Eruca sativa seed napin structural insights and thorough functional characterization

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC–MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

www.nature.com/scientificreports/ antimicrobial compounds are of high clinical value for the treatment of bacterial infections and infections caused by several fungi as well 12 . Specifically, some napins possess cytotoxic effects, whereby they can be applied in the development of new anti-cancer drugs 13 . Napin genes are being used in the development of transgenic plants expressing higher levels of napins, making them more pathogen resistant supporting a reduction of yield losses in the agriculture sector 14 . In short, the promising bioactivities of napins make them suitable candidates to act against a number of human pathogens 15,16 . Rocket Salad (Eruca sativa Miller), locally known as Taramira; is an annual herb and belongs to the family Brassicaceae (Cruciferae). It is grown in different parts of the Indo-Pak subcontinent and in the Middle East. Eruca sativa is a minor oil crop; widely used as culinary and for medicines as remedies for different diseases. There is only sporadic information available about phytochemistry and bioactivity of this oily crop 17 . The regular consumption of E. sativa has been associated with the prevention of cardiovascular diseases and reduction in cancer risk 18,19 . It is known to have diuretic and anti-inflammatory activities 20 . Eruca seeds possess various proteins, glucosinolates, vitamins A and C, flavonoids, erucic acid and a relatively high oil content 17,21 . It is commonly used as animal feed in Asia, particularly in India and Pakistan. In view of its potential medicinal uses, it is hypothesized that Rocket Salad might have antimicrobial proteins/peptides in their seeds/leaves which can be exploited for the development of anti-cancerous drug after detailed understanding of its structure with subsequent characterization. Present study describes the structural insights and thorough functional characterization of a napin, which was identified and purified from seeds of E. sativa.

Results
Napin purification. Napin was precipitated by ammonium sulfate from the crude extract of Eruca sativa (Fig. 1A). Napin protein remained in supernatant after 50% (w/v) (NH 4 ) 2 SO 4 saturation constant while subsequent 70% precipitated the protein in pellet. The dissolved pellet was extensively dialyzed to remove any further salt traces and subjected to size-exclusion chromatography to obtain the highly purified protein fractions. Ultimately, an optimized combination of ammonium sulfate precipitation along with chromatographic steps provided a > 95% pure napin solution from seeds of Eruca sativa, as judged by SDS-PAGE analysis. The gel filtration chromatogram showed two absorbance peaks and the corresponding SDS-PAGE showed that first peak contained high molecular weight cruciferins while EsNap was found in the second peak (Fig. 1B,C). EsNap fractions  www.nature.com/scientificreports/ with maximum purity were stored at 4 °C. Further SDS-PAGE analysis showed the splitting of napin (16 kDa) into two daughter fragments of 11 and 5 kDa upon addition of DTT confirming the quaternary structure as well as the presence of inter-chain disulfide bonds within the structure (Fig. 1D). The purification strategy resulted in purification of 13-fold with a 7.5% yield (Table 1) from one gram of E. sativa seed powder.
Protein identification. LC-MS/MS identified two peptides (IYQTATHLPK 10 , QQQGQQGQQLQQVISR 16 ) (peak raw data is shown in Fig. S1: Supplementary material) which showed 100 and 85% sequence identity with napin-3 (UniProtKB ID: P80208) and embryo-specific napin (UniProtKB ID: P09893) from Brassica napus, respectively. The fragmented sequence of EsNap was used for multiple sequence alignment with napin-3 and embryo-specific napin of Brassica napus (Fig. 2). The alignment analysis showed that EsNap is more identical to B. napus napin. Secondary structure determination of EsNap. The circular dichroism (CD) spectrum (Fig. 3) showed predominantly α-helical structure, as indicated by two distinct ellipticity minima 22 , as well as some flexible loops. The CD spectrum corresponds to approx. 38% α-helix, 9% β-sheet, 19% turn and 34% random coil structure DLS analysis and assessment of tertiary and quaternary structure by small-angle X-ray scattering. Monodispersity and homogeneity of EsNap solution was verified by applying DLS calculation. A hydrodynamic radius of 2.4 ± 0.2 nm is ( Fig. 4) indicating that the protein is monomeric in solution. The averaged scattering amplitudes of EsNap are indicating an R g of 1.96 ± 0.01 nm according to the Guinier approximation determined by AUTORG, which is implemented in PRIMUS 24 . The P(R) function is indicative for an oblate particle with a maximum diameter of 5.6 nm. According to the volume of correlation, the molecular weight of napin is approximately 16 kDa. Considering P1 symmetry of a monomer, an ab initio model was calculated using GASBOR, with a corresponding molecular weight of 15 kDa. The particle shape is rather oblate with extended C-and N-terminus that may