Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa

Two general-odorant binding proteins in Spodoptera litura are differentially tuned to sex pheromones and plant odorants Nai-Yong Liu a,b, Ke Yang a, Yan Liu a, Wei Xu b, Alisha Anderson b, Shuang-Lin Dong a,⁎ a b

Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, China CSIRO Ecosystem Sciences, Black Mountain, Australian Capital Territory, Australia

a r t i c l e

i n f o

Article history: Received 1 August 2014 Received in revised form 27 September 2014 Accepted 3 November 2014 Available online 10 November 2014 Keywords: Spodoptera litura General-odorant binding protein Gene structure qPCR Fluorescence binding assay Molecular docking

a b s t r a c t Moths have evolved a sensitive and sophisticated olfactory system to sense a variety of semiochemicals from the external environment. In chemosensory processes, the odorant binding protein (OBP) is an essential element for filtering, binding and transporting hydrophobic odorant molecules to the specific receptors. Here focusing on a major sub-class of lepidopteran OBPs, general-odorant binding proteins (GOBPs), we explored the relationship and functional difference between two GOBP members from a noctuid species Spodoptera litura. Using genomic DNA as the template, we demonstrated that SlitGOBP2 and three SlitPBPs are clustered on the same chromosome within a close proximity. qPCR results showed that two SlitGOBPs were primarily expressed in antennae at similar levels between females and males, but GOBP2 displayed much higher expression than GOBP1. Binding studies revealed that both SlitGOBP1 and 2 strongly bound C14–C16 alcohol-pheromone analogs with high affinities (Ki b 1.0 μM). However, SlitGOBP2 also strongly bound most acetate- and aldehyde-sex pheromone components and analogs, while SlitGOBP1 could not. For tested plant odorants, SlitGOBP1 showed a relatively broad ligand-binding spectrum with moderate affinities, while SlitGOBP2 was tuned to some compounds with strong binding activities (Ki b 5.0 μM). Finally, by molecular docking we explored the differences in protein structures and potential key residues in the binding pockets between the two SlitGOBPs. Taken together, our study strongly suggests that SlitGOBP2 and SlitPBPs evolved by gene duplication events, and two SlitGOBPs have functionally differentiated in odorant recognition. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Olfaction plays critical roles in insect physiology and behavior. Moths have evolved a highly sensitive and sophisticated olfactory system, by which they are capable of detecting thousands of external volatile compounds. These compounds include plant odorants and sex pheromones that are used to seek host plants or con-specific partners, and locate oviposition sites or avoid predators (Hansson and Stensmyr, 2011). Deciphering the molecular mechanisms underlying insect olfactory coding will greatly enhance our knowledge of the insect olfactory system, and facilitate the design and development of novel pest control strategies. In the first step of odorant recognition, it is believed that a small molecular weight (12–17 kDa) and water-soluble protein, namely odorant binding proteins (OBPs), contributes to the binding and transport of external odorant molecules that enter into the antennal sensillar lymph (Vogt and Riddiford, 1981; Pelosi et al., 2006). The complex of OBP-odorant passes through the aqueous sensillar lymph to specific odorant receptors (Leal, 2007, 2013). The first ⁎ Corresponding author. Tel./fax: +86 25 84399062. E-mail address: [email protected] (S.-L. Dong).

http://dx.doi.org/10.1016/j.cbpa.2014.11.005 1095-6433/© 2014 Elsevier Inc. All rights reserved.

insect OBP was identified from Antheraea polyphemus antennae (Vogt and Riddiford, 1981), and based on its bound ligand (sex pheromones) and the sensilla distribution, this OBP was named pheromone binding protein (PBP). The PBP is a specific sub-class of OBPs for sex pheromone detection in Lepidoptera (Vogt and Riddiford, 1981; Kaissling, 2007). Subsequently, another key sub-class of general-odorant binding protein (GOBP) was found in Lepidoptera (Vogt et al., 1991a). In general, lepidopteran GOBPs contain two conserved members and are clustered into two distinct groups, GOBP1 and GOBP2 (Vogt et al., 1991a,b), suggesting, to some extent, different functional roles in odorant recognition. Both GOBPs are highly expressed in antennae of both sexes with around equal abundance, differentiating them from the PBPs (Vogt et al., 1991a; Krieger et al., 1996). Previously, moth GOBP expression was found to be limited to general-odorant sensitive basiconic sensilla housed on the antennae, and thus was thought to be only responsible for the detection of food and host odorants (Vogt et al., 1991a,b; Laue et al., 1994; Steinbrecht et al., 1995). However, with more available lepidopteran GOBPs, later studies have demonstrated that these GOBPs were also detected in trichoid sensilla that are sensitive to sex pheromones. For example, in the noctuid moth Mamestra brassicae MbraGOBP2 was

