World J Microbiol Biotechnol (2010) 26:545–554 DOI 10.1007/s11274-009-0204-2

ORIGINAL PAPER

Expression and characterization of two domains of ternata agglutinin (PTA), a agglutinin from Pinellia ternata with antifungal activity

Li-Jun Ling • Yan-Zhuo Yang • Yu-Rong Bi

Received: 16 July 2009 / Accepted: 28 September 2009 / Published online: 9 October 2009 Ó Springer Science+Business Media B.V. 2009

Abstract Pinellia ternata agglutinin (PTA) from P. ter- defined simple sugar and/or complex carbohydrates nata f. angustata is a two-domain GNA-related lectin. The (Fouquaert et al. 2009). Monocot mannose-binding lec- current study indicates that the PTA gene encodes a pre- tins is one of major plant lectin families that have been cursor consisting of two tandemly arrayed domains, reported. The monocot mannose-binding lectins have N-terminal domain (PTA-DOM1) and C-terminal domain been widely found in higher in last decade with (PTA-DOM2). Both domains and the precursor without most of them playing significant roles in defense against signal peptide (PTA-P) present different number of activity foreign microorganisms or plant predators due to their mannose-binding sites which play key roles for the lectin ability to recognize high-mannose-type glycans. Because function. Analyses of the three fusion proteins, PTA- the monocot mannose-binding lectins do not only occur DOM1, PTA-DOM2 and PTA-P, expressed in Escherichia in the monocot plant families but also in other species coli revealed that one mannose-binding site the aggluti- such as liverwort Marchantia polymorpha, gymnosperm nation activity while the additional sites do not possess Taxus media and fish Fuga rubripes, the monocot such activity. However, the number of carbohydrate-bind- mannose-binding lectins have been generally designated ing sites suggests some significant properties on the anti- as ‘‘GNA (Galanthus nivalis agglutinin)-related lectin’’ fungal effectiveness. In addition, each of the PTA domains (Van Damme et al. 2007). has the same function when compared with the natural GNA was the first monocot mannose-binding lectin PTA (N-PTA). The information on PTA gene obtained in isolated and characterized (Van Damme et al. 1987). this study will served as baseline information in developing Native GNA is a homotetramer compose of four non- this protein as a form of transgenic plant protection. covalently associated 12 kDa monomers. The GNA tetra- mer contains 12 apparently functional mannose-binding Keywords Plant lectin Domain Mannose-binding site sites. Other monocot mannose-binding lectins possess a Antifungal protein Expression P. ternata similar three-dimensional structure (Chandra et al. 1999; Wood et al. 1999). Recent investigations of the two- domains of GNA-related lectins revealed that some of Introduction these lectins have dual specificity and interact with both high-mannose and complex N-glycans, and that some have Lectins or agglutinins are a heterogeneous group of complex specificity. For instance, lectin from Xanthosema carbohydrate-binding proteins classified together on the sagittifolium (XSA) has two different types of carbohy- basis of their ability to bind in a reversible way to well- drate-binding sites recognizing oligomannoses and com- plex N-linked carbohydrates, respectively (Mo et al. 1999). On the contrary, lectin from Arum maculatum (AMA) has L.-J. Ling Y.-Z. Yang Y.-R. Bi (&) an apparently complex specificity due to its two distinct Key Laboratory of Arid and Grassland Agroecology (Ministry binding sites (Van Damme et al. 1995). of Education), School of Life Sciences, Lanzhou University, 730000 Lanzhou, People’s Republic of Some plant lectins have potent antifungal activities, e-mail: [email protected] such as, Amaranthus viridis lectin (AVL) which exert 123 546 World J Microbiol Biotechnol (2010) 26:545–554 antifungal activity against phytopathogenic fungi Botrytis was a generous gift from Gansu Academy of Agriculture cinerea and Fusarium oxysporum (Kaur et al. 2006). Sciences. Similarly, two chitin-binding lectins from seeds of Arto- carpus genus inhibited the growth of Fusarium monili- Isolation of the cDNA clone encoding PTA forme and Saccharomyces cerevisiae (Trindade et al. 2006) from P. ternata f. angustata while the protein obtained from Gastrodia elata confer antifungal activity against Botrytis cinerea, Gibberella For 30 Rapid amplification of cDNA ends (RACE), 1.0 ll zeae, Ganoderma lucidum, Rhizoctonia solani and Valsa total RNA was reversely transcribed with the 30 RACE ambiens (Wang et al. 2001). Adaptor. The specific primer P1 for the 30-RACE system Previous studies demonstrated that the function of lec- was designed and synthesized (TaKaRa, Japan) according tins depends on their specific carbohydrate binding activi- to the conserved amino acid sequence of the monocot ties (Trigue´ros et al. 2001). For example the specific mannose-binding site (MQGDCNL). 30 RACE Outer Pri- carbohydrate-binding capacities of lectins involve the mer and P1 were used for PCR in the 30 RACE. PCR extensive use of water bridges, post-translation modifica- reaction for 30 RACE and 50 RACE was the same and tion and oligomerisation. Exogenous saccharides including performed under the following condition: cDNA was glycoproteins on surface of fungi, plant viruses, the midgut denatured at 94°C for 3 min following by 35 cycles of epithelial membrane of insects and plant predators are the amplification (94°C for 30 s, 55°C for 30 s, 72°C for most possible receptors of lectins (De Hoff et al. 2009). 1 min) and 72°C for a 10-min extension. Based on the Crop losses due to fungal diseases pose a serious threat sequence of the 30-RACE product, specific primers P2 and to global food security. In addition, certain groups of fungi P3 were designed to amplify the 50 end of PTA. The Outer can produce highly toxic and carcinogenic mycotoxins in PCR was performed with primer P2 and 50 RACE Outer infected crops, posing a direct health hazard to humans and Primer. The Inner PCR was carried out with primer P3 and animals. Some fungi, such as Alternaria alternata, Bipo- 50 RACE Inner Primer. By comparing the 30-RACE and 50- laris sorokiniana and Curvularia lunata, cause wilt disease RACE sequences, the full-length cDNA of PTA was in important crop plants, such as wheat and potato, obtained using gene-specific primers P4 and P5. All the resulting in poor yield and quality. The main control primers used above are listed in Table 1 and the PCR methods for fungal pathogens in agricultural crops involve products were purified and cloned into pMDTM 18-T (Ta- chemicals which, although effective, have serious ecolog- KaRa) vector and sequenced. 30 RACE Adaptor, 30 RACE ical and social consequences due to the toxic residues that Outer Primer, 50 RACE Outer Primer and 50 RACE Inner remain after their use. The ability to control fungal Primer used in this assay provided by Kit (30 Full RACE pathogens through transgenic expression of a lectin such as Core Set, 50 Full RACE Core Set kits, TaKaRa). PTA would be a useful and preferable alternative to toxic chemicals. Bioinformatics analysis of PTA gene and structure We report in this paper the cloning, expression, purifi- analysis of PTA cation and characterization of an agglutinin from P. ter- nata, designated as PTA, a traditional Chinese medicinal The open reading frame (ORF) of the PTA gene was plant species belong to to explore its potential as searched using the ORF finder program (http://www.ncbi. plant defense system against fungal pathogens of agricul- nlm.nih.gov/gorf/gorf.html). The theoretical molecular tural crops. mass and isoelectric points of the proteins were calculated using the ProtParam tool (http://us.expasy.org/tools/prot param.html). Using GNA as the model protein, molecular Materials and methods modeling was carried out using the Swiss-Model program according to (Schwede et al. 2003). Amino acid sequences Plant material and fungal strains of lectins from different plant species were aligned using the ClustalX program. Pinellia ternata f. angustata was collected from the prov- ince of Gansu, China while A. alternata, B. sorokiniana, Plasmid constructs for recombinant proteins and C. lunata were obtained from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. To obtain clones merely with the mature peptide coding The strains A. alternata (No. 3.5373), B. sorokiniana (No. region of PTA-DOM1, PTA-DOM2 and PTA-P, primers 3.2881), and C. lunata (No. 3.1866) have been deposited at (Table 1) with corresponsive restriction enzyme sites were the China General Microbiological Culture Collection designed based on the mature peptide domains sequences of Centre (CGMCC). Natural PTA isolated from P. ternata the target clone obtained above (PTA-DOM1, P6 and P7; 123 World J Microbiol Biotechnol (2010) 26:545–554 547

