| The tarantula Chilobrachys jingzhao is one of the most venomous spiders in the south of China.The elucidation of both the molecular diversity of the venoms as well as the underlying genetic mechanisms of toxin diversification is of great importance.Although about 60 peptides from this spider were purified and sequenced fully or partially by Edman degradation,the full-lenth precursors and thus the peptide sequences of most of the venom components are still unknown.To obtain a comprehensive view of function of its venom gland we constructed a non-normalized cDNA library of the venom gland and generated 788 expressed sequence tags(ESTs).All ESTs were assembled into 356 non-redundancy sequences including 85 clusters and 271 singlets,of which 31.4%of total unique sequences belong to secretion protein coding sequences including cystine knot toxins(29.1%) and other secretion proteins(2.3%);54.0%are similar to common cellular transcripts;and 14.6%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:EU233831-EU233934,FE530957-FE531203). Cystine knot toxins(CKTs) in spider venoms represent a rich source of novel ligands for varied ion channels.Phylogenetics analyses of these 104 putative CKTs precursors revealed one orphan family and six families with sequence similarity to known toxins.In order to further investigate the relationships of their structure,functions and evolution, we assayed 10 representative toxins for their effects on ion channels, structure model comparisons,evolution analysis and their relative abundances in the venom.This study revealed two major types of CKTs: pore-blocking toxins and gating modifier toxins.A few blockers were observed with relatively high abundance and wide distribution,which may be a category of original toxins that block channels conserved in various preys with relatively high specificity.The gating modifier families contain advanced toxins,usually have many members and interact with diverse regulatory components of channels.With respect to potential pharmacological applications,these peptides provide promising new templates to further explore.On the other hand,annotation of these common cellular transcripts revealed some novel possible venom components and cellular processes important for venom gland functions, including protein posttranslation processing,cell motility,protein synthesis,energy supply,etc.Transcriptome analysis and gene cataloguing of the venom gland of C.jingzhao represent a general panorama of the physiological events of the very specialized secretory tissue.Jingzhaotoxin-Ⅲ(JZTX-Ⅲ) is a peptide toxin isolated from the venom of the tarantula C.jingzhao that inhibits Nav channels of rat cardiac myocytes by modifying voltage-dependent gating.Different from scorpionβ-toxins,it caused a 10-mV depolarizing shift in the channel activation threshold.The binding site for JZTX-Ⅲon VGSCs is further suggested to be site 4.JZTX-Ⅲshows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs,and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.However,the surfaces that are critical for interacting with voltage-gated sodium channels are poorly defined.In this thesis we describe a development of a successful expression system for JZTX-Ⅲ. The expressed JZTX-Ⅲhad similar biological activity as those of the native toxin isolated from the spider venom.We also investigated the bioactive surface by site-directed mutagenesis the toxin and characterizing the interaction of each mutant with Nav1.5.Examination of the concentration dependence for inhibition identified 13 mutants with little effect on the concentration dependence for toxin inhibition of the Nav1.5,and 7 mutants that display moderate to dramatic perturbations. Intriguingly,the active surface of the toxin is largely composed of hydrophobic and anionic residues,but not basic residues.Moreover,the mutant of Arg-13 to Glu significantly inhibits Nav1.5 with an IC50 value of 31.9 nM,which is ten-fold active then the native JZTX-Ⅲ.Mapping of these results onto the structure of JZTX-Ⅲindicates a hydrophobic surface and two anionic residues arranged in N-terminus are particularly critical,which leads us to speculate that the gate modifier,JZTX-Ⅲ, might inhibit activation of sodium channels of cardiac myocites through a mechanism similar toβ-scorpion by trapping theⅡS4 voltage sensor in the resting conformation.
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