Shunyi Zhu

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 Ca2+‐activated K+ channels (BmP02 and BmP03) and two novel putative long chain K+ channel‐blocking peptides (named BmTXKβ and BmTXKβ2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKβ2 showed a high similarity with AaTXKβ, while BmTXKβ was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKβ 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 α‐helix and a three‐stranded β‐sheet.

Cloning and characterization of a cDNA sequence encoding the precursor of a chlorotoxin-like peptide from the Chinese scorpion Buthus martensii Karsch☆

A full-length cDNA sequence encoding the precursor of a venom peptide with homology to chlorotoxin (named BmKCT) was isolated from a cDNA library made from the venom glands of the Chinese Scorpion Buthus martensii Karsch. The encoded precursor of BmKCT was 59 amino acid residues long including a signal peptide of 24 residues and a mature toxin of 35 residues with four disulfide bridges. The sequence of BmKCT is similar (68% identities) to that of chlorotoxin isolated from Leiurus quinguestriatus quinquestriatus. BmKCT is the first report of the cDNA sequences encoding four-disulfide-bridged short-chain toxins from Buthus martensuii Karsch so far.

Molecular cloning and characterization of four scorpion K+-toxin-like peptides: A new subfamily of venom peptides (α-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.

Nine novel precursors of Buthus martensii scorpion α-toxin homologues.

Abstract The cDNAs encoding nine novel α-toxin homologues were isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). They are rich in AAAA and TTTT elements at the 5′ UTRs. The flanking region of the translation initiation codon ATG is AAAATGAA, which is highly conserved in scorpion Na+, K+ and Cl− channel toxin genes. These putative scorpion α-toxins shared 45.5–98.4% homology with the characterized BmK α-toxins, and were completely conserved in the positions of all eight cysteines. This showed, together with higher homology at nucleotide level than that at amino acid level, that these toxins may originate from a common ancestor. The discovery of a series of homologues of scorpion α-toxin with a different degree of natural mutation in the primary structure will provide us with a valuable system for studying the structure–function relationship of scorpion toxins.

Evidence for the existence of insect defensin-like peptide in scorpion venom.

Insect defensin refers to a group of antibacterial peptides derived from a variety of insect species as well as from scorpion and possessing a three‐dimensional structure highly similar to that of scorpion toxins. A full‐length cDNA encoding an insect defensin‐like peptide was isolated from the venom gland cDNA library of the Chinese scorpion

Precursor of a Novel Scorpion Venom Peptide (BmKn1) with no Disulfide Bridge from Buthus martensii Karsch

A full‐length cDNA sequence encoding the precursor of a novel venom peptide (named BmKn1) with no disulfide bridge was first isolated from the venom gland cDNA library of Chinese scorpion Buthus martensii Karsch. The encoded precursor consisted of 70 amino acid residues including two parts: a signal peptide of 23 residues, and a putative mature venom peptide (BmKn1) of 47 residues. The sequence of BmKn1 showed no similarity to those of other scorpion venom peptides. BmKn1 may be the first member of a new venom peptide family from scorpion. Future research will be interesting to unravel further the pharmacological function of this novel scorpion venom peptide.

A naturally occurring non-coding fusion transcript derived from scorpion venom gland: implication for the regulation of scorpion toxin gene expression

Scorpion venom glands synthesize and secrete a great number of low molecular mass toxic peptides for prey and defense. Many cDNAs and genomic genes encoding these toxins have been isolated and sequenced. However, their expression regulation mechanism is not yet known at present. During screening of a cDNA library prepared from venom glands of the scorpion Buthus martensii Karsch, we isolated a natural fusion cDNA composed of the 5′‐untranslated region (UTR) and upstream coding sequence of a long‐chain toxin transcript and the downstream coding sequence and 3′‐UTR of a short‐chain toxin transcript. The junction site is just the overlapping region of 11 nucleotides (GGCAAGGAAAT) between the two wild transcripts, and thus leads to the formation of an early stop codon, which will cause premature translation. Based on the above observations, combined with the genomic data, we proposed a characteristic regulation mechanism of scorpion toxin genes, in which trans‐splicing and nonsense mediated mRNA decay are involved.

