Isolation and characterization of novel defensin from Fabaceae seed and preliminary studies of mechanism of action

Abstract

An emergence of drug resistant infection has led to increase in both morbidity and mortality rate. Besides, resistance to cancer drugs and the tumor heterogeneity are the serious obstacle for treatment and make it more disease complicated. Currently, several researches have focused on the discovery of effective agents to overcome these burdens. Defensins are classified as of antimicrobial peptide isolated from plant and are extremely high biodiversity. The previous studies reported that plant defensins exhibited broad spectrum antimicrobial activity against various pathogens and also represented anticancer activity in vitro. In this study, the isolation of defensin gene from five Fabaceae seeds was evaluated using 3’RACE-PCR and cDNA cloning. Nucleotide sequencing analysis revealed the identified cDNAs was ranged from 234 to 437 bp in length encoding for a protein of 75 amino acids with 28 residues N-terminal signaling sequence. Multiple sequences alignment indicated that novel defensins had overall identity over 92% as compared to previously reported plant defensins and were clustered to be subfamilies of Papilionoideae. In silico analysis demonstrated that the molecular weight of defensins were 5.4 to 5.6 kDa, net positive charge of +1 to +2 with the estimated pI of 7.72 to 8.22. The putative peptides were predicted to be antimicrobial peptide by j using bioinformatics as tools of data analysis. One of putative peptides, javanicin, was selected as a candidate defensin for functional studies in this study. Gene encoded for javanicin was fused with intein and expressed in E. coli Origami2 (DE3). This expression system facilitated one step purification of peptide through the affinity chromatography. After purification, a single band of approximately 6 kDa analyzed by SDS-PAGE analysis was apparent. The final recovery yield was 2.5 mg/L with purity greater than 90%. The recombinant javanicin was further assessed its biological activities of antimicrobial and anticancer activity. The results indicated that it exhibited antifungal activity against Candida albicans and the fluconazole-resistant strain, as well as Cryptococcus neoformans and filamentous fungi, Trichophyton rubrum. The preliminary mode of action of antifungal peptide was studied. The inhibitory effect of peptide by internalizing into the fungal cells without inducing morphological change was observed. Proteomic studies revealed that its action may involve in cellular process of carbohydrate metabolism/energy. Meanwhile, anticancer activity of javanicin was evaluated and it was implied that the peptide could diminish cell proliferation of both phenotypic difference of human breast cancer, MCF-7 and MDA-MB-231 cells. Together with the result of FACs analysis, the inhibitory effect of peptide against MCF-7 cells was presumably associated with late apoptosis. The biocompatibility of javanicin against human red blood cells and mammary epithelial MCF-10A cells was determined, the results showed that peptide can induce toxic effect toward both human red blood cells and mammary epithelial MCF-10A cells at the concentration nearly comparable to the effective dose. Therefore, further investigation of javanicin needs to be performed for selectively improving or delivery modification before efficacy determination and application in vivo.