Accepted Manuscript Spider acetylcholine binding proteins: An alternative model to study the interaction between insect nAChRs and neonicotinoids Haibo Bao, Xiangkun Meng, Zewen Liu PII:
S0965-1748(17)30158-3
DOI:
10.1016/j.ibmb.2017.09.014
Reference:
IB 3000
To appear in:
Insect Biochemistry and Molecular Biology
Received Date: 15 June 2017 Revised Date:
27 September 2017
Accepted Date: 27 September 2017
Please cite this article as: Bao, H., Meng, X., Liu, Z., Spider acetylcholine binding proteins: An alternative model to study the interaction between insect nAChRs and neonicotinoids, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/j.ibmb.2017.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Spider acetylcholine binding proteins: an alternative model to study the interaction between insect nAChRs and neonicotinoids
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Haibo Baoa, Xiangkun Mengb, Zewen Liua, *
Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of
Education), College of Plant Protection, Nanjing Agricultural University, 1 Weigang,
b
SC
Nanjing 210095, China.
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou,
M AN U
225009, China.
*Corresponding author: Zewen Liu, [email protected]. College of Plant Protection, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China. Tel/
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Fax: +86-25-84399051.
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ACCEPTED MANUSCRIPT Abstract
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Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of
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nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on
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nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to
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difficulties in their heterologous expression have benefitted from the discovery of
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AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which
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have shown low sensitivity to neonicotinoids, the insecticides targeting insect
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nAChRs. However, AChBPs were also found in spiders based on the sequence and
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tissue expression analysis. Here, we report five AChBP subunits in Pardosa
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pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits
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shared higher sequence similarities with nAChR subunits of both insects and
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mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P.
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pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from
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Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand
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site per subunit was present at each interface between two adjacent subunits.
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Neonicotinoids had higher affinities (7.9–18.4 times based on Kd or Ki values) for
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Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed
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higher affinities for Ls-AChBP. These results indicate that spider AChBP could be
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used as an alternative model to study the interaction between insect nAChRs and
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neonicotinoids.
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Key words: acetylcholine binding proteins, Pardosa pseudoannulata, neonicotinoid,
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binding affinity
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ACCEPTED MANUSCRIPT 1. Introduction
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As the receptors to acetylcholine, the most important neurotransmitter in invertebrates,
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nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic
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transmission in invertebrate central nervous system (CNS). Because of the abundance
27
and importance of nAChRs in the insect CNS, nAChRs have been used as molecular
28
targets to develop several classes of insecticides (Matsuda et al., 2001; Millar and
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Denholm, 2007). Neonicotinoids are nAChR agonists that have significantly higher
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affinities for insect nAChRs than towards mammalian nAChRs, thus offering the
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advantage of selectivity between insects and mammals (Tomizawa and Casida, 2000).
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nAChR subunits in insects and their predator enemy spiders share many similarities
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(Meng et al., 2015b), thus understanding the selectivity of neonicotinoids between
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insects and spiders is of particular importance due to the use of spiders in the
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biological control of insect pests to protect agricultural crops. However, studies of this
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type have been stalled by difficulties with the heterologous expression of insect and
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spider nAChRs (Meng et al., 2015c; Millar and Lansdell, 2010). The commonly used
38
methods for heterologous protein expression, including co-expression with the rat β2
39
subunit, co-immunoprecipitation with native nAChRs and RNAi in living cells (Li et
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al., 2010; Meng et al., 2015a; Sun et al., 2017), have not been successful.
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Acetylcholine binding proteins (AChBPs) in aquatic mollusks could be appropriate
42
models for studying invertebrate nAChR (Brejc et al., 2001; Celie et al., 2005).
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AChBPs from several aquatic mollusks share high amino acid identities with nAChR
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extracellular domains, which form ligand-binding pockets on the interfaces between
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two adjacent subunits (Brejc et al., 2001; Smit et al., 2001). Mollusk AChBPs are
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soluble proteins lacking transmembrane regions that form ion channels on plasma
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on their structures and accurately analyze AChBP–ligand interaction (Billen et al.,
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2012; Brejc et al., 2001; Celie et al., 2005; Hansen et al., 2004). However, the affinity
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of imidacloprid, a representative neonicotinoid, for mollusk AChBPs is significantly
51
lower than for insect nAChRs. For example, imidacloprid binds Lymnaea stagnalis
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AChBP with a Kd of 1.57 µM (Ihara et al., 2008) and Aplysia californica AChBP with
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a Kd of 68 nM (Talley et al., 2008), while the binding affinities of imidacloprid
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binding sites in insects were 0.004–4.8 nM (Li et al., 2010; Lind et al., 1998; Wiesner
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and Kayser, 2000).
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Recently, one AChBP subunit was reported in the American wandering spider,
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Cupiennius salei, after transcriptome analysis and in situ hybridization (Liu et al.,
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2017; Torkkeli et al., 2015); this finding increased the possibilities of studying the
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interaction between neonicotinoids and nAChRs in spiders and insects. Prior to these
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studies, it is critical to understand the number of AChBP subunits in spider species
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and to determine whether spider AChBPs are more suitable than mollusk AChBPs to
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study the neonicotinoids–nAChRs interaction. In this study, we report the
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identification of five AChBP subunits in the wolf spider, Pardosa pseudoannulata, a
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predator enemy against several insect pests on rice. We expressed one of the AChBPs
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(Pp-AChBP1) in Sf9 cells and analyzed its pharmacological properties. Our results
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indicate that this AChBP could be a useful alternative model to study the interaction
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between neonicotinoids and insect/spider nAChRs due to its high affinity for
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neonicotinoids.
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2.1. Spiders and compounds
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The wolf spider, P. pseudoannulata, was collected from a paddy field in Nanjing City
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(China) in June 2016. [3H]epibatidine ([3H]Epi) [250 µCi (30–70Ci /mmol)] and
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[3H]α-bungarotoxin ([3H]α-Bgt) [50 µCi (40–105 Ci/mmol)] were purchased from
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PerkinElmer Inc. (Downers Grove, IL, USA). [3H]imidacloprid ([3H]Imi) [120 µCi
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(32 Ci/mmol)] was generously provided by Syngenta Ltd. (Guildford, UK). Other
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compounds and insecticides were purchased from Sigma–Aldrich (St. Louis, MO,
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USA).
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2.2. cDNA cloning and sequence analysis
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By searching the P. pseudoannulata transcriptome data (accession number:
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GCKE00000000) (Guo et al., 2015) in GenBank, we found five putative AChBP
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subunit sequences. Full-length cDNA of these sequences were then obtained by
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performing 3′ RACE and 5′ RACE using the SMART RACE kit (Clontech, San Jose,
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CA, USA). The amplified products were then validated by PCR with gene-specific
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primers and further sequencing. Multiple sequence alignments of the putative amino
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acids sequences were performed by GeneDoc and the phylogenetic tree was
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constructed using the Neighbor-Joining method.
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2.3. Protein expression and purification
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Recombinant Pp-AChBP1 subunit with 6×His-tag in the C-terminus was subcloned
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into the pFastbac Ⅰ vector and expressed using the Bac-to-Bac baculovirus
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expression system in Sf9 insect cells (Invitrogen) following the manufacturer’s
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instructions. The Pp-AChBP protein was purified 72 h after transfection using a
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ACCEPTED MANUSCRIPT His-tag antibody column followed by elution with the 6×His-tag peptide (Invitrogen).
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Yield of the purified protein was between 1.2–2.0 µg/mL. In parallel, Lymnaea
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stagnalis AChBP (Ls-AChBP) was also expressed and purified following the same
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procedure to compare the two proteins.
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2.4. Titration of ligand stoichiometry
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Equilibrium fluorescence was monitored in 96-well UV plates using a Tecan Safire
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fluorescence plate reader (Tecan, Männedorf, Switzerland). After exciting at 280 nm,
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the emission intensity of AChBP was recorded at 340 nm using the
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excitation/emission slit width of 7.5 nm at room temperature (Hansen et al., 2002).
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Then, based on the calculated molar masses of a single subunit, 500 nM subunit
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(Pp-AChBP1 or Ls-AChBP) solutions were prepared and used for tryptophan
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quenching through titration using the high affinity ligand, epibatidine.
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2.5. Analysis of the α-Bgt and Pp-AChBP complex
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Complex formation between Pp-AChBP1 subunit and α-Bgt was determined based on
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a previously described protocol (Bourne et al., 2005). In brief, increasing molar ratios
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of α-Bgt (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 per binding site calculated from the
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titration of ligand stoichiometry) was added to 50 nM solution of Pp-AChBP1 and
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incubated for 2 h at room temperature. Then, the mixture was subjected to native
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PAGE (15 % homogenous gels) and the binding complex was detected by Western
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blotting
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Staining/De-Staining Kit M335 (Amresco, Solon, OH, USA). The final stoichiometry
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of α-Bgt binding was analyzed by recording the progressive shift of the intermediate
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complexes toward the cathode on the native PAGE.
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using
His-tag
antibody
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silver
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the
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Total protein was extracted from the culture medium of Sf9 cells transfected with the
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Pp-AChBP1 subunit and protein concentration was determined by using the Bradford
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method (Bradford, 1976) and bovine serum albumin (BSA) as the standard. Radio
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ligand binding assays were performed as described previously (Li et al., 2010; Liu et
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al., 2005) in a total volume of 100 ml incubation buffer (0.05 mM Tris, 0.12 mM
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NaCl, 100 mM EDTA, pH 7.4), AChBP (50 mg protein per assay), and one of the
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radio ligands ([3H]epibatidine, [3H]α-Bgt or [3H]imidacloprid) and incubated at 4°C
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for 2 h. Then, samples were filtered using Whatman GF/B filters presoaked in 0.5%
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polyethylenimine followed by rapid washing with ice-cold saline (pH 7.4, 20 mM
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Na2HPO4, 0.15 M NaCl, 0.2% BSA). The filters were then transferred into
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scintillation vials and the radioactivity remaining on the filter was assayed after
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overnight incubation in 3 mL scintillation cocktail (OptiPhase Supermix, PerkinElmer,
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USA) on a LS6500 Liquid Scintillation Counter (Beckman Coulter, Fullerton, CA,
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USA). Specific binding was defined as the difference in radioactivity in the presence
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and absence of 1000-fold molar excess of the unlabeled ligand compared with radio
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ligand.
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Competitive binding assay was performed by including different concentrations of
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neonicotinoid in the incubation mixture containing 5.0 nM [3H]imidacloprid and 50
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mg AChBP protein per assay.
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2.7. Statistical analysis
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For radio ligand binding, nonlinear regression analysis was used to determine the
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dissociation constant (Kd) and maximal binding capacity (Bmax) from double
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(Bowen and Jerman, 1995).
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Significant differences between samples were analyzed by one-way ANOVA with at
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least three replicates. Multiple comparisons between groups were performed using
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LSD pair wise comparison. The level of significance for results was set at P >0.01 or
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0.05.
