Biotechnol Lett DOI 10.1007/s10529-014-1744-6
ORIGINAL RESEARCH PAPER
Isolation, structure modeling and function characterization of a trypsin inhibitor from Cassia obtusifolia Zubi Liu • Qiankun Zhu • Juanjuan Li • Gan Zhang • Aerguli Jiamahate • Jiayu Zhou Hai Liao
•
Received: 13 October 2014 / Accepted: 26 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A trypsin inhibitor gene (CoTI1) from Cassia obtusifolia was isolated and the deduced amino acid sequence was attributed to the Kunitz-type trypsin inhibitor. The recombined CoTI1, expressed in E. coli, exhibited strong inhibitory effect on bovine trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura. CoTI1 thus presents insecticidal properties that may be useful for the genetic engineering of plants. Leu84, Arg86 and Thr88 were predicted as three key residues by molecular modeling in which Arg86, inserted into the substrate pocket of trypsin, interacted directly with residue Asp189 of trypsin causing the specific inhibition against trypsin. The predicted results were confirmed by site-directed mutagenesis with L84A,
Zubi Liu and Qiankun Zhu have equal contribution
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1744-6) contains supplementary material, which is available to authorized users. Z. Liu J. Li G. Zhang A. Jiamahate J. Zhou (&) H. Liao (&) School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China e-mail: [email protected] H. Liao e-mail: [email protected] Q. Zhu College of Life Sciences, Zhejiang University, Hangzhou 310058, China
R86A and T88A, respectively. The substantial changing expression level of CoTI1 under salt, drought and abscisic acid treatment suggested that CoTI1 might play important role in the resistance against abiotic stress. Keywords Cassia obtusifolia Insectcidal trypsin inhibitor Lepidopterous pests Molecular cloning Molecular modeling Trypsin inhibitor Trypsin-like proteases
Introduction Trypsin inhibitors, which play an important role in controlling activity of proteolytic enzymes, are present in significant quantities in seeds and storage organs of plants (Nixon and Wood 2006; Dreon et al. 2010). Most plant trypsin inhibitors are similar to the Kunitztype trypsin inhibitor family, which are mainly found in the seeds of leguminous plants (Rawlings et al. 2004). The sequences of many members of this protein family have high homologies and their structures reveal several conserved features (Dreon et al. 2010; Zhou et al. 2013). Kunitz-type trypsin inhibitors inhibit not only mammalian trypsin but also trypsinlike proteases in the midgut of lepidopterous pests (Nixon and Wood 2006; Oliveira et al. 2009). For this reason, Kunitz-type trypsin inhibitors can act as a defense factor to protect plants against lepidopterous
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pests. Over-expression of Kunitz-type trypsin inhibitors in transgenic plants confers increasing resistance to lepidopterous pests (Nixon and Wood 2006; Oliveira et al. 2009). Cassia obtusifolia L. (Caesalpinioideae, Leguminosae), an important herb commonly found in subtropical areas, has attracted attention as its seed can be used to produce foods, drinks, pesticides, and anticancer medicines (Sob et al. 2010; Kim et al. 2011). Previously, we purified a Kunitz-type trypsin inhibitor from the seeds of C. obtusifolia that showed strong resistance against the midgut trypsin-like protease of Pieris rapae (Liao et al. 2007). We also obtained the transcriptome from the seeds of C. obtusifolia (Liu et al. 2014). Based on function annotations, we found a unigene (BMK_unigene 30808) of Kunitz-type trypsin inhibitor that had the highest homology with Inga laurina trypsin inhibitor (Accession JF766936). It was named as C. obtusifolia trypsin inhibitor 1 (CoTI1). It had a fragment of 221 bp length containing 16 nucleotides upstream of the initial ATG codon but an incomplete ORF. Therefore, our main aim in this paper was the full-length molecular cloning of CoTI1 from the seed and its expression in E. coli. In addition, after homology modeling and site-mutagenesis of CoTI1, the in vitro activities of the recombinant and mutated CoTI1 against trypsin and midgut trypisn-like proteases from three pests were evaluated. Finally, the expression pattern of CoTI1 under various stresses was investigated. This gene may therefore be useful for the transgenic engineering to improve the resistance of plants against these pests.
primer (Supplementary Table 1). 30 -RACE was performed by Ex Taq (Takara, Japan) using DSP and 3RP primers (Supplementary Table 1). The full-length cDNA was amplified using two primers (CDSFP and CDSRP, Supplementary Table 1). All resulting products were subcloned into pMD19-T vectors (Takara) and sequenced (Shanghai Songon, China). The specific primers were designed with Primer Premier 5.0 software. Alignments of amino acid sequences were carried out with the sequence analysis software DNAMAN. Molecular modeling of CoTI1 According to the NCBI BLAST research against Protein Data Bank (PDB), Enterolobium contortisiliquum trypsin inhibitor EcTI (PDB ID: 4J2K) (Zhou et al. 2013) and Glycine max trypsin inhibitor GmTI (PDB ID: 1BA7) (De Meester et al. 1998) showing 33 and 34 % identity with CoTI1, respectively, were selected as the templates to construct the CoTI1 structure by MODELER (Sali 1995). The initial CoTI1 model was optimized by energy minimization (EM) and molecular dynamics (MD) simulation (Zhu et al. 2014). The quality of CoTI1 structure was checked using PROCHECK, PROSA and Verify-3D (Zhu et al. 2014). For the purpose of inhibitor-protein complex formation, the structure trypsin was copied from the 4J2K crystal complex to the model of CoTI1. The CoTI1-trypsin complex was subjected to EM and MD simulation (Zhu et al. 2014). Construction of expression plasmids
Materials and methods Molecular cloning of CoTI1 gene Cassia obtusifolia was grown in a greenhouse at 25 °C with 50 % humidity and 16 h light/8 h dark. The seeds of C. obtusifolia on day 30 after flowering are collected and frozen in liquid N2 and stored at -80 °C for RNA isolation. Total RNA was isolated using the RNA plant-plus reagent (Omega, USA). After being treated with DNase I (Takara, Japan) to remove contaminant DNA, total RNA was used to synthesize the first-strand cDNA by M-MLV reverse transcriptase (Takara, Japan) using oligo dT-AP
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The coding region of CoTI1 gene was amplified by PCR using the forward primer ExFP containing the XhoI site and the reverse primer ExRP containing the EcoRI site (Supplementary Table 1). The fragment was cloned into a pMD19-T vector for sequencing to confirm that no substitutions or deletions occurred. The insert was digested with XhoI and EcoRI and gelpurified with a Gel Extraction Kit (BioTech, China), and then integrated into a purified pET28a vector (Amersham) digested with the same enzymes, resulting in a recombinant plasmid pET28a-CoTI11. The recombinant plasmid pET28a-CoTI1 and empty vector pET28a were transformed into E. coli Rosetta (DE3).
