Venomic And Biochemical Properties Study Of Venom From The Spider Araneus Ventricosus

The Araneus ventricousus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and mass spectrometry showed that the venom contained aboundant peptides and proteins with molecular weights ranging from4kDa to45kDa. Injection of the venom in cockroaches Periplaneta americana gave rise to obvious poisoned symptoms, with LD50value of30.73μg/g. Electrophysiological experiments showed that the venom could not block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. Enzymatic analysis indicated that the venom possessed activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrate that A. ventricosus venom is rich in bioactive components targeting insects, the spider natural preys, and the content of neurotoxins to mammals, if present, is low. The electrical stimulation method to collect the venom has the advantages of avioding contamination and repeated use of the valuable A. ventricosus venom resoures.In order to clone the genes encoding the proteins and peptides from the venom glands, a directional cDNA library of A. ventricosus venom glands was firstly constructed using the expressed sequence tag (EST) strategy. Clones from original cDNA library were randomly selected to sequence. Eight handreds and eighty-six ESTs from cDNA sequencing were grouped into49clusters and197singletons. All ESTs were translated into protein sequences using software Emboss. According to the usual properties of animal toxins, the parameters of toxin filter were set for:(1)4or more cysteines;(2) contain signal peptide. The result indicated that68.7%of total ESTs belong to toxin-like genes,20.2%are cellular transcripts and11.1%have no significant similarity to any known genes. Two handreds unique propeptides were produced by translating all ESTs and nonredundancing. These propeptides were grouped into15superf ami lies which contain30families from AVTX-I to AVTX-XXX. All precursors were processed to achieve150mature peptides with abundant cysteines. However, the number of cysteine is diversified from4to20among these families. And these propeptides were not identified by any database. Moreover, the gene ecoding chitinase was found in the cDNA library. All results demonstrated that these protein toxins from A. ventricosuswere some novel toxins which may be give novel model for understanding action mechanism of spider toxin.In order to analyze systematically the components of A. ventricosus venom, in this paper, combinative proteomic strategies were employed to analyze the venom collected from living spider A. ventricosus by electrostimulation. The combinative proteomic strategies including the following two different approaches:(i) one-dimensional SDS/PAGE plus nanoLC-MS/MS,(ii)2DE plus nanoLC-MS/MS. The images show that most proteins visualized on the gel have molecular masses in the range of10-45kDa. And three major positions correspondings to molecular weight of about10to20kDa,26kDa and43kDa, there were high abundance alkaline proteins. Peptides from in-gel digestion were analyzed by nanoLC-MS/MS. A total of71venomous proteins were identified using cross-species searching. Besides typtical spdier hemocyanins, several hydrolases were found in the venom, which were speculated to be related to insecticidal activity. In addition, beyond half of high-quality MS/MS spectra gave no signification search results, most likely due to the lack of specific database for spider venom.In this paper, we employ de novo sequencing to combine the proteomic and transcriptomic data. The gel spots from2-DE were carefully excised and digested with trypsin. Then the tryptic peptides were analyzed by nanoLC-MS/MS. The spectral data were searched against an online Mascot database. Then these data with no signification search results were sequenced using the software DataAnlysis4.0from Bruker company. The mass accuracy better than±0.015Da is required for decreasing the false positive rate. About2000sequences were produced by de novo sequencing. And about200unique sequences were collected by removing the duplication. These sequences were assigned against protein sequences from cDNA library. Efficient results must fill these parameters:(1) Mass accuracy of peptide is better than±0.5Da between theoretical and measurement value;(2) the peptide can be achieved by tryptic digestion. The results indicated that twenty of thirty families from cDNA library were matched by sequences from de novo sequncing. The identification rate is66.67%. Interestingly, abundant mutants and methyl esterification of glutamic acid from the venom were identificated by the method. The method gives a new approach for indentif ication of proteins from an organism whose genome is not known.