Yongyi XU Yanling LIAO Qiqi GUO, Ming LI Jinxing FU, Bingmiao GAO
Abstract ［Objectives］This study was conducted to synthesize sea anemone peptide toxin Ap-TxI and investigate its insecticidal activity.
［Methods］ The sea anemone linear peptide toxin Ap-TxI was synthesized by the solid-phase peptide synthesis (SPPS), and six cysteines were oxidized to form three disulfide bonds by a three-step directional oxidation method. Then, purification by high performance liquid chromatography (HPLC) and mass spectrometry identification were performed. Finally, the insect cytotoxicity and insecticidal activity of Ap-TxI were studied by the MTT method and insect injection method.
［Results］ The oxidized peptide Ap-TxI with three disulfide bonds in natural configuration was successfully synthesized by the SPPS method, and its purity was >90% by HPLC analysis. The results of the MTT method showed that Ap-TxI was active on the growth of insect cells sf9, with a half effective dose of 0.2 nM; and the results of the mealworm injection test showed that the polypeptide Ap-TxI had high insecticidal activity with a median lethal dose of 11.7 nM.
［Conclusions］ The sea anemone peptide toxin Ap-TxI with high insecticidal effect was obtained, laying a foundation for the development of new, efficient and safe biological insecticides.
Key words Sea anemone peptide toxin; Solid-phase synthesis; Oxidative folding; Insecticidal activity
Received: April 20, 2022 Accepted: June 21, 2022
Supported by Natural Science Foundation of Hainan Province (820RC636); Undergraduate Innovation and Enterpreneurship Training Program of Hainan Province (X202011810003); Special Fund for Academician Innovation Platform in Hainan Province (YSPTZX202132).
Yongyi XU (2002-), male, P. R. China, major: pharmacy.
*Corresponding author. E-mail: gaobingmiao@qq.com.
With the widespread abuse of pesticides, the problem of agricultural pollution is imminent, directly threatening agricultural ecological security and sustainable agricultural development. Therefore, food safety issues seriously affect human health［1］. Solving security problems of agriculture and ecology has become a general trend, and the demand for new, efficient and safe biological pesticides is very urgent［2-3］. At present, animals that feed on insects can generally secrete some peptide toxins, which play a poisonous role through specific ion channels or receptors of insects to capture food, but these toxins have little or no toxic side effects to mammals［4-5］. Among them, sea anemones, cone snails, scorpions, spiders and predatory mites have been reported to be capable of secreting peptide toxins［6-9］. Sea anemones, as primitive metazoans, are rich in a variety of bioactive peptide neurotoxins. These peptides have been used in neuroscience research tools or directly developed as marine drugs. So far, more than 1 100 species of sea anemones have been reported, but only 5% of them have been used for the isolation and identification of sea anemone peptide neurotoxins［10］. The tentacles of sea anemones have stinging cells that can secrete venom to prey on fish, shellfish, copepods, crustaceans and worms. Its venom is rich in various peptide toxins and is an important marine drug resource［11-14］. Chinese Materia Medica and Chinese Medicinal Animals record that sea anemones have the functions of inducing astringency, removing dampness and destroying parasites, and are used in traditional Chinese medicine to treat prolapse of anus, hemorrhoids, etc.［18］. According to research and analysis in modern pharmacology, sea anemone peptide toxin has parasite-destroying, antitumor, antihypertensive, antibacterial, analgesic and neurosuppressive effects［15-17］. Therefore, there is an urgent need to discover and study sea anemone peptide neurotoxins more systematically, and search for peptides with insecticidal activity. A previous study discovered from Aiptasia pallida by high-throughput transcriptomics, a sea anemone peptide toxin, Ap-TxI, the sequence of which is SGACAEICVHRCIPSCNFACCVAR, with six cysteines forming three disulfide bonds. In this study, linear peptide Ap-TxI was synthesized by the solid-phase peptide synthesis (SPPS), and oxidized by a directed oxidation method to obtain the oxidized peptide Ap-TxI containing three disulfide bonds (disulfide bond connection modes C1-C6, C2-C5, C3-C4), which was then purified by high performance liquid chromatography and identified by mass spectrometry. The synthetic peptide Ap-TxI was tested for its inhibitory effect on insect cells by the MTT method, and its insecticidal effect on mealworms by the insect injection method.
Materials and Methods
Experimental materials
Chromatographic grade trifluoroacetic acid (TFA) and chromatographic grade acetonitrile (ACN), purchased from Thermo Fisher Scientific; ninhydrin, methanol and NH4HCO3, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; DCM, purchased from Xilong Science Co., Ltd.; DMF, purchased from Shanghai Yien Chemical Technology Co., Ltd.; Vydac analytical C18 column (5 μm, 4.6 mm×250 mm) and preparative C18 column (10 μm, 22 mm×250 mm), purchased from Shanghai Chenqiao Bioscience Co., Ltd.; conventional molecular biology reagents such as plasmid extraction kits, purchased from Tiangen Bioengineering Co., Ltd.