Biotechnol Lett
Protein expression and purification To express CoTI11 fusion protein, the transformed cells were grown at 37 °C in 500 ml LB liquid medium containing 0.1 mg kanamycin ml-1. When OD600 reached 0.6, IPTG was added to 1 mM and the culture was further incubated for 6 h at 22 °C and 160 rpm. The cells were harvested by centrifugation for 20 min at 4 °C and 5,0009g. The sediment was suspended in lysis buffer (20 mM Tris/HCl, pH 8.0, containing 0.1 % (w/v) lysozyme and 150 mM NaCl) and sonicated four times in 20 s bursts at medium intensity. The resulting suspension was centrifuged at 4 °C and 15,0009g for 20 min and the supernatant was collected and loaded onto a Ni-agarose column are equilibrated with buffer (20 mM Tris/HCl (pH 8.0), 150 mM NaCl) following the manufacturer’s instructions. The Ni-agarose column loaded recombinant protein was rinsed with buffer (20 mM Tris/HCl (pH 8.0), 150 mM NaCl, and 10 mM imidazole). The recombinant protein was eluted with elution buffer (20 mM Tris/HCl (pH 8.0), 150 mM NaCl, and 60–300 mM imidazole). 10 lg of crude protein extract and 10 lg purified protein were analyzed using 12.5 % SDS-PAGE followed by Coomassie Blue staining. Enzyme inhibition assay The midguts of the larvaes of Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura at fourth instars were removed in cold 150 mM NaCl, homogenized, and centrifuged for 10 min at 4 °C and 10,0009g. The supernatants were used as midgut proteases and were stored at -20 °C. Protein concentration was determined by Bradford method using bovine serum albumin as standard. The activities of bovine trypsin (Simga, USA) and midgut trypin-like proteases of pests were determined using the synthetic substrate, N-benzoyl-DL-argininep-nitroaniline (BAPNA), as described by Erlanger et al. (1961). After inhibitor samples were incubated with enzymes at 37 °C in 100 mM Tris/HCl, pH 8.0 and 10 mM CaCl2 for 10 min, the BAPNA solution was then added to start the reaction. After 10 min, the reaction was stopped with 33 % (w/v) acetic acid. One unit of trypsin or pest proteases activity was defined as the increase of 0.1 absorbance units at 410 nm and one
unit of inhibitory activity (UI) was defined as the decrease of 0.1 absorbance units at 410 nm. Site-directed mutagenesis The codon AGG (Arg86), CTC (Leu84) and ACT (Thr88) were changed to GCG (Ala), respectively, using three primer pairs (L84AFP-L84ARP, R86AFPR86ARP, T88AFP-T88ARP, Supplementary Table 1). The mutagenesis was performed using Point Mutation Kit (Tiandz, China). The pET28a-CoTI1 was used as template to produce the mutated recombinant constructs pET28a-CoTI1R86A, pET28a-CoTI1L84A and pET28aCoTI1T88A. The transformation, expression, purification and enzyme inhibition assay of mutated proteins were performed using the same methods as those used for pET28a-CoTI1. Expression pattern analysis of CoTI1 under stresses The sterile seedlings (7 days after germination) cultured on MS solid medium at 25 °C with 16 h light/8 h dark were used in the stress experiments. For cold treatment, seedlings were placed on MS liquid medium at 4 °C for 0, 1, 6, 12, 24 and 48 h, respectively. For drought, salt and growth hormone treatments, the seedlings were placed on the MS culture medium supplemented with PEG 6,000 (100 mg ml-1), NaCl (200 mM) or with abscisic acid (ABA, 100 lM) for 0, 1, 6, 12, 24 and 48 h, respectively. The total RNA of cotyledons of all samples was extracted and subjected to cDNA synthesis with the same method used for 30 RACE. The quantitative analysis of CoTI1 expression was estimated by real-time PCR. Gene-specific primer pairs for real-time PCR of CoTI1 (TIqFP and TIqRP in Supplementary Table 1) and reference gene elongation factor 1a (EF1aqFP and EF1aqRP in Supplementary Table 1) were designed using Primer Premier 5.0 based on the full-length cDNA. The Real-time PCR was performed by SYBR Premix Ex Taq II (Takara, Japan) with LightCycler 96 system (Roche Diagnostics, Germany) based on 1 ll cDNA template in 10 ll reaction buffer using Gene-specific primer pairs under the following conditions: pre-denaturation at 95 °C for 1 min, followed by 40 cycles of 10 s at 95 °C, 10 s at 60 °C, and 20 s at 72 °C. The expression level of CoTI1 was analyzed using DDCT-method in LightCycler 96 software.
