Study on Chemical Synthesis and Insecticidal Activity of Peptide κ-CTx-btg02

Qin CHEN Jinxing FU Min XU Bingmiao GAO

Abstract [Objectives] This study was conducted to investigate the chemical synthesis of peptide κ-CTx-btg02.

[Methods]Linear peptide κ-CTx-btg02 was synthesized by solid phase peptide synthesis （SPPS）. After oxidation and folding of the linear peptide， mass spectrometry identification and high performance liquid chromatography （HPLC） purification were performed. Then， the MTT method and insect injection method were applied to study its insecticidal activity.

[Results] The peptide κ-CTx-btg02 was successfully synthesized by the SPPS method， and identified for the formation of disulfide bonds by mass spectrometry identification， and the purity after HPLC separation was greater than 95%. The MTT experiment showed that the peptide κ-CTx-btg02 could inhibit the growth of insect cells sf9， with a half effective dose of 0.13 nM. The insect injection experiment showed that the peptide κ-CTx-btg02 could effectively kill Tenebrio molitor， with a half lethal dose of 15.6 nM. The results of the electrophysiological experiment showed that 10 μM peptide κ-CTx-btg02 had no blocking activity on murine acetylcholine α2β3 and α3β4. Therefore， the peptide κ-CTx-btg02 had a good inhibitory effect on the growth of insect cells， highly effective insecticidal activity and weak mammalian toxicity.

[Conclusions]This study lays a foundation for the development of new， safe and efficient peptide biological insecticides.

Key words Peptide; Solid phase peptide synthesis; Oxidative folding; Insecticidal activity; Electrophysiology

Received： May 3， 2021  Accepted： July  2021

Supported by Natural Science Foundation of Hainan Province （820RC636）.

Qin CHEN （1983-）， female， P. R. China， lecturer， devoted to research about marine resource development.

*Corresponding author. E-mail： gaobingmiao@hainmc.edu.cn.

In recent years， with the abuse of chemical pesticides， the problem of agricultural pollution has become more and more serious， which directly threatens the safety of agricultural ecology and the sustainable development of agriculture， which in turn brings food safety problems to human health[1]. How to solve the agricultural environmental crisis and ecological security issues is imminent， so the demand for new， efficient and safe biological pesticides is very urgent[2-3]. At present， animals that prey on insects can usually secrete peptide toxins， which can kill and capture prey through the ion channels or receptors of specific insects， but their toxicity to mammals is very small or has no toxic side effects[4-5]. It has been reported in literatures that cone snails， sea anemones， scorpions， spiders and predatory mites can secrete peptide toxins[6-9]. Therefore， the search for insecticidal active ingredients from marine or terrestrial animal peptide toxins is currently one of the hotpots in the research of safe pesticides. In this study， the linear peptide κ-CTx-btg02 was synthesized by solid phase peptide synthesis （SPPS）， and oxidized by the two-step oxidation method to oxidized folded peptide κ-CTx-btg0 which was then purified by high performance liquid chromatography for identification by mass spectrometry， and the insecticidal activity of the peptide was tested by the MTT method and insect injection method， laying a foundation for the development of new， efficient and safe biological pesticides.

Materials and Methods

Experimental materials

Peptide synthesis-related amino acid activation reagents were purchased from ABI; chromatographic-grade trifluoroacetic acid （TFA） and chromatographic-grade acetonitrile （Acetonitrile， ACN） were purchased from Thermo Fisher Scientific; Vydac analytical C18 column （5 μm， 4.6 mm×250 mm） and preparative C18 column （10 μm， 22 mm×250 mm） were purchased from Shanghai Sun Year Experimental Instruments Co.， Ltd.）; plasmid midi preparation kit， restriction endonucleases， molecular weight standards and other conventional molecular biological reagents were purchased from TaKaRa Dalian Bao Biological Engineering Co.， Ltd.）; cRNA synthesis kit mMESSAGE mMACHINE SP6， T7 Kit， and MEGA clear kit were purchased from American Ambion company; and other commonly used biochemical reagents were all domestically pure.

Experimental instruments

CEM automatic microwave peptide synthesizer （LibertyBlue， USA）; high performance liquid chromatograph （Waters2535， USA）; electrospray ionization mass spectrometry （Shimadzu， Japan）; freeze dryer （Christ， Germany）; microplate reader （MR-96A， Shenzhen Mindray）; patch clamp system （Axon900A， USA）.

