£]-Bungarotoxin (£]-Bgt), a presynaptic phospholipase A2 (PLA2) neurotoxin isolated from the venom of Bungarus multicinctus (Taiwan banded krait), consists of A chain and B chain, cross-linked by an interchain disulfide bond. A chain is structurally homologous with phospholipase A2 (PLA2) enzymes, while the sequence of B chain is homologous to the Kunitz-type protease inhibitor and dendrotoxin. In addition to PLA2 activity, £]-Bgt blocked the neurotransmission at the neuromuscular junction by selectively inhibiting certain voltage-sensitive potassium channels. The present studies investigated the B chain of £]-bungarotoxin and B chain homologous proteins in evolutionary relationship and cytotoxic mechanism.
Eight A chain cDNAs and three B chain cDNAs have been cloned from B. multicinctus venom glands. Random combination of the A and the B chains should produce a number of £]-Bgt isotoxins. There are at least 16 isoforms of £]-Bgt were been isolated. Previous studies indicate that A and B chains are encoded separately by different genes, and the A and B chain genes do not originate from a common ancestor. These findings suggest that the intact £]-Bgt molecules should be derived from the pairing of A and B chains after their mRNAs are translated. And, our recent studies show that B chain genes and Naja naja atra chymotrypsin inhibitor (NACI) share the same genomic organization and high sequence identity. Alternatively, limited studies on the evolutionary divergence of B chain gene and its homologous have been reported.
In the first part, the structural organization of the genes encoding B2, B4, B5 and B6 chains of £]-Bgt are reported. These genes shared virtually identical overall organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 and B2 chains were absent in that of B4, B5 and B6 chains. RT-PCR analyses indicated that Bungarus multicinctus venom gland, heart, liver and muscle expressed the RNA transcripts showing sequence similarity with the intronic segment being deleted in B4, B5 and B6 chain genes. This reflects that the ancestral gene of the intronic segment might insert in multiple loci of B. multicinctus genome. Comparative analyses of B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that intron insertions or deletions occur with the evolution of B chains, and that accelerated evolution may diversify the protein-coding sequence of B chain genes same as snake phospholipase A2, neurotoxin and cardiotoxin genes.
The second part is to explore the functional contribution of the two subunits to the toxicity of £]-Bgt. £]-Bgt was found to induce apoptotic death of SK-N-SH cells via elevating intracellular Ca2+ and intracellular ROS production. Moreover, an activation of p38 MAPK was associated with the cytotoxicity of £]-Bgt. SB202190( p38 MAPK inhibitor), N-acetylcysteine (antioxidant reagent), 1,2-bis (2-amino-phenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca2+ chelator) and the inhibitors of Ca2+ release from intracellular depots (ruthenium red and 2-aminoethoxydiphenyl borate) effectively attenuated the cytotoxicity of £]-Bgt. In sharp contrast to the inability of A chain, B chain was able to induce cytotoxic effects on SK-N-SH cells as £]-Bgt did. Abolishment of PLA2 activity did not significantly alter the cytotoxic activity of £]-Bgt. MK801 (an NMDA receptor antagonist), antibodies against NMDA receptor and 4-aminopyridine (a potassium channel blocker) markedly reduced the cytotoxic effects of £]-Bgt, B chain and catalytically inactivated £]-Bgt. Moreover, antibodies against NMDA receptor blocked the binding of rhodamine-labeled £]-Bgt to SK-N-SH cells. Taken together, our data indicate that B chain is a functional subunit responsible for the cytotoxicity of £]-Bgt, and suggest that the cytotoxicity of £]-Bgt is mediated by NMDA receptor and potassium conductance.
Homologous proteins of the B chain, namely, protease inhibitor-like protein-1 (PILP-1), PILP-2, and PILP-3, were successfully cloned from the Bungarus multicinctus genome. The 3 cloned genes each comprised 3 exons and 2 introns, similar to the genes of the B chain of £]-bungarotoxin and NACI. Based on the Ka/Ks values of the gene sequences of PILPs, the B1 chain, and NACI, the PILPs may have undergone adaptive evolution. The cDNAs of the PILPs were recombined by PCR, and the recombinant proteins were successfully expressed and purified. PILPs induced cytotoxicity in U937 cells, but PILP-3 reduced viability only slightly. Induction of cell death by PILP-1 and PILP-2 was by apoptosis that occurred in a caspases-dependent manner. PILP-1 and PILP-2 activated p38 MAPK and downregulated ERK1/2; however, PILP-3 caused no such effects. PILP-1 increased the population of sub-G1 cells and caused mitochondrial damage. PILP-1-treated cells were observed to demonstrate loss of mitochondrial membrane potential, release of cytochrome C, and a decrease in the level of Bcl-2 and pro-caspase 9. Pretreatment of U937 cells with either SB202190 or a caspase 8 inhibitor significantly prevented the PILP-1- and PILP-2-induced degradation of caspase 8 or 3. PILP-1 and PILP-2 increased the level of TNFRII, which was suppressed by the p38 MAPK inhibitor. The transient transfection of PILP-1- and PILP-2-treated cells with pCMV-MEK1 efficiently increased their survival and phosphorylation of ERK1/2 but reduced the level of TNFRII. After PILP-1 and PILP-2 induced TNFRII, the excess TNF caused a significant reduction in cell viability. TNFRI levels were not significantly changed in PILP-1- and PILP-2-treated U937 cells. These results suggest that PILP-1 and PILP-2 induce apoptosis via an increase in the level of TNFRII and p38 MAPK activity and the suppression of ERK activity.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0124108-173924 |
| Date | 24 January 2008 |
| Creators | Cheng, Yun-Ching |
| Contributors | Chun-chang Chang, Long-sen Chang, Wen-chun Hung |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0124108-173924 |
| Rights | campus_withheld, Copyright information available at source archive |
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