Table 1 Primers used in this Primer Sequence study P1 50-ATGCAGGGCGACTGCAACCT-30 P2 50-ACCCTGCTTGGAGCTGGACTG-30 P3 50-CAGCCGTACTTGCCGCCGT-30 P4 50-ACACAAAGCCGCGAACACGTATTC-30 P5 50-GGGAGCCATATTTATTTATTCAAGGG-30 P6 50-GCTAGC GCAGTGGGCACCAACTATCTGCTGTCC-30 (NheI) P7 50-AAGCTTGGGGACCCAAGGGTTGATCTTGAAGAC-30 (HindIII) P8 50-GCTAGCATCAACCCTTGGGTCCCCGGCCTCAAC-30 (NheI) P9 50-AAGCTTCGTCTCCTGAGCAGCAACGGAGCGC-30 (HindIII) Engineered restriction sites are ActinF 50-CCTCCAATCCAGACACTGTA-30 underlined. All primers were ActinR 50-AACTGGGATGATATGGAGAA-30 synthesized by TaKaRa

PTA-DOM2, P8 and P9; PTA-P, P6 and P9). The follow dialysis against 20 mM PBS for 12 h. Desalted sample was assays of construction were performed as previous descri- adjusted to *2 mg/ml and stored at -20°C for the coming bed (Li et al. 2007). PCR reaction was as follows: 94°C for use. Primary antibodies against purified antigens were 30 s, 60°C for 45 s, 72°C for 30 s (30 cycles), and finally, produced by a commercial vendor (IGDB, CAS, China) 72°C for a 10-min extension. The amplified production was according to their standard protocol, using 0.5 mg of the cloned into pMDTM 18-T vector and sequenced. The purified protein as antigen. Groups of New Zealand white recombinant vectors were digested with NheI and HindIII, rabbits were immunized subcutaneously with fusion pro- and the resulting fragments were cloned into the right sites teins and emulsified in Montanide ISA-720 (Seppic Inc., of the expression vector pET28a(?), which was designated Paris, France) as described previously (Dutta et al. 2002). cloned into the NheI and HindIII sites of the expression vector, which were designed pET28a(?)-PTA-DOM1, Hemagglutinating activity and saccharide inhibition pET28a(?)-PTA-DOM2 and pET28a(?)-PTA-P. They assays were introduced into E. coli BL21 cells. The empty vector was also introduced into BL21 as a control. Hemagglutinating activity and saccharide inhibition assays were performed in microtiter V-plates (50 ll) adapted from Purification of the expressed His-Tag protein Wang et al. (2004). Agglutination of a 2% (v/v) suspension and antiserum production of rabbit red blood cells in 20 mM phosphate buffered saline (pH 7.2) and inhibition of hemagglutination by Expression and purification of proteins were carried out as various carbohydrates were carried out by a twofold serial described by Kim et al. (2006). The expression plasmids dilution technique. Agglutination was controlled visually were introduced into E. coli BL21 cells. Cells from a after 1 h at room temperature. 1,000 ml culture were overexpressed by isopropylthio-b- galactoside (IPTG) induction, and harvested by centrifu- Assay for antifungal activity gation, resuspended in 40 ml binding buffer (20 mM PBS pH 7.8, 500 mM NaCl) containing 1% (v/v) Triton X-100 Three fungal species, A. alternata, B. sorokiniana, and and 1% (w/v) protease inhibitor (Sigma–Aldrich), lysed on C. lunata were included in this assay adapted from Wang ice via sonication using a Branson Sonifier 450 (Branson et al. (2004). To determine the IC50 values for the anti- Ultrasonic Corporation, USA), and subsequently centri- fungal activity of the PTA, three doses (0.3, 1.56, and fuged at 3,000 g for 30 min at 4°C. The pellet was washed 3.13 lM) each of PTA-DOM1 and PTA-DOM2 peptides three times with binding buffer and centrifuged under the were added to 4 ml potato dextrose agar at 45°C. The same condition. Then, the pellet was dissolved in 40 ml solution were rapidly mixed and poured into Petri dishes. binding buffer containing urea (final 8 M). The resulting After the agar had cooled down, same quantity of mycelia lysate was filtered through a 0.45 lM filter, loaded onto was added to each plate. Buffer only without the antifungal Ni–NTA resin (Invitrogen, USA) equilibrated with binding peptide served as a control. After incubation at 23°C for buffer. The His-tagged proteins were eluted in a 150 mM 72 h, the area of the mycelial colony was measured and the imidazole in washing buffer (20 mM PBS pH 6.0, 500 mM inhibition (%) of fungal growth determined. The concen- NaCl) containing urea (final 8 M). The obtained protein tration of the antifungal peptide that produces 50% was confirmed by SDS–PAGE, and then desalted by reduction in the area of the mycelial colony was considered

123 548 World J Microbiol Biotechnol (2010) 26:545–554 as the IC50 (Wang and Ng 2007). To examine the ther- contains an 807 bp open reading frame (ORF) with a cal- mostability, lectin solution (1 mg/ml) prepared in phos- culated molecular mass of 29.22 kDa and an isoelectric point phate buffer (10 mM, pH 7.2) was incubated at different (pI) of 7.00. According to the rule reported by Von Heijne temperatures ranging from 20 to 100°C for 10 min, with (1986), the deduced amino acid sequences of PTA include a

