of rLj-112 was more active than rLj-RGD3 on anti-angiogenesis indicate that rLj-112 was likely with histidine-rich glycoprotein（HRG）,and the factor of sequence homologous between rLj-RGD3 and HRG cannot enhance antiangiogenic activities of rLj-RGD3,the signal pathway of anti-angiogenesis of rLj-RGD3 and rLj-112 are differently.