Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

Toxicon 40 (2002) 1719–1722 www.elsevier.com/locate/toxicon

Short Communication

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karschq Xian-Chun Zeng1, San-Xia Wang, Wen-Xin Li* Department of Biotechnology, Institute of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China Received 30 January 2002; accepted 22 April 2002

Abstract A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensii Karsch by cDNA library screening combined with 50 -race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Venom peptide; Toxin; Scorpion; Buthus martensii Karsch; BmKAPi; cDNA

During more than 400 million years of evolution, scorpions have evolved a variety of venom peptides to interfere with the normal functions of cellular ion channels or other receptors of their enemy and prey species (Possani et al., 1999, 2000). The most important components in scorpion venoms are small peptides with 13 – 76 amino acid residues, which can be classified into two main types: toxins with three or four disulfide bridges, and venom peptides with no disulfide bridge (Zeng et al., 2000a, 2001a). Four different q

The nucleotide sequence of BmKAPi reported in this paper has been submitted to GenBank Database under the accession number: AF 462308. * Corresponding author. Tel.: þ86-27-87682831; fax: þ 86-2787882661. E-mail addresses: [email protected] (W.X. Li), [email protected] (X.C. Zeng). 1 Present address: National Heart, Lung and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA. Fax: þ1-301-402-1519.

families of scorpion toxins have been described, which specifically act on Naþ, Kþ, CI2, or Ca2þ channels (Possani et al., 1999, 2000; Tytgat et al., 1999; Debin et al., 1993; Zamudio et al., 1997). Although their primary structures can be different, they share a common, dense core formed by an a-helix and two to three b-strands, stabilized by three or four disulfide bridges. As for the venom peptides with no disulfide bridge, at least five polypeptides have been characterized (Torres-Larios et al., 2000; Dai et al., 2001; Zeng et al., 2000a, 2001a). Their pharmacological functions are proved to be different from those of the disulfide-bridged scorpion toxins. Scorpion toxins are valuable tools used in the identification, purification and functional characterization of ion channels or other receptors, as well as in the new drug development (Garcia et al., 2001). So, the search for new scorpion toxins has become an active and interesting area of investigation. In order to search and identify new type of scorpion venom peptides from Chinese scorpion Buthus martensii Karsch, we have screened the venom

0041-0101/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 2 ) 0 0 1 3 4 - 4

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Fig. 1. Nucleotide sequence of the cDNA encoding precursor of BmKAPi from Chinese scorpion B. martensii Karsch. The encoded peptide is shown below its corresponding nucleotide sequence, and is numbered on top from the N-terminal amino acid residue of the peptide; the putative signal peptide is underlined; the two potential polyadenylation signals (AATAAA) are italicized.

gland cDNA library by ‘size-selective’ sequencing. Combined with the 50 -race, we isolated a full-length cDNA sequence encoding a novel type of scorpion venom peptide (named BmKAPi) cross-linked by five disulfide bridges. A scorpion venom gland cDNA library of B. martensii Karsch was constructed as described previously (Zeng et al., 2001b). PCR amplification was used to determine the insert size of the clones from the cDNA library. The forward primer was T7 promoter primer; and the reverse primer was 50 -AGCGGCCGCCCT(15)-30 , corresponding to a partial sequence of the pSPORT1 vector and 30 -terminal poly(A) sequence. The PCR products were analyzed by electrophoresis on 2.0% agarose gel. The clones with insert size between 350 and 450 BP were subject to sequencing. We obtained an interesting patial cDNA sequence of 350 BP encoding part of a novel scorpion venom peptide (named BmKAPi). A 50 -race method was carried out to get the fulllength cDNA sequence. The gene-specific primer used was 50 -AGCATTCAGTTTTGCAAAACGCA TCTTCC-30 , corresponding to the C-terminal region of BmKAPi. Total RNA of scorpion venom gland was prepared by TRIZOL LS Reagent (GIBCO/BRL). The SMART RACE kit (Clontech,