20°C interval. To examine the pH-stability, the purified signal peptide that may be cleaved between amino acids A23 fusion protein was incubated in a water bath at 20°C for and V24. The P. ternata f. angustata lectin domains were 30 min at various pH values ranging from 1 to 14. synthesized in one large precursor and post-translationally Microspectrophotometry (OD 595) was performed to cleaved into mature lectin polypeptides, based on the data- measure microbial growth (Thevissen et al. 1997). base search with Blast RPS on NCBI (http://www.ncbi.nlm. nih.gov/Structure/cdd/wrpsb.cgi). The current result con- Semiquantitative RT-PCR assays firms that the precursor contain two tandem-arrayed poly- peptides, designated as PTA-DOM1 and PTA-DOM2, with Semiquantitative RT-PCR was carried out as described 43.5% sequence identity. previously (Lin et al. 2005). 2 lg total RNA was used to synthesize the first strand cDNA with ExScript RT reagent Molecular modeling of PTA Kit (TaKaRa) following the supplier’s instructions. The Semiquantitative PCR (20 ll total volume) was done using To assess whether both the overall folding of the two PTA 0.5 units of Premix Taq (Ex Taq Version, TaKaRa), and the domains and the structure of the carbohydrate-binding sites PCR mix prepared according to manufacturer’s instruc- resemble those of GNA, molecular modeling was carried tions. For normalization of signal strength, the RT-PCR out using GNA as a model protein. Although PTA-DOM1 reaction for the 18 s rRNA using specific primers ActinF and PTA-DOM2 shared only moderate identity (41.7 and and ActinR (Table 1) were also performed to serve as 42.5%, respectively), the deduced three-dimensional control. PCR products were analyzed on 1% agrose gels. models of either PTA domains coordinated well with that Three separate runs were conducted. of GNA. The two PTA domains exhibit the same b-prism structure as the GNA monomer (Fig. 1a). Both the PTA- Western-blot analysis DOM1 and PTA-DOM2 monomers consisted of three bundles of b-sheets oriented perpendicularly to the axis of Crude extracts were obtained by grinding , petioles, the monomers. These b-sheets formed sub-domains, and roots, bulbils and tubers of P. ternata f. angustata in extract the three sequential sub-domains were connected by loops buffer (10 mM Hepes–KOH, 10 mM MgCl ,330mM 2 to form a 12-stranded b-barrel. The b-barrel contained Sorbitol, 2 mM PMSF, pH 7.5). The extracts were centri- three putative carbohydrate-binding sites located in the fuged and the proteins in the supernatants were separated clefts formed by the three b-sheet bundles (Fig. 1b). by SDS–PAGE and electroblotted on a nitrocellulose membrane (Millipore, USA). The blot was first blocked for 1 h with 5% non-fat dry milk in TTBS (10 mM Tris, Bacterial expression of fusion PTA-DOM1, 150 mM NaCl, and 0.1% (v/v) Triton X-100, pH 7.4), and PTA-DOM2 and PTA-P followed by incubation for 1 h with rabbit primary anti- bodies (Anti-DOM1, Anti-DOM2). After washing with When induced by IPTG, the E. coli BL21 transformed with TTBS, the blot was incubated with goat anti-rabbit anti- pET28a(?)-PTA-DOM1 and pET28a(?)-PTA-DOM2 body conjugated to horseradish peroxidase. The detected could express His-tag fusion PTA-DOM1, PTA-DOM2 protein was visualized by incubating the blot with chemi- and PTA-P proteins. The sizes of the predicted fusion luminescence’s substrate and exposing to X-ray film. proteins PTA-DOM1 and PTA-DOM2 are 16 kDa, while PTA-P is 30 kDa, which roughly corresponds to the molecular size of the expressed proteins on SDS–PAGE. Results The amount of the fusion proteins could reach up to about 12% of total BL21 proteins. Via purification through the Cloning and characterization of the full-length resin with affinity to the proteins tagged with His, the cDNA of PTA fusion proteins with more than 98% purity were obtained. The same polypeptide bands (*16 or 30 kDa) were pro- RACE-PCRs were performed based on the sequences of the duced in the fusion proteins reduced with b-mercap- conserved region of mannose-binding lectins from other toethanol, as well as unreduced one. These results suggest plant species. A 1,331 bp cDNA sequence was cloned and that the protein subunits are not linked by a disulphide deposited in GenBank (EU199445). The fragment of PTA bond (Fig. 2). 123 World J Microbiol Biotechnol (2010) 26:545–554 549