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3.1. Identifying AChBP in P. pseudoannulata
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We identified five putative AChBP subunit sequences in the brain transcriptome of the
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wolf spider, P. pseudoannulata, (Fig. 1). All putative amino acids sequences have
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typical motifs including the six loops (single lines) that form the ligand binding
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pocket at the interface of two adjacent subunits and the Cys-loop (double line) formed
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by two cysteines. These motifs are similar to the extracellular region of the nAChR
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subunits (Tcas α1 in the alignment) and AChBPs of aquatic mollusks. However, while
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all nAChR subunits have 13 amino acids between the two cysteines in the Cys-loop,
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the AChBP subunits have more or less than 13. For example, 16 and 11 amino acid
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residues are present between the cysteines in Pp-AChBP1-4 and Pp-AChBP5,
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respectively. The results indicate that these five sequences are AChBP subunits rather
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than the alternative splice forms of nAChR subunits.
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3.2. Heterologous expression of a spider AChBP
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In order to test whether these Pp-AChBP subunits are functional, Pp-AChBP1 subunit
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was selected for heterologous expression in Sf9 cells. SDS–PAGE and Western blot
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analysis of the Sf9 cell culture medium using the His-tag antibody indicated the
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presence of a specific band with a molecular mass of 37 kDa (Fig. 2A and 2C), which
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was absent in the control without Pp-AChBP1 transfection. On native-PAGE, a band
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with molecular mass more than 220 kDa was detected (Fig. 2B and 2D), and further
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analysis using mass spectrometry confirmed it to be Pp-AChBP1 (Table S1). These
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data showed the successful construction of recombinant AChBP for heterologous
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protein expression.
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ACCEPTED MANUSCRIPT 3.3. Stoichiometry of ligand binding sites in Pp-AChBP
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Titration of epibatidine binding to Pp-AChBP1 was performed and compared with the
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binding to Ls-AChBP (Fig. 3A) using the tryptophan fluorescence method (Hansen et
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al., 2002). For the 500 nM Pp-AChBP1 subunit, the detected ligand sites were 490
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nM and the calculated molar ratio was 0.98 sites per subunit. On the other hand, the
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molar ratio for Ls-AChBP was 1.06 sites per subunit (530 nM sites for 500 nM
173
Ls-AChBP subunit), which is consistent with the 0.97 sites per subunit reported in the
174
previous study (Hansen et al., 2004). The results indicated the existence of one ligand
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site between two adjacent subunits in each interface in both Pp-AChBP1 and
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Ls-AChBP.
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Furthermore, the Pp-AChBP1 and α-Bgt complex formation was analyzed by
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monitoring the migration of the complex toward the cathode on native PAGE (Bourne
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et al., 2005). To optimize occupancy of all binding sites on Pp-AChBP1, molar ratios
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of α-Bgt and AChBP were carefully adjusted (Fig. 3B). The binding of α-Bgt to
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Pp-AChBP1 reduced the mobility of Pp-AChBP1 on native PAGE, when compared
182
with unliganded Pp-AChBP1. Pp-AChBP1 mobility was reduced in the ratio range of
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0.2–1.0 α-Bgt per subunit in a ratio-dependent manner, while migration was
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unaffected when the ratio was above 1.0 α-Bgt per subunit. These results showed that
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there was likely only one ligand binding site per subunit in the recombinant
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Pp-AChBP1.
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3.4. Radio ligand binding to recombinant AChBPs
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The
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[3H]imidacloprid, to recombinant Pp-AChBP1 was determined and compared with
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specific
binding
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[3H]epibatidine,
[3H]α-Bgt
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Ls-AChBP than to Pp-AChBP1 (Fig. 4A, 4B) with Kd ratios of Pp-AChBP1 and
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Ls-AChBP being 8.07 and 11.31, respectively (Table 1). In contrast, [3H]imidacloprid
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showed significantly higher affinity to Pp-AChBP1 (Kd=2.43±0.36 nM) than to
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Ls-AChBP (44.72±5.64 nM) with a Kd ratio of 0.05.
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3.5. Neonicotinoids displace [3H]imidacloprid binding to AchBPs
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The affinity of other neonicotinoids to recombinant Pp-AChBP1 was evaluated by
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competitive binding assays. Imidacloprid and acetamiprid were able to better displace
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the binding of [3H]imidacloprid binding to Pp-AChBP1 than to Ls-AChBP (Fig. 5A),
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indicating that both neonicotinoids had higher binding affinities to Pp-AChBP1 than
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to Ls-AChBP. For all test neonicotinoids, Ki values on Ls-AChBP were 7.9–11.7
201
times of those on Pp-AChBP1 (Table 1).
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In the competitive binding assays, different binding affinities were observed among
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neonicotinoids based on Ki values. Acetamiprid, clothianidin, nitenpyram, thiacloprid
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and thiamethoxam exhibited comparable affinities to Pp-AChBP1 with Ki values of
205
1.5–3.5 nM (Fig. 5B, 5C), although displacement of [3H]imidacloprid was not
206
complete even at higher concentrations of thiamethoxam (1 µM). In contrast,
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dinotefuran and sulfoxaflor showed much lower affinities to Pp-AChBP1 with Ki
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values of 11.8 nM and 14.1 nM, respectively when compared with other tested
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neonicotinoids (Fig. 5B, 5C).
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Discussion
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Studies on AChBPs from aquatic mollusks have accelerated our understanding of the
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interaction between nAChRs and their specific ligands with respect to their structural
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and functional roles (Sine and Engel, 2006). The relatively low affinity of
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neonicotinoids to mollusk AChBPs is the primary motivating factor driving studies to
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find new AChBPs in other invertebrates more closely related to insects, such as
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spiders, which belong to the order Arthropoda with insects.
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Neonicotinoids have selective toxicity against insects; imidacloprids have shown
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much higher affinities (Kd range of 0.004–4.8 nM) for insect nAChRs (Li et al., 2010;
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Lind et al., 1998; Wiesner and Kayser, 2000) than for mollusk AChBPs (Ihara et al.,
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2008; Talley et al., 2008). Here, we showed that [3H]imidacloprid bound P.
221
pseudoannulata AChBP (Pp-AChBP1) with a high affinity of Kd 2.43±0.36 nM,
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which was in the Kd range of imidacloprid binding to native insect nAChRs and
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significantly higher than its binding to L. stagnalis AChBP (Ls-AChBP, Kd
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44.72±5.64 nM). Other neonicotinoids also showed higher affinities for Pp-AChBP
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than for Ls-AChBP in their displacement of [3H]imidacloprid binding. In contrast,
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[3H]epibatidine and [3H]α-Bgt bound Pp-AChBP with much lower affinities than
227
Ls-AChBP. These data showed that neonicotinoids were highly selective for
228
Pp-AChBP or Ls-AChBP.
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Sequence analysis of AChBP subunits (from spiders and aquatic mollusks) and
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nAChR subunits (from insects, mammals and spiders) could also provide clues for the
231
differences in neonicotinoid selectivity between Pp-AChBP and Ls-AChBP.
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Pp-AChBP1 showed genetic distances of 0.9607–1.1710 and 1.0263–1.1737 from the
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extracellular domains of insect and mammalian nAChR α subunits, respectively,
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ACCEPTED MANUSCRIPT while Ls-AChBP had values of 0.9916–1.2709 and 1.1895–1.2546, respectively (Fig.
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S1). However, this comparison between AChBP and nAChR α subunits did not
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provide evidence for sequence inclination, such as insect vs. mammal or Pp-AChBP
237
vs. Ls-AChBP. However, such sequence inclination was found in the comparison of
238
AChBP and nAChR β subunits (Fig. S2). Pp-AChBP1 showed genetic distances of
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0.9867–1.1442 and 1.1227–1.2383 from insect and mammal β subunits, respectively,
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and Ls-AChBP showed 1.1616–1.2021 and 1.0402–1.1852, respectively. These
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results clearly indicated that Pp-AChBP1 was much more closely related to insect
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nAChR β subunits than their mammalian counterparts, while Ls-AChBP was more
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closely related to mammalian nAChR β subunits than their insect counterparts.
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In nAChRs, six loops (loops A, B and C from the α subunits and loops D, E and F
245
from the β subunits) contribute to the ligand-binding pockets of acetylcholine as well
246
as neonicotinoid insecticides (Sine and Engel, 2006). The amino acid residues that
247
differ among the subunits often play important roles in neonicotinoid selectivity
248
between insects and mammals (Shimomura et al., 2006; Shimomura et al., 2004; Yao
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et al., 2008). The amino acids in loops A, B and C of AChBPs and nAChR α subunits
250
(Fig. S3) were identical or more similar to insect and mammal nAChR subunits than
251
to those of Ls-AChBP, such as L117A (Pp-AChBP1 vs. Ls-AChBP), D123K, Y180H
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and G182S. These differences indicate that the ligand-binding pocket in Pp-AChBP1
253
may be more similar to those in nAChRs compared with Ls-AChBP, although this
254
barely supports the selectivity of neonicotinoids between Pp-AChBP1 and Ls-AChBP.
255
The proline residue at position 221 in loop C plays an important role in the
256
imidacloprid selectivity in both insects and mammals (Shimomura et al., 2004). In
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Pp-AChBP1, proline is present at this position, while serine was found in Ls-AChBP
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Differences in loops D, E and F between Pp-AChBP1 and Ls-AChBP compared with
260
nAChR β subunits may provide further evidence for the neonicotinoid selectivity
261
differences between Pp-AChBP1 and Ls-AChBP (Fig. S4). R81T and R81Q
262
(corresponding to position 82 in AChBP reported in this study) mutations have been
263
reported to reduce the neonicotinoid potency of insect nAChRs (Bass et al., 2011;
264
Shimomura et al., 2006; Song et al., 2009). Here, we found that Q82 was in loop D of
265
Ls-AChBP, while M82 instead of R82 was at this site in Pp-AChBP1. At position 83,
266
leucine was present in both Pp-AChBP1 and insect β subunits, while threonine was
267
found in Ls-AChBP. Similar observations were also made for N139D and V194S (Fig.
268
S4). These sequence comparisons indicated that Pp-AChBP1 could have
269
neonicotinoid binding pockets with higher binding affinities for insect nAChRs than
270
for Ls-AChBP, which makes Pp-AChBP1 a more suitable model to study the
271
interaction between insect nAChRs and neonicotinoids.
272
Mollusk AChBPs have been reported to be involved in the regulation of cholinergic
273
synaptic signaling (Smit et al., 2001) as well as non-synaptic cholinergic
274
communication (Banks et al., 2009). Similar roles have been suggested for AChBPs in
275
the spider, C. salei, although this conclusion was inferred from the tissue-specific
276
expression profile (Liu et al., 2017). When neonicotinoids bind AChBPs and weaken
277
their possible effects on cholinergic synaptic signaling among nAChRs, synaptic
278
signaling transmission among neurons may be altered. However, the effects may not
279
be as direct as targeting neurons via neonicotinoids. Neonicotinoid toxicity on spider
280
AChBPs may be slower than the acute toxicity that may occur by their direct action
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on neural nAChRs. However, it is likely that sublethal effects may also occur.
