A Study Of The Site-directed Mutant Of Adinbitor. A Disintegrin From Gloydius Blomhoffi Brevicaudus And Theries Biological Activities

Adinbitor was cloned from Gloydius blomhoffi brevicaudus and character- rized as a novel disintergrin. Total RNA was extracted from venom gland and RT-PCR was used to generate a cDNA, which was 219 bp long. The sequence encoded a polypeptide with 73 amino acids including 12 cysteines and an RGD motif, the signature motif of disintergrin. Recombinant Adinbitor (rAdinbitor) was expressed in E.coli and purified by using the His-Bind affinity chromatography. In previous study, Adinbitor could dose-dependently inhibit ADP-induced human platelet aggregation in human platelet-rich plasma (PRP) system. The IC50 value of Adinbitor for platelet aggregation was 6μmol/L. Furthermore, Adinbitor significantly inhibited angiogenesis both in vivo and in vitro. Taken together, these results suggested that Adinbitor had typical functions of disintegrins.In our previous study, we constructed the site-directed mutagenesis of RGD motif of Adinbitor to KGD (we called it KGD mutant for short). We found that the KGD mutant could inhibit platelet aggregation but couldn't inhibit angiogenesis in CAM model. In the present study, we constructed the site-directed mutagenesis of Adibibitor. The sequences RIARGD of Adinbitor was mutated to RPTRGD (we called it RPT mutant for short) by the method based on PCR mutagenesis. We evaluated the platelet aggre- gation of the RPT mutant Adinbitor, the results suggested that it had lost the function of inhibiting platelet aggregation. In order to elucidate the molecular mechanism of three kinds of Adinbitor for platelet aggregation, we evaluated the rate of CD41 binding to GPⅡb/Ⅲa by flow cytometry. Our data demonstrated that both the wild type Adinbitor and KGD mutant could inhibit the binding of anti-CD41 antibody toαⅡbβ3 in a dose-dependent manner with an IC50 of 1.72μmol/L and an IC50 of 1.72μmol/L. The effect of RPT mutant Adinbitor on the subsequent binding of anti-CD41 antibody was negligible at the same concentration. We believed that both the wild type Adinbitor and KGD mutant could competitively occupy the binding site of CD41 which then did not allow the binding for CD41 antibody. By mutating the amino acid residues flanking the RGD motif to RPTRGD, a conforma- tional change in the spatial structure of this motif could be suggested, and which resulted in the remarkable decrease in the affinity for integrinαⅡbβ3.Angiogenesis, the formation of new capillaries from pre-existing vessels, is involved in the pathogenesis of inflammatory diseases as well as in tumor progression. The CAM model provides an excellent method to investigate the role of a wide spectrum of pro- or antiangiogenic substances. In our study, the RPTRGD Adinbitor mutant inhibited angiogenesis in the CAM model significantly versus the control group, p<0.05. The ratio of the vessel numbers to the total CAM area exposed of the control group was 14.4%, the ratio of 5μg, 20μg and 50μg group were 11.4%, 9.4%, and 6.3%, respectivaly.The results demonstrated that the mutant Adinbitor had maintained the biological function of inhibiting angiogenesis while it was no longer to inhibit platelet aggregation. This strategy may represent a novel potential anti-tumor agent devoid of the potential side effect on inhibiting blood clotting.We also explored the effects of three kinds of Adinbitor on cell proliferation, apoptosis, migration, invasion and adhesion. MTT assay was used to detect the effect of the Adinbitors on proliferation. SSMC7721 cells were incubated with different concentrations of Adinbitor and stained by fluorescence dye Hoechst 33258 to observe apoptosis .Transwell was used to explore the effect of three kinds of Adinbitor on cell migration. Transwell and matrigel were used to observe the effect of Adinbitor on cell invasion. Our results showed that three kinds of Adinbitor could inhibit cell proliferation and enhance cell apoptosis. All of them could inhibit cell migration and invasion significantly. In order to elucidate the mechanism of three kinds of Adinbitor for apoptosis, the amount of caspase-3 was detected by Western blotting. Spectrophotometric method was selected to explore the activity of caspase-3. The results showed that the amounts of caspase-3 in cytoplasm were decreased. The activity of caspase-3 was increased remarkably.In order to elucidate some of the potential underlying mechanism by which Adinbitor inhibited angiogenesis, we explored the effects of the mutant Adinbitor on the signaling molecules Akt, p-Akt, NF-κB, IκB-α, RelA ( p65 ) NF-κB by Western blotting. NF-κB had previously been demonstrated to have critical role for endothelial cell migration, angio- genesis, and tumor metastasis. The results indicated that the phosphory- lation of Akt was inhibited, the expression of IκB-αin cytoplasm increased, the expression of RelA(p65)in nucleus decreased significantly. Our results suggested that these effects of both the wild type and RPT mutant Adinbitor were at least through Akt, which activated NF-κB. These results should provide clues for a better understanding of the mechanisms underlying angiogenesis.In summary, the mutation of RIA residues at the flank of RGD to RPT decreased the binding affinity for integrinαⅡbβ3. Wild type Adinbitor's function of inhibiting platelet aggregation had been eliminated in the mutant Adinbitor by this same mutation. However, the RPT mutant Adinbi- tor could still inhibit angiogenesis possibly through an Akt and NF-κB dependant pathway. Adinbitor as a potential anti-tumor agent would always suffer from the potential undesirable side effect of platelet aggregation inhibition. This experiment lays a ground work for understanding the influence between primary protein structure and the effects on spatial structure in proteins, and the interactions between proteins. In future studies, it may be possible to obtain molecules with a higher specificity for integrinαvβ3 or for integrinαⅡbβ3 by further mutagenesis.