CTLGA9 Interacts with ALP1 and APN Receptors To Modulate

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Biotechnology and Biological Transformations

CTLGA9 interacts with ALP1 and APN receptors to modulate Cry11Aa toxicity in Aedes aegypti Khadija Batool, Intikhab Alam, Liang Jin, Jin Xu, Chenxu Wu, Junxiang wang, Enjiong Huang, Xiong Guan, Xiao-Qiang Yu, and Lingling Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01840 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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Journal of Agricultural and Food Chemistry

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CTLGA9 interacts with ALP1 and APN receptors to modulate Cry11Aa

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toxicity in Aedes aegypti

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Khadija Batool 1*, Intikhab Alam 2*, Liang Jin 1, Jin Xu 1, Chenxu Wu 1, Junxiang Wang 1, Enjiong Huang 3,

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Xiong Guan 1, Xiao-Qiang Yu 4, and Lingling Zhang 1* *

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1

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Key Lab of Biopesticides and Chemical Biology, MOE, Fujian Agriculture and Forestry University, 350002

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Fuzhou, Fujian, PR China.

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2

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College of Crop Science, Fujian Agriculture and Forestry University, 350002 Fuzhou, Fujian, People’s

State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences,

Key Laboratory of Genetics, Breeding and Comprehensive Utilization of Crops, Ministry of Education,

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Republic of China.

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3 Fujian

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International Travel Healthcare Center, 350001 Fuzhou, Fujian, People’s Republic of China.

Division of Cell Biology and Biophysics, University of Missouri - Kansas City, MO 64110, USA.

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* These authors contributed equally to this work.

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** Correspondence: [email protected]; Tel.: +86-591-8378-9258

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ABSTRACT: The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans,

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including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus

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(CHIKV). Bacillus thuringiensis (Bt) is widely used as biopesticide, which produces Cry toxins for mosquito

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control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and

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aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in

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response to Cry toxins. Our results showed that CTLTGA9 protein interacted with brush border membrane

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vesicles (BBMVs) of A. aegypti larvae, and with ALP1, APN and Cry11Aa proteins by Far-Western blot and

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ELISA methods. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for

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binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the

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larvicidal activity of Cry toxins.Based on the results of molecular docking, CTLGA9 may compete with

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Cry11Aa for binding to ALP1 and APN receptors, and thus decreases the mosquitocidal toxicity of Cry11Aa.

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Our results provide further insights into better understanding the mechanism of Cry toxin and help improve the

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Cry toxicity for mosquito control.

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KEYWORDS: Aedes aegypti; CTLGA9; Bacillus thuringiensis; Cry11Aa; Toxicity

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INTRODUCTION

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Mosquitoes fulfill their nutritional needs for reproduction through frequent blood feeding and this feeding

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behavior makes them efficient vectors for human dangerous diseases.1 For example, the mosquito Aedes

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aegypti transmits dengue fever,2 a serious outbreak that affects about hundred million people yearly, and it is

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also responsible for transmission of Zika virus (ZIKV) that has become a significant health issue worldwide.3,4

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Due to lack of efficient vaccines, rapid increase in pesticide resistance, and socio-economic conditions in

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endemic countries, it is very important to investigate practical strategies to limit the mosquito-borne diseases.

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Nowadays, the main approach to limit mosquito-borne diseases is through vector control.5 Bacillus

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thuringiensis subsp. israelensis (Bti) has been used in the control of vector-borne diseases.6-9 Bti produces four

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main crystalline inclusions during sporulation phase: Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa.10 Except for

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Cyt1Aa, the three Cry toxins have been proved as active primary mosquitocidal toxins.11, 12 Among the active

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toxins in Bti, Cry11Aa is widely used for the eradication of A. aegypti due to its higher toxicity,13 with a high

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binding affinity to A. aegypti brush border membrane vesicles (BBMV).14 After ingestion of Cry toxins, the

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insect larval gut solubilized the crystal proteins in the alkaline pH environment, producing Cry and Cyt

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protoxins that are activated to toxins by gut proteases, thus toxin oligomerization and insertion into midgut

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epithelial cells ultimately cause pore formation in midgut eventually leading to cell death.15,16 In this toxicity

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mechanism, the main step is the interaction between Cry toxins and their putative receptors on BBMVs.8,17 For

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example, Cry11Aa binds to different protein receptors with high affinities such as aminopeptidase-N (APN),

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alkaline phosphatase (ALP), cadherin (CAD), and ATP-binding cassette (ABC) transporters.14,18-20 Though the


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interaction of Cry toxins with the specific receptor is well known, interactions between Cry toxins and other

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midgut proteins, which may alter the toxicity, are still elusive.