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P. pseudoannulata as well as other spiders, in order to reduce any adverse impacts of
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neonicotinoids on natural spider enemies.
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Acknowledgement This work was supported by National Natural Science Foundation of China
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(31601662) and National Key Technology Research and Development Program
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(2012BAD19B01).
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ACCEPTED MANUSCRIPT References
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Bass, C., Puinean, A.M., Andrews, M., Cutler, P., Daniels, M., Elias, J., Paul, V.L., Crossthwaite, A.J., Denholm, I., Field, L.M., Foster, S.P., Lind, R., Williamson, M.S., Slater, R., 2011. Mutation of a nicotinic acetylcholine receptor beta subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae. BMC Neurosci. 12, 11.
297 298 299 300
Billen, B., Spurny, R., Brams, M., van Elk, R., Valera-Kummer, S., Yakel, J.L., Voets, T., Bertrand, D., Smit, A.B., Ulens, C., 2012. Molecular actions of smoking cessation drugs at alpha4beta2 nicotinic receptors defined in crystal structures of a homologous binding protein. Proc Natl Acad Sci U S A 109, 9173-9178.
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Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake α-neurotoxins and nicotinic receptors. The EMBO Journal 24, 1512-1522.
304 305
Bowen, W.P., Jerman, J.C., 1995. Nonlinear regression using spreadsheets. Trends Pharmacol. Sci. 16, 413-417.
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Brejc, K., van Dijk, W.J., Klaassen, R.V., Schuurmans, M., van Der Oost, J., Smit, A.B., Sixma, T.K., 2001. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269-276.
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Celie, P.H., Klaassen, R.V., van Rossum-Fikkert, S.E., van Elk, R., van Nierop, P., Smit, A.B., Sixma, T.K., 2005. Crystal structure of acetylcholine-binding protein from Bulinus truncatus reveals the conserved structural scaffold and sites of variation in nicotinic acetylcholine receptors. J Biol Chem 280, 26457-26466.
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Guo, B., Zhang, Y., Meng, X., Bao, H., Fang, J., Liu, Z., 2015. Identification of key amino acid differences between Cyrtorhinus lividipennis and Nilaparvata lugens nAChR α8 subunits contributing to neonicotinoid sensitivity. Neurosci Lett 589, 163-168.
316 317 318
Hansen, S.B., Radic, Z., Talley, T.T., Molles, B.E., Deerinck, T., Tsigelny, I., Taylor, P., 2002. Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein. J Biol Chem 277, 41299-41302.
319 320
Hansen, S.B., Talley, T.T., Radic, Z., Taylor, P., 2004. Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica. J Biol Chem 279, 24197-24202.
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Ihara, M., Okajima, T., Yamashita, A., Oda, T., Hirata, K., Nishiwaki, H., Morimoto, T., Akamatsu, M., Ashikawa, Y., Kuroda, S., Mega, R., Kuramitsu, S., Sattelle, D.B., Matsuda, K., 2008. Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin. Invert Neurosci 8, 71-81.
325 326 327
Li, J., Shao, Y., Ding, Z., Bao, H., Liu, Z., Han, Z., Millar, N.S., 2010. Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid. Insect Biochem Mol Biol 40, 17-22.
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Lind, R.J., Clough, M.S., Reynolds, S.E., Earley, F.G.P., 1998. [3H]Imidacloprid Labels High- and Low-Affinity Nicotinic Acetylcholine Receptor-like Binding Sites in the Aphid Myzus persicae (Hemiptera: Aphididae). Pestic Biochem Physiol 62, 3-14.
331 332 333
Liu, H., French, A.S., Torkkeli, P.H., 2017. Expression of Cys-loop receptor subunits and acetylcholine binding protein in the mechanosensory neurons, glial cells, and muscle tissue of the spider Cupiennius salei. J Comp Neurol 525, 1139-1154.
334 335
Liu, Z., Williamson, M.S., Lansdell, S.J., Denholm, I., Han, Z., Millar, N.S., 2005. A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens
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Meng, X., Li, C., Bao, H., Fang, J., Liu, Z., Zhang, Y., 2015a. Validating the importance of two acetylcholinesterases in insecticide sensitivities by RNAi in Pardosa pseudoannulata, an important predatory enemy against several insect pests. Pestic Biochem Physiol 125, 26-30.
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Meng, X., Zhang, Y., Bao, H., Liu, Z., 2015b. Sequence Analysis of Insecticide Action and Detoxification-Related Genes in the Insect Pest Natural Enemy Pardosa pseudoannulata. PLoS ONE 10.
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Meng, X., Zhang, Y., Guo, B., Sun, H., Liu, C., Liu, Z., 2015c. Identification of key amino acid differences contributing to neonicotinoid sensitivity between two nAChR α subunits from Pardosa pseudoannulata. Neurosci Lett 584, 123-128.
349 350
Millar, N.S., Denholm, I., 2007. Nicotinic acetylcholine receptors: targets for commercially important insecticides. Invertebr Neurosci 7, 53-66.
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Millar, N.S., Lansdell, S.J., 2010. Characterisation of insect nicotinic acetylcholine receptors by heterologous expression. Adv Exp Med Biol 683, 65-73.
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Shimomura, M., Yokota, M., Ihara, M., Akamatsu, M., Sattelle, D.B., Matsuda, K., 2006. Role in the Selectivity of Neonicotinoids of Insect-Specific Basic Residues in Loop D of the Nicotinic Acetylcholine Receptor Agonist Binding Site. Mol Pharmacol 70, 1255-1263.
356 357 358
Shimomura, M., Yokota, M., Matsuda, K., Sattelle, D.B., Komai, K., 2004. Roles of loop C and the loop B–C interval of the nicotinic receptor α subunit in its selective interactions with imidacloprid in insects. Neurosci Lett 363, 195-198.
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Sine, S.M., Engel, A.G., 2006. Recent advances in Cys-loop receptor structure and function. Nature 440, 448-455.
361 362 363
Smit, A.B., Syed, N.I., Schaap, D., van Minnen, J., Klumperman, J., Kits, K.S., Lodder, H., van der Schors, R.C., van Elk, R., Sorgedrager, B., Brejc, K., Sixma, T.K., Geraerts, W.P., 2001. A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature 411, 261-268.
364 365 366
Song, F., You, Z., Yao, X., Cheng, J., Liu, Z., Lin, K., 2009. Specific loops D, E and F of nicotinic acetylcholine receptor beta 1 subunit may confer imidacloprid selectivity between Myzus persicae and its predatory enemy Pardosa pseudoannulata. Insect Biochem Mol Biol 39, 833-841.
367 368 369
Sun, H., Liu, Y., Li, J., Cang, X., Bao, H., Liu, Z., 2017. The potential subunits involved in two subtypes of α-Bgt-resistant nAChRs in cockroach dorsal unpaired median (DUM) neurons. Insect Biochem Mol Biol 81, 32-40.
370 371 372
Talley, T.T., Harel, M., Hibbs, R.E., Radic, Z., Tomizawa, M., Casida, J.E., Taylor, P., 2008. Atomic interactions of neonicotinoid agonists with AChBP: molecular recognition of the distinctive electronegative pharmacophore. Proc Natl Acad Sci U S A 105, 7606-7611.
373 374
Tomizawa, M., Casida, J.E., 2000. Imidacloprid, Thiacloprid, and Their Imine Derivatives Up-Regulate the α4β2 Nicotinic Acetylcholine Receptor in M10 Cells. Toxicol. Appl. Pharmacol. 169, 114-120.
375 376 377
Torkkeli, P.H., Liu, H., French, A.S., 2015. Transcriptome Analysis of the Central and Peripheral Nervous Systems of the Spider Cupiennius salei Reveals Multiple Putative Cys-Loop Ligand Gated Ion Channel Subunits and an Acetylcholine Binding Protein. PLoS ONE 10, e0138068.
378 379 380
Wiesner, P., Kayser, H., 2000. Characterization of nicotinic acetylcholine receptors from the insects Aphis craccivora , Myzus persicae , and Locusta migratoria by radioligand binding assays: Relation to thiamethoxam action. Journal of Biochemical & Molecular Toxicology 14, 221-230.
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Yao, X., Song, F., Chen, F., Zhang, Y., Gu, J., Liu, S., Liu, Z., 2008. Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor beta subunits influence neonicotinoid selectivity. Insect
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Figure legends
385
Figure 1. Amino acids sequence alignment of insect AChBPs and a nAChR subunit
386
ligand-binding domain. Ac, Aplysia californica (Ac-AChBP, NP_001191488); Bt,
387
Bulinus
388
Cupiennius salei (Cs-AChBP, ALE66018.1); Ct, Capitella teleta (Ct-AChBP,
389
ELT90066); Ls, Lymnaea stagnalis (Ls-AChBP, AAK64377.1); Pp, Pardosa
390
pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2, KY765676; Pp-AChBP3,
391
KY765674; Pp-AChBP4, KY765675; Pp-AChBP5, KY765677); Tcas, Tribolium
392
castaneum (Tcasα1, ABS86902). Asterisk indicates the presence of only the nAChR
393
α1 subunit ligand-binding domain. Double lines indicate the Cys-loop formed by two
394
cysteines.
395
Figure 2. Detecting Pp-AChBP1 expression in Sf9 cells. (A) SDS-PAGE analysis of
396
the transfected Sf9 cells' supernatant. Lane 1, prestained protein standard, Broad
397
range (New England Biolabs Inc.), Lane 2, transfected Sf9 cells' supernatant; Lane 3,
398
negative control without the subunit. The gel was stained with Coomassie brilliant
399
blue G 250. (B) Native-PAGE analysis of transfected Sf9 cells' supernatant. Lane 1,
400
NativeMark™ Unstained Protein Standard (Novex®, Life technologies); Lane 2,
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transfected Sf9 cells' supernatant; Lane 3, negative control without the subunit. The
402
gel was stained with Coomassie brilliant blue G 250. (C) Western blotting of
403
SDS-PAGE. Lane 1, MagicMark™ XP Western Protein Standard; Lane 2, transfected
404
Sf9 cells' supernatant; lane 3, negative control without the subunit. (D) Western blot
405
of Native-PAGE. Lane 1, MagicMark™ XP Western Protein Standard; Lane 2,
406
transfected Sf9 cells' supernatant; Lane 3, negative control without the subunit.
407
Although the protein standard, MagicMark™ XP Western Protein Standard (Novex®,
(Bt-AChBP1,
BD358768;
Bt-AChBP2,
BD358769);
Cs,
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truncates
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ACCEPTED MANUSCRIPT Life technologies; indicated by asterisk) is not suitable for native Western Blot, we
409
could not find a better standard at present.