Experimental instruments
Reversed-phase high-performance liquid chromatography (Agilent, USA); triple quadrupole liquid chromatography-mass spectrometer (Shimadzu, Japan); refrigerated high-speed centrifuge (CR22GIII, Hitachi, Japan); desktop freeze dryer (Safer, China ); carbon dioxide incubator (SI19, UK); inverted microscope (OLYMPUs, China); microplate reader (MR-96A, Shenzhen Mindray).
Experimental methods
Synthesis of sea anemone peptide toxin Ap-TxI
① First, 2 g of Fmoc-Arg(pbf)-oh-wang resin (SD=0.3 mmol/g) was weighed and soaked in 20 ml of DCM for 10 min.
② Next, 20% piperidine was added to remove the Fmoc protecting group of amino acids through 20 min of reaction. DMF was used to wash the resin 5 times. A small amount of resin was added to a detection tube, which was then added with 2 drops of ninhydrin detection reagent (5 g of ninhydrin was dissolved in 100 ml of ethanol) and 2 drops of pyridine. The resin was heated at 100 ℃, and if the color was blue, the protecting group Fmoc had been removed. Then, the resin was washed with DMF 5 times, 1 min each time.
③ Next, 2 mmol of Fmoc-Ala-OH, 2 mmol of HOBT and 2 mmol of DIC were added, as well as 10 ml of DMF and 2 ml of DIC, for 1 h of reaction. After the reaction, the resin was washed 4 times with DMF, and a small amount of resin was detected using ninhydrin to detect for the result of colorless. According to the sequence of Ap-TxI, the cysteine was protected by different protecting groups, and steps 2 and 3 were repeated continuously until the end of the last amino acid. The resin peptide obtained by the reaction was SGAC(acm)AEIC(trt)VHRC(mmt)IPSC(mmt)NFAC(trt)C(acm) VAR-WANG RESIN.
④ The resin obtained above was cut with 6% TFA+94% DCM for 3 times, 10 min each, to obtain SGAC(acm)AEIC(trt)VHRCIPSCNFAC(trt)C(acm)VAR-WANG RESIN.
⑤ The resin obtained in step 4 was soaked with DMF, adjusted to pH 8 with triethylamine, and stirred overnight for oxidation.
⑥ The resin obtained in step 5 was washed with methanol and drained, put into a 50 ml centrifuge tube, and added with 40 ml of cutting solution (95% TFA+5% H2O). The mixture was cut with shaking in a shaker for 2-3 h, and then added with 40 ml of ice ether. After shaking evenly, centrifugation was performed at 12 000 r/min for 2 min. The oxidation product, which had formed the first disulfide bond C3-C4, was obtained after sedimentation.
⑦ The thing obtained in step 6 was lyophilized with a freeze dryer, and 200 mg was weighed and dissolved into 200 ml of (20% ACN+H2O). The obtained liquid was adjusted to pH 8 with NH4HCO3 and oxidized overnight to form the second pair of disulfide bond C2-C5.
⑧ The thing obtained in step 7 was weighed, and 100 mg was dissolved into 100 ml of (20% ACN+H2O). High-concentration iodine (300 mg iodine dissolved in 50 ml methanol) was added dropwise to the liquid, and the formation of the last pair of disulfide bond was confirmed by mass spectrometry. The product was purified by HPLC to obtain a pure product, which was lyophilized into powder.
MTT method
Insect cells sf9 were used for the MTT test, and the specific steps were referred to the literature［6］. Specifically, the insect cells sf9 were cultured to the logarithmic growth phase and collected when the density reached 80%-90%. The cells were seeded into 96-well plates, about 103 per well, cultured for 4 h and added with 0.1, 0.25, 0.5, 0.75, and 1.0 nM of peptide Ap-TxI, and three replicate wells were set for each concentration. The blank experimental group was used as a negative control. The cells were incubated in a cell incubator for 48 h, and after MTT was added, the incubation was continued for 4 h. Dimethyl sulfoxide was added, and the absorbance of each well was measured with a microplate reader after shaking well on a shaker.
Insect injection
Mealworms with a body weight of about 180 mg was used for insect injection. The specific steps were referred to the literature［6］. In specific, the peptide Ap-TxI was dissolved to 2.5, 5, 10, 15 and 20 nM with 0.7% NaCl, and 5 μl of the peptide Ap-TxI was injected into the lower abdomen of mealworms. Ten individuals were injected for each group, and three replicates were set for each concentration. Mealworms free of injection were used as a blank control, and 5 μl of 0.7% NaCl solution was injected into mealworms as a negative control group. The death of mealworms was observed in the later stage.