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C-type lectins (CTLs) are a large family of pattern recognition receptors (PRRs) that can bind to galactose

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and mannose type carbohydrates with the help of calcium ions.21 CTLs were firstly reported in plants, later

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discovered in invertebrates and vertebrates.22 CTLs also regulate the developmental systems and immune

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functions in invertebrates.23-27 These lectins help in the elimination of invading microorganisms.28

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Furthermore, as compared to the vertebrates, the invertebrate C-type lectins are more abundant and multi-

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functional.29 Insect CTLs play a crucial role in innate immunity.30 Recently, A. aegypti CTL-20 that was up-

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regulated after feeding Bt toxins, was found to compete with Cry11Aa for binding to ALP1 receptor, and then

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increased the survival of mosquito larvae.31 However, it is not clear whether other C-type lectins expressed in

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the midgut of the mosquito have similar functions.

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In the present study, the recombinant CTLGA9 (C-type lectin with predicted galectose binding) protein was

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expressed and purified, the interaction of CTLGA9 with Cry toxin, ALP1 and APN was confirmed by Western

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blot, Far-Western, and ELISA methods. Molecular docking showed that CTLGA9 might compete with

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Cry11Aa for binding to ALP1 and APN receptor, which may result in diminishing the toxicity. Additionally,

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results from insect feeding assays showed that mortality of A. aegypti fed with Cry11Aa+CTLGA9 was

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significantly decreased compared to the control group (Cry11Aa+thioredoxin). These results provide useful

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information for better understanding the mechanism of Cry toxins in mosquito control.

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MATERIALS AND METHODS

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Mosquito strain

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A. aegypti Haikou strain was provided by the Fujian international travel health care center and was

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reared in the laboratory at 28°C temperature with 75-80% relative humidity (RH). Photoperiod condition was

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controlled with 14 h light and 10 h dark. Midguts from the fourth instar A. aegypti larvae were dissected and

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immediately soaked in the TRIzol Reagent (Invitrogen Life Technologies, Thermofisher Scientific, USA), and



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then stored at −80°C until use. Plasmid DNAs for expression of recombinant Cry11Aa, ALP1 and APN were

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kindly provided by Dr. Sarjeet Gill, University of California Riverside, CA, USA.

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Bioinformatics

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The deduced CTLGA9 protein sequence was analyzed using DNAMAN (Lynnon Corporation,

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Quebec, QC, Canada) software. The CTLGA9 protein sequence was submitted and analyzed by SMART

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(http://smart.embl-heidelberg.de) to confirm the existence of carbohydrate recognition domain (CRD). Using

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the ProtParam tool (http://web.expasy.org/protparam/), the size and isoelectronic point of CTLGA9 were also

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analyzed. For phylogenetic relationship with other closely related CTLs, CTLGA9 homologous sequences

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were identified and obtained from NCBI (http://www.ncbi.nlm.nih.gov/). The retrieved CTL proteins

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sequences were aligned by using the program Clustal W and MUSCLE module. With the Neighbor-joining

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method in the Molecular Evolutionary Genetics Analysis (MEGA) software version 6.06 and 1000 replicate

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bootstrap analysis, the phylogenetic tree was constructed in this study.32

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Cloning of CTLGA9

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Total RNA extraction was carried out with TRIzol Reagent following the company’s instructions, and the

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single-stranded cDNA was obtained using the PrimeScript™ RT Kit (TaKaRa, Dalian, China). The A. aegypti

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CTLGA9 (Genbank accession number #AAEL014385) was then amplified by the polymerase chain reaction

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(PCR) using gene-specific forward primer: 5'-CAT GCC ATG GAA GCA TGG AGA CTG TG-3' and reverse

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primer: 5'-CCC AAG CTT TTA CGA CCA GTG TGG ACT GA-3'. The amplified fragment was then cloned

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to the vector pMD18-T (TaKaRa, Dalian, China) with competent cell E.coli DH5α strain. The recombinant

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positive clones were selected, recombinant plasmids were prepared and digested by specific restriction

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enzymes Nco I/Hind III, and the sequence of the insert was confirmed by DNA sequencing (Biosune

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Company, Beijing, China).