410
Figure 3. Analysis of α-Bgt-AChBP complex formation. (A) Titration of epibatidine
411
stoichiometry with AChBPs from two biological species (L. stagnalis and P.
412
pseudoannulata; (B) Analysis of α-Bgt-AChBP complex formation on native PAGE
413
by Western blotting and silver staining.
414
Figure 4. Analysis of radio ligand binding to AChBPs. Equilibrium binding of
415
[3H]epibatidine (A), [3H]α-Bgt (B) and [3H]imidacloprid (C) to two recombinant
416
AChBPs. Data are mean±SEM of 3–5 independent experiments.
417
Figure 5. Competitive binding of recombinant AChBPs against [3H]imidacloprid (5
418
nM). (A) Competitive binding of imidacloprid and acetamiprid to Pp-AChBP1 and
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Ls-AChBP compared with [3H]imidacloprid; (B) Competitive binding of clothianidin,
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dinotefuran and nitenpyram to Pp-AChBP1 compared with [3H]imidacloprid; (C)
421
Competitive binding of sulfoxaflor, thiacloprid and thiamethoxam to Pp-AChBP1
422
compared with [3H]imidacloprid. Data are mean±SEM of 3 – 5 independent
423
experiments.
424
Figure S1. The phylogenetic tree of AChBP and nAChR α subunit (extracellular
425
domains) sequences constructed using the Neighbor-Joining method. (Ric)-3
426
(resistance to inhibitors of cholinesterase) from Drosophila melanogaster was used as
427
the outgroup. (Dm-Ric3, CAP16652; Dmα1, P09478; Dmα2, P17644; Dmα3, O18394;
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Dmα4,
429
AAK64377.1); Ac, Aplysia californica (Ac-AChBP, NP_001191488); Bt, Bulinus
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Q9NFR5;
Dmα5,
Q8T5F5;);
21
Ls,
Lymnaea
stagnalis
(Ls-AChBP,
ACCEPTED MANUSCRIPT truncates (Bt-AChBP1，BD358768; Bt-AChBP2，BD358769); Cs, Cupiennius salei
431
(Cs-AChBP, ALE66018.1); Pp, Pardosa pseudoannulata (Pp-AChBP1, KY765678;
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Pp-AChBP2, KY765676; Pp-AChBP3, KY765674; Pp-AChBP4, KY765675; Ppα1,
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D6QYZ4; Ppα8, D6QYZ5); Rn, Rattus norvegicus (Rnα2, P12389; Rnα4, P09483);
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Hs, Homo sapiens (Hsα2, Q15822; Hsα4, P43681); Mp, Myzus persicae (Mpα1,
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P91765; Mpα2, P91764; Mpα3, Q9U941; Mpα4, Q9U940; Mpα5, Q5F2K3 A8DIP3);
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Tcas, Tribolium castaneum (Tcasα1, A8DIP3; Tcasα2, A8DIQ7; Tcasα3, A8DIR0;
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Tcasα4, A8DIR5; Tcasα5, A8HTE9; Tcasα8, C3TS54). Asterisks indicates removal of
438
the transmembrane region of nAChR subunits from the analysis.
439
Figure S2. The phylogenetic tree of AChBP and nAChR β subunit (extracellular
440
domains) sequences constructed using the Neighbor-Joining method. (Ric)-3
441
(resistance to inhibitors of cholinesterase) from Drosophila melanogaster was used as
442
the outgroup. Dm, Drosohila melanoganster (Dm-Ric3, CAP16652; Dmβ1, P04755;
443
Dmβ2,
444
pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2, KY765676; Pp-AChBP3,
445
KY765674; Pp-AChBP4, KY765675; Ppβ1, C7EA46; Ppβ2, D6QYZ6); Ls, Lymnaea
446
stagnalis
447
NP_001191488); Bt, Bulinus truncates (Bt-AChBP1，BD358768; Bt-AChBP2，
448
BD358769); Cs, Cupiennius salei (Cs-AChBP, ALE66018.1); Hs, Homo sapiens
449
(Hsβ2, P17787; Hsβ4, P30926); Rn, Rattus norvegicus (Rnβ2, P12390; Rnβ4,
450
P12392).
451
Figure S3. Alignment of the amino acid sequences from loops A, B and C in AChBP
452
and nAChR α subunits. Numbers on the top indicate the positions of amino acids in
Mp,
Myzus
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P25162);
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AAK64377.1);
Ac,
(Mpβ1,
Aplysia
Q9NFX8);
californica
Pp,
Pardosa
(Ac-AChBP,
AC C
(Ls-AChBP,
persicae
22
ACCEPTED MANUSCRIPT Pp-AChBP1. Hs, Homo sapiens (Hsα2, Q15822; Hsα4, P43681); Rn, Rattus
454
norvegicus (Rnα2, P12389; Rnα4, P09483); Ls, Lymnaea stagnalis (Ls-AChBP,
455
AAK64377.1); Pp, Pardosa pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2,
456
KY765676; Pp-AChBP3, KY765674; Pp-AChBP4, KY765675); Ppα1, D6QYZ4);
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Dm, Drosophila melanogaster (Dmα1, P09478; Dmα2, P17644); Mp, Myzus persicae
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(Mpα1, P91765; Mpα2, P91764); Loc, Locusta migratoria (Locα1, W6ECM5; Locα2,
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W6E9A5).
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Figure S4. Alignment of amino acid sequences of loops D, E and F in AChBP and
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nAChR β subunits. Numbers on the top indicate the positions of amino acids in
463
Pp-AChBP1. Hs, Homo sapiens (Hsβ2, P17787); Rn, Rattus norvegicus (Rnβ2,
464
P12390); Mm, Mus musculus (Mmβ2, Q9ERK7); Ls, Lymnaea stagnalis (Ls-AChBP,
465
AAK64377.1); Pp, Pardosa pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2,
466
KY765676; Pp-AChBP3, KY765674; Pp-AChBP4, KY765675; Ppβ1, C7EA46; Ppβ2,
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D6QYZ6); Loc, Locusta migratoria (Locβ1, W6EL29); Mp, Myzus persicae (Mpβ1,
468
Q9NFX8); Nl, Nilapavarta lugenns (Nlβ1, B6VAH8); Dm, Drosophila melanogaster
469
(Dmβ1, P04755); Tcas, Tribolium castaneum (Tcasβ1, A8DIV7).
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0.75±0.09 3.48±0.46 2.49±0.35 11.84±2.18 3.13±0.52 14.08±3.19 1.53±0.34
12.48±2.13 33.73±5.44 31.17±4.28 133.46±30.31 27.52±6.60 120.71±24.65 22.03±4.68
Thiamethoxam
3.01±0.86
M AN U
30.86 30.36 54.81 16.43 14.11 20.64 20.06
SC
Imidacloprid Acetamiprid Clothianidin Dinotefuran Nitenpyram Sulfoxaflor Thiacloprid
23.67±7.42
9 10 11 9 9 9 9
11
for P-value 0.00016 0.00009 0.00002
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Table 1. Summary of Kd values (nM) for radio ligands and Ki values (nM) neonicotinoids binding to recombinant Pp-AChBP1 and Ls-AChBP F crit Insecticide Pp-AChBP1 Ls-AChBP df F (α=0.01) 3 [ H]Epibatidine 1.21±0.17 0.15±0.02 9 44.37 11.26 3 [ H]α-Bgt 24.09±3.14 2.13±0.29 9 51.10 11.26 3 [ H]Imidacloprid 2.43±0.36 44.72±5.64 10 69.24 10.56
9.71
11.26 10.56 10.04 11.26 11.26 11.26 11.26 10.04 4.96*
0.00054 0.00038 0.00002 0.00367 0.00557 0.00189 0.00206 0.01095
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For radio ligands, saturation binding was determined to calculate the Kd values. For unlabeled neonicotinoids, the competition binding to displace specific [3H]imidacloprid (5 nM) on recombinant AChBPs was performed to determine Ki values. Data are mean±SEM of 3–5 independent experiments. Significant differences were analyzed by one-way ANOVA with at least three repeats. Multiple comparisons between the groups were performed using LSD pair wise comparison. For each compound (radio ligand or neonicotinoid), the values between Pp-AChBP1 and Ls-AChBP were significantly different at 0.01 level, with the exception of thiamethoxam that was significantly different only at 0.05 level. * indicates F(df1=1, df2=10) crit for α=0.05.
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Table S1. Mascot search results of peptide sequences by the mass spectrometric analysis.
918464064
ALA09394
915293426
XP_013309012
928193524
ALE66018
938149245
ALJ10921
597873494
EYC22868
597873492
EYC22866
341900060
EGT55995
1134468548
JAV23811
NO. of Unique peptides
5027.46
28338.18
296
82567.56
7
416.73
RI PT
AAG44630
Protein Mass
SC
12005809
acetylcholine-binding protein 1 [Pardosa pseudoannulata] 90-kDa heat shock protein HSP83 [Spodoptera frugiperda] heat shock protein 70 A1, partial [Spodoptera frugiperda] hypothetical protein NECAME_16129 [Necator americanus]
Protein Score
19550.98
4
130.71
8924.47
1
acetylcholine binding protein [Cupiennius salei]
122.36
28870.71
6
acetylcholine receptor [Dolomedes sulfureus]
86.62
28407.27
2
82.58
47957.54
3
70.23
58377.72
3
CBN-LGC-10 protein [Caenorhabditis brenneri]
51.37
55914.14
1
putative thioredoxin-dependent peroxidase [Culex tarsalis]
43.90
73542.27
1
hypothetical protein [Ancylostoma ceylanicum] hypothetical protein [Ancylostoma ceylanicum]
183.05
M AN U
ARU12133
Y032_0016g2995
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1199050132
Description
Y032_0016g2995
EP
Accession NO.
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Gi NO.
Only top 10 results were provided with the MASCOT minimal protein score above 40.
ACCEPTED MANUSCRIPT Table S2. The peptide sequences matched to Pp-AChBP1 and their hit numbers.
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EP
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9 10 11 12 13 14 15
Hit Number
19-27 35-42 35-48 50-57 58-66 72-91 92-104
923.83 970.90 1663.78 785.90 985.16 2543.83 1623.53
26 2 33 17 5 25 43
105-153
5515.94
9
160-174 167-174 175-183 184-193 184-207 194-207 219-237
1745.80 967.08 1016.00 1149.29 2965.97 1834.70 2226.28
23 14 17 5 14 36 27
RI PT
8
SAVNGDAYK DIFNGYDK DIFNGYDKLVRPVK ATTAVPVK ISISPLSLR NQIISLEAWMMLTWMDEYLR WDPSDYDNITELR ISPTEIWKPDIALYTASPDTSLFPVVH TEAIVYNNGMIIWVPPFTINSR SYVSVSRTFVDCNIR TFVDCNIR MGSWTYSGK MVDLQLSTDK MVDLQLSTDKVDLTNFQDYNYEWK VDLTNFQDYNYEWK LYPCCADEYPHVNFNVTLK
Fragment Mass
M AN U
1 2 3 4 5 6 7
Sequence Range
SC
Peptide Sequence
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ACCEPTED MANUSCRIPT Highlights Five acetylcholine binding proteins (AChBPs) were identified in the spider Pardosa pseudoannulata.
