Molecular Diversification Of Peptide Toxins From The Tarantual Haplopelma Hainanum (Ornithoctonus Hainana) Venom

Spider venoms contain a mixture of toxins with different biological activity. The majority of spider toxins are peptide toxins with 3-5 disulfide bonds which display various structures and functions. Spider peptide toxins have proved to be powerful tools for the study of voltage-gated and ligand-gated ion channels and have potential applications as novel pharmaceutical drugs. The tarantula Haplopelma hainanum, which is similar to the spider Ornithoctonus huwena in morphology, is a very venomous spider distributed in the hilly areas of Hainan province in southern China. In our previous work, several neurotoxins which are the major components of the venom from H. hainanum have been purified and characterized. These results provide a tantalizing glimpse of pharmaceutical components in this venom and stir up new interest in systematic research on venom peptides. In the present study, we used a venomic strategy for large-scale identification of tarantula-venom peptides from H. hainanum. This strategy includes three different approaches:(ⅰ) transcriptomics, namely, EST-based cloning and PCR-based cloning plus DNA sequencing; (ⅱ) peptidomics, namely, off-line multiple dimensional liquid chromatography coupled with mass spectrometry (MDLC-MS) plus peptide sequencing (direct Edman sequencing and bottom-up mass spectrometric sequencing); (ⅲ) genomics, namely, genomic DNA cloning plus DNA sequencing. About 420 peptide toxins were detected by mass spectrometry, and 272 peptide precursors were deduced from cDNA and genomic DNA sequences. After redundancy removal,192 mature sequences were identified by three approaches. This is the largest number of peptide toxin sequences identified from a spider species so far.On the basis of precursor sequence identity, peptide toxins from the tarantula H. hainanum venom can be classified into 11 superfamilies, and then several superfamilies are further divided into distinct families. Phylogenetic analysis showed that all peptide precursors originated from two different clades. Members of superfami lies A-I belong to one clade, and those of the remaining superfamilies belong to the other clade. Almost all precursors from toxin superfamilies contain a signal peptide, a propeptide and a mature peptide. However, there is an evident diversity of molecular characters in the superfamilies (and related families). The application of a venomic strategy derived from transcriptomics, peptidomics, and genomics has demonstrated the enormous molecular diversity from tarantula-venom peptides. Multiple isoforms with high homology have been found in the superfami lies from the tarantula H. hainanum venom, suggesting that gene duplication and hypermutation may be responsible for the molecular diversity. Furthermore, we found that there is C-terminal amidation at mature peptides of several families, and post-translational modifications such as C-terminal amidation may be the other potential mechanism of toxin diversification. The current work is an initial overview for the study of tarantula-venom peptides in parallel transcriptomic, peptidomic, and genomic analyses. It is hoped that this work will also provide an effective guide for high-throughput identification of peptide toxins from other spider species, especially tarantula species.On the other hand, a detailed analysis for cDNA library of H. hainanum venom glands was described. All ESTs were assembled into 344 non-redundant sequences, of which 45% of total unique sequences belong to toxin transcripts; 43% are similar to common cellular transcripts; 12% have no significant similarity to any known sequences. In this study, we used the annotation of the eukaryotic orthologous group (KOG) for cellular transcripts, and the annotated gene products were classified into 22 functional categories. The annotation of the KOG for cellular transcripts revealed that their gene products (proteins or enzymes) participates in some important cellular processes such as transcription and translation, posttranslation processing, metabolism pathway and energy supply, etc.