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Prokaryotic expression and purification of recombinant CTLGA9


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Prokaryotic expression was performed with pET-series expression vector pET32a. The DNA fragment

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digested by Nco I/Hind III was further ligated to pET32a vector and transformed to BL21 (DE3) competent

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cells. Positive clones were picked, analyzed and DNA sequence was subsequently confirmed by sequencing

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(Biosune Company, Beijing, China). Then, the confirmed positive clones were grown in LB medium

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containing 0.1% ampicillin at 37°C with constant agitation of 220 rpm until the OD600 of culture reached 0.6-

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0.8. Expression of recombinant protein was induced with isopropyl-β-D-thiogalactosidase (IPTG) in a final

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concentration of 0.5 mM. After induction at 16°C for 12 h, bacterial cells expressing recombinant CTLGA9

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and the control protein thioredoxin (pET32a-Trx) were harvested and sonicated in the binding buffer (0.02M

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Na3PO4.12H2O, 0.5M NaCl and pH: 7.4). The soluble proteins in the supernatants were separated and purified

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by Ni-NTA His-tag chromatography (Transgen Biotech, Beijing, China). The resulting recombinant proteins

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were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained

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with Coomassie brilliant blue R-250. A. aegypti receptors ALP1 and APN were also expressed and purified by

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His-tag Chromatography as reported previously.14

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Cry11Aa toxin preparation and in vitro processing

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The bacterial strain PCG6 harboring Cry11Aa was cultured in nutrient-rich sporulation medium that

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contains antibiotic erythromycin in the concentration of 25 mg/ml at 30°C with constant shaking incubator for

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2 to 3 d until reaching the complete sporulation stage.33 Bacterial growth was monitored by microscopy.

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Crystal inclusions were collected, prepared and purified according to previous reports.14,20,33 The purified

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crystal protoxin was further activated with trypsin at a ratio of 20:1 (w/w) and incubated in water bath at 37°C

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for 2 h.34 The insoluble fraction was eliminated by centrifugation at 15,800 × g for 5 min, and the activated

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Cry11Aa toxins were collected and stored at −20°C until use. According to the manufacturer’s instruction, the

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activated Cry11Aa was labeled with NHS-biotin and purified with a Sephadex G25 column (GE Healthcare

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Life Science, Pittsburgh, PA).

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Toxicity analysis



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Bioassays were performed with 3rd instar larvae of A. aegypti according to the previously reported

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method.31 Test larvae were prepared in 30 mL distilled water containing purified Cry11Aa toxin (LC50: 0.85

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µg/mL) and recombinant CTLGA9 protein with different concentrations (0, 0.15, 1.5, 15 μg/mL). Control

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larvae in 30 mL distilled water were treated with Cry11Aa toxin and control thioredoxin (Trx) protein (0, 0.15,

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1.5, 15 μg/mL). Twenty-five larvae were added in each cup, and each treatment was repeated three times,

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respectively. Survival rate was analyzed after feeding at 12 and 24 h. GraphPad Prism was used to graph the

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data and bars represent the means ± SD of three replicates.

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Isolation of A. aegypti Midgut BBMVs

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Late fourth instar larvae of A. aegypti were dissected to collect the midguts. Differential magnesium

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precipitation method reported by Wolfersberger35 was used to prepare BBMV in the presence

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of protease inhibitors (Roche protease inhibitor in the concentration of 1 mM PMSF). The homogenizing

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buffer A (300 mM mannitol, 17 mM Tris-HCl, 5 mM EGTA) was used to homogenize the midgut tissue. Then,

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the same volume of 24 mM MgCl2 solution was added to the homogenate, and the mixture was kept on ice for

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15 min. After centrifuging for 15 min at 4500 rpm at 4 °C, the supernatant was transferred into a new EP-tube.

