A catalog for transcripts in the venom gland of the Agkistrodon acutus: identification of the toxins potentially involved in coagulopathy

Agkistrodon acutus is a special agkistrodon halys, only distributed in Southern China, with a few exceptions in Vietnam. It is a cherished element used in traditional Chinese medicine. In order to produce a global panorama of gene expression in the Agkistrodon acutus venom gland, a non-normalized cDNA library was constructed, and 8696 high quality 5' end expressed sequenced tags (ESTs) were sequenced and analyzed. The initial sequences were assembled into 2855 clusters. Of these clusters, only 45.60% clusters matched known sequence and 54.40% had no match to any known sequence in GenBank. Except for putative cellular proteins (1184 clusters), the remaining 118 clusters (40.16% of all ESTs) corresponded to sequences associated with diverse toxin function. According to expression abundance, the major toxin components were metalloproteinases (32.08%) and C-type lectin (5.22%), and other components including bradykinin-potentiating peptide (0.90%), serine proteases (0.51%), nucleotidase and nuclease (0.41%), phospholipase A2 (0.30%), disintegrin (0.05%), cytokine-like molecules (0.06%), and other proteins (0.63%). The majority of these components are thought to be responsible for coagulopathy after A. acutus bites. We have therefore generated a comprehensive catalog of the A. acutus venom gland described so far. Gene expression from the very specialized secretory tissue, especially for those involved in coagulopathy, can be surveyed and provide important information in finding novel toxins.     

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genusLoxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.