Fig. 1 Diagrams generated based on models of GNA, PTA- DOM1, and PTA-DOM2 (a). The b-sheet strands (indicated by gray arrows) associate in three-four-stranded bundles, forming the b-prism fold. ‘‘N’’ indicates the N-terminal extra loop present in the models. Each model has three carbohydrate- binding sites (I, II, and III). Comparison of the amino acid sequences of two lectin polypeptides encoded by PTA and GNA (b). The sequences were aligned by introducing gaps () to maximize identity; black bars indicate the carbohydrate sites. Identical amino acids for all aligned sequences are shown in gray. GNA (AAA33346)

Hemagglutination and hemagglutination-inhibition monosaccharides, disaccharides, and oligosaccharides, assays only Mannan and LacNAc and partial Mannose have inhibitory effects on agglutination. The agglutination Hemagglutination assay as carried out using the fusion activity of the lectins was also inhibited by Asialofetuin at lectins and native lectin. The minimal concentration of low concentrations. Asialofetuin is a serum desialyated purified fusion proteins required to completely agglutinate glycoprotein possessing exposed terminal T-disaccharide rabbit erythrocytes were PTA-P 5.08 lg/ml, PTA-DOM1 and LacNAc residues. Inhibition of agglutination by sugar 5.54 lg/ml and PTA-DOM2 5.19 lg/ml. Under identical and glycoprotein showed that the recombination proteins condition, the minimal concentration of native protein for have similar specificity and almost at the same level with completely agglutination was 3.97 g/ml. The carbohydrate- native PTA. Inhibition of agglutination further showed that binding specificity assay revealed that, among all tested the fusion proteins have similar specificity to the native one (Table 2).

Antifungal activity of PTAs

Both PTA domains and the precusor exhibited potent antifungal activity toward A. alternata, B. sorokiniana, and C. lunata. The antifungal activity of PTA-DOM2 was obviously stronger than that of PTA-DOM1. When the activities of the three confusion proteins were compared, PTA-P showed antifungal activity stronger than any of two domains (Table 3). Among the tested fungi, B. sorokiniana

appeared most sensitive to PTA-DOM2, with an IC50 of 0.67 lM. Additionally, A. alternata and B. sorokiniana were more sensitive than C. lunata to three fusion proteins (Fig. 3a, b). C. lunata showed morphological toxicity responses to PTA-DOM1 and PTA-DOM2 including an increase in mycelial apex offshoot, distortion, tumescence Fig. 2 SDS–PAGE of purified proteins. Both b-mercaptoethanol- reduced and unreduced lectins demonstrated the same result. and rupture after treatment with 8.0 lM proteins for 24 h 1 N-PTA, 2 PTA-DOM1, 3 PTA-DOM2, M protein marker (Data not shown). Thermal denaturation of the fusion 123 550 World J Microbiol Biotechnol (2010) 26:545–554

Table 2 Minimum concentration of sugars or glycoprotein required to cause inhibition of haemagglutination by native and fusion agglutinins Sugar Concentration for minimal inhibition N-PTA PTA-P PTA-DOM1 PTA-DOM2

D-Mannose 71 mM 61 mM 59 mM 63 mM D-Mannan 0.14 mM 0.14 mM 0.13 mM 0.14 mM Mannotriose 17 mM 15 mM 14 mM 14 mM Asialofetuin 1.2 nM 1.2 nM 1.1 nM 1.1 nM LacNAc 21 mM 23 mM 23 mM 22 mM