harbor a certain degree of flexibility, as indicated by superimposition (Fig. 5 25 . Two disulfide bridges (CYs10-Cys62 and Cys23-Cys51) are formed between the smaller and longer chains in Fig. 6(2A). H3 and H4 are almost antiparallel to each other and are connected by two short β-sheets, which are constituted by residues Gln76-Gln91, known as the hypervariable region in 2S albumins [26][27][28] , because of the high variability in length and sequence composition as shown in Fig. 6(1A). The scattering amplitudes of EsNap processed by PRIMUSQT were compared to the predicted 3D model of EsNap using the program CRYSOL, as shown in Fig. 6B with a calculated minimized χ 2value of 1.87. The manual superimposition well confirms the conclusion that the structures are widely similar, including the molecular weight comparison with the ab initio model. Lys 9, Arg 11, Lys 12 (blue) and Lys 105 (red) residues form flexible N and C-terminal of EsNap ab initio model are as shown in Fig. 6B. These residues are involved in the antifungal and anticancer activity of EsNap. The in silico model of EsNap was aligned with the    (Table 1). Similarly, the total numbers of counted pupae and adults were strongly reduced at all concentrations of EsNap and a maximum was recorded in the control with no EsNap treatment (86.6 ± 10.5 and 41.0 ± 8.0) (P < 0.0, F = 22.5). In parallel, average larval, pupal and adults populations were decreased with increasing concentration, which means the smallest populations were observed at 3 mg/ml concentration followed by 2 mg/ml and 1 mg/ml of EsNap. The ratio of male and female pupae as well as adults was also recorded;   www.nature.com/scientificreports/ the male population was significantly larger in number as compared to the female population for all EsNap concentrations ( Table 2).

Discussion
Napins are present in leaves, seeds, roots and stems of a number of plant species belonging to cereals and crucifers. Napins are synthesized as larger precursors, which have a post-translational N-terminal signal peptide and a C-terminal precursor peptide. Napin from the seeds of Taramira (Eruca sativa), a member of the family Brassicaceae, has been isolated and characterized. Eruca sativa napin (EsNap) has a molecular mass of around 16 kDa, as determined by SDS-PAGE. Most of the napins known today have molecular masses in the range of 16 kDa and showed relatively high levels of sequence similarity. Napins are polypeptides containing S-S bonds that are formed under reducing conditions. Four S-S bonds existed in native napin structures, two between the chains and two within the larger chain [30][31][32] . Napin family is typically rich in arginine, lysine, and cysteine residues and have a strong antimicrobial activity 33 . EsNap protein has basic properties due to high level of arginine and lysine compared to other amino acids. The IEF of EsNap showed the pI value of approximately 8.00. In 1981, Crouch and Sussex indicated that napins have basic pI of 11 which is verified due to high number of arginine, lysine and histidine residues 34 . CD showed that EsNap has highly helical secondary structure content. In this context, Sharma et al. 35 reported that 2S Albumin (napin) from seeds of Wrightia tinctoria has a high content of α-helices. Previous studies have reported 40-45% helices and 16-20% β-sheets, 25% α-helix and 38% β-sheets for napin. The high content of α-helix in napins may promote some toxic biological activities against pathogens and facilitate the dynamic association of the protein with membranes, as summarized for some peptides by Bechinger 36 . Additional structural information about EsNap was obtained by SAXS analysis which strongly indicated that EsNap is globular with slightly elongated native form in solution and exists in a monomeric state. The shape factor, R g divided by R H as determined by DLS, has a value of 0.8, which is indicative for a globular and slightly elongated shape of EsNap particles in agreement with the displayed ab initio model shown in Fig. 5C. N and C-terminal basic residues (Lysine and Arginine) of EsNap protein formed the flexible part of ab initio model in Fig. 6B and responsible for antifungal and anticancer activity in EsNap due to the distribution of cationic charge.
EsNap 3D structure has α-helix dominant secondary element as well as possesses high lysine and arginine contents and these properties together are responsible for antifungal and anti-cancerous activities. It has already been reported that an amphipathic conformation and high cationic charge distribution are responsible for antifungal activity of napins 37 . EsNap 3D structure showed strong amphipathic behavior parallel to their high lysine and arginine content, and thus fully comply with these two requirements. Amphipathic α-helix structure of EsNap may be involved in the CaM (Calmodulin) antagonist and formation of pores in membranes. This is probably because CaM and two subunits of EsNap contain similar α-helical conformations. Neumann and his colleagues reported that amphipathic α-helix structure of napins showed the CaM (Calmodulin) antagonist activity and may be involved in development of membranous pores 38 .