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One of them, Pp-AChBP1, was expressed in Sf9 cells and characterized. Pp-AChBP1 had a much higher affinity to imidacloprid compared to an AChBP from the snail Lymnaea stagnalis.
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Spider AChBPs can provide a suitable alternate model to insect nAChRs.
S0965-1748(17)30158-3
DOI:
10.1016/j.ibmb.2017.09.014
Reference:
IB 3000
To appear in:
Insect Biochemistry and Molecular Biology
Received Date: 15 June 2017 Revised Date:
27 September 2017
Accepted Date: 27 September 2017
Please cite this article as: Bao, H., Meng, X., Liu, Z., Spider acetylcholine binding proteins: An alternative model to study the interaction between insect nAChRs and neonicotinoids, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/j.ibmb.2017.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Spider acetylcholine binding proteins: an alternative model to study the interaction between insect nAChRs and neonicotinoids
a
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Haibo Baoa, Xiangkun Mengb, Zewen Liua, *
Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of
Education), College of Plant Protection, Nanjing Agricultural University, 1 Weigang,
b
SC
Nanjing 210095, China.
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou,
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225009, China.
*Corresponding author: Zewen Liu, [email protected]. College of Plant Protection, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China. Tel/
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Fax: +86-25-84399051.
1
ACCEPTED MANUSCRIPT Abstract
2
Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of
3
nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on
4
nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to
5
difficulties in their heterologous expression have benefitted from the discovery of
6
AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which
7
have shown low sensitivity to neonicotinoids, the insecticides targeting insect
8
nAChRs. However, AChBPs were also found in spiders based on the sequence and
9
tissue expression analysis. Here, we report five AChBP subunits in Pardosa
10
pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits
11
shared higher sequence similarities with nAChR subunits of both insects and
12
mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P.
13
pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from
14
Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand
15
site per subunit was present at each interface between two adjacent subunits.
16
Neonicotinoids had higher affinities (7.9–18.4 times based on Kd or Ki values) for
17
Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed
18
higher affinities for Ls-AChBP. These results indicate that spider AChBP could be
19
used as an alternative model to study the interaction between insect nAChRs and
20
neonicotinoids.
21
Key words: acetylcholine binding proteins, Pardosa pseudoannulata, neonicotinoid,
22
binding affinity
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24
As the receptors to acetylcholine, the most important neurotransmitter in invertebrates,
25
nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic
26
transmission in invertebrate central nervous system (CNS). Because of the abundance
27
and importance of nAChRs in the insect CNS, nAChRs have been used as molecular
28
targets to develop several classes of insecticides (Matsuda et al., 2001; Millar and
29
Denholm, 2007). Neonicotinoids are nAChR agonists that have significantly higher
30
affinities for insect nAChRs than towards mammalian nAChRs, thus offering the
31
advantage of selectivity between insects and mammals (Tomizawa and Casida, 2000).
32
nAChR subunits in insects and their predator enemy spiders share many similarities
33
(Meng et al., 2015b), thus understanding the selectivity of neonicotinoids between
34
insects and spiders is of particular importance due to the use of spiders in the
35
biological control of insect pests to protect agricultural crops. However, studies of this
36
type have been stalled by difficulties with the heterologous expression of insect and
37
spider nAChRs (Meng et al., 2015c; Millar and Lansdell, 2010). The commonly used
38
methods for heterologous protein expression, including co-expression with the rat β2
39
subunit, co-immunoprecipitation with native nAChRs and RNAi in living cells (Li et
40
al., 2010; Meng et al., 2015a; Sun et al., 2017), have not been successful.
41
Acetylcholine binding proteins (AChBPs) in aquatic mollusks could be appropriate
42
models for studying invertebrate nAChR (Brejc et al., 2001; Celie et al., 2005).
43
AChBPs from several aquatic mollusks share high amino acid identities with nAChR
44
extracellular domains, which form ligand-binding pockets on the interfaces between
45
two adjacent subunits (Brejc et al., 2001; Smit et al., 2001). Mollusk AChBPs are
46
soluble proteins lacking transmembrane regions that form ion channels on plasma
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48
on their structures and accurately analyze AChBP–ligand interaction (Billen et al.,
49
2012; Brejc et al., 2001; Celie et al., 2005; Hansen et al., 2004). However, the affinity
50
of imidacloprid, a representative neonicotinoid, for mollusk AChBPs is significantly
51
lower than for insect nAChRs. For example, imidacloprid binds Lymnaea stagnalis
52
AChBP with a Kd of 1.57 µM (Ihara et al., 2008) and Aplysia californica AChBP with
53
a Kd of 68 nM (Talley et al., 2008), while the binding affinities of imidacloprid
54
binding sites in insects were 0.004–4.8 nM (Li et al., 2010; Lind et al., 1998; Wiesner
55
and Kayser, 2000).
56
Recently, one AChBP subunit was reported in the American wandering spider,
57
Cupiennius salei, after transcriptome analysis and in situ hybridization (Liu et al.,
58
2017; Torkkeli et al., 2015); this finding increased the possibilities of studying the
59
interaction between neonicotinoids and nAChRs in spiders and insects. Prior to these
60
studies, it is critical to understand the number of AChBP subunits in spider species
61
and to determine whether spider AChBPs are more suitable than mollusk AChBPs to
62
study the neonicotinoids–nAChRs interaction. In this study, we report the
63
identification of five AChBP subunits in the wolf spider, Pardosa pseudoannulata, a
64
predator enemy against several insect pests on rice. We expressed one of the AChBPs
65
(Pp-AChBP1) in Sf9 cells and analyzed its pharmacological properties. Our results
66
indicate that this AChBP could be a useful alternative model to study the interaction
67
between neonicotinoids and insect/spider nAChRs due to its high affinity for
68
neonicotinoids.
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70
2.1. Spiders and compounds
71
The wolf spider, P. pseudoannulata, was collected from a paddy field in Nanjing City
72
(China) in June 2016. [3H]epibatidine ([3H]Epi) [250 µCi (30–70Ci /mmol)] and
73
[3H]α-bungarotoxin ([3H]α-Bgt) [50 µCi (40–105 Ci/mmol)] were purchased from
74
PerkinElmer Inc. (Downers Grove, IL, USA). [3H]imidacloprid ([3H]Imi) [120 µCi
75
(32 Ci/mmol)] was generously provided by Syngenta Ltd. (Guildford, UK). Other
76
compounds and insecticides were purchased from Sigma–Aldrich (St. Louis, MO,
77
USA).
78
2.2. cDNA cloning and sequence analysis
79
By searching the P. pseudoannulata transcriptome data (accession number:
80
GCKE00000000) (Guo et al., 2015) in GenBank, we found five putative AChBP
81
subunit sequences. Full-length cDNA of these sequences were then obtained by
82
performing 3′ RACE and 5′ RACE using the SMART RACE kit (Clontech, San Jose,
83
CA, USA). The amplified products were then validated by PCR with gene-specific
84
primers and further sequencing. Multiple sequence alignments of the putative amino
85
acids sequences were performed by GeneDoc and the phylogenetic tree was
86
constructed using the Neighbor-Joining method.
87
2.3. Protein expression and purification
88
Recombinant Pp-AChBP1 subunit with 6×His-tag in the C-terminus was subcloned
89
into the pFastbac Ⅰ vector and expressed using the Bac-to-Bac baculovirus
90
expression system in Sf9 insect cells (Invitrogen) following the manufacturer’s
91
instructions. The Pp-AChBP protein was purified 72 h after transfection using a
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ACCEPTED MANUSCRIPT His-tag antibody column followed by elution with the 6×His-tag peptide (Invitrogen).
93
Yield of the purified protein was between 1.2–2.0 µg/mL. In parallel, Lymnaea
94
stagnalis AChBP (Ls-AChBP) was also expressed and purified following the same
95
procedure to compare the two proteins.
96
2.4. Titration of ligand stoichiometry
97
Equilibrium fluorescence was monitored in 96-well UV plates using a Tecan Safire
98
fluorescence plate reader (Tecan, Männedorf, Switzerland). After exciting at 280 nm,
99
the emission intensity of AChBP was recorded at 340 nm using the
100
excitation/emission slit width of 7.5 nm at room temperature (Hansen et al., 2002).
101
Then, based on the calculated molar masses of a single subunit, 500 nM subunit
102
(Pp-AChBP1 or Ls-AChBP) solutions were prepared and used for tryptophan
103
quenching through titration using the high affinity ligand, epibatidine.
104
2.5. Analysis of the α-Bgt and Pp-AChBP complex
105
Complex formation between Pp-AChBP1 subunit and α-Bgt was determined based on
106
a previously described protocol (Bourne et al., 2005). In brief, increasing molar ratios
107
of α-Bgt (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 per binding site calculated from the
108
titration of ligand stoichiometry) was added to 50 nM solution of Pp-AChBP1 and
109
incubated for 2 h at room temperature. Then, the mixture was subjected to native
110
PAGE (15 % homogenous gels) and the binding complex was detected by Western
111
blotting
112
Staining/De-Staining Kit M335 (Amresco, Solon, OH, USA). The final stoichiometry
113
of α-Bgt binding was analyzed by recording the progressive shift of the intermediate
114
complexes toward the cathode on the native PAGE.
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using
His-tag
antibody
and
6
silver
staining
using
the
Silver
ACCEPTED MANUSCRIPT 2.6. Radio ligand binding analysis
116
Total protein was extracted from the culture medium of Sf9 cells transfected with the
117
Pp-AChBP1 subunit and protein concentration was determined by using the Bradford
118
method (Bradford, 1976) and bovine serum albumin (BSA) as the standard. Radio
119
ligand binding assays were performed as described previously (Li et al., 2010; Liu et
120
al., 2005) in a total volume of 100 ml incubation buffer (0.05 mM Tris, 0.12 mM
121
NaCl, 100 mM EDTA, pH 7.4), AChBP (50 mg protein per assay), and one of the
122
radio ligands ([3H]epibatidine, [3H]α-Bgt or [3H]imidacloprid) and incubated at 4°C
123
for 2 h. Then, samples were filtered using Whatman GF/B filters presoaked in 0.5%
124
polyethylenimine followed by rapid washing with ice-cold saline (pH 7.4, 20 mM
125
Na2HPO4, 0.15 M NaCl, 0.2% BSA). The filters were then transferred into
126
scintillation vials and the radioactivity remaining on the filter was assayed after
127
overnight incubation in 3 mL scintillation cocktail (OptiPhase Supermix, PerkinElmer,
128
USA) on a LS6500 Liquid Scintillation Counter (Beckman Coulter, Fullerton, CA,
129
USA). Specific binding was defined as the difference in radioactivity in the presence
130
and absence of 1000-fold molar excess of the unlabeled ligand compared with radio
131
ligand.