24

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

detected in the pheromone-sensitive long trichoid sensilla of male moths (Jacquin-Joly et al., 2000). Similar results were obtained in Heliothis virescens and Spodoptera littoralis (Zhang et al., 2001). In non-noctuid moths, Manduca sexta MsexGOBP1 was expressed in long type-I trichoid sensilla of male antennae that respond to sex pheromones (Nardi et al., 2003). Bombyx mori BmorGOBP1 was also expressed in male long trichoid sensilla, although exhibited very weak expression (Maida et al., 2005). These studies suggest that GOBPs may also be involved in binding and transport of sex pheromones, in addition to the general odorants. Furthermore, studies have indicated that the GOBP2 can strongly bind their respective sex pheromones in several moth species, including Chilo suppressalis (Gong et al., 2009), B. mori (Zhou et al., 2009), Amyelois transitella (Liu et al., 2010), Orthaga achatina (Liu et al., 2012b) and Spodoptera exigua (Liu et al., 2014). Studies have rarely explored the relationship of GOBPs and PBPs in the noctuid species, and particularly their genomic organization on chromosome(s), which is helpful for understanding the functional similarity and differentiation between the two OBP sub-classes. In addition, comparative studies on the differences between GOBP1 and GOBP2, in terms of protein structure, expression profile and binding specificity, will be significant to deepen our understanding of the evolution of GOBPs. The common cutworm (Spodoptera litura Fabricius, 1775; Lepidoptera: Noctuidae) is a destructive agricultural pest in Asia and other countries (CABI, 1967) with the larvae feeding a variety of crops. In this study, we identified the genomic assembly on the chromosome of SlitGOBP2 and three SlitPBPs, investigated the expression profile and ligand-binding specificity of two SlitGOBPs, and finally conducted a molecular docking analysis of the two SlitGOBPs to explore key amino acid residues involving in the ligand-binding. Our study provides the first evidence that SlitGOBP2 and SlitPBPs possibly occurred via gene duplications in noctuid species, and reveals that the two SlitGOBPs are differentially tuned to odorant molecules of specific structural characteristics. 2. Materials and methods 2.1. Insects S. litura larvae were reared on an artificial diet (Huang et al., 2002) in the laboratory under 14 h light:10 h dark cycle at 25 ± 1 °C and 65 ± 5% humidity. Pupae were sexed, and females and males were kept separately in cages after eclosion. The moths were provided 10% honey solution. All adult tissues were collected from 3-day-old female and male moths. A single 4th instar larva was used for genomic DNA extraction. All these tissues were immediately transferred to 1.5 mL Eppendorf tubes immersed in liquid nitrogen and were stored at −70 °C until use.

volumes of chilled ethanol were added to the aqueous supernatant for DNA precipitation. The pellets were washed using 70% ethanol, briefly vacuum-dried and the DNA was re-dissolved in 50 μL TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0; 100 mg/mL RNase A). All the products were stored at −20 °C until use.

2.3. Identification and analysis of genomic DNA sequence SlitGOBP1 (GenBank accession number: EF159978) and SlitGOBP2 (GenBank accession number: EF159979) open reading frame (ORF) sequences were downloaded from National Center for Biotechnology Information (NCBI). For intron identification, we first ascertained the possible sites of SlitGOBPs based on lepidopteran GOBP exon/intron splice sites known, and then designed the gene-specific primers (Table S1). Next, touchdown PCR was performed: 94 °C for 3 min; 5 cycles at 94 °C for 50 s, 61 °C for 50 s and 72 °C for 3 min with a decrease of the annealing temperature by 1 °C per cycle, and followed by 35 cycles of 94 °C for 50 s, 55 °C for 50 s and 72 °C for 3 min and final extension for 10 min at 72 °C. For genomic arrangement of SlitGOBPs and SlitPBPs, multiple primers' combinations from different genes (Table S1) were carried out: 1) GOBP1-forward or reverse primer & PBPs (PBP1, PBP2 and PBP3)-forward primers or reverse primers; 2) GOBP2-forward or reverse primer & PBPs (PBP1, PBP2 and PBP3)-forward primers or reverse primers; 3) PBP1-forward or reverse primer & PBP2 and PBP3forward or reverse primers; and 4) PBP2-forward or reverse primer & PBP3-forward or reverse primers. The PCR reaction was run using KOD Hot Start DNA Polymerase (Novagen, Germany) according to the protocol provided. Briefly, PCR procedures were: 95 °C for 2 min, 40 cycles of 95 °C for 20 s, 52 °C for 10 s, 70 °C for 10 min and final extension for 10 min at 70 °C. PCR products were analyzed by 1.0% (w/v) agarose gels. The purification was performed using Wizard® SV Gel and PCR Clean-Up System (Promega, USA). Purified PCR products were sub-cloned into pEASY-T3 vector (TransGen, Beijing, China), and positive clones were verified using T7 and SP6 primers and sequenced by ABI 3730 sequencer in GenScript Biology Company (Nanjing, China). Genomic databases for B. mori (Xia et al., 2004), Danaus plexippus (Zhan et al., 2011) and Heliconius melpomene (Dasmahapatra et al., 2012) were downloaded from the Ensembl Genomes FTP Server (http://metazoa.ensembl.org/info/data/ftp/index.html). Plutella xylostella genomic database was downloaded from DBM-DB (http://iae.fafu.edu. cn/DBM/) (You et al., 2013). Gene structure was aligned and analyzed using a GeneWise program with the Modelled splice site. Phylogenetic tree was constructed using MEGA5 under Jones-Taylor-Thornton (JTT) model with 1000 replicates (Tamura et al., 2011). Phylogenetic raw data was found in Supplemantary material 1.