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Results and discussion
In silico analysis on structure and trypsin binding of CoTI1
Cloning and sequence analysis of CoTI1 gene The full-length cDNA of CoTI1 was obtained by RACE and sequenced as 830 bp, containing an entire ORF of CoTI1 cDNA (Supplementary Fig. 1). The genomic DNA of CoTI1 ORF had 100 % identity with its cDNA, which showed that CoTI1 had no intron in the coding region (Supplementary Fig. 1). The translated polypeptide of 209 amino acids included a signal peptide of 23 amino acid (Fig. 1) and showed 30–46 % identity with plant Kunitz-type trypsin inhibitors. COTI1 had the highest identity of 46 % with Inga laurina trypsin inhibitor. Kunitz-type trypsin inhibitors have a typical active motif (P30 -P20 -P1-P2-P3). Usually, Arg or Lys residue in the P1 site of trypsin inhibitor is important for the formation of trypsin-inhibitor complex and specific inhibition. The active motif (P30 -P20 -P1-P2-P3) of CoTI1 was located from L84 to T88 (Fig. 1). CoTI1 had an Arg86 in the P1 site and the Inga laurina trypsin inhibitor contained a Lys 64 in this position (Fig. 1). Moreover, CoTI1 contained four cystseine residues (Cys 64, 108, 156 and 164) that might be involved to form disulfide bonds. The nucleotide sequence of COTI1 cDNA was submitted to GenBank databases with the accession number of KM670436.
The constructed 3D structure of CoTI1 was predominantly organized in 11 sheets and 12 loops (Fig. 2a). There was a disulfide bond between Cys156 and Cys164 (Figs. 1, 2a). The active loop (L84-V85-R86-P87-T88), located between S4 and S5, inserted into the substrate pocket of trypsin (Fig. 2a). The side chains of Leu84, Arg86 and Thr88 stretched out of the loop and bound to trypsin, while those of Val85 and Pro87 located inside the loop (Fig. 2b, c). The side chain of P1 residue Arg86 reached the bottom of trypsin substrate pocket surrounded by His57, Asp189, Ser190, Cys191, Gln192, Gly193, Asp194, Ser195, Ser214, Gly216, Ser217, Gly219, Cys220, Gly226 of trypsin and formed hydrogen bonds with S1 residue Asp189 and Gly193 of trypsin, causing the specific inhibition. The side chain of P30 residue Leu84 was close to Leu89, Ser214, and Trp215 of trypsin and might form hydrophobic interaction. P3 residue Thr88 was bound to His40 of trypsin and was close to Phe41 and Tyr161 of trypsin. Therefore, Leu84 and Thr88 were suggested to anchor trypsin at the gate of the substrate pocket of trypsin and prevent the substrate to move into the substrate pocket. According to the homology modeling results, Leu84, Arg86 and Thr88 were regarded as the key residues of CoTI1 and were selected for the further site-directed mutagenesis.
Fig. 1 Sequence alignment of CoTI1 with relative Kunitz-type trypsin inhibitors. IlTI, EcTI, GmTI were trypsin inhibitors from Inga laurina, Enterolobium contortisiliquum and Glycine max, respectively. The accession numbers were presented in brackets. Similar and identical amino acids residues in whole
sequences were outlined. The active motif was shown with yellow. The number sign indicated the key residues selected for mutagenesis by molecular modeling and docking. Asterisk indicated the Cys. ‘‘S–S’’ indicated disulfide bond that was also presented in Fig. 2a
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Fig. 3 The inhibitory activities of recombinant CoTI1 and mutants CoTI1R86A, CoTI1L84A and CoTI1T88A on the bovine trypsin and trypisn-like proteases from H. armigera, S. exigua and S. litura
Fig. 2 In silico analysis on structure and trypsin binding of CoTI1. a The ribbon structure of CoTI1-trypsin complex. ‘‘S–S’’ indicated disulfide bond. b The binding residues of CoTI1 with the surface model of trypsin. The arrow indicated the trypsin substrate pocket. c The key residues (Leu84, Arg86 and Thr88) in CoTI1
Expression and enzyme inhibition assay of recombinant CoTI1 Bhattacharjee et al. (2014) reported there was no signal peptide in the mature Kunitz-type trypsin inhibitors and prokaryotic expression of the mature Kunitz-type trypsin inhibitors (without signal peptide) would not decrease the inhibitory activities. Therefore, in our
study, the amplification product using ExFP and ExRP primers did not include the N-terminal signal peptide of 23 amino acids. The recombinant CoTI1 was successfully expressed in transformed E. coli Rosetta (DE3) with a molecular weight of 24 kDa (Supplementary Fig. 2). Its molecular weight was higher than that of the predicted mass of 20.31 kDa due to the His-tag in the N-terminal fusion peptide. The recombinant CoTI1 was further purified by Ni-agarose column (Supplementary Fig. 2). The purified COTI1 showing a single band in SDSPAGE (Supplementary Fig. 2, Lane 5) was subjected to enzyme inhibition assay. The expressed CoTI1 showed prominent inhibition against trypsin with inhibitory activity of 2,490 UI mg-1. In addition, CoTI1 could inhibit the trypsin-like proteases from H. armigera, S. exigua and S. litura with inhibitory activities of 1,770, 1,210 and 1,510 UI mg-1, respectively (Fig. 3). The substantial inhibition of proteases from three lepidopterous pests suggested that CoTI1 might affect the growth and/or survival of these pests when incorporated into their diet. Future studies of CoTI1 are required to confirm its anti-pest potential as a defense agent. Site-directed mutagenesis To confirm whether the Leu84, Arg86 and Thr88 were the key residues of CoTI1 to bind trypsin, the three residues were subjected to mutagenesis. The codons AGG (Arg86), CTC (Leu84) and ACT (Thr88) were changed to GCG (Ala), respectively (Supplementary Fig. 3). As shown in Fig. 3, the fully active CoTI1 became almost
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the photosynthesis and organ growth, were degraded due to the cleavage of endogenous trypsin-like proteases in plant under abiotic stresses (Callis 1995; Srinivasan et al. 2009). Therefore, the increased expression level of CoTI1 under salt, drought and ABA treatment suggested that COTI1 might reduce the plant damage under abiotic stresses by inhibiting the endogenous trypsin-like proteases in plants. In addition to the potential defense against the above pests, CoTI1 might also play important role in the resistance to abiotic stresses.