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After centrifuging again for 30 min at 30,000 g, the supernatant was discarded and the pellet was resuspended

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in half volume of buffer A containing protease inhibitors, and stored at –80 °C until use. The protein

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concentration was then analyzed using bovine serum albumin (BSA) as the standard.36

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Western blot and Far-Western blot analysis

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Protein analysis and protein-protein interactions were examined by Western blot and Far-Western blot

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techniques as reported previously.31 Quality of recombinant proteins was firstly analyzed by Western blot

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analysis. Recombinant CTLGA9, ALP1, APN and Cry11Aa proteins were separated on 10% polyacrylamide

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gel (SDS-PAGE) and then transferred to PVDF membranes of pore size 0.2 μm. The membranes were blocked

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with 5% skimmed milk dissolved in PBS (pH: 7.4) buffer for 2 h at 37°C, washed with PBST (PBS buffer

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containing 0.1% Tween-20) and PBS buffer. The membranes were incubated with primary rabbit polyclonal

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antibody (1:3000 dilution) for 2 h, washed with PBST and PBS buffer again, followed by incubation with


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secondary goat anti-rabbit antibody (1:3000). Finally, binding results were envisioned by a color reaction using

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BCIP/NBT Alkaline Phosphatase (AP) Color Development kit (Beyotime biotech, Shanghai, China) following

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the manufacturer’s protocol.

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Protein interactions were carried out by Far-Western blot analysis as described previously.31 A.

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aegypti CTLGA9, activated Cry11Aa, ALP1 or APN protein was separated on 10% SDS-PAGE and

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transferred to PVDF membrane by semi-dry method. The membrane was blocked with 5% skim milk in PBS

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buffer (pH 7.4). After washing with PBS and PBST buffer, the membrane was probed with purified biotin

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labeled CTLGA9, Cry11Aa, ALP1 or APN protein in PBS (pH 7.4), respectively, for 2 h at 37°C. Finally,

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after washing with PBS and PBST, the membrane was incubated with biotin specific antibody Streptavidin/AP

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(1:3000) for 2 h and washed with PBST and PBS buffers. The binding of interacting protein was determined

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using BCIP/NBT alkaline phosphatize (AP) color development kit (Beyotime biotech, Shanghai, China).

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ELISA and competitive ELISA assays

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Interaction among CTLGA9, ALP1, APN and Cry11Aa, and binding of these proteins to isolated

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BBMVs were determined by ELISA and competitive ELISA assay as reported previously.37 The 96 wells

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microtiter plates were coated with 4 μg/well of purified BBMVs, ALP1 or APN protein diluted in 50 mM

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Na2CO3 at 4°C overnight, then blocked with PBST buffer for 2 h at 37°C. After washing with PBS,

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biotinylated Cry11Aa or CTLGA9 protein (0 to 80 nM) in PBST buffer was transferred to the coated plates

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and placed at 37°C for 2 h. The unbound proteins were removed, and the plate was washed with PBST.

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Streptavidin HRP antibody (1:3000 dilutions) (SA-HRP, Pierce) was applied to the plate and incubated for 1.5

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h at 37°C. Afterward, HRP chromogenic agent tetramethylbenzidine solution (TMB-ELISA, Thermo Fisher

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Scientific, Inc.) was applied in dark condition to each well and kept at room temperature for 15 min for color

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development. Termination agent (2 M H2SO4) was added to stop the reaction, and Multiskan™GOMicroplate

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Spectrophotometer (Thermo180 Scientific™) was then used to determine the absorbance at 450 nm.

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The competitive ELISA assays were performed to detect the ability of CTLGA9 to compete with

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Cry11Aa for binding to ALP1 and APN receptors. Briefly, increasing concentrations of unlabeled Cry11Aa or



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CTLGA9 (0-500 nM) was mixed with 10 nM biotin-labeled CTLGA9 or Cry11Aa at room temperature for 1

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h, and then applied to (100 μl/well) ELISA plates coated with ALP1 or APN protein. The assays were

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performed the same as described above. All the binding data were graphed using GraphPad Prism software.

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Model structure and molecular docking of proteins

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The three-dimensional (3D) model structures of Cry11Aa, ALP1, APN and CTLGA9 were obtained using Bt

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δ-endotoxin (PDB: 1DLC), crystal structure of shrimp alkaline phosphatase (PDB: 1K7H), Anopheles gambiae

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APN (PDB: 4WZ9) and human carbohydrate binding protein (PDB: 5E4L) as templates, respectively, with the

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Phyre2 software (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index).38 Molecular docking of protein

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complexes was obtained by using GRAMM-X Docking Web engine v.1.2.0 (http://vakser.compbio.