2.2. Nucleic acid extraction and first-strand cDNA synthesis 2.4. Quantitative real-time PCR (qPCR) Total RNA was extracted from adult tissues using SV 96 Total RNA Isolation System (Promega, Madison, WI, USA), according to the manufacturer's instructions. Briefly, genomic DNA was digested by DNaseI at RT for 15 min. Next, first-strand cDNA synthesis was performed with Oligo (dT)18 primer, using M-MLV reverse transcriptase (TaKaRa, Dalian, China) at 42 °C for 1 h and then the reaction was stopped by heating at 70 °C for 15 min, according to the protocol provided. Genomic DNA was prepared from a single 4th instar larva. The larva was disrupted by grinding in liquid nitrogen and homogenizing with 300 μL DNA extraction buffer (100 mM Tris-HCl, pH 8.0; 50 mM EDTA; 200 mM NaCl; 1% SDS). The homogenate was transferred to a clean tube, mixed with 20 μL Proteinase K (20 mg/mL) and incubated at 56 °C for 3 h. Genomic DNA was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) followed by an equal volume of chloroform:isoamyl alcohol (24:1). Two-and-one-half

qPCR was performed on an ABI 7500 (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq™ (TaKaRa, Dalian, China) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank accession number: HQ012003) as a reference gene (Shen et al., 2011). The specificity and amplification efficiency for each pair of primers were assessed by a five-fold serial dilution of cDNA. Each reaction well had a total volume of 20 μL, containing 10 μL of SYBR Green PCR Master Mix, 0.2 μM of each primer, 0.4 μL of ROX Reference Dye II, 2 μL of cDNA template and 6.8 μL of nuclease-free water. Each sample was run with three technical replicates (individual reaction wells) on three independent biological pools using the following PCR conditions: 1 cycle of 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. All primers are available in Table S1. Candidate gene expression was normalized to the control gene SlitGAPDH using the Q-GENE statistical analysis package (Muller et al., 2002; Simon, 2003).

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

2.5. Construction of recombinant expression vector

3. Results

Signal peptides were predicted and removed according to SignalP4.1 Server (Petersen et al., 2011). Both SlitGOBPs were amplified by genespecific primers with protective bases and restriction sites (GOBP1: BamHI and XhoI; GOBP2: BamHI and HindIII) (Table S1). Purified PCR products were sub-cloned into pEASY-T3 vector, and positive clones were sequenced. The plasmids containing the correct inserts were digested by FastDigest® restriction sites (GOBP1: BamHI and XhoI; GOBP2: BamHI and HindIII). The expected band was purified from agarose gels and ligated into expression vector pET-30a (+) (Novagen, Darmstadt, Germany), previously digested by the same enzymes. The constructed recombinant expression vectors were further confirmed by sequencing.

3.1. Gene structure of S. litura GOBPs and PBPs

2.6. Expression and purification of the protein The recombinant plasmid was transferred into Escherichia coli BL21 (DE3) competent cells, and expression and purification were performed according to a previously reported protocol (Liu et al., 2012a, 2013). The crude protein fractions were purified using an affinity chromatography XK 16/20 column filled with Ni Sepharose High Performance (GE Healthcare, Little Chalfont, Buckinghamshire, UK), along with the manufacture's protocols. To avoid the effects of His-tag on subsequent experiments, the Histag was removed by digestion with recombinant enterokinase (rEK) (GenScript, Nanjing, China). A second purification was performed by affinity chromatography. The purified protein fractions were analyzed by SDS-PAGE. 2.7. Fluorescence binding assay The binding assays were conducted following our previous studies (Liu et al., 2012a, 2013). First, we tested the binding of a fluorescent probe N-Phenyl-1-naphthylamine (1-NPN) to the proteins. Next, the compounds were measured in fluorescence competitive binding assays using 1-NPN as the fluorescent reporter (2 μM), and 0.25–4.0 μM or 2–20 μM for each competitor. In binding assays, we selected 58 chemicals of different structural characteristics including plant odorants, sex pheromones and pheromone analogs. Then, we picked up these ligands with 1-NPN fluorescence b 75% at 20 μM, and further measured their affinities with at least two technical replicates. The binding data were analyzed along with previous methods (Ban et al., 2003; Liu et al., 2012a, 2013).

2.8. Structural modeling and molecular docking The crystal structure of B. mori GOBP2 (PDB: 2WCK) (Zhou et al., 2009) was used as the template. The three-dimensional (3D) models of SlitGOBPs were constructed using MODELER (Sali and Blundell, 1993) in Discovery Studio 3.5 (Accelrys Software Inc.) and the lowest Probability Density Function (PDF) energy was retained for subsequent studies. Minimization was performed on the structure using CHARMm fore-field (Brooks et al., 1983). The Profile-3D (Luthy et al., 1992) was used to calculate the compatibility score of each residue in the structure. Using the built models, potential binding sites of SlitGOBPs to ligands were defined and edited according to the complex of BmorGOBP2 with bombykol. The 3D structures of all chemicals were sketched and refined using CHARMm force-field in Discovery Studio 3.5. Then, a CHARMm-based molecular dynamics scheme CDOCKER (Wu et al., 2003) was employed to dock ligands into the protein binding site. The top 10 hits ranked by CDOCKER energy were further analyzed to find the most optimal binding mode.