132
Competitive binding assay was performed by including different concentrations of
133
neonicotinoid in the incubation mixture containing 5.0 nM [3H]imidacloprid and 50
134
mg AChBP protein per assay.
135
2.7. Statistical analysis
136
For radio ligand binding, nonlinear regression analysis was used to determine the
137
dissociation constant (Kd) and maximal binding capacity (Bmax) from double
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139
(Bowen and Jerman, 1995).
140
Significant differences between samples were analyzed by one-way ANOVA with at
141
least three replicates. Multiple comparisons between groups were performed using
142
LSD pair wise comparison. The level of significance for results was set at P >0.01 or
143
0.05.
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145
3.1. Identifying AChBP in P. pseudoannulata
146
We identified five putative AChBP subunit sequences in the brain transcriptome of the
147
wolf spider, P. pseudoannulata, (Fig. 1). All putative amino acids sequences have
148
typical motifs including the six loops (single lines) that form the ligand binding
149
pocket at the interface of two adjacent subunits and the Cys-loop (double line) formed
150
by two cysteines. These motifs are similar to the extracellular region of the nAChR
151
subunits (Tcas α1 in the alignment) and AChBPs of aquatic mollusks. However, while
152
all nAChR subunits have 13 amino acids between the two cysteines in the Cys-loop,
153
the AChBP subunits have more or less than 13. For example, 16 and 11 amino acid
154
residues are present between the cysteines in Pp-AChBP1-4 and Pp-AChBP5,
155
respectively. The results indicate that these five sequences are AChBP subunits rather
156
than the alternative splice forms of nAChR subunits.
157
3.2. Heterologous expression of a spider AChBP
158
In order to test whether these Pp-AChBP subunits are functional, Pp-AChBP1 subunit
159
was selected for heterologous expression in Sf9 cells. SDS–PAGE and Western blot
160
analysis of the Sf9 cell culture medium using the His-tag antibody indicated the
161
presence of a specific band with a molecular mass of 37 kDa (Fig. 2A and 2C), which
162
was absent in the control without Pp-AChBP1 transfection. On native-PAGE, a band
163
with molecular mass more than 220 kDa was detected (Fig. 2B and 2D), and further
164
analysis using mass spectrometry confirmed it to be Pp-AChBP1 (Table S1). These
165
data showed the successful construction of recombinant AChBP for heterologous
166
protein expression.
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168
Titration of epibatidine binding to Pp-AChBP1 was performed and compared with the
169
binding to Ls-AChBP (Fig. 3A) using the tryptophan fluorescence method (Hansen et
170
al., 2002). For the 500 nM Pp-AChBP1 subunit, the detected ligand sites were 490
171
nM and the calculated molar ratio was 0.98 sites per subunit. On the other hand, the
172
molar ratio for Ls-AChBP was 1.06 sites per subunit (530 nM sites for 500 nM
173
Ls-AChBP subunit), which is consistent with the 0.97 sites per subunit reported in the
174
previous study (Hansen et al., 2004). The results indicated the existence of one ligand
175
site between two adjacent subunits in each interface in both Pp-AChBP1 and
176
Ls-AChBP.
177
Furthermore, the Pp-AChBP1 and α-Bgt complex formation was analyzed by
178
monitoring the migration of the complex toward the cathode on native PAGE (Bourne
179
et al., 2005). To optimize occupancy of all binding sites on Pp-AChBP1, molar ratios
180
of α-Bgt and AChBP were carefully adjusted (Fig. 3B). The binding of α-Bgt to
181
Pp-AChBP1 reduced the mobility of Pp-AChBP1 on native PAGE, when compared
182
with unliganded Pp-AChBP1. Pp-AChBP1 mobility was reduced in the ratio range of
183
0.2–1.0 α-Bgt per subunit in a ratio-dependent manner, while migration was
184
unaffected when the ratio was above 1.0 α-Bgt per subunit. These results showed that
185
there was likely only one ligand binding site per subunit in the recombinant
186
Pp-AChBP1.
187
3.4. Radio ligand binding to recombinant AChBPs
188
The
189
[3H]imidacloprid, to recombinant Pp-AChBP1 was determined and compared with
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specific
binding
of
radio
ligands,
10
[3H]epibatidine,
[3H]α-Bgt
and
ACCEPTED MANUSCRIPT Ls-AChBP. Both [3H]epibatidine and [3H]α-Bgt showed higher affinities to
191
Ls-AChBP than to Pp-AChBP1 (Fig. 4A, 4B) with Kd ratios of Pp-AChBP1 and
192
Ls-AChBP being 8.07 and 11.31, respectively (Table 1). In contrast, [3H]imidacloprid
193
showed significantly higher affinity to Pp-AChBP1 (Kd=2.43±0.36 nM) than to
194
Ls-AChBP (44.72±5.64 nM) with a Kd ratio of 0.05.
195
3.5. Neonicotinoids displace [3H]imidacloprid binding to AchBPs
196
The affinity of other neonicotinoids to recombinant Pp-AChBP1 was evaluated by
197
competitive binding assays. Imidacloprid and acetamiprid were able to better displace
198
the binding of [3H]imidacloprid binding to Pp-AChBP1 than to Ls-AChBP (Fig. 5A),
199
indicating that both neonicotinoids had higher binding affinities to Pp-AChBP1 than
200
to Ls-AChBP. For all test neonicotinoids, Ki values on Ls-AChBP were 7.9–11.7
201
times of those on Pp-AChBP1 (Table 1).
202
In the competitive binding assays, different binding affinities were observed among
203
neonicotinoids based on Ki values. Acetamiprid, clothianidin, nitenpyram, thiacloprid
204
and thiamethoxam exhibited comparable affinities to Pp-AChBP1 with Ki values of
205
1.5–3.5 nM (Fig. 5B, 5C), although displacement of [3H]imidacloprid was not
206
complete even at higher concentrations of thiamethoxam (1 µM). In contrast,
207
dinotefuran and sulfoxaflor showed much lower affinities to Pp-AChBP1 with Ki
208
values of 11.8 nM and 14.1 nM, respectively when compared with other tested
209
neonicotinoids (Fig. 5B, 5C).
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ACCEPTED MANUSCRIPT 4.
Discussion
211
Studies on AChBPs from aquatic mollusks have accelerated our understanding of the
212
interaction between nAChRs and their specific ligands with respect to their structural
213
and functional roles (Sine and Engel, 2006). The relatively low affinity of
214
neonicotinoids to mollusk AChBPs is the primary motivating factor driving studies to
215
find new AChBPs in other invertebrates more closely related to insects, such as
216
spiders, which belong to the order Arthropoda with insects.
217
Neonicotinoids have selective toxicity against insects; imidacloprids have shown
218
much higher affinities (Kd range of 0.004–4.8 nM) for insect nAChRs (Li et al., 2010;
219
Lind et al., 1998; Wiesner and Kayser, 2000) than for mollusk AChBPs (Ihara et al.,
220
2008; Talley et al., 2008). Here, we showed that [3H]imidacloprid bound P.
221
pseudoannulata AChBP (Pp-AChBP1) with a high affinity of Kd 2.43±0.36 nM,
222
which was in the Kd range of imidacloprid binding to native insect nAChRs and
223
significantly higher than its binding to L. stagnalis AChBP (Ls-AChBP, Kd
224
44.72±5.64 nM). Other neonicotinoids also showed higher affinities for Pp-AChBP
225
than for Ls-AChBP in their displacement of [3H]imidacloprid binding. In contrast,
226
[3H]epibatidine and [3H]α-Bgt bound Pp-AChBP with much lower affinities than
227
Ls-AChBP. These data showed that neonicotinoids were highly selective for
228
Pp-AChBP or Ls-AChBP.
229
Sequence analysis of AChBP subunits (from spiders and aquatic mollusks) and
230
nAChR subunits (from insects, mammals and spiders) could also provide clues for the
231
differences in neonicotinoid selectivity between Pp-AChBP and Ls-AChBP.
232
Pp-AChBP1 showed genetic distances of 0.9607–1.1710 and 1.0263–1.1737 from the
233
extracellular domains of insect and mammalian nAChR α subunits, respectively,
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ACCEPTED MANUSCRIPT while Ls-AChBP had values of 0.9916–1.2709 and 1.1895–1.2546, respectively (Fig.
235
S1). However, this comparison between AChBP and nAChR α subunits did not
236
provide evidence for sequence inclination, such as insect vs. mammal or Pp-AChBP
237
vs. Ls-AChBP. However, such sequence inclination was found in the comparison of
238
AChBP and nAChR β subunits (Fig. S2). Pp-AChBP1 showed genetic distances of
239
0.9867–1.1442 and 1.1227–1.2383 from insect and mammal β subunits, respectively,
240
and Ls-AChBP showed 1.1616–1.2021 and 1.0402–1.1852, respectively. These
241
results clearly indicated that Pp-AChBP1 was much more closely related to insect
242
nAChR β subunits than their mammalian counterparts, while Ls-AChBP was more
243
closely related to mammalian nAChR β subunits than their insect counterparts.
244
In nAChRs, six loops (loops A, B and C from the α subunits and loops D, E and F
245
from the β subunits) contribute to the ligand-binding pockets of acetylcholine as well
246
as neonicotinoid insecticides (Sine and Engel, 2006). The amino acid residues that
247
differ among the subunits often play important roles in neonicotinoid selectivity
248
between insects and mammals (Shimomura et al., 2006; Shimomura et al., 2004; Yao
249
et al., 2008). The amino acids in loops A, B and C of AChBPs and nAChR α subunits
250
(Fig. S3) were identical or more similar to insect and mammal nAChR subunits than
251
to those of Ls-AChBP, such as L117A (Pp-AChBP1 vs. Ls-AChBP), D123K, Y180H
252
and G182S. These differences indicate that the ligand-binding pocket in Pp-AChBP1
253
may be more similar to those in nAChRs compared with Ls-AChBP, although this
254
barely supports the selectivity of neonicotinoids between Pp-AChBP1 and Ls-AChBP.
255
The proline residue at position 221 in loop C plays an important role in the
256
imidacloprid selectivity in both insects and mammals (Shimomura et al., 2004). In
257
Pp-AChBP1, proline is present at this position, while serine was found in Ls-AChBP
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ACCEPTED MANUSCRIPT (Fig. S3); this difference could contribute to their different imidacloprid affinities.
259
Differences in loops D, E and F between Pp-AChBP1 and Ls-AChBP compared with
260
nAChR β subunits may provide further evidence for the neonicotinoid selectivity
261
differences between Pp-AChBP1 and Ls-AChBP (Fig. S4). R81T and R81Q
262
(corresponding to position 82 in AChBP reported in this study) mutations have been
263
reported to reduce the neonicotinoid potency of insect nAChRs (Bass et al., 2011;
264
Shimomura et al., 2006; Song et al., 2009). Here, we found that Q82 was in loop D of
265
Ls-AChBP, while M82 instead of R82 was at this site in Pp-AChBP1. At position 83,
266
leucine was present in both Pp-AChBP1 and insect β subunits, while threonine was
267
found in Ls-AChBP. Similar observations were also made for N139D and V194S (Fig.