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ku.edu/resources /gramm/grammx) and displayed in Discovery studio with different parameters, including

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ribbon protein selection, orthographic with line width 1.60, color adjusting and lightening. The binding

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interface of the docked proteins was analyzed by root mean square deviation (RMSD) in Discovery Studio 2.5

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software.

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RESULT

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Characterization of A. aegypti CTLGA9

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The A. aegypti lectin gene CTLGA9 (AAEL014385) containing single CRD domain was amplified by gene-

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specific primers through PCR, the PCR product was purified, cloned and sequenced. Sequence analysis

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showed that CTLGA9 contained an ORF of 354 bp, encoding 118 amino acid residues with calculated

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molecular weight of 33 kDa together with the tag of 20 kDa, and isoelectric points (pI) of 5.40. According to

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the instability index (II), the CTLGA9 has a score of 29.01 and is classified as a stable protein.

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To gain insight in to the evolutionary relationship of CTLGA9 with homologous CTLs, CTLs from other

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insect species, including flies and mosquitoes, were obtained from NCBI and a phylogenetic tree was

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generated. The result showed that CTLGA9 was clearly clustered with mosquito CTLs as compared to CTLs

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from other species (Figure 1).


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Figure 1. Phylogenetic tree of A. aegypti CTLGA9 and homologous CTLs in other insect species. A Neighbor-

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joining (NJ) phylogenetic tree was generated through MEGA 6.06 with a bootstrap analysis of 1000 replicates. For

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evolutionary distances, p-distance method was used. The genus and species were indicated in the parenthesis .

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Binding of CTLGA9 to ALP1, APN and Cry11Aa toxin

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The CTLGA9 cDNA was ligated with expression vector pET32α and the ligation product was

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transformed into E. coli BL21 competent cells. Recombinant CTLGA9 protein was purified by ProteinISO Ni-

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NTA Resin. Quality of recombinant CTLGA9 was checked by 12% SDS-PAGE with a molecular weight of 33

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kDa compared to the control thioredoxin (Trx) protein of 20 kDa (Figure 2A-a). Western blot analysis showed

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that the band of 33 kDa recombinant CTLGA9 protein was specifically recognized by rabbit polyclonal

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antibodies to CTLGA9 (Figure 2A-b). Recombinant Cry11Aa, ALP1 and APN proteins were also purified and

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confirmed SDS-PAGE and Western blot analyses. The results showed 70 kDa band of Cry11Aa protoxin

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(Figure 2B-a) and the trypsin activated Cry11Aa fragments of 32 and 36 kDa (Figure 2B-b) on SDS-PAGE.

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Activated Cry11Aa fragments were also detected by specific polyclonal antibodies (Figure 2B-c). ALP1

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protein showed a band of 65 kDa (Figure 2C-a) on SDS-PAGE and the protein was recognized by ALP1



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antibody (Figure 2C-b). APN protein showed a band of 73 kDa in the gel (Figure 2D-a), which was recognized

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by APN specific antibodies (Figure 2D-b). Then, binding of CTLGA9 to ALP1, APN and Cry11Aa was

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analyzed by far Western blot technique. The 33 kDa CTLGA9 band was detected by antibodies specific for

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Cry11Aa, ALP1 and APN after the membrane was probed with Cry11Aa, ALP1 and APN, respectively

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(Figure 2B-d, 2C-c, 2D-c). Similarly, the 32 kDa and 36 kDa Cry11Aa fragments, 65 kDa ALP1 and 73 kDa

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APN were detected by antibodies specific to CTLGA9 when the membranes were probed with CTLGA9

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(Figure 2B-e, 2C-d, 2D-d)).

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Figure 2. The interactions among recombinant CTLGA9, Cry11Aa, ALP1 and APN proteins. (A) Purified

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recombinant CTLGA9 protein analyzed by SDS-PAGE and Western blot. a). Lane M: marker; lane 1: Purified

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recombinant thioredoxin (Trx) control protein; lane 2: total bacterial lysates containing CTLGA9; lane 3:

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Supernatant proteins containing CTLGA9; lane 4: Unbound proteins after resins binding; lane 5: Purified

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recombinant CTLGA9 protein; b). Detection of biotin labeled CTLGA9 protein by anti-biotinantibody (B)

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Purification, activation and binding analysis of recombinant Cry11Aa protein. a). Purified Cry11Aa protoxin