Data processing
The data were analyzed and processed by the software GraphPad Prism6. The data between the control group and the experimental groups were analyzed by t test, with * indicating a significant difference (P<0.05), and ** indicating an extremely significant difference (P<0.01).
Results and Analysis
Synthesis and oxidative folding of peptides
The synthesized resin peptide was stirred and oxidized in DMF solution (adjusted to pH 8 using triethylamine) overnight, and then the linear resin peptide was cleaved with 95% TFA and 5% H2O for 2-3 h. The peptide with disulfide bond C3-C4 (Ap-TxI-C3-C4) was sedimented with ice ether and centrifuged, and the molecular weight was identified to be 2 653.992 Da by mass spectrometry (Fig. 1). Then, 200 mg of Ap-TxI-C3-C4 was weighed and dissolved into 200 ml of (20% ACN and H2O), and the liquid was adjusted to pH 7-8 with ammonium bicarbonate, and oxidized overnight to form the second pair of disulfide bond C2-C5 (Ap-TxI-C2-C5). The product was identified to have a molecular weight of 2 651.992 Da by mass spectrometry (Fig. 2). Next, 100 mg of Ap-TxI-C2-C5 was weighed and dissolved into 100 ml of (20% ACN+H2O), and high concentration iodine (300 mg of iodine dissolved in 50 ml of methanol), and added dropwise into the liquid. The formation of the last pair of disulfide bond C1-C6 (Ap-TxI-C1-C6) was confirmed by mass spectrometry, and the molecular weight of the product was 2 507.992 Da (Fig. 3). Next, the obtained product was purified by HPLC and lyophilized into powder. The theoretical molecular weight of the linear peptide Ap-TxI was 2 513.992 Da, and the molecular weight of the oxidized peptide was 2 507.992 Da, so the difference between the two was 6 Da, proving that three disulfide bonds were correctly formed.
Separation and purification of oxidized peptide
The oxidized peptide Ap-TxI was separated and purified by preparative HPLC, and then analyzed by analytical HPLC. The results are shown in Fig. 4. The elution time of the oxidized peptide Ap-TxI was 10.492 min, and its purity was calculated to be 90.688% according to the peak area.
MTT method
The inhibitory effect of polypeptide Ap-TxIon insect cells sf9 was tested by the MTT method. The experimental results (Fig. 5) showed that the experimental groups had significant differences from the control group (0.7% NaCl solution). The inhibitory effect of sea anemone polypeptide Ap-TxIon insect cells had a dose effect, and the calculated half effective dose was 0.2 nM.
Insect injection
The experimental results are shown in Fig. 6. The mortality of mealworms in the blank control and the negative control group was 0, indicating that it is feasible to use the injection method to evaluate the insecticidal effect of peptide Ap-TxI. The mortality of mealworms increased with the increase of the dose of peptide Ap-TxI, and there were significant differences from the control group. The mortality rate of high-dose 20 nM peptide Ap-TxI to insects reached 71.7%, and the median lethal dose calculated by software was 11.7 nM.
Conclusions and Discussion
In modern times, agricultural pests have become one of the main reasons for crop yield reduction, and the extensive use of chemical pesticides has threatened environmental pollution and human health. Therefore, it is particularly urgent to study safe and efficient green pesticides. Yang Lin et al.［21-22］ isolated 7 peptide toxins from yellow sea anemones that had rapid lethal effects on mealworms and Exopalaemon carinicauda, but weaker toxicity to mice. The action mechanism of sea anemone peptide neurotoxin is that it can specifically act on nerves and muscles, excite specific nerve receptors or ion channels on the cell membrane, and exert insecticidal effects by changing the function of key targets［20］. Therefore, it is possible to screen sea anemone peptide toxins with efficient and specific insecticidal activity from sea anemones, which can be prepared into effective and safe insecticides. In the early stage, the transcriptome sequencing of Aiptasia pallida was carried out, and the sea anemone peptide toxin Ap-TxI was found. In this study, the sea anemone peptide toxin Ap-TxI with natural structure was synthesized by the SPPS method and directional oxidative folding. The results of the MTT method showed that Ap-TxI was active on the growth of insect cells sf9, with a half effective dose of 0.2 nM; and the results of the mealworm injection test showed that the peptide Ap-TxI had high insecticidal activity with a median lethal dose of 11.7 nM. Therefore, the sea anemone peptide toxin Ap-TxI with high insecticidal effect was obtained in this study, laying a foundation for the development of new, efficient and safe biological insecticides.
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