Anti-angiogenic And Anti-fungal Activities Of The Lj-RGD3Toxin Protein And Its’ Mutations

Lj-RGD3is a RGD toxin protein with3RGD motifs from the saliva gland of Lampetrajaponica, it shares homologous with a Histidine-rich glycoprotein (HRG). In this study, weshow both HRG-like anti-angiogenesis and anti-bacterial function of Lj-RGD3. Wesynthesized the RGD and Histidine defective mutations: the mutation Lj-26deleted all3RGDmotifs and all Histidine, the mutation Lj-27kept all3RGD motifs and deleted all Histidine,the mutation Lj-112deleted all three RGD motifs of Lj-RGD3, Lj-113kept the first RGDmotif, Lj-114kept the second RGD motif, and Lj-115kept the third RGD motif. Weconstructed it to the plasmid pET23b, and expressed the recombinant proteins in Escherichiacoli BL21. Then the recombinant proteins expression was induced by IPTG and purified byHis-affinity chromatography.Methods and results of anti-angiogenic activities：The MTT assay showed that, exceptmutation protein rLj-26, the five forms of mutation protein inhibited bFGF(basic fibroblastgrowth factor) induced proliferation of HUVEC (Human Umbilical Vein Endothelial Cells) ina dose-dependent manner. The effect of rLj-RGD3and the six mutation proteins onangiogenesis in vivo was tested in the chicken CAM assay. Treatment of the CAM with allabove proteins led to an an efficient suppression of newly formed vessels, especially rLj-112and rLj-27, but rLj-26didn’t. A common feature of the anti-angiogenic molecules is theirability to inhibit migration and invasion of endothelial cells in vitro. We used Transwellcontaining insert filter to determine the effect of rLj-RGD3and the six mutation proteins onHUVEC cells migration and invasion toward bFGF. In the invasion assay, the Matrigel wasused to imitate environment in vivo. All the proteins except rLj-26, showed significantinhibition on HUVEC cells migration and invasion toward bFGF, the inhibition rates ofmutation proteins rLj-112and rLj-27were higher than others. Taken together, these resultssuggest that all of above proteins have the activity of anti-angiogenesis, with the exception ofrLj-26. rLj-112is a HRG-like protein, which may involve in growth factor signaling pathway,while rLj-27is a RGD toxin protein,which may inhibit angiogenesis because of the integrinsignaling pathway. The effect of the three RGDs is not enhanced.Methods and results of anti-fungal activities: To elucidate whether rLj-RGD3and themutation proteins possess antibacterial activity, we initially investigated effects onGram-positive species E. Coli, Gram-negative S. aureus and the fungus C. albicans. rLj-RGD3as well as the mutation proteins were antifungal in Oxford cup Assay, but notantibacterial. Noteworthy, of all the proteins, rLj-112yielded a larger inhibition zone againstC. albicans than the others. And its activity of inhibition of C. albicans is dose-dependently.In viable-count assays, rLj-112showed80–100%killing at7.7μM（the MIC of rLj-112is7.7μM）, while positive control ketoconazole is15μM. Time-kill kinetics against C. albicansdemonstrated that rLj-112was fungicidal at1×MIC for12h,2×MIC for6h, but at0.5×MIC,rLj-112can’t kill C. Albicans completely for24h. To examine whether rLj-RGD3and themutation proteins interacts with and generates breaks in plasma membranes, C. albicans wasincubated with rLj-RGD3, rLj-112and rLj-26at the half of required fungicidal concentrations,and analyzed by electron microscopy. There were clear differences in the morphology ofprotein-treated fungus comparing with the control, rLj-112caused local perturbations andbreaks along C. albicans plasma membranes, and intracellular material was foundextracellularly. The antifungal activity of rLj-26which deleted all Histidine was attenuated.Therefore we speculate rLj-112can effectively inhibit C. albicans in a unique mechanism,and the activity of rLj-RGD3and other mutation proteins is weeker than rLj-112. It will needto be further research to determine which part of rLj-112that was responsible for antifungalactivity.