Experimental methods

Synthesis of linear peptide

The SPPS method was used to synthesize the peptide κ-CTx-btg02 （SECCIRNFLCC-NH2）， and the specific method referred to the literature [10]. Cys 1 and Cys 3 were protected by the protecting group S-acetamidomethyl （Acm）. Resin peptides were cut with cutting solution （TFA∶TIPS∶DODT∶H2O=92.5%∶2.5%∶2.5%∶2.5%） at 42 ℃ for 30 min. After suction filtration， 5 times the volume of dry ice pre-cooled ether was used for precipitation and centrifugation to recover the crude peptide. Then， the linear peptide was purified by preparative HPLC with Vydac C18 column using mobile phase A （H2O， 0.1% TFA）， mobile phase B （ACN， 0.1% TFA） at a flow rate of 15 ml/min. Linear gradient elution was performed for 60 min， during which B phase changed from 0% to 60%， and the detection wavelength was 214 nm. The purity of the linear peptide was more than 90%， and it was freeze-dried and stored after being identified correct by mass spectrometry.

Two-step oxidation folding

The oxidation of the linear peptide adopted a two-step oxidation method， and the specific method referred to literature[6]. In the first step of oxidation， the purified linear peptide was dissolved in 50% methanol solution at 10 mg/ml， then diluted to 1 mg/ml with acetic acid， and added with methanol iodine solution （10 mg/ml） dropwise gradually while keeping stirring. When the yellow color did not disappear under the condition of stirring， dropping was stopped， and the first disulfide bond （Cys2 and Cys4） had formed. A small amount of the solution was taken out for mass spectrometry identification. In the second step of oxidation， hydrochloric acid solution （50 mM HCl /50% methanol） was first added to remove the protective group Acm， and 10 times the amount of peptide iodine solution was then added. Stirring was kept for 1 h. Ascorbic acid was added until the color of the solution disappeared， and the solution was identified by mass spectrometry for the formation of the second disulfide bonds （Cys1 and Cys3）.

MTT method

The insect cell Sf9 was used for MTT experiment， and the specific steps referred to literature[6]. First， 100 μl of Sf9 （about 103 cells） grown in logarithmic phase was inoculated into a 96-well plate， and cultured until the cells were fully attached. Then， conotoxin ImI （positive control） and pure κ-CTx-btg02 were added at concentrations of 0.1 nM， 0.5 nM， and 1.0 nM， respectively， with 3 replicate wells for each concentration， and the experimental group without the addition of the peptide was used as a control. The cells were incubated at 27 ℃ for 48 h， and 10 μl of 3-（4，5-dimethylthia zol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） at a concentration of 5 mg/ml was added to each well， and the culture was continued for 4 h. The culture medium was removed， and 100 μl of dimethyl sulfoxide （DMSO） was added to each well， followed by shaking at room temperature for 15 min at a low speed. Finally， the absorbance was measured at 490 nm on a microplate reader.

Inset injection method

Tenebrio molitor of about 180 mg was weighed， and fed with chicken feed normally before the experiment， and the insect injection method referred to literature[6]. On the day of the experiment， 0.7% NaCl was used to dissolve the conotoxin ImI （positive control） and κ-CTx-btg02 to required concentrations， respectively， and 5 μl of different concentrations of conotoxin ImI （positive control） and κ-CTx- btg02 were drawn with micro syringes and injected to the abdomen of T. molitor. Each concentration was injected to 10 individuals， and each concentration was repeated three times. After injection， 15 μl of 0.7% saline was used to flush the residual drug in the dead space of the catheter. T. molitor was injected with 0.7% NaCl solution as a negative control group， and T. molitor without injection of any liquid was used as a blank control. After 48 h， the death of T. molitor in the experimental groups and the control group was observed.

Electrophysiological method

For electrophysiology， frog egg patch clamp was used. The specific steps referred to literature[11]. The plasmid containing nAChRs and other subunit genes was transcribed in vitro to obtain the corresponding cRNA. The cRNA was mixed according to species and then injected into oocytes of Xenopus laevis. The oocytes were cultured at 17 ℃ in a physiological fluid ND96 with freshly supplemented antibiotics for 3 to 5 d， and detected for the expression of receptors using an Axon900A patch clamp system. The peptide with a concentration of 10 μmol/L was added to cell tanks to investigate the interaction between the peptide and various targets.

Data processing

The data of MTT and injection method were processed by the software GraphPad Prism6. The data between the control group and the experimental group 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 peptide

The linear peptide κ-CTx-btg02 has a theoretical molecular weight of 1 290.6 Da. After solid-phase synthesis and cutting， the linear peptide κ-CTx-btg02 was oxidized and folded by the iodine two-step oxidation method. After oxidative folding， κ-CTx-btg02 was identified by mass spectrometry. The molecular weight was 1 285.56 Da （Fig. 1）， which was about 4 Da different from the molecular weight of its linear peptide of 1 290.6 Da， which proves that its oxidative folding had successfully formed two disulfide bonds.