EsNap exhibited antifungal activity against Fusarium graminearum at 30-100 µg quantity. Initially, (48-72 h) as shown in figure S4, the fungal growth was promoted by the napin protein itself which is presumably due to the fact that the fungus partly metabolized the protein and used it as a carbon or nitrogen nutrition source. It is well known that nitrogen is an essential requirement for growth, and the ability to metabolize a wide variety of nitrogen sources enables fungi to colonize different environmental niches and survive nutrient limitations 39 . However, later on 96-120 h, napin-treated samples showed a significant reduction in growth compared to BSAtreated-samples (Fig. S4). Tomar and his colleagues reported that pumpkin 2S albumin inhibited the growth of Fusarium oxysporum, Phanerochaete chrysosporium and Aspergillus flavus grown in PDA medium at 50 and 100 µg protein dissolved in a similar culture volume 40 . Napin (PR protein-13) from Pennisetum glaucum (pearl millet) inhibited the growth of Sclerospora graminicola spores at a quantity of 100 µg 41 . Wheat puronapins showed antifungal activity against Rhizoctonia solani by membrane permeabilization, responsible for significant crop losses and rice sheath blight 42  Due to the alarming situation regarding the impact of conventional insecticides on human health and the surroundings, the search for novel molecules with insecticidal activity with minimal adverse effects has become paramount 43 . Purified EsNap produced strong negative effects on all life stages of stored grain insect pest T. castaneum. The plant extracted protein PA1b from Pisum sativum was first reported to act against stored grain insect pests especially on cereal weevils and was found to be a valuable naturally occurring biopesticide 44,45 . Many toxic metabolites with antimicrobial activity released by plants are currently commercially available as they also show an individual level of toxicity towards insect pests. Consequently, entomo-toxic plant compounds are an appreciated starting point to further develop bio-insecticides against stored product insect pests 46 on a long-term scale after carefully verifying their respective persistency and toxicity spectrum. Muench et al. 47  www.nature.com/scientificreports/ albumin PA1b and mapped its binding site on insect vacuolar ATPase. Its interaction is influencing the toxicity and a similar mechanism is conceivable in case of napins resulting in reduced T. castaneum populations, as also observed by Da Silva and his group in 2012. The saponin 3-GlcA-28-AraRhaxyl-medicagenate from Medicago truncatula seeds due to its high toxicity against Sitophilus oryzae (Da Silva., 2012). This 3-GlcA-28-AraRhaxylmedicagenate exhibits repellent properties and has a CMC of about 0.6 mM. The exposed protein treatment to the insect is actually 0.4 mg/g of the flour which is not that high and review of literature supports our treatment values. However, there could be many reasons for toxicity of napins against T. castaneum as described previously [47][48][49][50] . Experiments applying napin of Pyrularia, which is hemolytic, cytotoxic and neurotoxic, suggest that negatively charged membrane lipids are targeted directly by conserved basic amino acids 51 . Consequently, it is concluded that napins commonly do not possess receptor specificity but induce the formation of oligomeric protein-lipid complexes and ion permeable membrane pores 52,53 . This mechanism would target a broad spectrum of species and is in agreement with the observed toxicity of EsNap towards the species that were selected for this study. Nonetheless, an improved understanding of the structure-function relationship and mode of action is essential for understanding the ecological mechanisms promoted by plant napins as well as utilizing napins for more biotechnological and medical applications. Mass spectrometric analysis. Gel bands stained with colloidal Coomassie were cut out and reduced and alkylated with DTT (10 mM, 56 °C, 30 min.) and Iodoacetamide (IAA, 55 Mm, room temperature in dark), respectively. The protein in the gel was digested with trypsin (conditions: 5 ng trypsin/µl (sequencing grade modified trypsin, Promega, Madison, USA) in 50 mM NH 4 HCO 3 , 37 °C, 16 h). After digestion, the gel pieces were repeatedly extracted (65% acetonitrile/5% formic acid) the combined extracts were dried in a vacuum concentrator and redissolved in 20 µl 0.1% formic acid. LC-MS/MS measurements were performed by injecting the samples into a nano liquid chromatography system (Dionex UltiMate 3000) coupled via electrospray-ionization (ESI) to an orbitrap mass spectrometer (Orbitrap Fusion, Thermo Scientific, Bremen, Germany). The samples were loaded (3 μl/min) onto a trapping column (Acclaim PepMap μ-precolumn, C18; buffer A: 0.1% formic acid in H2O; buffer B: 0.1% formic acid in acetonitrile) with 2% buffer B, washed for 5 min with 2% buffer B (3 μl/min) and the peptides were eluted (300 nl/min) onto the separation column (Acclaim PepMap 100, C18, 75 μm × 250 mm, gradient: 2-30% B in 35 min). Mass spectrometric analysis was performed in positive ion mode. LC-MS/MS analysis was carried out in data dependent acquisition mode (DDA). MS ions were detected in orbitrap at 120 k resolution while MS/MS spectra were recorded in the ion trap as detector. LC-MS raw data were processed with Proteome Discoverer 2.0 (Thermo Scientific, Bremen, Germany). For identification, MS/MS spectra were searched with Sequest HT against the Arabidopsis and the plant Uniprot database (https:// www. unipr ot. org, downloaded November 10, 2019). The searches were performed using the following parameters: precursor mass tolerance 10 ppm, fragment mass tolerance 0.5 Da, two missed cleavages allowed, carbamidomethylation of cysteine residues as fixed modification, oxidation of methionine residues as a variable modification. Identifications were validated manually.