The following sugars or glycoprotein were not inhibitory at a final concentration of 200 mM: Maltose, Sucrose, D-fructose, D-fucose, D-glucose, lactose, N-acetylglucosamine, N-acetyl-galactosamine, N-acetyl-D-glucosamine, Maltohexaose proteins showed that the antifungal activities of PTA- Discussion DOM1, PTA-DOM2 were extremely stable between 20 and 80°C. Even after heating at 100°C for 10 min, only 25% of Following the rule described previously (Von Heijne 1986), the original activity of the samples was lost (Fig. 3c). In the deduced amino acid sequence of PTA includes a signal addition, the fusion proteins were also active across a broad peptide that may be cleaved between amino acids A23 and pH range, i.e., from pH 4.0 to pH 9.0, with optimal activity V24. The P. ternata f. angustata lectin domains were syn- at pH 7.8 (Fig. 3d). thesized in one large precursor and post-translationally cleaved into mature lectin polypeptides, based on the data- Expression profiling of PTA in various tissues base search with Blast RPS on NCBI (http://www.ncbi.nlm. nih.gov/Structure/cdd/wrpsb.cgi). The results are good To investigate the expression pattern of PTA in various agreement with lectins of other Araceae species, such as tissues of P. ternata f. angustata, total RNA was isolated Pinellia pedatisecta (Lin et al. 2006) and Typhonium diva- from leaves, petioles, roots, bulbils and tubers, respec- ricatum (Luo et al. 2007). Previous study of Chandra et al. tively, and subjected to semi-quantitative RT-PCR analy- (1999) showed that lectin domains undergo several steps of sis. As a result, PTA was expressed in all tissues tested but post-translational modification to produce the mature poly- at slight different levels. High expression was found in peptides, which is consistent with what we found in the bulbils and tubers, while low expression was found in current study for PTA. leaves, petioles and roots (Fig. 4). Whatever functions lectins play in nature, their carbo- Correspondingly, differential tissues expression was hydrate-binding properties are usually the key determinants analyzed using Western-blot. Rabbit primary antibodies of their roles in different organisms. Previous studies have against purified antigen (Anti-PTA-DOM1 against purified indicated that each subunit of two-domain GNA-related antigen protein PTA-DOM1, and Anti-PTA-DOM2 against lectins contains one to three active mannose-binding sites, purified antigen protein PTA-DOM2.) were used to detect which may be the carbohydrate binding sites that perform PTA proteins in the tissues of P. ternata f. angustata. The the carbohydrate recognition function (Chandra et al. 1999; results showed that PTA was most abundant in the bulbils Wood et al. 1999; Ramachandraiah and Chandra 2000; and tubers at the same level, but in the leaves, petioles and Shridhar et al. 2009). In the case of the sweet protein roots no labeling above background was observed. A single curculin, all three sites of the domain are inactive and specific band with an approximate molecular weight of hence the protein is completely devoid of sugar binding 12 kDa was obtained (Fig. 5). These data further show that activity (Barre et al. 1996). Previous studies have shown the *46 kDa full-length PTA preproprotein is converted that among the two-domain GNA-related identified lectins: to *12-kDa mature subunits after synthesis. DOM1 of Allium sativum agglutinin I (Smeets et al. 1997) contains two active mannose-binding sites; both domain of Arum maculatum agglutinin (Van Damme et al. 1995), Table 3 Determination of IC50 (lM) value of antifungal activity similar to DOM2 of Colocasia esculenta agglutinin (Hirai A. altanata B. sorokiniana C. lunata et al. 1993), have one active mannose-binding site; how- ever, DOM2 of Crocus vernus agglutinin and Hippeastrum PTA-DOM1 1.86 ± 0.05 2.19 ± 0.05 2.80 ± 0.06 hybrid agglutinin has no active mannose-binding site PTA-DOM2 1.41 ± 0.04 0.67 ± 0.02 0.90 ± 0.03 (Kaku et al. 1996; Van Damme et al. 2000). By contrast, PTA-P 0.79 ± 0.03 0.21 ± 0.01 0.44 ± 0.02 the full-length amino acid sequence of the PTA precursor Results are expressed as the average of three experiments ± SE contains three active mannose-binding sites (QDNVY), 123 World J Microbiol Biotechnol (2010) 26:545–554 551

Fig. 3 Antifungal activity of PTA-DOM1 toward A. alternata (a) and PTA-DOM2 toward B. sorokiniana (b); effect of temperature (c) and pH (d) on the antifungal activity of PTA-DOM1 and PTA-DOM2 toward C. lunata. a Control: 15 ll 50 mM MES buffer (pH 7.2); b 90 lg peptide in 15 ll MES buffer; c 180 lg peptide in 15 ll MES buffer

