The effect of intron location on the splicing of BmKK2 in 293T cells

Previously reported results showed that the BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found in the second exon. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression level of the toxin-GFP fusion protein (Cao et al., J Biochem Mol Toxicol 2006;20:1-6). In this investigation, the BmKK2's intron with 79 nucleotides length was artificially shifted from the 49th nt (the 17th Gly codon between the first base and the second base) to the 100th nt (the 34th Gly codon between the first base and the second base). Based on the constructed intron-splicing system, the results of RT-PCR and the western blotting analysis showed that the BmKK2's shifted-intron (named BmKK2-s) was not recognized and spliced correctly, but the cryptic splicing site of BmKK2 gene was still spliced in the second exon, which possibly indicated that locations of introns were very important to the recognition and splicing of introns, and splicing of introns was very much associated with the corresponding upstream and downstream exons. This result possibly provides evidence for splice-site recognition across the exons.     

Adaptive evolution after gene duplication in alpha-KT x 14 subfamily from Buthus martensii Karsch

A series of isoforms of alpha-KT x 14 (short chain potassium channel scorpion toxins) were isolated from the venom of Buthus martensii Karsch by RACE and screening cDNA library methods. These isoforms adding BmKK1--3 and BmSKTx1--2 together shared high homology (more than 97%) with each other. The result of genomic sequence analysis showed that a length 79 bp intron is inserted Ala codes between the first and the second base at the 17th amino acid of signal peptide. The introns of these isoforms also share high homology with those of BmKK2 and BmSKT x 1 reported previously. Sequence analysis of many clones of cDNA and genomic DNA showed that a species population or individual polymorphism of alpha-KT x 14 genes took place in scorpion Buthus martensii Karsch and accelerated evolution played an important role in the forming process of alpha-KT x 14 scorpion toxins subfamily. The result of southern hybridization indicated that alpha-KT x 14 toxin genes existed in scorpion chromosome with multicopies. All findings maybe provided an important evidence for an extensive evolutionary process of the scorpion "pharmacological factory": at the early course of evolution, the ancestor toxic gene duplicated into a series of multicopy genes integrated at the different chromosome; at the late course of evolution, subsequent functional divergence of duplicate genes was generated by mutations, deletions and insertion.     

Molecular cloning and characterization of four scorpion K(+)-toxin-like peptides: a new subfamily of venom peptides (alpha-KTx14) and genomic analysis of a member.

Four full-length cDNAs encoding the precursors of four K(+)-toxin-like peptides (named BmKK(1), BmKK(2), BmKK(3) and BmmKK(4), respectively) were first isolated from a venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The deduced precursors of BmKK(1), BmKK(2) and BmKK(3) are all made of 54 amino acid residues including a signal peptide of 23 residues, and a mature toxin of 31 residues with three disulfide bridges. The precursor of BmKK(4) is composed of 55 amino acid residues including a signal peptide of 23 residues, a mature toxin of 30 residues cross-linked by three disulfide bridges, and an extra Gly-Lys tail which should be removed in the processing step. The four peptides displayed 24-97% sequence identity with each other, and less than 27% homology with any other scorpion toxins described. However, they shared a common disulfide bridge pattern, which was consistent with that of most short-chain K(+)-toxins, suggesting they represent a new class of scorpion toxins and their target receptors may be a subfamily of K(+) channels. We classified the BmKK toxin subfamily as alpha-KTx14 according to the classification rules. The genomic sequence of BmKK(2) was also cloned and sequenced. It consisted of two exons, disrupted by an intron of 79 bp inserted in the region encoding the C-terminal part of the signal peptide. This structure was very similar to that of other K(+)-toxins described previously.     

Splicing of intron 3 of human BACE requires the flanking introns 2 and 4

Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer’s disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.     

Solution structure of BmKK2, a new potassium channel blocker from the venom of chinese scorpion Buthus martensi Karsch

A natural K+ channel blocker, BmKK2 (a member of scorpion toxin subfamily alpha-KTx 14), which is composed of 31 amino acid residues and purified from the venom of the Chinese scorpion Buthus martensi Karsch, was characterized using whole-cell patch-clamp recording in rat hippocampal neurons. The three dimensional structure of BmKK2 was determined with two-dimensional NMR spectroscopy and molecular modelling techniques. In solution this toxin adopted a common alpha/beta-motif, but showed distinct local conformation in the loop between alpha-helix and beta-sheet in comparison with typical short-chain scorpion toxins (e.g., CTX and NTX). Also, the alpha helix is shorter and the beta-sheet element is smaller (each strand consisted only two residues). The unusual structural feature of BmKK2 was attributed to the shorter loop between the alpha-helix and beta-sheet and the presence of two consecutive Pro residues at position 21 and 22 in the loop. Moreover, two models of BmKK2/hKv1.3 channel and BmKK2/rSK2 channel complexes were simulated with docking calculations. The results demonstrated the existence of a alpha-mode binding between the toxin and the channels. The model of BmKK2/rSK2 channel complex exhibited favorable contacts both in electrostatic and hydrophobic, including a network of five hydrogen bonds and bigger interface containing seven pairs of inter-residue interactions. In contrast, the model of BmKK2/hKv1.3 channel complex, containing only three pairs of inter-residue interactions, exhibited poor contacts and smaller interface. The results well explained its lower activity towards Kv channel, and predicted that it may prefer a type of SK channel with a narrower entryway as its specific receptor.     