Fig. 4 The relative expression level of CoTI1 under ABA, cold, salt and drought treatment
inactive with respect to its trypsin inhibitory activity by a point mutation on its Arg86 residue. Compared with the recombinant CoTI1, CoTI1R86A, CoTI1L84A and CoTI1T88A lost its 90, 54 and 53 % inhibitory activity against trypsin, respectively. This almost complete loss of functionality of mutated CoTI1R86A confirmed the Arg86 as the P1 residue of CoTI1. Furthermore, the residual inhibitory activities of the mutated CoTI1s against the trypsin-like proteases from three pests were investigated. Compared with the recombinant COTI1, CoTI1R86A lost most of its inhibitory activity (89, 90 and 91 %) against trypsinlike proteases from H. armigera, S. exigua and S. litura. Interestingly, the lost inhibitory activities of CoTI1T88A (64, 73 and 72 %) were higher than those of CoTI1 L84A (51, 50 and 46 %), suggesting Thr88 might play more important role in the inhibition against trypsin-like proteases from pests. Expression pattern of CoTI1 under stresses To understand the metabolism related to CoTI1 under stresses, the sterile seedlings were treated with different stresses and then used as samples to analyze the expression pattern of CoTI1 by real-time PCR. As shown in Fig. 4, the expression level of CoTI1 was relative stable under cold stress, whereas it was unstable under salt, drought and ABA treatment. The expression level of CoTI1 went up continuously under salt/drought stresses, whereas it went up before 24 h and then went down under ABA treatment. It was known that abiotic stresses could lead to severe plant damage, and several peptides, which were involved in
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Conclusion This is the first report of the cloning of the CoTI1 gene and its expression. The expressed CoTI1 showed dominant inhibitory activities against trypsin and trypsin-like proteases from H. armigera, S. exigua and S. litura. Leu84, Arg86 and Thr88 were identified as the key residues in which Arg86 was the P1 residue and determined the inhibitory specificity of CoTI1. In addition, the substantial increased expression level of CoTI1 under salt, drought and ABA treatment suggested that CoTI1 might also play important role in the resistance to abiotic stress. Hopefully, COTI1 could be used to construct transgenic plants with resistance to pests and tolerance to abiotic stress in the future. Acknowledgments This work was supported by the Grant (no. 31371232) from National Natural Science Foundation of China and Grant (no. SWJTU11CX114) from Fundamental Research Funds for the Central Universities of China. Supporting information Supplementary Table 1 Primers used in this paper. Supplementary Figure 1 Alignment of genomic DNA, cDNA and the translated polypeptide of CoTI1. (A) Alignment of genomic DNA and cDNA of COTI1. (B) Translated polypeptide from cDNA. Supplementary Figure 2 The expression and purification of recombinant CoTI1. ‘‘M’’ indicated protein maker. Lanes 1–3 indicated collection solution of lysate, penetration and rinse, respectively. Lanes 4–6 indicated the collection solution eluted with 60, 100 and 300 mM imidazole by turns. Supplementary Figure 3 Sequencing results of CoTI1 and mutants CoTI1R86A, CoTI1L84A and CoTI1T88A.
