Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins.

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.

A unique approach for high level expression and production of a recombinant cobra neurotoxin in Escherichia coli.

In this report, we describe a simple approach to produce a large quantity of a recombinant cobra neurotoxin containing four pairs of disulfide bonds. A cDNA encoding the toxin was fused, in frame, to the carboxyl termini of thioredoxin via a linker sequence encoding two amino acids, Asp and Pro. Due to the presence of thioredoxin, a soluble form of the fusion protein was expressed in a compartment, sensitive to osmotic pressure, in Escherichia coli. The fusion protein was released into the solution with low ionic strength under an osmotic shock treatment, and purified in a single step using an ion exchange chromatography column. The purified protein was treated in diluted hydrochloric acid to induce hydrolysis of the protein at the Asp-Pro linker site. Then, the recombinant neurotoxin was purified by gel filtration of the acid-treated sample. When the biological activity of the purified toxin was assayed, it was as potent as the natural toxin. Using this protocol, approximately 12 mg of pure recombinant neurotoxin can be produced from one liter of bacterial culture. More importantly, this protocol can be easily used for the production of the toxin at a larger scale with low cost. The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.

Apoptosis of human hepatocellular carcinoma cell (HepG2) induced by cardiotoxin III through S-phase arrest.

Cytotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, have potential therapeutic activity in tumor therapy. However, the therapeutic effect in solid tumor treatment with CTX III are still largely unknown. In the present study, we investigated whether CTX III affects cell growth and cell cycle progression of hepatocellular carcinoma cell (HepG2). We found that the proliferation of HepG2 cell was inhibited by CTX III, to some extent, in a time- and dose-dependent manner (IC50 2.58microg/ml at 24h). Flow cytometric analysis and annexin V labeling also demonstrated that CTX III increased the percentage of apoptotic cells being associated with cell cycle arrest at S-phase. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot revealed that cyclin D1, cyclin A and cyclin E, which involved in cell apopotosis and cell cycle progression, were down regulated both at transcription and translation levels. CTX III-induced caspase-8, -9 and caspase-3 activation, generation of truncated Bid, releasing of cytochrome c and the change of Bcl-2/Bax ratio on protein and mRNA levels. These findings demonstrated that cyclin D1, cyclin B and cyclin A down-regulation, change of Bcl-2/Bax ratio and caspase-8 and -9 activation contribute to CTX III-induced HepG2 cell apoptosis.

Involvement of p38 and c-Jun N-terminal protein kinase in cardiotoxin III-induced apoptosis of K562 cells.

Cardiotoxin III (CTX III), a 60-amino acid basic polypeptide isolated from Naja venom, showed potential therapeutic activity toward cancer cells. Here we report that CTX III inhibited proliferation of human leukemia K562 cells by G2/M phase arresting and apoptosis which was associated with the activation of caspase-8 and cytochrome c release as well as the p38 and c-Jun N-terminal protein kinase (JNK) phosphorylation signaling pathway. We further demonstrated that daily administration of CTX III for 2 d to chicken chorioallantoic membrane (CAM) bearing tumours derived from the CAM at E10 administration of K562 cells resulted in inhibition of the tumours in vivo. Importantly, this in vivo inhibition was also associated with caspase-8 activation and cytochrome c release. Our results suggest that CTX III-induced apoptosis is mediated via the p38 and JNK pathway as well as the caspase-8-dependent Bid-Bax pathway in human K562 cells.

Thrombin-like enzymes from venom gland of Deinagkistrodon acutus: cDNA cloning, mechanism of diversity and phylogenetic tree construction.

AIM: To clone cDNAs of thrombin-like enzymes (TLEs) from venom gland of Deinagkistrodon acutus and analyze the mechanisms by which their structural diversity arose. METHODS: Reverse transcription-polymerase chain reaction and gene cloning techniques were used, and the cloned sequences were analyzed by using bioinformatics tools. RESULTS: Novel cDNAs of snake venom TLEs were cloned. The possibilities of post-transcriptional recombination and horizontal gene transfer are discussed. A phylogenetic tree was constructed. CONCLUSION: The cDNAs of snake venom TLEs exhibit great diversification. There are several types of structural variations. These variations may be attributable to certain mechanisms including recombination.

cDNA cloning, sequence analysis, and recombinant expression of akitonin beta, a C-type lectin-like protein from Agkistrodon acutus.

AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template. The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin beta was found and accepted by GenBank (accession number AF387100). Akitonin beta consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin beta expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.