Purification, characterization and sequence determination of BmKK4, a novel potassium channel blocker from Chinese scorpion Buthus martensi Karsch

The scorpion neurotoxin BmKK4 was purified from the venom of the Chinese scorpion Buthus martensi Karsch by a combination of gel-filtration, ion exchange and reversed phase chromatography. The primary sequence of BmKK4 was determined using the tandem MS/MS technique and the cDNA database searching as followings: ZTQCQ SVRDC QQYCL TPDRC SYGTC YCKTT (NH(2)). BmKK4 is the first isolated member of a new subfamily alpha-KTx17 of scorpion K(+) toxins.     

一个东亚钳蝎蝎毒素BmTXKβ基因的克隆及其基因表达调控

从东亚钳蝎 (ButhusmartensiiKarsch ,BmK)毒腺组织cDNA文库中分离的长链钾通道毒素BmTXKβcDNA序列 ,克隆了BmTXKβ基因组序列 .BmTXKβ基因含有一个长度为 886bp的内含子 ,定位于BmTXKβ成熟肽中 ,与其它蝎毒素基因内含子定位于信号肽的基因结构不同 .并且 ,BmTXKβ基因的内含子特征也与其它蝎毒素基因不同 .研究结果从基因水平上证实了BmTXKβ是一个新的蝎毒素样肽 .以BmTXKβcDNA序列为探针与蝎基因组DNASouthern杂交出现 2条特异性杂交带 .杂交结果为蝎毒素基因可能通过DNA重排、多拷贝或多基因家族来调控基因表达提供了证据 .     

Molecular cloning and sequencing of two 'short chain' and two 'long chain' K(+) channel-blocking peptides from the Chinese scorpion Buthus martensii Karsch.

Five full-length cDNAs encoding the precursors of two 'short chain' scorpion non-toxic peptides active on Ca(2+)-activated K(+) channels (BmP02 and BmP03) and two novel putative long chain K(+) channel-blocking peptides (named BmTXKbeta and BmTXKbeta2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKbeta2 showed a high similarity with AaTXKbeta, while BmTXKbeta was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKbeta expands the scorpion long chain K(+) channel-blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an alpha-helix and a three-stranded beta-sheet.     

BmP09, a "long chain" scorpion peptide blocker of BK channels

A novel "long chain" toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na(+) channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na(+) channel blocker toaK(+) channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca(2+)- and voltage-dependent K(+) channels (BK) but not Na(+) channels at a range of 100 nm concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 alpha-subunits in Xenopus oocytes. The half-maximum concentration EC(50) of BmP09 was 27 nm, and the Hill coefficient was 1.8. In outside-out patches, the 100 nm BmP09 reduced approximately 70% currents of BK channels without affecting the single-channel conductance. In comparison with the "short chain" scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic pi -pi interactions around the center.     

Solution structure of BmP08, a novel short-chain scorpion toxin from Buthus martensi Karsch

A novel short-chain scorpion toxin BmP08 was purified from the venom of the Chinese scorpion Buthus martensi Karsch by a combination of gel-filtration, ion exchange, and reversed-phase chromatography. The primary sequence of BmP08 was determined using the tandem MS/MS technique and Edman degradation, as well as results of NMR sequential assignments. It is composed of 31 amino acid residues including six cysteine residues and shares less than 25% sequence identity with the known alpha-KTx toxins. BmP08 shows no inhibitory activity on all tested voltage-dependent and Ca(2+)-activated potassium channels. The 3D-structure of BmP08 has been determined by 2D-NMR spectroscopy and molecular modeling techniques. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation and features a 3(10)-helix and a shorter beta-sheet. The unique structure is closely related to the distinct primary sequence of the toxin, especially to the novel arrangement of S-S linkages in the molecule, in which two disulfide bridges (C(i)-C(j) and C(i+3)-C(j+3)) link covalently the 3(10)-helix with one strand of the beta-sheet structure. The electrostatic potential surface analysis of the toxin reveals salt bridges and hydrogen bonds between the basic residues and negatively charged residues nearby in BmP08, which may be unfavorable for its binding with the known voltage-dependent and Ca(2+)-activated potassium channels. Thus, finding the target for this toxin should be an interesting task in the future.