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analyzed by SDS-PAGE; b). Activation of Cry11Aa protoxin by trypsin (1:20 w/w); c). Detection of the biotin

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labeled Cry11Aa activated fragments by anti-biotinantibody; d). Recombinant CTLGA9 protein in the membrane

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detected by Cry11Aa antibody after probing with Cry11Aa fragments; e). Activated Cry11Aa in the membrane

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detected by CTLGA9 antibody after probing with CTLGA9. (C) Purification and binding analysis of recombinant

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ALP1 protein. a). Purified ALP1 recombinant protein analyzed by SDS-PAGE; b). Detection of ALP1 protein with

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specific antibody against ALP1; c). Recombinant CTLGA9 protein detected by ALP1 antibody after probing with

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ALP1 protein; d). Recombinant ALP1 protein detected by CTLGA9 specific antibody after probing with CTLGA9

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protein. (D) Purification and binding analysis of recombinant APN protein. a). Purified APN recombinant protein

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analyzed by SDS-PAGE; b). Detection of APN protein with specific antibody against APN; c). Recombinant

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CTLGA9 protein detected by APN specific antibody after probing with APN protein; d). Recombinant APN protein

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detected by CTLGA9 specific antibody after probing with CTLGA9 protein.

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Binding affinity of CTLGA9 with ALP1, APN, Cry11Aa and BBMVs

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Plate ELISA assay was carried out to confirm the binding of CTLGA9 to midgut BBMVs, ALP1 and

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APN of A. aegypti larvae. The results revealed that CTLGA9-Trx fusion protein bound to BBMVs with one-

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site binding and a calculated Kd of 8.756 nM, whereas neglected amount of the control thioredoxin protein

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bound to BBMVs (Figure 3A). Moreover, we analyzed the binding activity of biotin-labeled CTLGA9-Trx to

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ALP1 and APN receptors, and the results clearly revealed that CTLGA9-Trx bound to the immobilized ALP1

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(Kd = 13.37 nM) and APN (Kd = 15.60 nM) with one-site binding (Figure 3B, C). We also determined the

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binding of Cry11Aa to the coated receptors ALP1 (Kd = 9.327 nM) and APN (Kd = 11.39 nM) with one-site

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binding (Figure 3D, E). The combined results revealed that CTLGA9 and Cry11Aa can bind to ALP1 and

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APN, and Cry11Aa has higher affinity than CTLGA9 for ALP1 and APN receptors.

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We then performed the competitive binding assays to further confirm that CTLGA9 and Cry11Aa can

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compete for binding to ALP1 and APN receptors. The results showed that increasing concentrations of

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unlabeled Cry11Aa (0-500 nM) competed with biotinylated CTLGA9 (10 nM) for binding to immobilized

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ALP1 and APN (Figure 3F, G), and similarly, increasing concentrations of unlabeled CTLGA9 (0-500 nM)

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competed with biotinylated Cry11Aa (10 nM) for binding to immobilized ALP1 and APN (Figure 3H, I).

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These results suggest that both Cry11Aa and CTLGA9 can compete for binding to ALP1 and APN receptors.



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Figure 3. Binding of CTLGA9 and Cry11Aa to BBMVs, ALP1 and APN of A. aegypti. CTLGA9-Trx binding to the

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coated BBMVs (A), ALP1 (B), and APN (C). Cry11Aa binding to the coated ALP1 (D) and APN (E). Biotinylated

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CTLGA9-Trx binding to immobilized ALP1 (F) and APN (G) in the presence of increasing concentrations of

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unlabeled Cry11Aa or thioredoxin, and biotinylated Cry11Aa binding to the coated ALP1 (H) and APN (I) in the

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presence of increasing concentrations of unlabeled CTLGA9 or thioredoxin.


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Three-dimensional model structures of proteins

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In order to investigate interaction of CTLGA9 with ALP1 and APN receptors of A. aegypti, molecular docking

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of CTLGA9 protein with the two receptors was performed using online software PHYRE 2 and GRAMM-X

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Protein-Protein Docking Web Server v.1.2.0. We first obtained the three-dimensional model structures of

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Cry11Aa (Figure 4A), ALP1 (Figure 4B), APN (Figure 4C) and CTLGA9 (Figure 4D) by using specific

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structure templates. Molecular docking results indicated that Cry11Aa and CTLGA9 can dock to ALP1

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receptor at two different but partially overlapped interfaces (Figure 5A, B), many residues in the overlapped

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interfaces of ALP1 participated in binding to both Cry11Aa and CTLGA9 (Figure 5C). Based on the

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interaction data, the area of contact interface between CTLGA9 and ALP1 was 121.2045 angstrom2, slightly

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higher than that (119.338 angstrom2) between Cry11Aa and ALP1. The binding free energy between CTLGA9

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and ALP1 was -652.4 KJ/mol, which is slightly higher than that (-700.6 KJ/mol) between Cry11Aa and ALP1.