USA) was used to synthesize the first-strand cDNA and prepare the 50 -race reaction according to the Product’s instruction. The PCR products were purified by QIAquick Spin kit (Qiagen GmbH, Germany) and cloned. Nucleotide sequences were determined with the universal M13 forward and reverse primers from both directions according to the method described elsewhere (Sanger et al., 1977). The full-length cDNA sequence of BmKAPi was completed by overlapping the two fragments obtained by selective sequencing and 50 -race, respectively. As shown in Fig. 1, it was composed of 50 -untranslated region (50 UTR), an open reading frame and 30 -untranslated region (30 UTR). The 50 UTR is only 5 bp probably due to incomplete elongation during the cDNA synthesis by reverse transcriptase. The flanking nucleotides of initiation codon ATG are ATACC which are similar to those of BmKK4 cDNA from B. martensii Karsch (Zeng et al., 2001b) and some cDNAs from vertebrates, but different from most other described scorpion toxin cDNAs from B. martensii Karsch (Zeng et al., 2000b, 2001b,c). The common feature is that the nucleotide at position 2 3 is an adenine. The 30 UTR is 178 BP long that is much longer than those of other scorpion toxin cDNAs

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Fig. 2. Sequence alignment of BmKAPi with those of other similar short-chain peptides by matching cysteine residues. Amino acids common to BmKAPi are indicated by ‘·’. Gaps are indicated by ‘-’. AMCI is from honeybee A. mellifera (Bania et al., 1999; Cierpicki et al., 2000); AcAP5 and AcAP6 from hookworm A. caninum (Stanssens et al., 1996); BST I from European frog B. bombina (Mignogna et al., 1996).

from B. martensii Karsch. Two putative polyadenylation signals (AATAAA) were found 16 and 22 nucleotides upstream from the poly(A)-tail, respectively, which may be used to generate different BmKAPi transcripts by selecting different tailing sites to produce different length of 30 UTR (Zeng et al., 2001b). The 30 UTR contains some AU-rich motifs that might be involved in the control of mRNA stability. The open reading frame of BmKAPi cDNA is 267 BP long encoding a precursor of 89 amino acid residues, including a signal peptide of 24 residues, a putative mature venom peptide of 64 residues and an extra basic residue at the C-terminus which might be removed in the posttranslational processing step (Possani et al., 1999). The signal peptide cleavage site is predicted to be between the residues S(21) and Y(þ1); moreover, the residues at position 22 and 2 3 are Q and S, respectively. It follows the rule of signal peptide cleavage described by Von Heijne (1983, 1986). Although the peptide length of BmKAPi is similar to the long-chain scorpion toxins, its amino acid sequence showed no homology to these toxins. A search for amino acid sequence homology indicated that BmKAPi just showed little similarity to the sequences of a serine protease inhibitor AMCI from honeybee Apis mellifera (Bania et al., 1999; Cierpicki et al., 2000), two anticoagulant peptides, AcAP5 and AcAP6, both from hookworm Ancylostoma

caninum (Stanssens et al., 1996), and a trypsin/thrombin inhibitor BST I from European frog Bombina bombina (Mignogna et al., 1996) (Fig. 2). Although their homology is very low (from 20 to 33%), these peptides showed a similar disulfide bridge pattern formed by 10 cysteines distributed in the following consensus: 50 -X1 – 5CX8CX3 – 5CX3 – 4CX6 – 0 11CX3 – 6CX3 – 5CX1CX11 – 13CX5CX0 – 8-3 (X: amino acid residue) (Fig. 2). It suggests they may share a consistent scaffold as AMCI that consists of two approximately perpendicular beta-sheets, several turns, and a long exposed loop. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment (Cierpicki et al., 2000). Therefore, the functions of BmKAPi might be related to inhibition of blood coagulation or protease activity. The scorpion venom of B. martensii Karsch has been used as a therapeautic antithrombotic agent for more than 2000 years in China. Further research will be interesting to unravel if BmKAPi has anti-thrombosis function. RT-PCR was performed to analyze expression level of BmKAPi mRNA at various time points after extraction of scorpion venom. An up-regulated gene expression pattern in response to venom extraction stimulus was observed (date not shown). After venom extraction, its expression level increased as time went on. On the second day, the highest

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level was observed. This finding suggested the production of BmKAPi mRNA could be induced by venom extraction, and it shared a common expression pattern with other scorpion toxins described (Alami et al., 2001). Therefore, BmKAPi should be a component of scorpion venom. BmKAPi is the first venom peptide with five disulfide bridges identified from scorpion.

Acknowledgments This work was partially supported by the National Medicine Creation Doctor-Foundation of China to Dr XianChun Zeng (No. 96-901-033), by the National Natural Science Foundation of China (No. 39970897) and by the Key Science and Technology Project of National Education Office of China, both to Dr Wen-Xin Li, as well as by the Zhiqiang Foundaton of Wuhan University to Dr Xian-Chun Zeng.

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