INVESTIGATING MULTIFUNCTIONAL PROPERTIES OF PUMPKIN 2S ALBUMIN AND IDENTIFICATION OF ITS POTENTIAL DNA/RNA BINDING SITE

Abstract

The plant 2S albumins have gained importance due to the biotechnologically exploitable properties having immense potential for use in medical and agriculture fields. Although the structure and function of many 2S albumins have been characterized, the molecular basis of many of these activities is still unknown. A prodigious amount of molecular-level research has been done on plant 2S albumin in the past few decades, focusing on various aspects, such as structure, synthesis control, mechanism of targetingiandideposition, functional properties, and applicationiinigeneticiengineering. Cumulatively, these studies pointed out a plethora of functions, including antimicrobial, anticancer, translational-inhibitory, DNase and RNase activities. Recently, the application of native pumpkin 2S albumin (nP2SA) was demonstrated in the agriculture field, where antimicrobial formulation containing Nano-Zinc Oxide and nP2SA significantly inhibited the growthiofiCandidatusiLiberibacteriasiaticus (CLas) in planta (Ghosh et al. 2018). CLas,itheifastidiousiphloem-restricted gram-negative bacterium vectoredibyitheiAsianicitrusipsyllid, causes one of the most devastating diseases of citrus known as Huanglongbing (HLB or citrus greening), affecting the citrus industry globally. As a result, biological control appears to be an environmentally sustainable and cost-effective way to handle this severe threat. In present study, we extent these results and then we next aimed to explore the efficacy of nP2SA against Xanthomonas oryzae (Xoo.), a causative agent of bacterial leaf blight (BLB) in Oryzae sp. Invivo experiment of nP2SA have the potential to overcome the leaf blight disease of rice caused by Xoo. Additionally, we found that treatment with the nP2SA also triggers metabolite production in Oryzae sp that speculate about initiating host immune response to eliminate the pathogen at the site of infection. The cloning of the gene and production of recombinant protein should be the next logical step in taking the research forward on this protein. Theipresentistudyi reportsitheicharacterizationi ofirecombinanti pumpkini2Sialbumini(rP2SA)iproteiniandiunravellingiofipotentialiDNA/RNAibindingisite.ii TheipurificationiandicharacterizationiofitheirP2SAiestablishedithatiitiretainsitheicharacteristi ciα-helicalistructureiandihasisimilaribiochemicalicharacteristicsiasinativeiform (Tomar, Nikhil, et al. 2014). The rP2SA protein exhibited comparable DNase, RNase, antifungal and anti-proliferative activities as native protein. A strong antiviral activity against chikungunya virus (CHIKV), an addition to hosts of functions, is also being reported for the first time for this protein. Inisilicoistudiesiandisite-directedimutagenesisiwere employed to unravel the binding site for DNA/RNA. The potential site, identified by Molecular Dynamic simulation and docking-based analysis revealed the presence of five arginine (R72, R76, R79, R84 and R91)residues.iTheimutatediformsiofirP2Saiproteini(R84AiandiR91A)ishowedireducedi Dnase/Rnasei activityisuggestingi theiripresencei atibindingi sitei andiinvolvementiinithei nucleasei activity.i TheipotentialiDNA/RNAi bindingisiteiwasi mappedibyi bioinformaticsi analysisi usingitheiresultsifromi mutationistudies.iAnalysisialsoishowediai metalibindingisitei adjacenti toi DNA/RNAibindingisite.ii The outline of Ph. D. thesis is divided into three chapters. A brief description of each chapter is mentioned below: Chapter 1: Introduction and literature review: In this chapter, the rationale behind the study will be described. A comprehensive and up-todate literature review related to each objective will be narrated. The chapter will also include the description of 2S albumin seed storage protein (with special preference to Pumpkin 2S albumin) and their structure and function properties. A detailed description of the following will also be included in this chapter: post-translation processing, understanding the structure of 2S albumin, different biological activities. Chapter 2: Materials and Methods: This section consists of a detailed description of the recipes for reagent, solution and experimental protocols performed to answer all the objectives. The protocol which will be described in detail is cloning, overexpression of protein in a bacterial system, SDS-PAGE gel electrophoresis, Western blotting, Circular Dichroism, Differential Scanning Calorimetry, Fluorescence Spectroscopy, DNase, RNase, antifungal, antiviral, antiproliferative assay against cancerous cell lines, site-directed mutation, antibacterial activity against CLas and Xanthomonas oryzae. Chapter 3: Results and Discussion The chapter includes objective wise significant findings of this study: The main objectives are 1. Assessment of antibacterial efficacy of nP2SA against CLas and Xanthomonas oryzae. 2. Characterization of biophysical and biochemical activity of rP2SA. 3. Deciphering the DNA Binding domain of rP2SA protein using insilico and Site- Directed Mutagenesis (SDM) studies.1. Assessment of antibacterial efficacy of nP2SA against CLas and Xanthomonas oryzae. 