25

Using genomic DNA as the template, the intron sequences of two SlitGOBPs were identified. The intron numbers and exon/intron splice sites are all conserved for the two SlitGOBPs, as well as the three SlitPBPs (Xiu et al., 2008; Yang et al., 2010) (Fig. 1A). In all five SlitPBP/GOBP genes, the first intron (Intron-1) is inserted between two amino acids and the second intron (Intron-2) is present within the codons. The Intron-1 of two SlitGOBPs exhibits a more similar length with 38.8% nucleotide identity (data not shown) compared to Intron-2, which is extremely different in length and identity. To determine whether SlitGOBPs and SlitPBPs loci are on the same chromosome in close vicinity to each other, PCR was performed using the specific primers from SlitPBP/GOBP genes (Fig. 1B). As a result, we demonstrated that SlitGOBP2 and the three SlitPBPs are on the same chromosome within a close proximity. We were unable to obtain the organizations of SlitGOBP1 and the other four SlitPBP/GOBPs. SlitGOBP2 is situated approximately 2.6 Kb upstream of the adjacent SlitPBP2, and is in the same orientation as SlitPBP2 and SlitPBP3 but not SlitPBP1 (Fig. 1C). The identified genomic DNA sequence from SlitGOBP2 to SlitPBP1 was deposited in NCBI (GenBank accession number: KJ956693). 3.2. Expression pattern of S. litura GOBPs To examine the expression patterns of the two SlitGOBPs in antennae and other tissues of both sexes, qPCR was performed. Results showed that both genes were highly expressed in adult antennae without obvious difference between females and males. However, SlitGOBP2 was expressed at much higher levels (~ 4.5-fold difference in female and male, respectively) than SlitGOBP1. Additionally, both SlitGOBPs showed extremely low or undetectable expression in other tested tissues (Fig. 2). 3.3. Bacterial expression and purification of recombinant S. litura GOBPs The protein was expressed in a bacterial system with a high yield of around 20 mg/L of culture. The two SlitGOBPs were present in inclusion bodies (Fig. 3A), and thus denaturation and renaturation were performed in accordance with previously reported protocols (Ban et al., 2003; Liu et al., 2012a). Using the affinity chromatography column XK 16/20, we obtained the recombinant proteins with His-tags, which are ~ 23 kDa for SlitGOBP1 and ~22 kDa for SlitGOBP2 (Fig. 3B). Next, to avoid a possible effect by the His-tag on subsequent experiments this tag was removed by digestion with rEK. Finally, a second purification was carried out and a single band of predicted size was observed (Fig. 3C). 3.4. Binding characterization of ligands to S. litura GOBPs We first monitored whether the fluorescent probe 1-NPN could be bound by SlitGOBPs. Like most OBPs, both SlitGOBPs could bind 1-NPN with a strong blue shift from around 460 nm to 400 nm as well as a significant increase in fluorescence intensity (data not shown). However, two SlitGOBPs exhibited different affinities to 1-NPN, with Ki value of 22.85 ± 2.68 μM for GOBP1 and 4.40 ± 0.15 μM for GOBP2 (Fig. 4A), suggesting possible different interactions to 1-NPN between the two SlitGOBPs. In order to investigate the functions of two SlitGOBPs in the detection of odorants, we used 1-NPN as the fluorescent reporter to evaluate binding affinities of SlitGOBPs to different compounds. As a result, four alcohol-pheromone analogs (Z9-14:OH, Z9-16:OH, Z11-16:OH and E11-16:OH) were the best ligands for both SlitGOBPs, with Ki values less than 1.0 μM. For acetate- and aldehyde-pheromones or analogs

26

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

Fig. 1. Gene structure and organization of SlitGOBPs. (A) Gene structure of two SlitGOBPs and three SlitPBPs. Intron-2 is boxed in red. (B) PCR analysis of SlitGOBP2 and/or SlitPBPs. PCR was performed using verified plasmids as the templates with the primers of PBP1-forward & PBP3-forward (lane 1), PBP2-forward & PBP3-reverse (lane 2) and GOBP2-forward & PBP2-reverse (lane 3). (C) Gene organization of SlitGOBP2 and three SlitPBPs on the chromosome. The orientation of gene transcription is represented by arrows. In S. litura, the distance between SlitGOBP2 and/or SlitPBPs is present on chromosome X. PBPs and GOBPs of other three species were named based on the identity and the phylogenetic tree with B. mori PBPs and GOBPs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. qPCR analysis of SlitGOBPs in antennae and other tissues. Expression level of two SlitGOBP genes was calculated relative to the reference gene SlitGAPDH using the Q-GENE methods. ♂AN: male antenna, ♀AN: female antenna, ♂WB: male whole body without antenna and ♀WB: female whole body without antenna. Bars represent standard errors of three independent biological pools.

with a double bond(s), SlitGOBP2 showed much stronger binding (Ki = 0.16–1.06 μM) than SlitGOBP1. For plant odorants tested, SlitGOBP1 could bind most ligands with moderate affinities, with 50–70% 1-NPN fluorescence calculated at the maximal ligand concentrations of 4 or 20 μM. In contrast, SlitGOBP2 was specially tuned to some ligands with higher affinities (Ki b 5.0 μM) compared to SlitGOBP1, such as (+)-cedrol, nerolidol, farnesol, farnesene, lauric acid, oleic acid, linoleic acid and 2-pentadecanone (Fig. 4B and Table S2). The two SlitGOBPs displayed different binding preferences for ligands of specific structures. Of -OH, -Ac and -Ald functional groups, SlitGOBP1 preferred to bind the ligands with a hydroxyl group, while SlitGOBP2 displayed no obvious discriminative ability between Z9-14: Ac and Z9-14:OH, Z11-16:OH and Z11-16:Ac, and among Z9-16:OH, Z9-16:Ac and Z9-16:Ald. In the carbon-chain length set, for C6-11 acetates tested SlitGOBP1 preferred to bind short-chain butyl acetate, while SlitGOBP2 showed a preference for the long-chain nonyl acetate. For C12–18 saturated fatty acids, SlitGOBP1 did not show a binding preference, but SlitGOBP2 exhibited a higher affinity to lauric acid