References Bhattacharjee N, Banerjee S, Dutta SK (2014) Cloning, expression and mutational studies of a trypsin inhibitor that
Biotechnol Lett retains activity even after cyanogen bromide digestion. Protein Expr Purif 96:26–31 Callis J (1995) Regulation of protein degradation. Plant Cell 7:845–857 De Meester P, Brick P, Lloyd LF, Blow DM, Onesti S (1998) Structure of the Kunitz-type soybean trypsin inhibitor (STI): implication for the interactions between members of the STI family and tissue-plasminogen activator. Acta Crystallogr D Biol Crystallogr 54:589–597 Dreon MS, Ituarte S, Heras H (2010) The role of the proteinase inhibitor ovorubin in apple snail eggs resembles plant embryo defense against predation. PLoS One 5:e15059 Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–280 Kim SJ, Kim KW, Kim DS, Kim MC, Jeon YD, Kim SG, Jung HJ, Jang HJ, Lee BC, Chung WS, Hong SH, Chung SH, Um JY (2011) The protective effect of Cassia obtusifolia on DSS-induced colitis. Am J Chin Med 39:565–577 Liao H, Ren W, Kang Z, Jiang JH, Zhao XJ, Du LF (2007) A trypsin inhibitor from Cassia obtusifolia seeds: isolation, characterization and activity against Pieris rapae. Biotechnol Lett 29:653–658 Liu Z, Song T, Zhu Q, Wang W, Zhou J, Liao H (2014) De novo assembly and analysis of Cassia obtusifolia seed transcriptome to identify genes involved in the biosynthesis of active metabolites. Biosci Biotechnol Biochem 78:791–799
Nixon A, Wood CR (2006) Engineered protein inhibitors of proteases. Curr Opin Drug Discov Dev 9:261–268 Oliveira AS, Migliolo L, Aquino RO, Ribeiro JK, Macedo LL, Bemquerer MP, Santos EA, Kiyota S, de Sales MP (2009) Two Kunitz-type inhibitors with activity against trypsin and papain from Pithecellobium dumosum seeds: purification, characterization, and activity towards pest insect digestive enzyme. Protein Pept Lett 16:1526–1532 Rawlings ND, Tolle DP, Barrett AJ (2004) Evolutionary families of peptidase inhibitors. Biochem J 378:705–716 Sali A (1995) Comparative protein modeling by satisfaction of spatial restraints. Mol Med Today 1:270–277 Sob SVT, Wabo HK, Tchinda AT, Tane P, Ngadjui BT, Ye Y (2010) Anthraquinones, sterols, triterpenoids and xanthones from Cassia obtusifolia. Biochem Syst Ecol 38:342–345 Srinivasan T, Kumar KR, Kirti PB (2009) Constitutive expression of a trypsin protease inhibitor confers multiple stress tolerance in transgenic tobacco. Plant Cell Physiol 50:541–553 Zhou D, Lobo YA, Batista IF, Marques-Porto R, Gustchina A, Oliva ML, Wlodawer A (2013) Crystal structures of a plant trypsin inhibitor from Enterolobium contortisiliquum (EcTI) and of its complex with bovine trypsin. PLoS One 8:e62252 Zhu Q, Zou J, Zhu M, Liu Z, Feng P, Fan G, Wang W, Liao H (2014) In silico analysis on structure and DNA binding mode of AtNAC1, a NAC transcription factor from Arabidopsis thaliana. J Mol Model 20:2117
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ORIGINAL RESEARCH PAPER
Isolation, structure modeling and function characterization of a trypsin inhibitor from Cassia obtusifolia Zubi Liu • Qiankun Zhu • Juanjuan Li • Gan Zhang • Aerguli Jiamahate • Jiayu Zhou Hai Liao
•
Received: 13 October 2014 / Accepted: 26 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A trypsin inhibitor gene (CoTI1) from Cassia obtusifolia was isolated and the deduced amino acid sequence was attributed to the Kunitz-type trypsin inhibitor. The recombined CoTI1, expressed in E. coli, exhibited strong inhibitory effect on bovine trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura. CoTI1 thus presents insecticidal properties that may be useful for the genetic engineering of plants. Leu84, Arg86 and Thr88 were predicted as three key residues by molecular modeling in which Arg86, inserted into the substrate pocket of trypsin, interacted directly with residue Asp189 of trypsin causing the specific inhibition against trypsin. The predicted results were confirmed by site-directed mutagenesis with L84A,
Zubi Liu and Qiankun Zhu have equal contribution
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1744-6) contains supplementary material, which is available to authorized users. Z. Liu J. Li G. Zhang A. Jiamahate J. Zhou (&) H. Liao (&) School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China e-mail: [email protected] H. Liao e-mail: [email protected] Q. Zhu College of Life Sciences, Zhejiang University, Hangzhou 310058, China
R86A and T88A, respectively. The substantial changing expression level of CoTI1 under salt, drought and abscisic acid treatment suggested that CoTI1 might play important role in the resistance against abiotic stress. Keywords Cassia obtusifolia Insectcidal trypsin inhibitor Lepidopterous pests Molecular cloning Molecular modeling Trypsin inhibitor Trypsin-like proteases
Introduction Trypsin inhibitors, which play an important role in controlling activity of proteolytic enzymes, are present in significant quantities in seeds and storage organs of plants (Nixon and Wood 2006; Dreon et al. 2010). Most plant trypsin inhibitors are similar to the Kunitztype trypsin inhibitor family, which are mainly found in the seeds of leguminous plants (Rawlings et al. 2004). The sequences of many members of this protein family have high homologies and their structures reveal several conserved features (Dreon et al. 2010; Zhou et al. 2013). Kunitz-type trypsin inhibitors inhibit not only mammalian trypsin but also trypsinlike proteases in the midgut of lepidopterous pests (Nixon and Wood 2006; Oliveira et al. 2009). For this reason, Kunitz-type trypsin inhibitors can act as a defense factor to protect plants against lepidopterous
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pests. Over-expression of Kunitz-type trypsin inhibitors in transgenic plants confers increasing resistance to lepidopterous pests (Nixon and Wood 2006; Oliveira et al. 2009). Cassia obtusifolia L. (Caesalpinioideae, Leguminosae), an important herb commonly found in subtropical areas, has attracted attention as its seed can be used to produce foods, drinks, pesticides, and anticancer medicines (Sob et al. 2010; Kim et al. 2011). Previously, we purified a Kunitz-type trypsin inhibitor from the seeds of C. obtusifolia that showed strong resistance against the midgut trypsin-like protease of Pieris rapae (Liao et al. 2007). We also obtained the transcriptome from the seeds of C. obtusifolia (Liu et al. 2014). Based on function annotations, we found a unigene (BMK_unigene 30808) of Kunitz-type trypsin inhibitor that had the highest homology with Inga laurina trypsin inhibitor (Accession JF766936). It was named as C. obtusifolia trypsin inhibitor 1 (CoTI1). It had a fragment of 221 bp length containing 16 nucleotides upstream of the initial ATG codon but an incomplete ORF. Therefore, our main aim in this paper was the full-length molecular cloning of CoTI1 from the seed and its expression in E. coli. In addition, after homology modeling and site-mutagenesis of CoTI1, the in vitro activities of the recombinant and mutated CoTI1 against trypsin and midgut trypisn-like proteases from three pests were evaluated. Finally, the expression pattern of CoTI1 under various stresses was investigated. This gene may therefore be useful for the transgenic engineering to improve the resistance of plants against these pests.