Fig. 5 Western-blot performed using Anti-PTA-DOM1 and Anti- PTA-DOM2. Equal amounts of protein (25 lg) were loaded in each lane. The assay performed using Anti-PTA-DOM1 resulted in bands of about 12 kDa in mass. The protein detected by Anti-PTA-DOM2 revealed the same result (data not shown)

one belongs to PTA-DOM1, and the other two belong to PTA-DOM2 (Table 4). According to the assay for lectin activity, the obtained fusion proteins demonstrated excellent hemagglutinating activity, indicating that the procedures of expression and purification of the proteins were effective. The presence of a typical secretion signal peptide is a common character- istic of plant lectins. In a previous study, researchers found Fig. 4 Expression profiles of PTA, as determined by RT-PCR. RT- PCR analysis of PTA expression. Semi-quantitative RT-PCR products that if a gene with its signal peptide coding region was separated in agarose gels and stained with ethidium bromide are inserted into a prokaryotic expression vector, no fusion shown on the bottom, and histograms depicting the band densities of protein could be induced, while a gene lacking that coding RT-PCR products representing PTA transcript levels are shown on the region was expressed (Li et al. 2007). The results of the top. Actin was used as an internal control. At least three reactions were performed with the primers, as described in the methods. Values current paper are consistent with this finding, as an E. coli are mean ± SD, P \ 0.05 system could not recognize the endogenous signal peptide 123 552 World J Microbiol Biotechnol (2010) 26:545–554

Table 4 Conservation/lack of conservation of the key amino acid was PTA-DOM1, which may be a consequence of its residues of the three mannose-binding sites of the GNA, PTA, and greater number of active carbohydrate-binding sites. other two-domain lectins Recent work suggested that protein stability is a key Lectin/domain Site I Site II Site III function of plant defense proteins given the hostile envi- ronment of the herbivore’s digestive system (Chen et al. GNA QDNVY QDNVY QDNVY 2007). The results of thermal denaturation studies for PTA CEA-N HE*R*VF THE*VN* QDNVY suggested that this lectin is significantly stable up to 80°C. CEA-C QDLVY NK*E*I.E* QDNVY This finding is consistent with similar studies of Gona- AMA-N HE*R*VY TFE*VK* QDNVY tanthus pumilus Agglutinin and Sauromatum guttatum AMA-C QDLIY TK*E*VK* QDNVY Agglutinin (Shangary et al. 1995). PTA activity also PTA-DOM1 HE*K*VY TRE*VN* QDNVY remained high at extreme pH values, with optimum activity PTA-DOM2 QDNVY NK*E*IK* QDNVY within a narrow pH range of pH 7.0–8.0. Conserved residues in the mannose-binding sites of PTA and other Using semi-quantitative RT-PCR analysis, the PTA monocot lectin protomers are indicated in bold. Charged residues that expression pattern in various tissues of P. ternata f. an- replace key residues are indicated by an asterisk (*), while neutral residues that replace key residues are underlined gustata showed that PTA was expressed in all tested tissues with slight differences. Thus, PTA was considered be a constitutively expressed gene. By Western blot, both of PTA. Therefore, the partial PTA-DOM1 and PTA- rabbit primary antibodies were detected only in the bul- DOM2, corresponding to the mature peptides, were inser- bils and tubers at the same location. Nevertheless, in the ted into the expression vector, and this allowed the pro- leaves, petioles and roots, no labeling above background duction of functional fusion proteins in E. coli. was observed. The failure to detect the fragments of PTA Inhibition of agglutination by sugars and oligosaccha- in the vegetative tissues may be attributed to further rides showed that the fusion proteins have similar speci- proteolysis and/or transport to the storage tissues, such as ficity to native agglutinin, even though the latter is a bulbils and tubers, since it is a storage protein to help mixture of isoforms. Assays with animal glycoprotein plants survive periods of adverse conditions or to survive showed that the agglutination activity of the lectins was between growing seasons and provide nutrients to support also inhibited by low concentrations of Asialofetuin. This the growth of new plants as seedlings or shoots once it is observation is in agreement with the fact that Asiolofetuin produced in P. ternata cells. On the other hand, many contains several types of branched oligomannosedes and lectins are expressed upon application of well-defined indicates that, although the lectins have a weak monosac- stress conditions, such as plant hormone treatment (Jiang charide specificity toward Mannose, the carbohydrate- et al. 2007; Vandenborre et al. 2009), salt stress or binding site of lectin is able to accommodate more com- drought (Wu et al. 2007; Shao et al. 2009). It can be plex sugar moieties. The carbohydrate specificity of the concluded, plant lectins, such as PTA, may therefore have two forms of PTA indicated that the fusion proteins and defensive as well as storage roles. Previously report native PTA were similar to that of known monocot man- (Shewry 2003; Shao et al. 2008) also showed that some nose-binding lectins, including Allium ascalonicum plant lectins have antioxidant properties, with activity as agglutinin (Mo et al. 1993), Galanthus nivalis agglutinin dehydroascorbate reductase and mono-dehydroacscorbate (Van Damme et al. 1987), and Tulipa hybrid lectin (Oda reductase, and an ability to scavenge against both 1,1- and Minami1986). dipheny1-2-picrylhydrazl (DPPH) radicals and hydroxyl Some lectins, such as wheat germ agglutinin (Ciopragra radicals. et al. 1999), red kidney bean hemagglutinin (Ye et al. In conclusion, the two domains of PTA including the 2001), and lectin from stinging nettle (Does et al. precursor form were effectively expressed in E. coli, and 1999) have been shown to possess antifungal properties, the fusions proteins all have the same activity, similar to while others are devoid of these properties (Wang and Ng the native protein. To our knowledge, this is the first time 2007). Like other monocot lectins, PTA is likely to have this method has been used to study lectin domains sepa- anti-fungal properties as well, which makes studying on the rately. Furthermore, we determined that one carbohydrate- structure and function of PTA a potentially important binding site of PTA is sufficient for agglutinin activity and transgenic option for control of pests and fungal diseases in that the presence of additional sites may have no further plants. The fusion proteins expressed as PTA domains were effect on this activity. These conclusions could help effective against a variety of fungal species, including A. researchers to produce large amounts of PTA by expressing alternata, B. sorokiniana, and C. lunata. The PTA-DOM2 either one of its two domains in E. coli. This method may protein was more potent against these fungal species than help alleviate the shortage of native lectins.

