Toxin Identification And Study On The Mechanism Of The Neurotoxin PPTX-22 From Pardosa Pseudoannulata

Spiders are a group of animals with more than 300 million years of evolutionary history.The success of their evolution is largely due to their sophisticated application of silk and the successful evolution of venom.Except for predatory beetles,spiders are the most abundant terrestrial predators.Spiders mainly prey on arthropods,and some spiders can also prey on small vertebrates such as mice,birds and lizards.Venom is the main weapon for spiders to prey,and most spiders produce venom in a pair of venom glands.Spider venom is a mixture of various compounds.Based on the chemical composition and pharmacological diversity,spider venom could be divided into six categories:small molecule compounds,acylpolyamines,linear peptides,disulfide-rich peptide toxins,high molecular weight proteins and enzymes.The disulfide-rich peptide toxins are the main compounds in most spider venom and the main active substances for spiders to prey or defend.Most disulfide-rich spider toxins act on the peripheral neuromuscular junction or at the synapse of the insect central nervous system,targeting presynaptic ion channels or postsynaptic receptors.The targets of spider neurotoxins in insects mainly include voltage-gated calcium channels,voltage-gated sodium channels,voltage-gated potassium channels and glutamate receptors.These peptide toxins often cause prey paralysis by either attenuating or excessively activating the nervous system and eventually immobilize the attacked preys.In the project,we examined the composition of venom at the molecular leveland and identified the toxins.In addition,in order to further study the function of the neurotoxin,we have screened and established a system for the expression and purification of disulfide-rich peptide toxins.Using the established expression and purification system,the neurotoxin PPTX-22 was successfully expressed,and its insecticidal activity and target were studied.1.Toxin identification and analysis of Pardosa pseudoannulataA total of 75,980 transcripts were obtained from the transcriptome sequencing of the P pseudoannulata venom gland,and 43 disulfide-rich peptide toxins were identified in these transcripts,including 38 putative neurotoxins and 5 hypothetical non-neurotoxic toxins.Based on the analysis of cysteine alignment,phylogeny and functional domains,32 putative neurotoxind were divided into 6 families,while the other 6 neurotoxins were classified as a single group because they lack a similar sequence or the same cysteine pattern in the library.Based on the genomic results,we analyzed the gene structure of the neurotoxin genes of the P.pseudoannulata.The mature peptide sequences of neurotoxin in Family F contained 2-3 exons,and mature peptide sequences of the toxins from the remaining 5 families contained just one exon.In addition to the disulfide-rich peptide toxins,the venom of P.pseudoannulata has some other components,as mainly astacin-like metalloproteinases,Kunitz-type serine protease inhibitors,venom allergens and hyaluronidase.In order to explore the distribution and expression of the genes coding venom toxins in P.pseudoannulata,the venom gland,brain and fat body were selected for tissue expression profiling.The majority of the putative neurotoxin genes were mainly expressed in the venom gland at high levels.These results indicated that neurotoxins might be the main component of its venom.In addition,trypsin-like serine protease genes,hyaluronidase genes and venom allergen gene were specifically expressed in venom gland and has a high expression level.2.Establishment of the in vitro expression and purification system of disulfide-rich peptide toxinsIn order to establish an efficient expression system suitable for disulfide-rich spider toxins,we tested the vector pFastBacTM1 of the insect baculovirus expression system,the vectors pPICZ A and pPICZa A of the Pichia pastoris expression system,and the expression vectors pET32a and pLicC-MBP of the Escherichia coli expression system.The eukaryotic expression vectors pFastBacTM1 and pPICZa A could not meet the needs of subsequent experiments due to a low success rate.The eukaryotic expression vector pPICZ A resulted in non-target bands affecting the subsequent purification despite a high success rate.The prokaryotic expression vector pET32a was not suitable because of the severely misfolded fusion proteins most likely to be inactive.Fortunately,the prokaryotic expression of toxins with pLicC-MBP turned to be a good option with high yield,relatively high specificity beneficial for subsequent purification and correctly folded proteins with potential acitivity.Therefore,the expression of a disulfide-rich polypeptide toxin will be carried out using the prokaryotic expression vector pLicC-MBP in the subsequent experiment.Using a HisTrap HP column,a fusion protein can be obtained with a high purity.The molecular weight of the fusion tag MBP is about 42 kDa,and the molecular weight of a spidertoxin is usually less than 10 kDa.Therefore,the fusion tag needs to be removed from the toxin by TEV protease.The fusion protein can be subjected to a displacement buffer treatment using a HiPrep 26/10 desalting column providing the optimal conditions for the TEV protease.Ultrafiltration tubes,molecular sieves,HisTrap Excel chromatography columns,MBPTrap HP chromatography columns failed to effectively remove the fusion label.Finally,a combination of the chromatographic column MBPTrap HP and the column HisTrap HP could remove the the fusion label from the TEV enzymatic digestion solution and the flow through solution.So far,recombinant toxins were obtained with the purity satisfying the subsequent experiments.3.Study on the mechanism of neurotoxin PPTX-22P.pseudoannulata is one of the dominant natural enemies of agricultural pests in Asian rice fields,mainly preying on rice planthoppers and rice leafhoppers.The venom plays a very important role in predation,and the neurotoxins rich in disulfide bonds is a major component of the venom.PPTX-22 was a disulfide-rich neurotoxin in the venom of P.pseudoannulata.It contained a signal peptide,a propeptide and a mature peptide within 109 amino acid residues.The mature PPTX-22 peptide contained 69 amino acid residues and had 5 disulfide bonds formed by 10 cysteines.PPTX-22 was successfully expressed with the prokaryotic expression vector pLicC-MBP.The molecular weight of the heterologous expressed PPTX-22 was about 7.78 kDa after the excision of the fusion protein.The recombinant toxin PPTX-22 caused paralysis and even death when injected into Nilapavata lugens.The PD50 after 2 h was 1402 pmol/g with the 95%confidence interval 1297-1515 pmol/g,and R2 0.9795.Calcium ion imaging experiments have shown that PPTX-22 caused an increase in the intracellular calcium concentration regardless of whether the extracellular fluid contained calcium ions or not.In addition,PPTX-22 caused an icrease in the intracellular Ca2+ concentration in the case of blocking the cell membrane Ca2+channel.These results indicated that PPTX-22 did not act on the calcium channel on the cell membrane.Further experiments showed that PPTX-22 could cause an increase in intracellular calcium concentration after treatment of cells with the IP3 receptor inhibitor 2-APB,but the toxin could not cause the increase of the intracellular calcium any more when the ryanodine receptors(a Ca2+ channel)were blocked with high concentrations of ryanodine.Therefore,PPTX-22 could cause an increase in intracellular calcium ion concentration by activating the insect ryanodine receptors.In this study,a complete transcript sequence information was obtained by transcriptome sequencing of the venom gland from the Pardosa pseudoannulata,and the toxin was also systematically classified.The bioactive recombinant toxin was acquired by establishing the disulfide-rich peptide toxin expression and purification system.Besides,we first discovered the spider toxin that acted on insect ryanodine receptor through sequence analysis and functional verification.Pardosa pseudoannulata is an important natural enemy of agricultural pests and plays an important role in the agro-ecological environment.Therefore,the systematic study of its toxins can not only enrich the targets of spider toxins,but also provide a new perspective for pest control.