primer (Supplementary Table 1). 30 -RACE was performed by Ex Taq (Takara, Japan) using DSP and 3RP primers (Supplementary Table 1). The full-length cDNA was amplified using two primers (CDSFP and CDSRP, Supplementary Table 1). All resulting products were subcloned into pMD19-T vectors (Takara) and sequenced (Shanghai Songon, China). The specific primers were designed with Primer Premier 5.0 software. Alignments of amino acid sequences were carried out with the sequence analysis software DNAMAN. Molecular modeling of CoTI1 According to the NCBI BLAST research against Protein Data Bank (PDB), Enterolobium contortisiliquum trypsin inhibitor EcTI (PDB ID: 4J2K) (Zhou et al. 2013) and Glycine max trypsin inhibitor GmTI (PDB ID: 1BA7) (De Meester et al. 1998) showing 33 and 34 % identity with CoTI1, respectively, were selected as the templates to construct the CoTI1 structure by MODELER (Sali 1995). The initial CoTI1 model was optimized by energy minimization (EM) and molecular dynamics (MD) simulation (Zhu et al. 2014). The quality of CoTI1 structure was checked using PROCHECK, PROSA and Verify-3D (Zhu et al. 2014). For the purpose of inhibitor-protein complex formation, the structure trypsin was copied from the 4J2K crystal complex to the model of CoTI1. The CoTI1-trypsin complex was subjected to EM and MD simulation (Zhu et al. 2014). Construction of expression plasmids
Materials and methods Molecular cloning of CoTI1 gene Cassia obtusifolia was grown in a greenhouse at 25 °C with 50 % humidity and 16 h light/8 h dark. The seeds of C. obtusifolia on day 30 after flowering are collected and frozen in liquid N2 and stored at -80 °C for RNA isolation. Total RNA was isolated using the RNA plant-plus reagent (Omega, USA). After being treated with DNase I (Takara, Japan) to remove contaminant DNA, total RNA was used to synthesize the first-strand cDNA by M-MLV reverse transcriptase (Takara, Japan) using oligo dT-AP
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The coding region of CoTI1 gene was amplified by PCR using the forward primer ExFP containing the XhoI site and the reverse primer ExRP containing the EcoRI site (Supplementary Table 1). The fragment was cloned into a pMD19-T vector for sequencing to confirm that no substitutions or deletions occurred. The insert was digested with XhoI and EcoRI and gelpurified with a Gel Extraction Kit (BioTech, China), and then integrated into a purified pET28a vector (Amersham) digested with the same enzymes, resulting in a recombinant plasmid pET28a-CoTI11. The recombinant plasmid pET28a-CoTI1 and empty vector pET28a were transformed into E. coli Rosetta (DE3).
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Protein expression and purification To express CoTI11 fusion protein, the transformed cells were grown at 37 °C in 500 ml LB liquid medium containing 0.1 mg kanamycin ml-1. When OD600 reached 0.6, IPTG was added to 1 mM and the culture was further incubated for 6 h at 22 °C and 160 rpm. The cells were harvested by centrifugation for 20 min at 4 °C and 5,0009g. The sediment was suspended in lysis buffer (20 mM Tris/HCl, pH 8.0, containing 0.1 % (w/v) lysozyme and 150 mM NaCl) and sonicated four times in 20 s bursts at medium intensity. The resulting suspension was centrifuged at 4 °C and 15,0009g for 20 min and the supernatant was collected and loaded onto a Ni-agarose column are equilibrated with buffer (20 mM Tris/HCl (pH 8.0), 150 mM NaCl) following the manufacturer’s instructions. The Ni-agarose column loaded recombinant protein was rinsed with buffer (20 mM Tris/HCl (pH 8.0), 150 mM NaCl, and 10 mM imidazole). The recombinant protein was eluted with elution buffer (20 mM Tris/HCl (pH 8.0), 150 mM NaCl, and 60–300 mM imidazole). 10 lg of crude protein extract and 10 lg purified protein were analyzed using 12.5 % SDS-PAGE followed by Coomassie Blue staining. Enzyme inhibition assay The midguts of the larvaes of Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura at fourth instars were removed in cold 150 mM NaCl, homogenized, and centrifuged for 10 min at 4 °C and 10,0009g. The supernatants were used as midgut proteases and were stored at -20 °C. Protein concentration was determined by Bradford method using bovine serum albumin as standard. The activities of bovine trypsin (Simga, USA) and midgut trypin-like proteases of pests were determined using the synthetic substrate, N-benzoyl-DL-argininep-nitroaniline (BAPNA), as described by Erlanger et al. (1961). After inhibitor samples were incubated with enzymes at 37 °C in 100 mM Tris/HCl, pH 8.0 and 10 mM CaCl2 for 10 min, the BAPNA solution was then added to start the reaction. After 10 min, the reaction was stopped with 33 % (w/v) acetic acid. One unit of trypsin or pest proteases activity was defined as the increase of 0.1 absorbance units at 410 nm and one