1.i. Purification of nP2SA with the help of ion-exchange chromatography. The purification of a ~12ikDaiheterodimericiprotein,idesignatediasinP2SA, from the seeds of pumpkin wasiaccomplishediusingiDEAE-iSepharose, where the protein was found in flowthrough, CM-SepharoseiandiCibacronibluei3GAiwhereiitiwasibound and appeared as a single peakioniaisize-exclusionichromatographyicolumn. SDS-PAGE confirm the protein. 1.ii. Antibacterial activity of nP2SA against CLas. Recently, it was shown that nP2SA can be used in agriculture. An antimicrobial formulation containing Nano-Zinc Oxide and nP2SA significantly hindered the growth of CLas in planta (Ghosh et al. 2018). We extended this result with large citrus plants in agricultural field. 1.iii. Antibacterial activity of nP2SA against Xanthomonas oryzae. 2S albumin at a different concentration up to 240 μM was checked for antibacterial activity. We found at 200 μM a significant reduction of bacterial invasion. The treatment was carried for 15 days, where on alternative days, the treatment was performed. Post-treatment with nP2SA protein, infection of Xanthomonas oryzae is found to be reduced significantly. Leave morphology picture showed to overcome the effect of Xoo. Preliminary results show that the treatment of 2S albumin affected several metabolic pathways in the plant. Metabolic profiles describe aspartic acid, palmitic acid, and glutamic acid are most important compounds because these compounds involve in changing the bacterial infection. 2. Characterization of biophysical and biochemical activity of recombinant pumpkin 2S albumin (rP2SA). 2.i. Cloning, overexpression and purification of rP2SA: 2S albumin gene from pumpkin seed with ~318bp has been cloned and overexpressed in E. coli. The protein with a molecular mass of ~15kDa has been purified using a Ni-NTA affinity chromatography column. Protein concentration was determined using BCA kit and the purity was determined on a 15% SDS-PAGE. Western blotting is used to detect the rP2SA protein. 2. ii. Biophysical characterization of the rP2SA The secondary structure was identified by Circular Dichroism and analysis with the help of online software DichroWeb. CD showed that rP2SA has an alpha-helical secondary structure similar to its native form. And also shows the high-temperature stability up to 90ºC. Differential Scanning Calorimetry (DSC) shows similarity in melting temperature of native and the recombinant protein. Intrinsic fluorescence studies of rP2SA and rP2SA with different concentration of Mg2+ demonstrated the conformational stability of the protein. 2. iii. Biochemical characterization of rP2SA. The purified rP2SA exhibited antiviral, antiproliferative, DNase, RNase and antifungal activities. InvitroistudiesirevealedithatirP2SAiexhibitsipotent antiviral activity against the chikungunya virusi(CHIKV)iatiainontoxiciconcentrationiwithian IC50 of 114.5 mg/mL. the antiviral activity is certainly a new entrant in the plethora of functions exhibited by pumpkin 2S albumin. rP2SA showed a strong antiproliferative activity towards breast cancer (MCF-7), cervical cancer (HeLa) andihepatocellularicarcinomai(HepG2)icell lines. DAPI staining indicated that the cytotoxicieffectiofirP2SAiisimediatedithroughiinductioniof apoptosis confirmed by FACS data. rP2SA showed DNase and RNase activity against pBR322 and MCF- 7 RNA. The antifungal property of rP2SA was tested against Fusarium oxysporum. It exerted a similar effect as the native counterpart where an agar diffusion assay observed a clear zone of inhibition. 3. Deciphering the DNA Binding domain of rP2SA protein using in silico and Site- Directed Mutagenesis (SDM) studies. 3.i. In silico studies to predict the potential DNA/RNA binding site The three-dimensional structure of rP2SA was predicted, refined, energy minimized and validated. A potential DNA/RNA binding site was identified based on the electrostatic profile of the structure, where areas around helix 5 showed an abundance of positive charge due to arginine and lysine residues. Molecular docking of dsDNA with rP2SA and rP2SA mutants was carried out using HADDOCK. The generated clusters were inspected and visualized in PyMOL. Molecular dynamics simulation of rP2SA, rP2SA mutants, rP2SA-DNA and rP2SA mutants-DNA complexes was also performed using AMBER. The Molecular dynamics was done for 100 ns, and the binding affinity of the protein-DNA complex was determined using the molecular mechanicsigeneralizediBornisurfaceiarea. Molecular docking was done using AutoDock Tools to identify and assess the interactions of Mg2+ with rP2SA.3.ii. Site-Directed Mutagenesis (SDM) employeditoidecipheritheiDNA/RNAibinding site in rP2SA. Out of five, two single point mutations of the arginine residues to alanine (R84A; R91A) were analysed for DNase and RNase activities to validate the hypothesis. The results showed a drastic reductioniinibothiDNaseiandiRNaseiactivitiesiin two mutants, confirming our findings of in silico studies. The secondary structure analysis by CD demonstrated that the mutation did not affect the protein's overall structure.