123 World J Microbiol Biotechnol (2010) 26:545–554 553

Acknowledgments We gratefully acknowledge Dr. Lu-Guang Wu Lin J, Zhou X, Fei J, Liao Z, Jin W, Sun X, Tang K (2006) Genomic (The University of Queensland, Australia) for reading the manuscript. cloning and characterization of a PPA gene encoding a mannose- Special thanks are due to Dr. Jiang-Qi Wen (The Samuel Roberts binding lectin from Pinellia pedadisecta. Biocell 30:15–25 Noble Foundation, USA) for helpful suggestions, advice, and extre- Luo Y, Xu X, Liu J, Li J, Sun Y, Liu Z, Liu J, Van Damme EJM, mely valuable comments on the project and manuscript. We also Balzarini J, Bao J (2007) A novel mannose-binding tuber lectin thank Dr. Xiang-Qi Zhang (CAS, China) for his excellent assistance. from Typhonium divaricatum (L.) Decne (family Araceae) with antiviral activity against HSV-II and anti-proliferative effect on human cancer cell lines. J Biochem Mol Biol 40:358–367 Mo HO, Van Damme EJM, Peumans WJ, Goldstein IJ (1993) References Purification and characterization of a mannose-specific lectin from shallot (Allium ascalonicum) . Arch Biochem Biophys Barre A, Van Damme EJM, Peumans WJ, Rouge´ P (1996) Structure– 306:431–438 function relationship of monocot mannose-binding lectins. Plant Mo H, Rice KG, Evers DL, Winter HC, Peumans WJ, Van Damme Physiol 112:1531–1540 EJM Goldstein IJ (1999) Xanthosoma sagittifolium tubers Chandra NR, Ramachandraiah G, Bachhawat K, Dam TK, Surolia A, contain a lectin with two different types of carbohydrate-binding Vijayan M (1999) Crystal structure of a dimeric mannose-specific sites. J Biol Chem 274:33300–33305 agglutinin from garlic: quaternary association and carbohydrate Oda Y, Minami K (1986) Isolation and characterization of a lectin from specificity. J Mol Biol 285:1157–1168 tulip bulbs, Tulipa gesneriana. Eur J Biochem 159:239–245 Chen H, Gonzales-Vigil E, Wilkerson CG, Howe GA (2007) Stability Ramachandraiah G, Chandra NR (2000) Sequence and structural of plant defense proteins in the gut of insect herbivores. Plant determinants of mannose recognition. Proteins 39:358–364 Physiol 143:1954–1967 Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an Ciopragra J, Gozia O, Tudor R, Brezuica L, Doyle RJ (1999) Fusarium automated protein homology-modeling server. Nucleic Acids sp. growth inhibition by wheat germ agglutinin. Biochim Biophys Res 31:3381–3385 Acta 1428:424–432 Shangary S, Singh J, Kamboj SS, Kamboj KK, Sandhu RS (1995) Does MP, Houterman PM, Dekker HL, Cornelissen BJC (1999) Purification and properties of four monocot lectins from the Processing, targeting and antifungal activity of stinging nettle family Araceae. Phytochemistry 40:449–455 agglutinin in transgenic tobacco. Plant Physiol 120:421–432 Shao HB, Chu LY, Lu ZH, Kang CM (2008) Primary antioxidant free De Hoff PL, Brill LM, Hirsch AM (2009) Plant lectins: the ties that radical scavenging and redox signaling pathways in higher plant bind in root symbiosis and plant defense. Mol Gen Genom cells. Int J Biol Sci 4:8–14 282:1–15 Shao HB, Chu LY, Jaleel A, Manivannan P, Panneerselvam R, Shao Dutta S, Lalitha PV, Ware LA, Barbosa A, Moch JK, Vassell MA, MA (2009) Understanding water deficit stress-induced changes Fileta BB, Kitov S, Kolodnv N, Heppner DG, Havnes JD, Lanar in the basic metabolism of higher plants—biotechnologically DE (2002) Purification, characterization, and immunogenicity of and sustainably improving agriculture and the ecoenvironment in the refolded ectodomain of the Plasmodium falciparum apical arid regions of the globe. Cri Rev Biotechnol 29:131–151 membrane antigen 1 expressed in Escherichia coli. Infect Immun Shewry PR (2003) Tuber storage proteins. Ann Bot (Lond) 91:755–769 70:3101–3110 Shridhar S, Chattopadhyay D, Yadav G (2009) Plecdom: a program Fouquaert E, Smith DF, Peumans WJ, Proost P, Balzarini J, Savvides for identification and analysis of plant lectin domains. Nucleic SN, Van Damme EJM (2009) Related lectins from snowdrop and Acids Res 37(Suppl 2):W452–W458 maize differ in their carbohydrate-binding specificity. Biochem Smeets K, Van Damme EJM, Verhaert P, Barre A, Rouge´ P, Van Biophys Res Commun 380:260–265 Leuven F, Peumans WJ (1997) Isolation, characterization and Hirai M, Nakamura K, Imai T, Sato T (1993) cDNAs encoding for molecular cloning of the mannose-binding lectin from leaves and storage proteins in the tubers of taro (Colocasia esculenta Schott). roots of Garlic (Allium sativum L.). Plant Mol Biol 33:223–234 Jpn J Genet 68:229–236 Thevissen K, Osborn WO, Acland DP, Broekaert WF (1997) Specific, Jiang Y, Yang B, Harris NS, Deyholos MK (2007) Comparative high affinity binding sites for an antifungal plant defensin on proteomic analysis of NaCl stress-responsive proteins in Ara- Neurospora crassa hyphae and microsomal membranes. J Biol bidopsis roots. J Exp Bot 58:3591–3607 Chem 272:32176–32181 Kaku H, Tanaka Y, Tazaki K, Minami E, Mizuno H, Shibuya N Trigue´ros V, Wang M, Pe`re D, Paquereau L, Chavant L, Fournier D (1996) Sialylated oligosaccharide-specific plant lectin from (2001) Modulation of a lectin insecticidal activity by carbohy- Japanese elderberry (Sambucus sieboldiana) bark tissue has a drates. Arch Insect Biochem Physiol 45:175–179 homologous structure to type II ribosome-inactivating proteins, Trindade MB, Lopes JLS, Soares-Costa A, Monteiro-Moreira AC, ricin and abrin. cDNA cloning and molecular modeling study. J Moreira RA, Oliva MLV, Beltramini LM (2006) Structural Biol Chem 271:1480–1485 characterization of novel chitin-binding lectins from the genus Kaur N, Dhuna V, Kamboj SS, Agrewala JN, Singh J (2006) A novel Artocarpus and their antifungal activity. Biochim Biophys Acta antiprolife and antifungal lectin from Amaranthus virids Linn 1764:146–152 seeds. Protein Pept Lett 13:897–905 Van Damme EJM, Allen AK, Peumans WJ (1987) Isolation and Kim HK, Chun DS, Kim JS, Yun CH, Lee JH, Hong SK, Kang DK characterization of a lectin with exclusive specificity towards (2006) Expression of the cationic antimicrobial peptide lactof- mannose from snowdrop (Galanthus nivalis) bulbs. FEBS Lett erricin fused with the anionic peptide in Escherichia coli. Appl 215:140–144 Microbiol Biotechnol 72:330–338 Van Damme EJM, Goossens K, Smeets K, Van Leuven F, Verhaert P, Li DH, Jian GL, Zhang YT, Ai TM (2007) Bacterial expression of a Peumans WJ (1995) The major tuber storage protein of Araceae Trichosanthes kirilowii defensin (TDEF1) and its antifungal activity species is a lectin: Characterization and molecular cloning of the on Fusarium oxysporum. App Microbiol Biotechnol 74:146–151 lectin from Arum maculatum L. Plant Physiol 107:1147–1158 Lin WH, Wang Y, Mueller Roeber B, Brearley CA, Xu ZH, Xue HW Van Damme EJM, Astoul CH, Barre A, Rouge´ P, Peumans WJ (2000) (2005) At5PTase13 modulates cotyledon vein development Cloning and characterization of a monocot mannose-binding through regulating auxin homeostasis. Plant Physiol 139:1677– lectin from Crocus vernus (family Iridaceae). Eur J Biochem 1691 267:5067–5077