unit of inhibitory activity (UI) was defined as the decrease of 0.1 absorbance units at 410 nm. Site-directed mutagenesis The codon AGG (Arg86), CTC (Leu84) and ACT (Thr88) were changed to GCG (Ala), respectively, using three primer pairs (L84AFP-L84ARP, R86AFPR86ARP, T88AFP-T88ARP, Supplementary Table 1). The mutagenesis was performed using Point Mutation Kit (Tiandz, China). The pET28a-CoTI1 was used as template to produce the mutated recombinant constructs pET28a-CoTI1R86A, pET28a-CoTI1L84A and pET28aCoTI1T88A. The transformation, expression, purification and enzyme inhibition assay of mutated proteins were performed using the same methods as those used for pET28a-CoTI1. Expression pattern analysis of CoTI1 under stresses The sterile seedlings (7 days after germination) cultured on MS solid medium at 25 °C with 16 h light/8 h dark were used in the stress experiments. For cold treatment, seedlings were placed on MS liquid medium at 4 °C for 0, 1, 6, 12, 24 and 48 h, respectively. For drought, salt and growth hormone treatments, the seedlings were placed on the MS culture medium supplemented with PEG 6,000 (100 mg ml-1), NaCl (200 mM) or with abscisic acid (ABA, 100 lM) for 0, 1, 6, 12, 24 and 48 h, respectively. The total RNA of cotyledons of all samples was extracted and subjected to cDNA synthesis with the same method used for 30 RACE. The quantitative analysis of CoTI1 expression was estimated by real-time PCR. Gene-specific primer pairs for real-time PCR of CoTI1 (TIqFP and TIqRP in Supplementary Table 1) and reference gene elongation factor 1a (EF1aqFP and EF1aqRP in Supplementary Table 1) were designed using Primer Premier 5.0 based on the full-length cDNA. The Real-time PCR was performed by SYBR Premix Ex Taq II (Takara, Japan) with LightCycler 96 system (Roche Diagnostics, Germany) based on 1 ll cDNA template in 10 ll reaction buffer using Gene-specific primer pairs under the following conditions: pre-denaturation at 95 °C for 1 min, followed by 40 cycles of 10 s at 95 °C, 10 s at 60 °C, and 20 s at 72 °C. The expression level of CoTI1 was analyzed using DDCT-method in LightCycler 96 software.
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Results and discussion
In silico analysis on structure and trypsin binding of CoTI1
Cloning and sequence analysis of CoTI1 gene The full-length cDNA of CoTI1 was obtained by RACE and sequenced as 830 bp, containing an entire ORF of CoTI1 cDNA (Supplementary Fig. 1). The genomic DNA of CoTI1 ORF had 100 % identity with its cDNA, which showed that CoTI1 had no intron in the coding region (Supplementary Fig. 1). The translated polypeptide of 209 amino acids included a signal peptide of 23 amino acid (Fig. 1) and showed 30–46 % identity with plant Kunitz-type trypsin inhibitors. COTI1 had the highest identity of 46 % with Inga laurina trypsin inhibitor. Kunitz-type trypsin inhibitors have a typical active motif (P30 -P20 -P1-P2-P3). Usually, Arg or Lys residue in the P1 site of trypsin inhibitor is important for the formation of trypsin-inhibitor complex and specific inhibition. The active motif (P30 -P20 -P1-P2-P3) of CoTI1 was located from L84 to T88 (Fig. 1). CoTI1 had an Arg86 in the P1 site and the Inga laurina trypsin inhibitor contained a Lys 64 in this position (Fig. 1). Moreover, CoTI1 contained four cystseine residues (Cys 64, 108, 156 and 164) that might be involved to form disulfide bonds. The nucleotide sequence of COTI1 cDNA was submitted to GenBank databases with the accession number of KM670436.
The constructed 3D structure of CoTI1 was predominantly organized in 11 sheets and 12 loops (Fig. 2a). There was a disulfide bond between Cys156 and Cys164 (Figs. 1, 2a). The active loop (L84-V85-R86-P87-T88), located between S4 and S5, inserted into the substrate pocket of trypsin (Fig. 2a). The side chains of Leu84, Arg86 and Thr88 stretched out of the loop and bound to trypsin, while those of Val85 and Pro87 located inside the loop (Fig. 2b, c). The side chain of P1 residue Arg86 reached the bottom of trypsin substrate pocket surrounded by His57, Asp189, Ser190, Cys191, Gln192, Gly193, Asp194, Ser195, Ser214, Gly216, Ser217, Gly219, Cys220, Gly226 of trypsin and formed hydrogen bonds with S1 residue Asp189 and Gly193 of trypsin, causing the specific inhibition. The side chain of P30 residue Leu84 was close to Leu89, Ser214, and Trp215 of trypsin and might form hydrophobic interaction. P3 residue Thr88 was bound to His40 of trypsin and was close to Phe41 and Tyr161 of trypsin. Therefore, Leu84 and Thr88 were suggested to anchor trypsin at the gate of the substrate pocket of trypsin and prevent the substrate to move into the substrate pocket. According to the homology modeling results, Leu84, Arg86 and Thr88 were regarded as the key residues of CoTI1 and were selected for the further site-directed mutagenesis.