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Together, these results suggest that Cry11Aa can bind to ALP1 slightly stronger than CTLGA9 does.

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Similarly, Cry11Aa and CTLGA9 were also docked to APN receptor and the results showed that the two

277

proteins bound to APN with two different but overlapped interfaces (Figure 6A, B), and many amino acid

278

residues in the overlapped interface of APN receptor are also involved in binding to both Cry11Aa and

279

CTLGA9 (Figure 6C). The interaction data showed the area of contact interface between CTLGA9 and APN

280

(105.2085 angstrom2) was much higher than that (13.676 angstrom2) between Cry11Aa and ALP1. Similarly,

281

the binding free energy between CTLGA9 and APN was -645 KJ/mol, which is also lower than that (-393.2

282

KJ/mol) between Cry11Aa and APN. Thus, the binding of CTLGA9 to APN is slightly stronger than Cry11Aa.

283

In a word, all the results suggest that both Cry11Aa and CTLGA9 bind to ALP1 and APN receptors, and the

284

amino acids in the overlapped interfaces of ALP1 and APN might be important for binding to CTLGA9 and

285

Cry11Aa.



286 287

Figure 4. Modeling structures of Cry11Aa, ALP1, APN and CTLGA9. Three-dimensional model structures of

288

Cry11Aa (template PDB: 1DLC) (A), ALP1 (template PDB: 1K7H) (B), APN (template PDB: 4WZ9) (C) and

289

CTLGA9 (template PDB: 5E4L) (D) were generated by PHYRE 2 Web engine.

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Figure 5. Molecular docking analysis of Cry11Aa and CTLGA9 with ALP1. Three-dimensional model structures

292

were generated by PHYRE 2 web server and molecular docking was carried out using Discovery Studio version 2.5.

293

Residues in yellow in Cry11Aa (A) and CTLGA9 (B) were predicted to be involved in interaction with ALP1

294

receptor. (C) Residues in ALP1 receptor that participate in interaction with Cry11Aa (green smudge), CTLGA9

295

(red), or both Cry11Aa and CTLGA9 (yellow).

296

297 298

Figure 6. Docking analysis of Cry11Aa and CTLGA9 with APN. Three-dimensional model structures were

299

generated by PHYRE 2 web server and molecular docking was carried out using Discovery Studio version 2.5.

300

Residues in yellow in Cry11Aa (A) and CTLGA9 (B) were predicted to participate in interaction with APN

301

receptor. (C) Residues in APN receptor that participate in interaction with Cry11Aa (green smudge), CTLGA9 (red),

302

or both Cry11Aa and CTLGA9 (yellow).

303

CTLGA9 inhibits Cry toxins toxicity

304

To further investigate whether CTLGA9 protein can change the Cry toxins activity, bioassay was performed

305

by feeding the 3rd instar larvae of A. aegypti Cry11Aa mixed with purified recombinant CTLGA9-Trx protein

306

or Trx control protein, and the survival rate of larvae was recorded. The results showed that feeding larvae

307

Cry11Aa mixed with CTLGA9-Trx protein significantly increased the survival rate of larvae at 12 h (Figure



308

7A) and 24 h (Figure 7B) after feeding compared to that of larvae fed Cry11Aa mixed with the control Trx

309

protein. These results suggest that CTLGA9 can decrease the toxicity of Cry toxins.

310 311

Figure 7. Bioassay of A. aegypti larvae fed with Cry11Aa toxin mixed with CTLGA9 recombinant protein. Third

312

instar A. aegypti larvae were treated with activated Cry11Aa (0.85 μg/mL) mixed with increasing concentrations of

313

the control Trx protein or CTLGA9 recombinant protein (0.15, 1.5, 15 μg/mL), then the survival rate of mosquito

314

larvae was recorded at 12 h (A) and 24 h (B) after feeding with three biological replicates. Significance of difference

315

among the concentrations of CTLGA9 was calculated by One way ANOVA followed by Tukey HSD test using IBM

316

SPSS (A and B), and significant difference was shown by different letters (p < 0.05). Unpaired t test was also used

317

to determine the Significance of difference between the control Trx and CTLGA9 protein treatments with the IBM

318

SPSS (*, p < 0.05).