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

27

Fig. 3. Expression and purification of recombinant SlitGOBPs. (A) Expression of pET/SlitGOBPs in a bacterial system. The crude bacterial extracts before (lane 1) and after (lane 2) IPTG induction, and the supernatant (lane 3) and pellet (lane 4) after sonication were analyzed by SDS-PAGE. (B) Eluted protein fractions of pET/SlitGOBPs with His-tags. The eluted protein fractions were collected using different tubes, and were further analyzed by SDS-PAGE (lanes 1–9). (C) Re-purification of SlitGOBPs after the removal of His-tags.

compared to the other acids. Similarly, between 2-tridecanone and 2pentadecanone, SlitGOBP2 strongly bound 2-pentadecanone, while SlitGOBP1 moderately bound both of them with no preference (Fig. 4B and Table S2). 3.5. Molecular docking of S. litura GOBPs and ligands To find the potentially key and different residues in the binding sites of SlitGOBPs to ligands with high affinities, we built 3D structural models of SlitGOBP1 and 2 using MODELER. The amino acid identities of SlitGOBP1 and 2 with BmorGOBP2 are 50.3% and 79.4%,

respectively (Fig. 5A). The verified scores of established SlitGOBP1 and 2 by Profile-3D are 65.68 (expected high score: 64.69 and expected low score: 29.11) and 64.36 (expected high score: 63.77 and expected low score: 28.70), respectively. These verified scores exceed the expected high scores, indicating that the quality of the models is reliable. Based on the 3D structural models, molecular dockings of SlitGOBP1 or 2 against the fluorescent probe 1-NPN and different ligands were performed under the same conditions. SlitGOBP1 and 2 exhibited different interactions to 1-NPN with − 24.33 and − 30.36 kcal/mol of energy values, respectively (Table 1). For different ligands, there are few

Fig. 4. Binding of selected ligands to SlitGOBPs. (A) Binding curves of 1-NPN to two SlitGOBPs and relative Scatchard plots. Dissociation constants (N = 3) were GOBP1: 22.85 μM (SEM 2.68) and GOBP2: 4.40 μM (SEM 0.15). (B) Binding curves of selected ligands to two SlitGOBPs. The binding data and abbreviations of all tested ligands are reported in Table S2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

28

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

Fig. 4 (continued).

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

29

Fig. 5. Structural modeling and molecular docking of SlitGOBPs to ligands. (A) 3D models of SlitGOBP1 and 2. Alignments of amino acid sequences for SlitGOBP1, SlitGOBP2 and BmorGOBP2 were performed using ClustalW. The disulfide bridges are numbered 1 to 3. N-terminus (Nt), C-terminus (Ct), disulfide bridge and alpha-helix (α) were labeled. (B) Molecular docking of SlitGOBP1 and 2 with Z9-16:OH, respectively. Amino acid residues (GOBP1: T9 and W37; GOBP2: E98 and R110) involved in the formation of hydrogen bond between SlitGOBPs and Z9-16: OH (green) are shown as stick styles with red or blue letters. The distance of hydrogen bond is represented in red or blue. All other residues in the binding site within 4 Å are present as line styles and names are listed in Table 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

differences in the amino acids in the binding pocket of SlitGOBP1 or 2 (Fig. 5 and Table 1). Moreover, most of these residues are hydrophobic. Some amino acids appeared to be crucial for the ligand-binding, such as threonine 9 (T9) and tryptophan 37 (W37) of SlitGOBP1, glutamic

acid 98 (E98) and arginine 110 (R110) of SlitGOBP2, which were involved in the formation of hydrogen bond (H-bond) between the SlitGOBPs and Z9-16:OH (Fig. 5B). In particular, the two residues of SlitGOBP2 are also present in the H-bond formation of other ligands

30

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

Table 1 The docking results of SlitGOBPs against ligands of different structures. Ligand

Structure

CDOCKER interaction energy (kcal/mol)†

Different residues within 4.0 Åa

Residues forming H-bond with ligand

GOBP1

GOBP2

GOBP1

GOBP2

GOBP1

GOBP2

1-NPN

−24.33

−30.46

M5, T9, M68, F76, A115, F118

L61

—

—

Z9-14:Ac

−41.54

−46.05

M5, T9, S56, S66, R67, M68, F76, I77, E98, R110, A115, F118, K119

M5, L61, S66, K119

—

—

Z9,E12-14:Ac

−41.85

−43.46

M5, T9, S56, S66, R67, M68, F76, I77, E98, R110, A115, F118, K119

M5, L61, S66, L86

—

T9 (2.855)

Z9-16:OH

−44.43

−44.54

M5, L61, S66, H95

Farnesol

−36.88

−40.15

M5, T9, S56, S66, R67, M68, I77, E98, R110 F43, S56, M68, F76, I77, E98, A115, F118

M5, S66

T9 (2.753) W37 (2.820) —

R110 (2.737) E98 (2.918) E98 (2.251)

Z9-16:Ald

−43.30

−46.42

M5, L61, S66

—

R110 (2.718)

Linoleic acid

−44.71

−50.38

M5, L61, H69, S66

—

R110 (2.792)

