Study On The Function And Mechanism Of Action Of The RLj-RGD3 Toxin Protein And The Mutations Of It

rLj-RGD3 is RGD toxin peptide from the oral gland secretion protein of Lampetra japonica containing three RGD?Arg-Gly-Asp?motifs and similar structural domains with a Histidine-rich glycoprotein?HRG?.The RGD toxin protein and HRG are both provided with the activity of inhibiting angiogenesis and anti-tumor,but by different targets.In order to research the relationship between function and the structure of the wild-type rLj-RGD3 with three RGD motifs and similar structural domains with a Histidine-rich glycoprotein?HRG?.Preliminary work showed that the wild-type rLj-RGD3 exhibited antitumor activity.we have synthesized the mutants which knockout of the RGD gene or His gene,rLj-112 was the mutation which knockout of all three RGD motifs of the wild-type rLj-RGD3,rLj-113was the mutation kept the first RGD motif of wild-type rLj-RGD3,rLj-114 was the mutation kept the second RGD motif of wild-type rLj-RGD3,rLj-114 was the mutation kept the second RGD motif of wild-type rLj-RGD3,rLj-26 was the mutation which knockout of all three RGD motifs and all the His gene of the wild-type rLj-RGD3,rLj-27 was the mutation which knockout of all the His gene of the wild-type rLj-RGD3.To analyze the relationship of its structure and function,biological activity has been made comparisons between the wild-type rLj-RGD3 and the mutations.The specific course of the study is as follows:Construct above genes to pET23b.Then transform the pET23b with purpose gene into E.coli BL21.After that,induce the expression of the recombinant proteins with IPTG and purify it by His-affinity chromatography.We have got the purpose soluble protein.Under the same conditions?0.22?g/?L?,study on the effects of the wild-type rLj-RGD3 and the mutations on cell proliferation,migration and invasion,apoptosis.The wild-type rLj-RGD3,rLj-27,rLj-112,rLj-113,rLj-114 and rLj-115 can inhibit bFGF-induced proliferation of Lewis cells and the inhibition effect is concentration-dependent.But rLj-26 is not able to inhibit bFGF-induced proliferation of Lewis cells obviously.The IC₅₀?half maximal inhibitory concentration of a substance?of the wild-type rLj-RGD3,rLj-27,rLj-112,rLj-113,rLj-114and rLj-115 are 20.16?mol/L,2.44?mol/L,2.91?mol/L,2.73?mol/L,2.75?mol/L,2.53?mol/L.Under the same concentration conditions?2.5?mol/L?,the wild-type rLj-RGD3,rLj-27,rLj-112,rLj-113,rLj-114 and rLj-115 can inhibit bFGF-induced migration of Lewis.Inhibition rates are 11%,57%,30%,42%,37%,20%.But inhibitory effect of rLj-26 is not obvious.The wild-type rLj-RGD3,rLj-27,rLj-112,rLj-113,rLj-114 and rLj-115 can also inhibit bFGF-induced invasion of Lewis.Inhibition rates are 11%,53%,35%,46%,35%,28%.Inhibitory effect of rLj-26 is not obvious.The results of the apoptosis research shows that the wild type rLj-RGD3,rLj-27,rLj-112,rLj-113,rLj-114,rLj-115 can induce apoptosis of Lewis cells but not obvious of rLj-26.Western blotting results showes that expression of caspase-3 in the cells which the wild type rLj-RGD3,rLj-27,rLj-112,rLj-113,rLj-114,rLj-115 effect on was increased.In addition,expression of Bcl-2 in the cells which the rLj-RGD3,rLj-27,rLj-112,rLj-113,rLj-114,rLj-115 effect on was reduced.Conclusion:With the reduction of the RGD motif,function of the histidine-rich?HRG?began to play.Inhibitory effect of RGD deletion mutant on Lewis cell proliferation,migration and invasion is much stronger than the wild-type.That seems to indicate that:?1?The RGD deletion mutant rLj-112 exercising anti-tumor function by class of histidine-rich;?2?The anti-tumor function of the wild-type rLj-RGD3 is not the superposition of the RGD-toxin function and class HRG,it seems that RGD motifs and class histidine-rich ran anti-tumor function through different pathways;?3?Three RGD motifs did not prove synergistic anti-tumor effects,if the only RGD motif in different positions lead to functional differences.