123 554 World J Microbiol Biotechnol (2010) 26:545–554

Van Damme EJM, Nakamura-Tsuruta S, Smith DF, Ongenaert M, mannose-binding proteins: a novel class of plant proteins with Winter HC, Rouge´ P, Goldstein IJ, Mo H, Kominami J, Culerrier antifungal properties. Plant J 25:651–661 R, Barre A, Hirabayashi J, Peumans WJ (2007) Phylogenetic and Wang H, Ng TB, Liu QH (2004) Alveolarin a novel antifungal specificity studies of two-domain GNA-related lectins: genera- polypeptide from the wild mushroom Polyporus al veolaris. tion of multispecificity through domain duplication and diver- Peptides 25:693–696 gent evolution. Biochem J 404:51–61 Wood SD, Wright LM, Reynolds CD, Rizkallah PJ, Allen AK, Vandenborre G, Miersch O, Hause B, Smagghe G, Wasternack C, Peumans WJ, Van Damme EJM (1999) Structure of the native Van Damme EJM (2009) Spodoptera littoralis-induced lectin (unligated) mannose-specific lectin from Scilla campanu- expression in Tabacco. Plant Cell Physiol 50:1143–1155 lata (bluebell) at 1.7 A˚ resolution. Acta Cryst 55:1264–1272 Von Heijne G (1986) A new method for predicting signal sequence Wu G, Zhang C, Chu LY, Shao HB (2007) Responses of higher plants cleavage sites. Nucleic Acids Res 14:4683–4690 to abiotic stresses and agricultural sustainable development. J Wang HX, Ng TB (2007) An antifungal peptide from red lectil seeds. Plant Interact 4:135–148 Peptides 28:547–552 Ye XY, Ng TB, Tsang PWK, Wang J (2001) Isolation of a homo- Wang X, Bauw G, Van Damme EJM, Peumans WJ, Chen Z, Van dimeric lectin with antifungal and antiviral activities from red Montagu M, Angenon G, Dillen W (2001) Gastrodianin-like kidney bean (Phaseolus vulgaris). J Protein Chem 20:367–375

123