2-Pentadecanone

−43.98

−43.31

M5, T9, S56, M68, F76, I77, A115, F118 M5, T9, F43, S56, S66, R67, M68, F76, I77, E98, R110, A115, F118, K119 M5, T9, S56, F76, I77, A115, F118

L61, S66

—

—

“—” means no hydrogen bond formation of SlitGOBPs and ligands. The value in bracket represents the distance of hydrogen bond between SlitGOBPs and ligands. A shorter distance indicates a stronger interaction between SlitGOBPs and ligands. † A lower value of CDOCKER interaction energy indicates a stronger binding between SlitGOBPs and ligands. a Common amino acid residues in the binding pockets of SlitGOBP1 or 2 with 4.0 Å are shown as follows: GOBP1: V8, F12, F33, F36, W37, I52, L61, L62, T73, M90, I94, I111 and V114; GOBP2: V8, T9, F12, F33, F36, W37, I52, S56, L62, R67, M68, M73, Y76, L90, I94, E98, R110, V111, V114, A115 and F118.

with different functional groups, suggesting that they are potentially key amino acid sites contributing to ligand-binding. In addition, all the docking interaction energies of SlitGOBPs and ligands are negative values and the distances of all H-bonds are less than 3 Å (Fig. 5B and Table 1), indicating a strong interaction between the ligand and protein. 4. Discussion This study is the first report of gene organization between GOBP2 and three PBPs in a noctuid species. Our result is well in line with previous studies in non-noctuid species that show the GOBP2 and PBPs are present on the same chromosome within a close proximity (Fig. 1C). We were unable to identify the positional relationship of SlitGOBP1 with the other four SlitPBP/GOBPs, possibly due to its far distance from these genes on the chromosome, as seen the case in B. mori, D. plexippus, H. melpomene and P. xylostella (Gong et al., 2009; Zhan et al., 2011; Dasmahapatra et al., 2012; You et al., 2013). A previous study has suggested that M. sexta GOBP2 and PBP1 occurred by gene duplications, as these two genes are tandemly located on the chromosome, and the physical proximity between these two genes is too close to have occurred by an arbitrary translocation event (Vogt et al., 2002). Similar results among different PBPs were obtained in Agrotis moths (Picimbon and Gadenne, 2002; Abraham et al., 2005). Therefore, our present study strongly suggests that S. litura GOBP2 and three PBPs occurred via gene duplications. In S. litura, the two SlitGOBPs are highly expressed in adult antennae, but no obvious expression level difference was observed between females and males, similar to other moths (Zhang et al., 2011; Liu et al., 2012b). This suggests that both SlitGOBPs play critical roles in olfactory relevant behaviors of both sexes. Although previous studies in M. sexta, A. polyphemus, B. mori and Antheraea pernyi showed that the GOBPs were only detected in general odorant-sensitive sensilla basiconica (Vogt et al., 1991a,b), later studies with more advanced techniques have clearly demonstrated that moth GOBPs were also expressed in other sensilla such as M. brassicae (Jacquin-Joly et al., 2000), M. sexta (Nardi et al., 2003) and B. mori (Maida et al., 2005). This diversity in sensilla distribution suggests functional divergence of

GOBPs. Indeed, our binding assays showed that the two SlitGOBPs could not only bind plant odorants but also sex pheromones and their analogs. In particular, SlitGOBP2 could strongly bind two sex pheromone components with even higher affinities than SlitPBP1 (Liu et al., 2012a), as seen the case of its sibling species S. exigua (Liu et al., 2014). Summarily, we suggest that SlitGOBP2 may carry sex pheromone molecules to the receptors. Different binding affinities between the two SlitGOBPs to most tested ligands as well as 1-NPN reflects their sequence and structural differences. SlitGOBP1 and 2 only share a moderate amino acid identity (53.1%). Our molecular docking results further indicate that the two SlitGOBPs have different energy values and key residues that interact with ligands. Particularly, SlitGOBP2 showed lower CDOCKER interaction energy values and more residues involved in the formation of hydrogen bond than SlitGOBP1. These results further support our binding studies where SlitGOBP2 displayed a stronger binding to these ligands compared to SlitGOBP1. To date, in Lepidoptera only B. mori GOBP2 crystal structure is available (Zhou et al., 2009), but GOBP1 is not. Thus, the predicted functional sites between GOBP1 and GOBP2, which are responsible for ligandbinding specificity and difference, need to employ other methods such as site-directed mutagenesis for future studies. In summary, our study demonstrated, for the first time, that SlitGOBP2 is situated very closely to three SlitPBPs on the chromosome in a noctuid moth, strongly suggesting gene duplication events occurred to SlitGOBP2 and SlitPBPs. Functional studies revealed different roles of the two SlitGOBPs in odorant recognition. In particular, SlitGOBP2, could strongly bind sex pheromones, and thus may act as a dual carrier of both sex pheromones and plant odorants. The molecular docking results further demonstrated the structural differences between the two SlitGOBPs, which will assist in determining functional binding sites of SlitGOBPs and ligands. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpa.2014.11.005. Acknowledgments We thank Cheng-Cheng Liu and Zhao-Qun Li for their help in qPCR experiments. This work was supported by National Natural Science

N.-Y. Liu et al. / Comparative Biochemistry and Physiology, Part A 180 (2015) 23–31