268
S4). These sequence comparisons indicated that Pp-AChBP1 could have
269
neonicotinoid binding pockets with higher binding affinities for insect nAChRs than
270
for Ls-AChBP, which makes Pp-AChBP1 a more suitable model to study the
271
interaction between insect nAChRs and neonicotinoids.
272
Mollusk AChBPs have been reported to be involved in the regulation of cholinergic
273
synaptic signaling (Smit et al., 2001) as well as non-synaptic cholinergic
274
communication (Banks et al., 2009). Similar roles have been suggested for AChBPs in
275
the spider, C. salei, although this conclusion was inferred from the tissue-specific
276
expression profile (Liu et al., 2017). When neonicotinoids bind AChBPs and weaken
277
their possible effects on cholinergic synaptic signaling among nAChRs, synaptic
278
signaling transmission among neurons may be altered. However, the effects may not
279
be as direct as targeting neurons via neonicotinoids. Neonicotinoid toxicity on spider
280
AChBPs may be slower than the acute toxicity that may occur by their direct action
281
on neural nAChRs. However, it is likely that sublethal effects may also occur.
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283
P. pseudoannulata as well as other spiders, in order to reduce any adverse impacts of
284
neonicotinoids on natural spider enemies.
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ACCEPTED MANUSCRIPT 285
Acknowledgement This work was supported by National Natural Science Foundation of China
287
(31601662) and National Key Technology Research and Development Program
288
(2012BAD19B01).
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ACCEPTED MANUSCRIPT References
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Celie, P.H., Klaassen, R.V., van Rossum-Fikkert, S.E., van Elk, R., van Nierop, P., Smit, A.B., Sixma, T.K., 2005. Crystal structure of acetylcholine-binding protein from Bulinus truncatus reveals the conserved structural scaffold and sites of variation in nicotinic acetylcholine receptors. J Biol Chem 280, 26457-26466.
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Guo, B., Zhang, Y., Meng, X., Bao, H., Fang, J., Liu, Z., 2015. Identification of key amino acid differences between Cyrtorhinus lividipennis and Nilaparvata lugens nAChR α8 subunits contributing to neonicotinoid sensitivity. Neurosci Lett 589, 163-168.
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Hansen, S.B., Radic, Z., Talley, T.T., Molles, B.E., Deerinck, T., Tsigelny, I., Taylor, P., 2002. Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein. J Biol Chem 277, 41299-41302.
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Hansen, S.B., Talley, T.T., Radic, Z., Taylor, P., 2004. Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica. J Biol Chem 279, 24197-24202.
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Ihara, M., Okajima, T., Yamashita, A., Oda, T., Hirata, K., Nishiwaki, H., Morimoto, T., Akamatsu, M., Ashikawa, Y., Kuroda, S., Mega, R., Kuramitsu, S., Sattelle, D.B., Matsuda, K., 2008. Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin. Invert Neurosci 8, 71-81.
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Li, J., Shao, Y., Ding, Z., Bao, H., Liu, Z., Han, Z., Millar, N.S., 2010. Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid. Insect Biochem Mol Biol 40, 17-22.
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Lind, R.J., Clough, M.S., Reynolds, S.E., Earley, F.G.P., 1998. [3H]Imidacloprid Labels High- and Low-Affinity Nicotinic Acetylcholine Receptor-like Binding Sites in the Aphid Myzus persicae (Hemiptera: Aphididae). Pestic Biochem Physiol 62, 3-14.
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Liu, H., French, A.S., Torkkeli, P.H., 2017. Expression of Cys-loop receptor subunits and acetylcholine binding protein in the mechanosensory neurons, glial cells, and muscle tissue of the spider Cupiennius salei. J Comp Neurol 525, 1139-1154.
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Liu, Z., Williamson, M.S., Lansdell, S.J., Denholm, I., Han, Z., Millar, N.S., 2005. A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens
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Matsuda, K., Buckingham, S.D., Kleier, D., Rauh, J.J., Grauso, M., Sattelle, D.B., 2001. Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors. Trends Pharmacol. Sci. 22, 573-580.
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Meng, X., Li, C., Bao, H., Fang, J., Liu, Z., Zhang, Y., 2015a. Validating the importance of two acetylcholinesterases in insecticide sensitivities by RNAi in Pardosa pseudoannulata, an important predatory enemy against several insect pests. Pestic Biochem Physiol 125, 26-30.
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Meng, X., Zhang, Y., Bao, H., Liu, Z., 2015b. Sequence Analysis of Insecticide Action and Detoxification-Related Genes in the Insect Pest Natural Enemy Pardosa pseudoannulata. PLoS ONE 10.
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Meng, X., Zhang, Y., Guo, B., Sun, H., Liu, C., Liu, Z., 2015c. Identification of key amino acid differences contributing to neonicotinoid sensitivity between two nAChR α subunits from Pardosa pseudoannulata. Neurosci Lett 584, 123-128.
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Millar, N.S., Denholm, I., 2007. Nicotinic acetylcholine receptors: targets for commercially important insecticides. Invertebr Neurosci 7, 53-66.
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Millar, N.S., Lansdell, S.J., 2010. Characterisation of insect nicotinic acetylcholine receptors by heterologous expression. Adv Exp Med Biol 683, 65-73.
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Shimomura, M., Yokota, M., Ihara, M., Akamatsu, M., Sattelle, D.B., Matsuda, K., 2006. Role in the Selectivity of Neonicotinoids of Insect-Specific Basic Residues in Loop D of the Nicotinic Acetylcholine Receptor Agonist Binding Site. Mol Pharmacol 70, 1255-1263.
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Shimomura, M., Yokota, M., Matsuda, K., Sattelle, D.B., Komai, K., 2004. Roles of loop C and the loop B–C interval of the nicotinic receptor α subunit in its selective interactions with imidacloprid in insects. Neurosci Lett 363, 195-198.
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Sine, S.M., Engel, A.G., 2006. Recent advances in Cys-loop receptor structure and function. Nature 440, 448-455.
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Smit, A.B., Syed, N.I., Schaap, D., van Minnen, J., Klumperman, J., Kits, K.S., Lodder, H., van der Schors, R.C., van Elk, R., Sorgedrager, B., Brejc, K., Sixma, T.K., Geraerts, W.P., 2001. A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature 411, 261-268.
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Song, F., You, Z., Yao, X., Cheng, J., Liu, Z., Lin, K., 2009. Specific loops D, E and F of nicotinic acetylcholine receptor beta 1 subunit may confer imidacloprid selectivity between Myzus persicae and its predatory enemy Pardosa pseudoannulata. Insect Biochem Mol Biol 39, 833-841.
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Sun, H., Liu, Y., Li, J., Cang, X., Bao, H., Liu, Z., 2017. The potential subunits involved in two subtypes of α-Bgt-resistant nAChRs in cockroach dorsal unpaired median (DUM) neurons. Insect Biochem Mol Biol 81, 32-40.
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Talley, T.T., Harel, M., Hibbs, R.E., Radic, Z., Tomizawa, M., Casida, J.E., Taylor, P., 2008. Atomic interactions of neonicotinoid agonists with AChBP: molecular recognition of the distinctive electronegative pharmacophore. Proc Natl Acad Sci U S A 105, 7606-7611.
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Tomizawa, M., Casida, J.E., 2000. Imidacloprid, Thiacloprid, and Their Imine Derivatives Up-Regulate the α4β2 Nicotinic Acetylcholine Receptor in M10 Cells. Toxicol. Appl. Pharmacol. 169, 114-120.
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Torkkeli, P.H., Liu, H., French, A.S., 2015. Transcriptome Analysis of the Central and Peripheral Nervous Systems of the Spider Cupiennius salei Reveals Multiple Putative Cys-Loop Ligand Gated Ion Channel Subunits and an Acetylcholine Binding Protein. PLoS ONE 10, e0138068.
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Wiesner, P., Kayser, H., 2000. Characterization of nicotinic acetylcholine receptors from the insects Aphis craccivora , Myzus persicae , and Locusta migratoria by radioligand binding assays: Relation to thiamethoxam action. Journal of Biochemical & Molecular Toxicology 14, 221-230.
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Yao, X., Song, F., Chen, F., Zhang, Y., Gu, J., Liu, S., Liu, Z., 2008. Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor beta subunits influence neonicotinoid selectivity. Insect
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Biochem Mol Biol 38, 834-840.
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Figure legends
385
Figure 1. Amino acids sequence alignment of insect AChBPs and a nAChR subunit
386
ligand-binding domain. Ac, Aplysia californica (Ac-AChBP, NP_001191488); Bt,
387
Bulinus
388
Cupiennius salei (Cs-AChBP, ALE66018.1); Ct, Capitella teleta (Ct-AChBP,
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ELT90066); Ls, Lymnaea stagnalis (Ls-AChBP, AAK64377.1); Pp, Pardosa
390
pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2, KY765676; Pp-AChBP3,
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KY765674; Pp-AChBP4, KY765675; Pp-AChBP5, KY765677); Tcas, Tribolium
392
castaneum (Tcasα1, ABS86902). Asterisk indicates the presence of only the nAChR
393
α1 subunit ligand-binding domain. Double lines indicate the Cys-loop formed by two
394
cysteines.
395
Figure 2. Detecting Pp-AChBP1 expression in Sf9 cells. (A) SDS-PAGE analysis of
396
the transfected Sf9 cells' supernatant. Lane 1, prestained protein standard, Broad
397
range (New England Biolabs Inc.), Lane 2, transfected Sf9 cells' supernatant; Lane 3,
398
negative control without the subunit. The gel was stained with Coomassie brilliant
399
blue G 250. (B) Native-PAGE analysis of transfected Sf9 cells' supernatant. Lane 1,
400
NativeMark™ Unstained Protein Standard (Novex®, Life technologies); Lane 2,
401
transfected Sf9 cells' supernatant; Lane 3, negative control without the subunit. The
402
gel was stained with Coomassie brilliant blue G 250. (C) Western blotting of
403
SDS-PAGE. Lane 1, MagicMark™ XP Western Protein Standard; Lane 2, transfected
404
Sf9 cells' supernatant; lane 3, negative control without the subunit. (D) Western blot
405
of Native-PAGE. Lane 1, MagicMark™ XP Western Protein Standard; Lane 2,
406
transfected Sf9 cells' supernatant; Lane 3, negative control without the subunit.
407
Although the protein standard, MagicMark™ XP Western Protein Standard (Novex®,
(Bt-AChBP1,
BD358768;
Bt-AChBP2,
BD358769);
Cs,
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ACCEPTED MANUSCRIPT Life technologies; indicated by asterisk) is not suitable for native Western Blot, we
409
could not find a better standard at present.