319

DISCUSSION

320

Although chemical insecticides are mainly used to control vector-borne diseases, excess applications of

321

insecticides have caused higher resistance in vectors.39 Bti plays an active role as a bio-control agent, and has

322

been increasingly used to limit the spreading of mosquito-borne diseases. Cry toxin domain II and the lectin

323

CRD domain both have a similar β-prism structure, 18, 40-44 with the exposed loop regions involved in receptor

324

binding. CTLs are a family of pattern recognition receptors (PRRs) that specifically bind to galactose and

325

mannose types glycans in the presence of Ca2+.21 Recently, we reported that A. aegypti CTL-20 and galectin-14,

326

which were expressed either highly or induced by Cry toxins in the midgut, can compete with Cry11Aa for binding


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to ALP1 receptor, and feeding mosquito larvae with Cry11Aa toxin mixed with CTL-20 and galectin-14 can

328

increase the survival of the mosquito larvae.31,45 Among the CTLs expressed in the midgut of A. aegypti, many

329

(such as CTLGA9) were not up-regulated by Cry toxins treatment. To further understand the function and

330

molecular mechanism of A. aegypti CTLs that are either expressed at low levels or not induced by Cry toxins,

331

CTLGA9, a single CRD domain lectin that was not up-regulated in the midgut of A. aegypti by Cry toxins, was

332

selected as a candidate gene in this study, to investigate the interaction of CTLGA9 with Cry11Aa and toxin

333

receptors.

334

In the current study, we showed that CTLGA9 and Cry11Aa both bound to toxin receptors ALP1 and

335

APN by Far-Western blot and ELISA assays. Molecular docking results also showed that Cry11Aa and

336

CTLGA9 docked to ALP1 and APN at two different but partially overlapped interfaces, with many amino

337

acids in the overlapped regions contributing to binding of the ALP1 and APN receptors to Cry11Aa and

338

CTLGA9. More importantly, the bioassay results demonstrated that CTLGA9 inhibited the larvicidal activity

339

of Cry11Aa, a function similar to CTL-20 and galectin-14. This is due to competitive binding of CTLGA9

340

with Cry11Aa for the ALP1 and APN receptors. Thus, CTLs and galectins in general can compete with Cry

341

toxins for binding to toxin receptors, resulting in decrease concentrations of active Cry toxin binding to

342

receptors, and finally inhibit the toxicity of Cry toxins.

343

Our results provide new evidence to understand the interaction between Cry toxins and toxin receptors. In

344

the midgut, many proteins are present and some proteins may interfere with the binding of Cry toxins to toxin

345

receptors, and thus can change the Cry toxins toxicity. Future research is required to identify proteins other

346

than CTLs and galectins in the midgut that may interfere with the binding of Cry toxins to receptors.

347 348

AUTHOR CONTRIBUTIONS: K.B. and L.Z. conceived and designed the experiments; K.B. and I.A.

349

conducted the experiments, processed the data and wrote the manuscript; J.W., J.X., L.J., E.H., X.G., X. Y.

350

and L.Z. revised and approved the paper.

351

Funding: Our work was funded by the National Program of China (2017YFE0122000 and

352

2017YFE0121700); the foreign cooperative project of Fujian province (2018I0023); the National Institute of



353

Health (AI117808); Science and Technology Project of Fuzhou (2018-G-70) and Open Project Funds of State

354

Key Laboratory of Pathogen and Biosecurity (SKLPBS1838).

355

ACKNOWLEDGMENTS

356

The authors are thankful to Dr. Sarjeet Gill at the University of California, Riverside, CA, USA, for providing

357

the recombinant Bt strains and the antibodies, and Dr. Liaoyuan Zhang at Fujian Agriculture and Forestry

358

University, Fuzhou, Fujian, China, for his help in molecular docking.

359

Conflicts of Interest: All the authors declare no conflicts of interest.

360 361

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CTLGA9 Interacts with ALP1 and APN Receptors To Modulate

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