Experimental
Protein identification. For protein identification, a search for sequence similarities was performed applying a BLAST tool through feeding of residual sequences https:// www. unipr ot. org/ blast. Homologous sequences were subsequently aligned using ClustalW https:// www. genome. jp/ tools-bin/ clust alw in the default set up and BoxShade server https:// embnet. vital-it. ch/ softw are/ BOX_ form. html.
Isoelectric focusing (IEF). IEF was performed using 17 cm long and 0.5 mm thick gel strips (pH 3-10, Sigma www.nature.com/scientificreports/ containing 8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte ampholytes and 0.001% bromophenol blue overnight. The pI markers proteins (Sigma) ranging from 3 to 10 were co-electrophoresed to estimate the pI of the proteins under investigation. Isoelectric focusing was performed in an IEF focusing cell (Bio-Rad). The voltage was increased stepwise starting from 250 V for 20 min, 10,000 V for 2.5 h and 10,000 V for 12 h. The gels were maintained at 28 °C during the run. After IEF, the proteins were stained by Coomassie blue.
Circular dichroism (CD) spectroscopy. CD spectroscopy experiments were performed to determine the secondary structure composition of napin applying a CD6 dichrograph instrument (Jobin Yvon, Longjumeau, France). Purified napin (0.2 mg/ml) was prepared in 25 mM phosphate buffer, pH 7.0 and the CD spectra of napin were recorded in the far-UV-range between 190 and 260 nm at 25 °C in a 1 mm path length quartz cell. A total of fifteen spectra were averaged after measuring the buffer separately. The percentage of secondary structure of napin was calculated by using Spectra manager™ software (Jasco).
Dynamic light scattering (DLS). Purified protein was analyzed by DLS using the SpectroLight 300 instrument (Xtal Concept, Germany) for confirming the monodispersity of the protein solution as well as the size distribution calculation of molecules.
Small-angle X-ray scattering (SAXS). Small-angle X-ray scattering data of napin at two different solution concentrations (3.2 and 6.5 mg/ml) were collected at EMBL beamline P12 55 at the storage ring PETRA III (DESY, Hamburg, Germany). At a sample-detector distance of 3.0 m and a wavelength of λ = 0.124 nm, scattering data were collected applying a 2D photon-counting Pilatus 2 M pixel detector (Dectris) with the momentum transfer ranging from 0.03 nm −1 < s < 4.80 nm −1 (s = 4π sinθ/λ, where 2θ is the scattering angle). To exclude significant radiation damage, 20 successive X-ray exposures of napin of 45 ms each were compared and no significant changes in the intensity pattern were observed over time. Data were normalized to the intensity of the transmitted beam and radially averaged. The scattering pattern of the buffer was subtracted, and the difference curves were scaled for protein concentration. The radius of gyration Rg along with the particle pair-distance distribution function p(r), which further provides the maximum dimension Dmax, were computed by the automated SAXS data analysis pipeline SASFLOW and verified via PRIMUS 56 . Low resolution ab initio shapes of napin were generated based on the composite scattering curves applying the program GASBOR 57 . It uses an assembly of interconnected dummy residue spheres to generate a chain-like ab initio protein model that fits the experimental scattering data. The molecular weight of EsNap was estimated by its excluded particle volume and further verified based on the forward scattering intensity of BSA (66 kDa; 5 mg/ml in 50 mM HEPES pH 7.5), which was measured in addition to verify beamline operation.
