Diversity And Function Analyses On Toxins From Latrodectus Tredecimguttatus Eggs

Latrodectus tredecimguttatus(L.tredecimguttatus)is a kind of black widow spider,belonging to Arthropoda,Arachnoidea,Araneida,Theridiidae,and Latrodectus.The spider is mainly distributed in Xinjiang,Yunnan,Inner Mongolia,Gansu and other places in China.Unlike other poisonous animals,L.tredecimguttatus has toxic components not only in its venomous glands but also other parts of its body,such as legs and abdomens,even in its newborn spiderlings and eggs.Studying the toxin components in the materials outside the venomous glands of L.tredecimguttatus not only can deepen our understanding of the mechanism of action of spider toxins,but also can broaden the fields of spider toxin research and screen out even more lead molecules for the development of reagent tools for neurophysiological research,clinical drugs and insecticides,thus having great theoretical and practical significance.The present study took L.tredecimguttatus eggs as the experimental materials,and first systematicaly investigated the gene expression profile and the toxin diversity of the eggs at transcriptome level,and then investigated the biological functions and the mechanism of action of the Ltroeggtoxin-V,a proteinaceous toxin mined from the transcriptome of the Latrodectus tredecimguttatus eggs.The high-throughput transcriptome sequencing of L.tredecimguttatus eggs with Illumina sequencing technology identified a total of 53,284 protein-coding unigenes,of which 14.185 unigenes,including 280 unigenes encoding proteins or peptides homologous to known proteinaceous toxins,produced significant hits in the available databases.GO annotation and KEGG pathway enrichment analyses of the 280 unigenes showed that 375 GO terms and 18 KEGG pathways were significantly enriched.Functional analysis indicated that these unigene-coded toxins have the bioactivities to degrade tissue proteins,inhibit ion channels,block neuromuscular transmission,provoke anaphylaxis,induce apoptosis and hyperalgesia,etc.,which suggested that the toxicity of eggs is based on the synergistic effect of various active ingredients.It is worthy of pointing out that no known typical proteinaceous toxins in Latrodectus spider venoms——Latrotoxins were identified,suggesting that the eggs have the toxicity mechanism different from that of the venom.Our present transcriptome analysis not only helps to reveal the gene expression profile and toxicity mechanism of the L.tredecimguttatus eggs,but also provides references for the further related researches.We mined a gene sequence encoding a proteinaceous toxin(named Latroeggtoxin-V)from the transcriptome data.After the gene of Latroeggtoxin-V was cloned and heterologously expressed,the biological activity and action mechanism of the recombinant Latroeggtoxin-V(r Latrodectus)were analyzed.The results indicated that the rLatroeggtoxin-V could selectively act on breast cancer line MDA-MB-231 cells,not only arresting their cell cycle,inhibiting their proliferation and migration,but also inducing their apoptosis.Bioinformatics analysis suggested that Latroeggtoxin-V belongs to the ATPase inhibitor protein family and the further activity assay showed that the rLatroeggtoxin-V inhibited the activity of the N a-/K--ATPase in MDA-MB-231 cells in a concentration-dependent manner,suggesting that the anticancer activity of Latroeggtoxin-V is based on its affecting the ion transport and receptor functions of Na-/K--ATPase.The present work not only laid the foundation for the utilization of Latroeggtoxin-V in the anticancer drug development and the related fields,but also provided a new paradigm for investigation of the low-abundent proteinaceous toxins under the direction of transcriptomics and bioinformatics.