The Construction Of RGD Motif Of Interleukin 18 And Study On Its Biological Activities

IL-18, previously known as interferon-(IFN-)-inducing factor, was from hepatocyte cDNA library of mice suffered from toxicopathic shock induced by infection of Propionibacterium acnes and subsequently challenged with lipopolysaccharide. The gene of IL-18 was screened from murine hepatocyte cDNA library. Molecular weight of IL18 is about 18.3kD. Nucleotide sequencing of murine IL-18 predicted a precursor polypeptide with 192 amino acids lacking a conventional signal peptide and a mature protein with 157 amino acids. Subsequent cloning of human IL-18 cDNA revealed that there was 65% homology compared with murine IL-18 and showed that both contain an unusual leader sequence consisted of 35 amino acids at their N terminus. IL-18 is a cytokine with many proinflammatory functions. IL-18 stimulates T cells and NK cells mediated Th1 activity, and promotes Th1 cell differentiation and immune responses. IL-18 can act in synergy with IL-12 in regulating IFN-. However, IL-18 also up-regulates other cytokines, such as Fas, TNF-, IL-1 . IL-18 has been recently suggested to inhibit angiogenesis and tumor growth in animal models. IL-18 has potential effects and regulatory roles on several human disease states including autoimmunity and infection. Based on the facts mentioned above, much attention was paid to IL18 on the treatment of diseases, among which cancer immunotherapy is focused. In this study we used mutagenesis and DNA recombinant technology, GGC fragment was inserted into human IL-18 cDNA. The mutated IL-18 cDNA was constructed into plasmid pPIC9K, then transformed into Pichia pastoris GS115 and efficiently expressed. A Glycine residue was inserted into the recombinant IL-18 between Arg39 and Asp40 to form a RGD motif. The mutated IL-18 was termed IL-18-RGD. The protein was purified with Sephadex G-100. The capacities of IL-18-RGD inducing the production of interferon γ(IFN-γ) in human peripheral blood monocyte (hPBMC) were examined for comparing the wild IL-18 with IL-18-RGD, the mutant. The Result indicated that the mutant still keep the bioactivities of wild IL-18. Human platelet aggregation was studied most thoroughly among all cell adhesion procedures. The main receptor protein of the membrane, Integrin GPⅡb-Ⅲa is essential for the aggregation procedures. The bind of RGD sequence to GPⅡb-Ⅲa is crucial step in platelet aggregation. The inhibitory activity of IL-18-RGD on ADP induced platelet aggregation and on the binding of GPⅡB-Ⅲαto fibronectin (Fn) were detected. The results showed that an obviously inhibitory activities of IL-18-RGD on ADP induced platelet aggregation (IC50=8.8μmol/L) and on the binding of GPⅡb-Ⅲa to Fn (IC50=8.00μmol/L) were observed by comparing with the wild IL-18. Disintegrins inhibit the binding of various ligands to integrins on the surface of cells. Since the active sequence of most disintegrins described to date contains the RGD motif, only those diseases or biological processes dealing with RGD-dependent integrins can be affected with these disintegrins. The α1β1 integrin is also known to be involved in angiogenesis and neovascularization, the two biological processes that occur during tumorigenesis. Inhibition of the α1β1 integrin at tumor sites with the compounds invention may thus limit tumor growth, metastasis, and vascularization regardless of whether the tumor cells express the α1β1 integrin. In our investigation, cultured melanoma B16 showed that IL-18-RGD efficiently inhibited the growth of cultured melanoma B16 cells, IC50 = 8.10μmol/L, while the inhibitory effect of IL-18 was not detected. Both IL-18-RGD and IL-18 showed inhibitory activities on mice loaded B16 in vivo, and the inhibitory effect of IL-18-RGD was stronger than that of wild IL-18. The inhibitory activities of IL-18-RGD and IL-18 on bFGF induced angiogenesis on chorioallantoic membranes were detected. Fibrinogen, which contains RGD motif, is a ligand of αⅡbβ3, IL18-RGD blocked αⅡbβ3 on the surface of endothelial cells, The cell culture of ECV304 showed that IL-18-RGD and IL18 ef...