Homology modeling and structure prediction. For the calculation of an EsNap homology model, fasta sequence of napin-3 from Brassica napus (UniProtKB ID: P80208) was consequently used for the 3D modeling of EsNap. Therefore, the primary sequence of napin-3 was subjected to model building via the Swiss-Model server [58][59][60] . The coordinate information of recombinant pronapin precursor, BnIb from B. napus (PDB-ID: 1SM7) was used as the most suitable template. The model was built based on the target-template alignment using ProMod3 61 . Coordinates of fragments with a conserved sequence comparing the target and the template were copied from the template to the model. Insertions and deletions were remodeled using a fragment library and side chains were then rebuilt. Finally, the geometry of the resulting model is regularized by using a force field. The images of the predicted model were prepared applying PyMOL 62 . The germination rate of the conidia (F. graminearum) was checked in 125 µl gene frame chamber (Thermofisher, Catalog, AB0578). Gene frame chamber is perfect for standard microscope slides and they prevent reagent loss during the longer time series. Gene frame chamber contained the conidia in minimal media, EsNap protein (100 µg), BSA (100 µg) and phosphate buffer.
Cell cytotoxicity assay. The cell survival and proliferation MTT (3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) assay kit (Millipore, USA) was used for rapid and perceptive quantification of cell proliferation and viability. Briefly, 100 µl (1 × 10 5 ) of Huh7 cells were cultured in a 96 wells plate using the Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C in a CO 2 incubator for 24 h. EsNap dilutions 3.12, 6.25, 12.5, 25 and 50 µM were added and the plate was incubated at 37 °C in a CO 2 incubator for another 24 h and three replications were performed and analyzed for each dilution. After 24 h the medium was removed and 100 µl freshly prepared medium was added along with 10 µl MTT solution ( www.nature.com/scientificreports/ plate was again incubated in a CO 2 incubator at 37 °C for 4 h and after this 0.1 ml DMSO was added to dissolve the formazan crystals in the wells. Mitochondrial succinic dehydrogenase in living cells converts the MTT substance in purple formazan crystals that are insoluble in water. The MTT formazan product was detected by measuring the optical density with a multi-channel plate reading photometer at a test wavelength of 570 nm and a reference wavelength of 620 nm 65 . Cell viability was attained by means of the following formula: The IC 50 (50% inhibitory concentration) value was calculated by non-linear regression analysis with GraphPad Prism software. The assay was conducted in triplicate. One way ANOVA was performed on data with a level of significance P < 0.05. Entomotoxicity assay. Entomotoxicity assays applying napin were performed in the Eco-toxicology laboratory, Faculty of Agriculture Sciences and Technology, Bahauddin Zakayria University Multan. Napin toxicity was determined for T. castaneum. A population of T. castaneum was collected from a local flour mill and was cultured on whole wheat flour with 5% brewer yeast 66 . To get an equal age insect population, the culture medium was complete wheat flour incubated at 60-90 °C for 60 min. One glass jar was used and filled with 500 g flour and 50 red flour beetles were added. For the oviposition beetles were left in the culture medium. After three days beetles were removed with the help of sieves and then added to a separate set of sterilized jars filled with 200 g flour for maintenance of the culture. Flour containing eggs was used as culture medium for obtaining adult beetles of a homogenous population 67,68 . The culture was maintained under optimum laboratory conditions at 30 °C with a relative humidity of 70%.
For the bioassays three different serial dilutions of napin were prepared in 100 mM phosphate buffer and 3, 2 and 1 mg/ml protein concentrations were used. A total of 450 g of wheat flour was pre-refrigerated at 4 °C to avoid any infestation. Each protein concentration was prepared in 100 ml buffer solution mixed with 150 g flour to form homogeneous dough. It was dried in the dark to form a hard pan and subsequently grinded with an electric grinder providing powder. Five replications using one fifth of the material each for all three concentrations and a control for comparison, i.e. buffer with no napin, were set up. Each replication was executed in an individual glass jar. Five males and five females of T. castaneum were released in each jar. After ten days, released adults were removed and interval data of larvae, male and female pupae including adults were recorded weekly.
Statistical analysis. The entomotoxin protein bioassay data were analyzed in one way ANOVA through the stat software "Statistix 8.1" and mean values were separated by a Tukey-HSD test with a level of significance of 0.05 (Analytical Software, 2005).

Conclusions
In conclusion, a napin protein was isolated and purified from Eruca sativa and contains disulfide bonds in a monomeric form. Furthermore, the napin inhibits the growth of F. graminearum at the stage of conidia and possesses cytotoxicity towards Huh7 cells. Based on its above-mentioned properties, different napins like EsNap may promote the resistance of plants against infections by parasitic fungi and likewise reduce the susceptibility towards other plant pathogens.