Does MMLV-RT lacking RNase H activity have the capability of switching templates during reverse transcription?

The reverse transcription reaction which transcribes mRNA into cDNA is catalyzed by an RNA-dependent DNA polymerase named reverse transcriptase (RT). This enzyme is involved in the infectious cycle of retroviruses and promotes RNA recombination by the strand transfer (also called strand switch) mechanism to expand the genetic variability of viruses. Research shows that the occurrence of strand transfer depends upon both activities carried by RT, that is, its RNase H and polymerase activities. Before switching templates, RNase activity is required for the removal of RNA fragments annealed to the nascent DNA strand, which makes the 3P end of this DNA available for hybridization to the homologous region of another template RNA [1]. In vitro experiments also suggest that homologous recombination between two distinct RNA templates promoted by RT needs the involvement of RNase H activity [2,3]. For example, Negroni et al. [3] found RNase Hþ RT may produce low recombination frequency (1.4U1033) from two distinct RNA templates, but RT lacking RNase H activity (RNase H RT) cannot recombine two RNA molecules. Moreover, to our knowledge, no one has reported that RNase H RT has the capability of switching templates during reverse transcription. Regarding the argument proposed by Zeng and Wang, the question is whether BmTXKL^BmKCT is an artifact or a naturally occurring transcript. First we must emphasize that the RT we used in the synthesis of the ¢rst strand cDNA is an engineered MMLV-RT provided by Gibco BRL (Superscript RT). This enzyme does not contain RNase H activity. Therefore, this excludes the possibility of template switching. Considering the fact that Zeng and Wang used the same RT as we did for the synthesis of the ¢rst strand cDNA, we do not know how they detected a 1% recombination rate in their experiment. And in fact, even with RNase Hþ RT, template switching in vitro is a low frequency event because the nascent DNA must bind to the free homologous region of the second RNA template. In other words, the acceptor RNA must remain uncomplexed to its cDNA during template switching [3]. Obviously, this scenario should be very rare in an in vitro system. Lastly, the only paper cited by Zeng and Wang for criticizing our work actually does not support their results. Perhaps they neglected the basic conclusion made by Ouhammouch et al., which is that RNase Hþ AMV-RT only forms fusion products at 37‡C for much longer incubation times (90 min) [5]. In fact, both Zeng and Wang and our laboratory used RNase H MMLV-RT to perform the ¢rst strand reaction at 37‡C for 60 min [4]. In conclusion, regardless of whether one argues on the basis of reaction parameters or enzymology, BmTXKL^BmKCT is a naturally occurring transcript, and not an artifact. We feel that Zeng and Wang’s argument lacks support in theory and overemphasizes cloning errors derived from RT.

Genomic Organization of BmTXKβ and BmTXKS2, Two Scorpion Venom Peptides From Buthus martensii Karsch

Toelucidate genomic organization of BmTXK βand BmTXKS2, two scorpion venom peptides from Chinese scorpion Buthus martensii Karsch(BmK) were first isolated and their genomic regions characterized using the PCR method. Analysis of nucleotide sequence shows that there exists different intron location in the venom genes. The region encoding mature peptide of BmTXK βis disrupted by an intron with 886 bp, whereas the intron of BmTXKS2 is located within its propeptide coding region, which is different from other scorpion toxin genes with their introns within the signal peptide coding region.

Molecular characterization of a K+ channel blocker in the scorpionButhus martensii Karsch

K+ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K+ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca2+-activated K+ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA, which is highly conserved in scorpion Na+ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is significantly different from that of the determined scorpion Na+ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined by primary structure analysis.