Separation and purification of oxidized peptide

The oxidized peptide κ-CTx-btg02 was purified by preparative HPLC， and then analyzed by analytical HPLC after purification. The results are shown in Fig. 2. The elution time of the oxidized peptide κ-CTx-btg02 was 8.570 min. According to the peak area， its purity was calculated to be 98.5%.

MTT method

The insecticidal activity of the peptide κ-CTx-btg02 was studied by the MTT method. The results are shown in Fig. 3. Compared with the negative control group， the experimental groups had the activity of inhibiting the growth of insect cells Sf9， and the peptide κ-CTx-btg02 had a significant difference from the negative control group at a concentration of 0.5 nM or more. The results of the study showed that the inhibitory effect of the peptide κ-CTx-btg02 on insect cells sf9 had a dose effect， and the half effective dose was calculated to be 0.13 nM.

When comparing the experimental groups with the negative control， * indicates a significant difference （**P<0.01）.

When comparing the experimental groups with the negative control， * indicates a significant difference （**P<0.01; ***P<0.001; ****P<0.000 1）.

Qin CHEN et al. Study on Chemical Synthesis and Insecticidal Activity of Peptide κ-CTx-btg02

Insect injection method

The results of the experiment are shown in Fig. 4. The death rate of T. molitor in the blank control and the negative control group were both 0， suggesting that it is feasible to evaluate the insecticidal effect of peptide κ-CTx-btg02 by injection. The death rates of T. molitor all increased with the increase of the dose of peptide κ-CTx-btg0 and there were significant differences from the control groups. The high-dose group of peptide κ-CTx-btg02 （20 nM） had a death rate of 70.0% on insects， and the calculated median lethal dose was 15.6 nM.

Electrophysiological activity

In this study， we tested the blocking activity of the peptide κ-CTx-btg02 on the 2 subtypes of murine acetylcholine receptors （nAChRs）. The results are shown in Fig. 5. It can be seen that 10 μmol/L peptide κ-CTx-btg01 had no effects on murine α3β4 and α2β3 nAChRs， revealing that the conotoxin κ-CTx-btg02 is less toxic to mammals.

Discussion

In recent years， the wanton abuse of chemical pesticides has led to environmental pollution by pesticide residues and pest resistance， and also poisoned the natural enemies of pests， thus destroying the ecological balance. Insect virus insecticides have the advantages of being safe against natural enemies of pests， being environmentally friendly and no production of drug resistance， so they are widely used[12]. However， insect virus insecticides have fatal disadvantages such as narrow insecticidal spectrum and slow insecticidal speed. With the development of molecular biology and genetic engineering， the use of exogenous genes to modify insect viruses has become an effective way to overcome its shortcomings[13-14]. Using insectivorous peptide genes as synergistic genes to construct recombinant baculoviruses can effectively solve the shortcomings of chemical pesticides and wild-type baculovirus insecticides， laying a foundation for the development of new， efficient and safe recombinant insect virus insecticides.

At present， it has been reported that peptide toxins of invertebrates such as cone snails， sea anemones， scorpions， spiders and predatory mites can bind to targets on insect nerve cell membranes and can specifically paralyze and poison insects[6-9]. Among them， previous studies have found that conotoxin can specifically act on insects and have insecticidal ability， and have little toxicity or no toxic side effects to mammals[4，6]. Conotoxin is mainly used in chronic pain， epilepsy and cancer. For example， MVIIA has been approved by the FDA as an analgesic for advanced cancer[15-18]. The peptide κ-CTx-btg022 is derived from the Hainan Conus betulinus. It has 2 disulfide bonds formed by 4 cysteines， and it is speculated through homology comparison that it can act on the acetylcholine receptor and has an insecticidal activity. In this study， the peptide κ-CTx-btg02 was synthesized by SPPS method and two-step oxidation folding method， and then the insecticidal activity was studied by MTT method， insect injection method and electrophysiology. The experimental results showed that the peptide κ-CTx-btg02 had the activity of inhibiting the growth of insect cells sf9， with a half effective dose of 0.13 nM; and it had insecticidal activity against T. molitor， with a half lethal dose of 15.6 nM. The electrophysiological results showed that the peptide κ-CTx-btg02 had no blocking effect on murine acetylcholine， suggesting that it has less toxicity to mammals， thus confirming that it has high safety characteristics for humans. Therefore， it was found from this study that the peptide κ-CTx-btg02 can specifically act on insects and has insecticidal ability， and has little toxicity or no toxic side effects to mammals. It will become the most potential insect baculovirus synergistic gene.

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