Foundation (31071978), a Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201203036) and the Academic Innovative Program of Jiangsu Higher Education Institutions (CXZZ13_0304), China. References Abraham, D., Lofstedt, C., Picimbon, J.F., 2005. Molecular characterization and evolution of pheromone binding protein genes in Agrotis moths. Insect Biochem. Mol. Biol. 35, 1100–1111. Ban, L., Scaloni, A., D'Ambrosio, C., Zhang, L., Yahn, Y., Pelosi, P., 2003. Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria. Cell. Mol. Life Sci. 60, 390–400. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., Swaminathan, S., Karplus, M., 1983. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217. CABI, 1967. Distribution maps of plant pests. Pest: Spodoptera litura (Fabricius), London. Series A, (Map no. 61). Dasmahapatra, K.K., Walters, J.R., Briscoe, A.D., Davey, J.W., Whibley, A., Nadeau, N.J., Zimin, A.V., Hughes, D.S.T., Ferguson, L.C., Martin, S.H., Salazar, C., Lewis, J.J., Adler, S., Ahn, S.-J., Baker, D.A., Baxter, S.W., 2012. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature 487, 94–98. Gong, Z.J., Zhou, W.W., Yu, H.Z., Mao, C.G., Zhang, C.X., Cheng, J.A., Zhu, Z.R., 2009. Cloning, expression and functional analysis of a general odorant-binding protein 2 gene of the rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). Insect Mol. Biol. 18, 405–417. Hansson, B.S., Stensmyr, M.C., 2011. Evolution of insect olfaction. Neuron 72, 698–711. Huang, C.X., Zhu, L.M., Ni, J.P., Chao, X.Y., 2002. A method of rearing the beet armyworm Spodoptera exigua. Entomol. Knowl. 39, 229–231. Jacquin-Joly, E., Bohbot, J., Francois, M.C., Cain, A.H., Nagnan-Le Meillour, P., 2000. Characterization of the general odorant-binding protein 2 in the molecular coding of odorants in Mamestra brassicae. Eur. J. Biochem. 267, 6708–6714. Kaissling, K.E., 2007. Structure and function of insect pheromone binding proteins. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 148 (Suppl. 1), S80. Krieger, J., von Nickisch-Rosenegk, E., Mameli, M., Pelosi, P., Breer, H., 1996. Binding proteins from the antennae of Bombyx mori. Insect Biochem. Mol. Biol. 26, 297–307. Laue, M., Steinbrecht, R.A., Ziegelberger, G., 1994. Immunocytochemical localization of general odorant-binding protein in olfactory sensilla of the silkmoth Antheraea polyphemus. Naturwissenschaften 81, 178–180. Leal, W.S., 2007. Rapid binding, release and inactivation of insect pheromones. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 148 (Suppl. 1), S81. Leal, W.S., 2013. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. Entomol. 58, 373–391. Liu, Z., Vidal, D.M., Syed, Z., Ishida, Y., Leal, W.S., 2010. Pheromone binding to general odorant-binding proteins from the navel orangeworm. J. Chem. Ecol. 36, 787–794. Liu, N.Y., He, P., Dong, S.L., 2012a. Binding properties of pheromone-binding protein 1 from the common cutworm Spodoptera litura. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 161, 295–302. Liu, S.J., Liu, N.Y., He, P., Li, Z.Q., Dong, S.L., Mu, L.F., 2012b. Molecular characterization, expression patterns, and ligand-binding properties of two odorant-binding protein genes from Orthaga achatina (Butler) (Lepidoptera: Pyralidae). Arch. Insect Biochem. Physiol. 80, 123–139. Liu, N.Y., Liu, C.C., Dong, S.L., 2013. Functional differentiation of pheromone-binding proteins in the common cutworm Spodoptera litura. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 165, 254–262. Liu, N.-Y., Yang, F., Yang, K., He, P., Niu, X.-H., Xu, W., Anderson, A., Dong, S.-L., 24 OCT 2014. Two sub-classes of odorant binding proteins in Spodoptera exigua display structural conservation and functional divergence. Insect Mol. Biol. http://dx.doi.org/10. 1111/imb.12143. Luthy, R., Bowie, J.U., Eisenberg, D., 1992. Assessment of protein models with threedimensional profiles. Nature 356, 83–85. Maida, R., Mameli, M., Muller, B., Krieger, J., Steinbrecht, R.A., 2005. The expression pattern of four odorant-binding proteins in male and female silk moths, Bombyx mori. J. Neurocytol. 34, 149–163. Muller, P.Y., Janovjak, H., Miserez, A.R., Dobbie, Z., 2002. Processing of gene expression data generated by quantitative real-time RT-PCR. BioTechniques 32, 1372–1374 (1376, 1378-1379). Nardi, J.B., Miller, L.A., Walden, K.K., Rovelstad, S., Wang, L., Frye, J.C., Ramsdell, K., Deem, L.S., Robertson, H.M., 2003. Expression patterns of odorant-binding proteins in antennae of the moth Manduca sexta. Cell Tissue Res. 313, 321–333.

