The Comparison Of Anti-angiogenic Activities Among The Lj-RGD3 Toxin Protein And Its' Five Forms Of Mutation Proteins

Lj-RGD3 was a RGD toxin protein with 3 RGD motifs from the saliva gland of Lampetra japonica, which shares homologous with a Histidine-rich glycoprotein (HRG), and both RGD-toxin proteins and HRG have antiangiogenic activities with different targets. In order to study the relationship between the function and the structure of Lj-RGD3, we synthesized the gene of the RGD delete mutations: the mutation which was deleted all three RGD motifs of Lj-RGD3 was named Lj-112, the mutation which kept the first RGD motif was named Lj-113, the mutation which kept the second RGD motif was named Lj-114, the mutation which kept the third RGD motif was named Lj-115, and the mutation which the all 3 RGD motifs were replaced by 3 KGD was named Lj-116. After constructed above genes to pET23b, we transformed the recombination plasmids into Ecoil BL21. Furthermore, the recombinant proteins expression was induced by IPTG and purified by His-affinity chromatography. The MTT assay showed that, both rLj-RGD3 and the five forms of mutation protein rLj-112, rLj-113, rLj-114, rLj-115 and rLj-116 inhibited bFGF-induced proliferation of ECV304 cells in a dose-dependent manner with IC50 at 0.889μmol/L, 0.160μmol/L, 0.215μmol/L, 0.143μmol/L, 0.150μmol/L and 0.529μmol/L, respectively. For in vivo assay, we examined the abilities of rLj-RGD3 and the five forms of mutation protein on anti-angiogenesis in chicken chorioallantoic membrane (CAM). The result demonstrated that all above proteins inhibited the angiogenesis in chick CAM, and rLj-112 was more active than others. In order to determine the effect of rLj-RGD3 and the five forms of mutation protein on ECV304 cells migration toward bFGF, we used Transwell containing insert filter. All of rLj-RGD3 rLj-112, rLj-113, rLj-114, rLj-115 and rLj-116 showed significant inhibition on ECV304 cells migration, the inhibition rates were 50%,63%,42%,65%,70% and 40%, respectively. In the invasion assay, the Matrigel and Transwell were used to imitate environment in vivo. The results of invasion assay revealed that, rLj-RGD3 and the five forms of mutation protein significantly inhibited bFGF induced invasion of ECV304 cells and rLj-112 was more active than others. ELISA assay of integrin-linked kinase (ILK) showed that, all of above proteins down-regulated ECV304 cell expression of ILK1. Taken together, these results suggested that all of above proteins have the activity of anti-angiogenesis. The fact of rLj-112 was more active than rLj-RGD3 in anti-angiogenesis indicate that, the function of rLj-112 are likely with HRG and the signal pathway of anti-angiogenesis of rLj-RGD3 and rLj-112 are differently.