Fig. 1 Sequence alignment of CoTI1 with relative Kunitz-type trypsin inhibitors. IlTI, EcTI, GmTI were trypsin inhibitors from Inga laurina, Enterolobium contortisiliquum and Glycine max, respectively. The accession numbers were presented in brackets. Similar and identical amino acids residues in whole
sequences were outlined. The active motif was shown with yellow. The number sign indicated the key residues selected for mutagenesis by molecular modeling and docking. Asterisk indicated the Cys. ‘‘S–S’’ indicated disulfide bond that was also presented in Fig. 2a
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Fig. 3 The inhibitory activities of recombinant CoTI1 and mutants CoTI1R86A, CoTI1L84A and CoTI1T88A on the bovine trypsin and trypisn-like proteases from H. armigera, S. exigua and S. litura
Fig. 2 In silico analysis on structure and trypsin binding of CoTI1. a The ribbon structure of CoTI1-trypsin complex. ‘‘S–S’’ indicated disulfide bond. b The binding residues of CoTI1 with the surface model of trypsin. The arrow indicated the trypsin substrate pocket. c The key residues (Leu84, Arg86 and Thr88) in CoTI1
Expression and enzyme inhibition assay of recombinant CoTI1 Bhattacharjee et al. (2014) reported there was no signal peptide in the mature Kunitz-type trypsin inhibitors and prokaryotic expression of the mature Kunitz-type trypsin inhibitors (without signal peptide) would not decrease the inhibitory activities. Therefore, in our
study, the amplification product using ExFP and ExRP primers did not include the N-terminal signal peptide of 23 amino acids. The recombinant CoTI1 was successfully expressed in transformed E. coli Rosetta (DE3) with a molecular weight of 24 kDa (Supplementary Fig. 2). Its molecular weight was higher than that of the predicted mass of 20.31 kDa due to the His-tag in the N-terminal fusion peptide. The recombinant CoTI1 was further purified by Ni-agarose column (Supplementary Fig. 2). The purified COTI1 showing a single band in SDSPAGE (Supplementary Fig. 2, Lane 5) was subjected to enzyme inhibition assay. The expressed CoTI1 showed prominent inhibition against trypsin with inhibitory activity of 2,490 UI mg-1. In addition, CoTI1 could inhibit the trypsin-like proteases from H. armigera, S. exigua and S. litura with inhibitory activities of 1,770, 1,210 and 1,510 UI mg-1, respectively (Fig. 3). The substantial inhibition of proteases from three lepidopterous pests suggested that CoTI1 might affect the growth and/or survival of these pests when incorporated into their diet. Future studies of CoTI1 are required to confirm its anti-pest potential as a defense agent. Site-directed mutagenesis To confirm whether the Leu84, Arg86 and Thr88 were the key residues of CoTI1 to bind trypsin, the three residues were subjected to mutagenesis. The codons AGG (Arg86), CTC (Leu84) and ACT (Thr88) were changed to GCG (Ala), respectively (Supplementary Fig. 3). As shown in Fig. 3, the fully active CoTI1 became almost
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the photosynthesis and organ growth, were degraded due to the cleavage of endogenous trypsin-like proteases in plant under abiotic stresses (Callis 1995; Srinivasan et al. 2009). Therefore, the increased expression level of CoTI1 under salt, drought and ABA treatment suggested that COTI1 might reduce the plant damage under abiotic stresses by inhibiting the endogenous trypsin-like proteases in plants. In addition to the potential defense against the above pests, CoTI1 might also play important role in the resistance to abiotic stresses.
Fig. 4 The relative expression level of CoTI1 under ABA, cold, salt and drought treatment
inactive with respect to its trypsin inhibitory activity by a point mutation on its Arg86 residue. Compared with the recombinant CoTI1, CoTI1R86A, CoTI1L84A and CoTI1T88A lost its 90, 54 and 53 % inhibitory activity against trypsin, respectively. This almost complete loss of functionality of mutated CoTI1R86A confirmed the Arg86 as the P1 residue of CoTI1. Furthermore, the residual inhibitory activities of the mutated CoTI1s against the trypsin-like proteases from three pests were investigated. Compared with the recombinant COTI1, CoTI1R86A lost most of its inhibitory activity (89, 90 and 91 %) against trypsinlike proteases from H. armigera, S. exigua and S. litura. Interestingly, the lost inhibitory activities of CoTI1T88A (64, 73 and 72 %) were higher than those of CoTI1 L84A (51, 50 and 46 %), suggesting Thr88 might play more important role in the inhibition against trypsin-like proteases from pests. Expression pattern of CoTI1 under stresses To understand the metabolism related to CoTI1 under stresses, the sterile seedlings were treated with different stresses and then used as samples to analyze the expression pattern of CoTI1 by real-time PCR. As shown in Fig. 4, the expression level of CoTI1 was relative stable under cold stress, whereas it was unstable under salt, drought and ABA treatment. The expression level of CoTI1 went up continuously under salt/drought stresses, whereas it went up before 24 h and then went down under ABA treatment. It was known that abiotic stresses could lead to severe plant damage, and several peptides, which were involved in
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Conclusion This is the first report of the cloning of the CoTI1 gene and its expression. The expressed CoTI1 showed dominant inhibitory activities against trypsin and trypsin-like proteases from H. armigera, S. exigua and S. litura. Leu84, Arg86 and Thr88 were identified as the key residues in which Arg86 was the P1 residue and determined the inhibitory specificity of CoTI1. In addition, the substantial increased expression level of CoTI1 under salt, drought and ABA treatment suggested that CoTI1 might also play important role in the resistance to abiotic stress. Hopefully, COTI1 could be used to construct transgenic plants with resistance to pests and tolerance to abiotic stress in the future. Acknowledgments This work was supported by the Grant (no. 31371232) from National Natural Science Foundation of China and Grant (no. SWJTU11CX114) from Fundamental Research Funds for the Central Universities of China. Supporting information Supplementary Table 1 Primers used in this paper. Supplementary Figure 1 Alignment of genomic DNA, cDNA and the translated polypeptide of CoTI1. (A) Alignment of genomic DNA and cDNA of COTI1. (B) Translated polypeptide from cDNA. Supplementary Figure 2 The expression and purification of recombinant CoTI1. ‘‘M’’ indicated protein maker. Lanes 1–3 indicated collection solution of lysate, penetration and rinse, respectively. Lanes 4–6 indicated the collection solution eluted with 60, 100 and 300 mM imidazole by turns. Supplementary Figure 3 Sequencing results of CoTI1 and mutants CoTI1R86A, CoTI1L84A and CoTI1T88A.
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