31

Pelosi, P., Zhou, J.J., Ban, L.P., Calvello, M., 2006. Soluble proteins in insect chemical communication. Cell. Mol. Life Sci. 63, 1658–1676. Petersen, T.N., Brunak, S., von Heijne, G., Nielsen, H., 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786. Picimbon, J.F., Gadenne, C., 2002. Evolution of noctuid pheromone binding proteins: identification of PBP in the black cutworm moth, Agrotis ipsilon. Insect Biochem. Mol. Biol. 32, 839–846. Sali, A., Blundell, T., 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815. Shen, Y., Gu, J., Huang, L.H., Zheng, S.C., Liu, L., Xu, W.H., Feng, Q.L., Kang, L., 2011. Cloning and expression analysis of six small heat shock protein genes in the common cutworm, Spodoptera litura. J. Insect Physiol. 57, 908–914. Simon, P., 2003. Q-Gene: processing quantitative real-time RT-PCR data. Bioinformatics 19, 1439–1440. Steinbrecht, R.A., Laue, M., Ziegelberger, G., 1995. Immunolocalization of pheromonebinding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx. Cell Tissue Res. 282, 203–217. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Vogt, R.G., Riddiford, L.M., 1981. Pheromone binding and inactivation by moth antennae. Nature 293, 161–163. Vogt, R.G., Prestwich, G.D., Lerner, M.R., 1991a. Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. J. Neurobiol. 22, 74–84. Vogt, R.G., Rybczynski, R., Lerner, M.R., 1991b. Molecular cloning and sequencing of general odorant-binding proteins GOBP1 and GOBP2 from the tobacco hawk moth Manduca sexta: comparisons with other insect OBPs and their signal peptides. J. Neurosci. 11, 2972–2984. Vogt, R.G., Rogers, M.E., Franco, M.D., Sun, M., 2002. A comparative study of odorant binding protein genes: differential expression of the PBP1-GOBP2 gene cluster in Manduca sexta (Lepidoptera) and the organization of OBP genes in Drosophila melanogaster (Diptera). J. Exp. Biol. 205, 719–744. Wu, G., Robertson, D.H., Brooks, C.L., Vieth, M., 2003. Detailed analysis of grid-based molecular docking: a case study of CDOCKER—a CHARMm-based MD docking algorithm. J. Comput. Chem. 24, 1549–1562. Xia, Q., Zhou, Z., Lu, C., Cheng, D., Dai, F., Li, B., Zhao, P., Zha, X., Cheng, T., Chai, C., Pan, G., Xu, J., Liu, C., Lin, Y., Qian, J., Hou, Y., Wu, Z., Li, G., Pan, M., Li, C., Shen, Y., Lan, X., Yuan, L., Li, T., Xu, H., Yang, G., Wan, Y., Zhu, Y., Yu, M., Shen, W., Wu, D., Xiang, Z., Yu, J., Wang, J., Li, R., Shi, J., Li, H., Su, J., Wang, X., Zhang, Z., Wu, Q., Li, J., Zhang, Q., Wei, N., Sun, H., Dong, L., Liu, D., Zhao, S., Zhao, X., Meng, Q., Lan, F., Huang, X., Li, Y., Fang, L., Li, D., Sun, Y., Yang, Z., Huang, Y., Xi, Y., Qi, Q., He, D., Huang, H., Zhang, X., Wang, Z., Li, W., Cao, Y., Yu, Y., Yu, H., Ye, J., Chen, H., Zhou, Y., Liu, B., Ji, H., Li, S., Ni, P., Zhang, J., Zhang, Y., Zheng, H., Mao, B., Wang, W., Ye, C., Wong, G.K., Yang, H., 2004. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306, 1937–1940. Xiu, W.M., Zhou, Y.Z., Dong, S.L., 2008. Molecular characterization and expression pattern of two pheromone-binding proteins from Spodoptera litura (Fabricius). J. Chem. Ecol. 34, 487–498. Yang, F., He, P., Dong, S.L., 2010. Molecular identification and evolutionary analysis of pheromone binding protein 3 gene in the common cutworm, Spodoptera litura (Fabricius). J. Nanjing Agric. Univ. 33, 65–70. You, M., Yue, Z., He, W., Yang, X., Yang, G., Xie, M., Zhan, D., Baxter, S.W., Vasseur, L., Gurr, G.M., Douglas, C.J., Bai, J., Wang, P., Cui, K., Huang, S., Li, X., Zhou, Q., Wu, Z., Chen, Q., Liu, C., Wang, B., Li, X., Xu, X., Lu, C., Hu, M., Davey, J.W., Smith, S.M., Chen, M., Xia, X., Tang, W., Ke, F., Zheng, D., Hu, Y., Song, F., You, Y., Ma, X., Peng, L., Zheng, Y., Liang, Y., Chen, Y., Yu, L., Zhang, Y., Liu, Y., Li, G., Fang, L., Li, J., Zhou, X., Luo, Y., Gou, C., Wang, J., Wang, J., Yang, H., Wang, J., 2013. A heterozygous moth genome provides insights into herbivory and detoxification. Nat. Genet. 45, 220–225. Zhan, S., Merlin, C., Boore, J.L., Reppert, S.M., 2011. The monarch butterfly genome yields insights into long-distance migration. Cell 147, 1171–1185. Zhang, S., Maida, R., Steinbrecht, R.A., 2001. Immunolocalization of odorant-binding proteins in noctuid moths (Insecta, Lepidoptera). Chem. Senses 26, 885–896. Zhang, T.T., Gu, S.H., Wu, K.M., Zhang, Y.J., Guo, Y.Y., 2011. Construction and analysis of cDNA libraries from the antennae of male and female cotton bollworms Helicoverpa armigera (Hubner) and expression analysis of putative odorant-binding protein genes. Biochem. Biophys. Res. Commun. 407, 393–399. Zhou, J.J., Robertson, G., He, X., Dufour, S., Hooper, A.M., Pickett, J.A., Keep, N.H., Field, L.M., 2009. Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components. J. Mol. Biol. 389, 529–545.