The Molecular Diversity And Function Study Of The Toxins From The Spider Lycosa Singoriensis

The wolf spider Lycosa singoriensis is a hunting spider with a widespread distribution in northwest China.The spider venom gland, which is a very specialized secretory tissue,can secrete abundant and complex toxin components.To extensively examine the transcripts expressed in the venom glands of Lycosa singoriensis,we generated 833 expressed sequence tags(ESTs) from a directional cDNA library.All ESTs were assembled into 331 non-redundancy sequences including 60 clusters and 271 singlets,of which toxin-like sequences accounts for 69.3%,17.3%are similar to the cellular transcripts and 13.4%have no significant similarity to any known sequences.All the cDNA sequences are available for public view in the GenBank database of NCBI (http://www.ncbi.nlm.nih.gov/entrez,accession numbers:EU925924-EU926146,EU247110- EU247223).Here,we identified 223 novel toxin-like sequences which can be classified into six different superfamilies(superfamilyâ… ,â…¡,â…¢,â…£,â…¤andâ…¥),that means a novel potential mine of ligands for varied ion channels was discovered.With the aid of Gene Ontology terms and homology to eukaryotic orthologous groups,the annotation of the cellular transcripts revealed some cellular processes important for the toxin secretion of venom gland including high protein synthesis,protein folding,tuned posttranslational processing and trafficking,and so on.Spider venoms are chemically complex cocktails,in which peptides are the principal constituents of most spider venoms.Here we investigated a new cytotoxic peptide with an average molecular mass of 7335.33 Da,named LSTX-A1,isolated from the venom of wolf spider Lycosa Singoriensis.LSTX-A1 contains 65 residues and possesses an amidated C-terminal phenylalanine-residue.In this paper we studied the cytolytic activity of LSTX-A1.Our results revealed that LSTX-A1 exerts cytotoxic and antitumor efficacy.It induced 42%hemolysis at a concentration of 100μM.And LSTX-A1 can inhibit the proliferation of human cervical carcinoma HeLa cells with an IC₅₀ values about 22μM.With the changement of the enviroment,species and quantites of spiders decrease sharply,precise analysis and application of peptide toxins from spiders are frequently restricted because of the difficulties of obtaining sufficient venom material.The expression of HWTX-XI was successful by using the plasmid pVT102U/αin the Saceharomyces cerevisiae strain S78,and the yield reached 12.5mg/L.In order to investigate whether this expression system is versatile to express the spider toxin genes,we clone 23 genes encoding toxin-like peptides.At last,6 toxin genes which have four or five disulfide bonds were successfully expressed.However,the functions of the expressed products are not found. In summary,by using molecular biology,bioinformactics, biochemistry,mass spectra and patch-clamp,etc,we have investigated transcriptomes of the L.singoriensis venom glands.And we isolated a new cytotoxic peptide from the venom of wolf spider Lycosa Singoriensis.We also have cloned and expressed some novel genes, which provid toxin source for the function study of the genes in the future.