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Figure 3. Analysis of α-Bgt-AChBP complex formation. (A) Titration of epibatidine
411
stoichiometry with AChBPs from two biological species (L. stagnalis and P.
412
pseudoannulata; (B) Analysis of α-Bgt-AChBP complex formation on native PAGE
413
by Western blotting and silver staining.
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Figure 4. Analysis of radio ligand binding to AChBPs. Equilibrium binding of
415
[3H]epibatidine (A), [3H]α-Bgt (B) and [3H]imidacloprid (C) to two recombinant
416
AChBPs. Data are mean±SEM of 3–5 independent experiments.
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Figure 5. Competitive binding of recombinant AChBPs against [3H]imidacloprid (5
418
nM). (A) Competitive binding of imidacloprid and acetamiprid to Pp-AChBP1 and
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Ls-AChBP compared with [3H]imidacloprid; (B) Competitive binding of clothianidin,
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dinotefuran and nitenpyram to Pp-AChBP1 compared with [3H]imidacloprid; (C)
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Competitive binding of sulfoxaflor, thiacloprid and thiamethoxam to Pp-AChBP1
422
compared with [3H]imidacloprid. Data are mean±SEM of 3 – 5 independent
423
experiments.
424
Figure S1. The phylogenetic tree of AChBP and nAChR α subunit (extracellular
425
domains) sequences constructed using the Neighbor-Joining method. (Ric)-3
426
(resistance to inhibitors of cholinesterase) from Drosophila melanogaster was used as
427
the outgroup. (Dm-Ric3, CAP16652; Dmα1, P09478; Dmα2, P17644; Dmα3, O18394;
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Dmα4,
429
AAK64377.1); Ac, Aplysia californica (Ac-AChBP, NP_001191488); Bt, Bulinus
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Q9NFR5;
Dmα5,
Q8T5F5;);
21
Ls,
Lymnaea
stagnalis
(Ls-AChBP,
ACCEPTED MANUSCRIPT truncates (Bt-AChBP1，BD358768; Bt-AChBP2，BD358769); Cs, Cupiennius salei
431
(Cs-AChBP, ALE66018.1); Pp, Pardosa pseudoannulata (Pp-AChBP1, KY765678;
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Pp-AChBP2, KY765676; Pp-AChBP3, KY765674; Pp-AChBP4, KY765675; Ppα1,
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D6QYZ4; Ppα8, D6QYZ5); Rn, Rattus norvegicus (Rnα2, P12389; Rnα4, P09483);
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Hs, Homo sapiens (Hsα2, Q15822; Hsα4, P43681); Mp, Myzus persicae (Mpα1,
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P91765; Mpα2, P91764; Mpα3, Q9U941; Mpα4, Q9U940; Mpα5, Q5F2K3 A8DIP3);
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Tcas, Tribolium castaneum (Tcasα1, A8DIP3; Tcasα2, A8DIQ7; Tcasα3, A8DIR0;
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Tcasα4, A8DIR5; Tcasα5, A8HTE9; Tcasα8, C3TS54). Asterisks indicates removal of
438
the transmembrane region of nAChR subunits from the analysis.
439
Figure S2. The phylogenetic tree of AChBP and nAChR β subunit (extracellular
440
domains) sequences constructed using the Neighbor-Joining method. (Ric)-3
441
(resistance to inhibitors of cholinesterase) from Drosophila melanogaster was used as
442
the outgroup. Dm, Drosohila melanoganster (Dm-Ric3, CAP16652; Dmβ1, P04755;
443
Dmβ2,
444
pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2, KY765676; Pp-AChBP3,
445
KY765674; Pp-AChBP4, KY765675; Ppβ1, C7EA46; Ppβ2, D6QYZ6); Ls, Lymnaea
446
stagnalis
447
NP_001191488); Bt, Bulinus truncates (Bt-AChBP1，BD358768; Bt-AChBP2，
448
BD358769); Cs, Cupiennius salei (Cs-AChBP, ALE66018.1); Hs, Homo sapiens
449
(Hsβ2, P17787; Hsβ4, P30926); Rn, Rattus norvegicus (Rnβ2, P12390; Rnβ4,
450
P12392).
451
Figure S3. Alignment of the amino acid sequences from loops A, B and C in AChBP
452
and nAChR α subunits. Numbers on the top indicate the positions of amino acids in
Mp,
Myzus
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P25162);
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AAK64377.1);
Ac,
(Mpβ1,
Aplysia
Q9NFX8);
californica
Pp,
Pardosa
(Ac-AChBP,
AC C
(Ls-AChBP,
persicae
22
ACCEPTED MANUSCRIPT Pp-AChBP1. Hs, Homo sapiens (Hsα2, Q15822; Hsα4, P43681); Rn, Rattus
454
norvegicus (Rnα2, P12389; Rnα4, P09483); Ls, Lymnaea stagnalis (Ls-AChBP,
455
AAK64377.1); Pp, Pardosa pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2,
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KY765676; Pp-AChBP3, KY765674; Pp-AChBP4, KY765675); Ppα1, D6QYZ4);
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Dm, Drosophila melanogaster (Dmα1, P09478; Dmα2, P17644); Mp, Myzus persicae
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(Mpα1, P91765; Mpα2, P91764); Loc, Locusta migratoria (Locα1, W6ECM5; Locα2,
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W6E9A5).
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Figure S4. Alignment of amino acid sequences of loops D, E and F in AChBP and
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nAChR β subunits. Numbers on the top indicate the positions of amino acids in
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Pp-AChBP1. Hs, Homo sapiens (Hsβ2, P17787); Rn, Rattus norvegicus (Rnβ2,
464
P12390); Mm, Mus musculus (Mmβ2, Q9ERK7); Ls, Lymnaea stagnalis (Ls-AChBP,
465
AAK64377.1); Pp, Pardosa pseudoannulata (Pp-AChBP1, KY765678; Pp-AChBP2,
466
KY765676; Pp-AChBP3, KY765674; Pp-AChBP4, KY765675; Ppβ1, C7EA46; Ppβ2,
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D6QYZ6); Loc, Locusta migratoria (Locβ1, W6EL29); Mp, Myzus persicae (Mpβ1,
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Q9NFX8); Nl, Nilapavarta lugenns (Nlβ1, B6VAH8); Dm, Drosophila melanogaster
469
(Dmβ1, P04755); Tcas, Tribolium castaneum (Tcasβ1, A8DIV7).
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0.75±0.09 3.48±0.46 2.49±0.35 11.84±2.18 3.13±0.52 14.08±3.19 1.53±0.34
12.48±2.13 33.73±5.44 31.17±4.28 133.46±30.31 27.52±6.60 120.71±24.65 22.03±4.68
Thiamethoxam
3.01±0.86
M AN U
30.86 30.36 54.81 16.43 14.11 20.64 20.06
SC
Imidacloprid Acetamiprid Clothianidin Dinotefuran Nitenpyram Sulfoxaflor Thiacloprid
23.67±7.42
9 10 11 9 9 9 9
11
for P-value 0.00016 0.00009 0.00002
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Table 1. Summary of Kd values (nM) for radio ligands and Ki values (nM) neonicotinoids binding to recombinant Pp-AChBP1 and Ls-AChBP F crit Insecticide Pp-AChBP1 Ls-AChBP df F (α=0.01) 3 [ H]Epibatidine 1.21±0.17 0.15±0.02 9 44.37 11.26 3 [ H]α-Bgt 24.09±3.14 2.13±0.29 9 51.10 11.26 3 [ H]Imidacloprid 2.43±0.36 44.72±5.64 10 69.24 10.56
9.71
11.26 10.56 10.04 11.26 11.26 11.26 11.26 10.04 4.96*
0.00054 0.00038 0.00002 0.00367 0.00557 0.00189 0.00206 0.01095
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Table S1. Mascot search results of peptide sequences by the mass spectrometric analysis.
918464064
ALA09394
915293426
XP_013309012
928193524
ALE66018
938149245
ALJ10921
597873494
EYC22868
597873492
EYC22866
341900060
EGT55995
1134468548
JAV23811
NO. of Unique peptides
5027.46
28338.18
296
82567.56
7
416.73
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AAG44630
Protein Mass
SC
12005809
acetylcholine-binding protein 1 [Pardosa pseudoannulata] 90-kDa heat shock protein HSP83 [Spodoptera frugiperda] heat shock protein 70 A1, partial [Spodoptera frugiperda] hypothetical protein NECAME_16129 [Necator americanus]
Protein Score
19550.98
4
130.71
8924.47
1
acetylcholine binding protein [Cupiennius salei]
122.36
28870.71
6
acetylcholine receptor [Dolomedes sulfureus]
86.62
28407.27
2
82.58
47957.54
3
70.23
58377.72
3
CBN-LGC-10 protein [Caenorhabditis brenneri]
51.37
55914.14
1
putative thioredoxin-dependent peroxidase [Culex tarsalis]
43.90
73542.27
1
hypothetical protein [Ancylostoma ceylanicum] hypothetical protein [Ancylostoma ceylanicum]
183.05
M AN U
ARU12133
Y032_0016g2995
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1199050132
Description
Y032_0016g2995
EP
Accession NO.
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Gi NO.
Only top 10 results were provided with the MASCOT minimal protein score above 40.
ACCEPTED MANUSCRIPT Table S2. The peptide sequences matched to Pp-AChBP1 and their hit numbers.
AC C
EP
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9 10 11 12 13 14 15
Hit Number
19-27 35-42 35-48 50-57 58-66 72-91 92-104
923.83 970.90 1663.78 785.90 985.16 2543.83 1623.53
26 2 33 17 5 25 43
105-153
5515.94
9
160-174 167-174 175-183 184-193 184-207 194-207 219-237
1745.80 967.08 1016.00 1149.29 2965.97 1834.70 2226.28
23 14 17 5 14 36 27
RI PT
8
SAVNGDAYK DIFNGYDK DIFNGYDKLVRPVK ATTAVPVK ISISPLSLR NQIISLEAWMMLTWMDEYLR WDPSDYDNITELR ISPTEIWKPDIALYTASPDTSLFPVVH TEAIVYNNGMIIWVPPFTINSR SYVSVSRTFVDCNIR TFVDCNIR MGSWTYSGK MVDLQLSTDK MVDLQLSTDKVDLTNFQDYNYEWK VDLTNFQDYNYEWK LYPCCADEYPHVNFNVTLK
Fragment Mass
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1 2 3 4 5 6 7
Sequence Range
SC
Peptide Sequence
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ACCEPTED MANUSCRIPT Highlights Five acetylcholine binding proteins (AChBPs) were identified in the spider Pardosa pseudoannulata.
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One of them, Pp-AChBP1, was expressed in Sf9 cells and characterized. Pp-AChBP1 had a much higher affinity to imidacloprid compared to an AChBP from the snail Lymnaea stagnalis.
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Spider AChBPs can provide a suitable alternate model to insect nAChRs.
