Gene Cloning, Expression And Function Study Of Toxins From The Spider Ornithoctonus Huwena

The Chinese bird spider Ornithoctonus huwena distributed in the hilly areas of Yunnan and Guangxi province in southern China is a venomous spider. The venom of O. huwena is a mixture of various components with different types of biological activities. In the previous work of this lab, using proteomic and peptidomic methods,90 proteins and 47 peptides have been identified from the spider venom, but usually only the most abundant components have been analyzed. In order to clone the genes encoding the proteins and peptides in the venom glands, a directional cDNA library of O. huwena venom glands was constructed by using the expressed sequence tag (EST) strategy and 468 ESTs were generated by a random sequencing. All ESTs were grouped into 24 clusters and 65 singletons, of which 68.0% of total ESTs belong to toxin-like genes,13.0% are cellular transcripts and 19.0% have no significant similarity to any known genes. Precursors of all toxin-like genes can be classified into eight superfamilies (HWTX-Ⅰsuperfamily, HWTX-Ⅱsuperfamily, HWTX-Ⅹsuperfamily, HWTX-ⅩⅣsuperfamily, HWTX-ⅩⅤsuperfamily, HWTX-ⅩⅥsuperfamily, HWTX-ⅩⅦsuperfamily, HWTX-ⅩⅧsuperfamily) except HWTX-Ⅺand HWTX-ⅩⅢ, according to the identity of their precursor sequences. All precursors were processed to achieve 43 mature peptides with abundant cysteines, named cystine knot toxins (CKTs), and 31 of them were novel CKTs. All cellular proteins or other possible venom components were annotated by KOG (eukaryotic orthologous group) and GO (gene ontology) terms. Comparison of the CKT repertoire and proteins revealed through a proteomic versus a transcriptomic approach, only 15 CKTs were identified by both approaches,29 transcripts coding for CKTs were found in the transcriptome but not as translated proteins in the venom proteome. However, no cellular proteins with identical molecular weights was identified by both approaches. In addition, a cellular transcript contig coding for an EF-hand protein (named HWEFHP1) had been identified for the first time from spider venom glands, which might be involved in the secretion of toxins from the gland into the venom.The genomic DNA of toxins from the spider O. huwena is seldom reported. To further investigate whether the genes encoding toxin-like peptides contain a common intronless feature, the genomic DNA encoding toxins of three superfamilies were cloned by using sequence specific or partially degenerate primers based on their cDNA sequences. An unexpected finding was that the intron was lacking in the genomic sequences of three superfamilies. In addition, we have cloned and analyzed 19 novel genes encoding toxin-like precursors by using the genomic DNA of the spider O. huwena. In order to indentify more HWTX-XI isoforms,38 cDNAs encoding HWTX-XI superfamily precursor were cloned by using partially degenerate primers and these precursors were processed to achieve 31 mature peptides. A phylogenetic tree was generated by using the neighbor-joining method, and HW11c21, HW11c25, HW11c40, HW11c50 and HW11c10 may play a very important role in the evolution relationship of HWTX-XI.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/a in the Saccharomyces 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 and express 20 genes encoding toxin-like peptides. At last,11 toxin genes were successfully expressed. The functions of these expressed products were also identified. The IC50 value of HW11c4 is 10.0μM for the Kv1.1 channel. HWTX-ⅩⅦb1 can reduce the peak currents of high voltage activated Ca2+ channels on adult rat dorsal root ganglion (DRG) neurons. HW11c4, HW11c27 and HW11c27m are potent trypsin inhibitors and can also inhibit the chymotrysin and thrombin. However, the functions of other expressed products are very low or are not found at all.In addition, in order to get the molecules from the venom glands of the spider O. huwena interacting with voltage-gated sodium channels, a yeat two-hybrid cDNA library and 4 bait plasmids of Nav1.8 were constructed respectively, and then were screened.In summary, by using molecular biology, bioinformactics, biochemistry, mass spectra and patch-clamp, etc, we have investigated transcriptomes and genomic DNAs of the O. huwena venom glands. We also have cloned and expressed some novel genes, and then analyzed the functions of the expressed products.