Eur. J. Biochem. 269, 2708–2715 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02940.x Cloning and characterization of novel snake venom proteins that block smooth muscle contraction Yasuo Yamazaki1, Hisashi Koike1, Yusuke Sugiyama1, Kazuko Motoyoshi1, Taeko Wada1, Shigeru Hishinuma2, Mitsuo Mita2 and Takashi Morita1 Departments of 1Biochemistry; and 2Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan In this study, we isolated a 25-kDa novel snake venom caffeine-stimulated contraction. Furthermore, we isolated protein, designated ablomin, from the venom of the Japan- three other proteins from snake venoms that are homolog- ese Mamushi snake (Agkistrodon blomhoffi). The amino-acid ous to ablomin and cloned the corresponding cDNAs. Two sequence of this protein was determined by peptide of these homologous proteins, triflin and latisemin, also sequencing and cDNA cloning. The deduced sequence inhibited high K+-induced contraction of the artery. These showed high similarity to helothermine from the Mexican results indicate that several snake venoms contain novel beaded lizard (Heloderma horridum horridum), which blocks proteins with neurotoxin-like activity. voltage-gated calcium and potassium channels, and ryano- Keywords: snake venom; neurotoxin; helothermine; cysteine- dine receptors. Ablomin blocked contraction of rat tail rich secretory proteins; ablomin. arterial smooth muscle elicited by high K+-induced depolarization in the 0.1–1 lM range, but did not block Over the past 30 years, a plethora of toxins have been homologous to Kunitz-type serine protease inhibitors and isolated from poisonous organisms, such as snakes, scorpi- act primarily by blocking neuronal K+ channels [30,31]. In ons, spiders, and micro-organisms. These natural toxins use contrast to the neurotoxin-rich venom from Elapidae a variety of approaches to arrest the homeostatic mecha- snakes, the venom from other deadly snakes, including nisms of other living organisms, including disruption of Viperidae and Colubridae snakes, contain surprisingly few intracellular signal transduction and cytoskeleton organiza- neurotoxins, although some neurotoxic phospholipases tion [1–4], and activation or inhibition of blood coagulation have been discovered [32–36]. factors [5–10]. Toxins that block synaptic transmission, In this report, we describe the isolation of a novel protein, called neurotoxins, are widely distributed in venoms. These ablomin, from the venom of the Japanese Mamushi snake toxins include the conotoxins from cone snails, agatoxins (Agkistrodon blomhoffi, a member of the Viperidae family). from spiders, and scorpion toxins [11–16]. These toxins exert When applied to arterial smooth muscle preparations from their potentially lethal effects by specifically and potently rat-tails, ablomin blocks K+-stimulated contraction. This blocking a variety of ion channels, including those that effect is similar to that resulting from application of conduct Na+,K+,andCa2+. Therefore, neurotoxins have calciseptine, a well-characterized neurotoxin from black been employed as useful tools to investigate the structure mamba (Dendroaspis polylepis polylepis).Calciseptineisa and function of these ion channels [17–20]. A large number known blocker of L-type Ca2+ channels, a property that of neurotoxin families have also been found in the venom of underlies its ability to block K+-induced contractions of Elapidae snakes. These toxins, the a-neurotoxins [21] aortic smooth muscle and spontaneous contractions of (represented by a-bungarotoxin [22,23], a-cobratoxin uterine smooth muscle [37]. Furthermore, we demonstrate [24–27], and erabutoxin [28,29])potently and specifically that several snake venoms contain ablomin-like proteins, prevent nicotinic acetylcholine receptor activation. A second which may constitute a novel venom protein family. family of snake venom neurotoxins, the dendrotoxins, are EXPERIMENTAL PROCEDURES Correspondence to T. Morita, Department of Biochemistry, Materials Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose, Tokyo 204-8588, Japan, The lyophilized venom of A. blomhoffi was a kind gift from Fax/Tel.: + 81 424 95 8479, S. Iwanaga (The Chemo-Sero-Therapeutic Research E-mail: [email protected] Institute, Kumamoto, Japan)[38]. Other snake venoms Abbreviations: CRISP, cysteine-rich secretory protein; HLTX, and venom glands were purchased from the Japan Snake helothermine; PsTx, pseudechetoxin; CAP, CRISPs Antigen 5 Institute (Gunma, Japan). Superdex 75 pg and 200 pg, proteins, and Pathogenesis-related proteins. SP–Sepharose High Performance, and Q-Sepharose Fast Note: the nucleotide sequences reported here have been submitted to Flow columns were from Amersham–Pharmacia Biotech. GenBank database (tigrin, AY093955; ablomin, AF384218; triflin, The Vydac Protein & Peptide C18 HPLC column and the AF384219; latisemin, AF384220). COSMOSIL 5C18 AR-300 HPLC column were the (Received 21 December 2001, revised 12 April 2002, products of JASCO (Tokyo, Japan)and Nacalai Tesque accepted 18 April 2002) (Kyoto, Japan), respectively. Endoprotease Lys-C was Ó FEBS 2002 Novel proteins in snake venoms (Eur. J. Biochem. 269)2709 purchased from Seikagaku Corporation (Tokyo, Japan). Amino-acid sequence analysis Other chemicals were of analytical grade (Sigma–Aldrich, Amersham–Pharmacia Biotech., Wako Pure Chemical Ind. Proteins were reduced for 3 h at room temperature with and Kanto Chemical Co.). 20 mM dithiothreitol in the presence of 0.5 M Tris/HCl, pH 8.5, 6 M guanidine hydrochloride, and 2 mM EDTA in a volume of 0.5 mL. Three microliters of 4-vinylpyridine Purification of proteins were then added, and alkylation was allowed to proceed Tigrin was isolated from the extract of Duvernoy’s glands of for 3 h at room temperature. The S-pyridylethylated Rhabdophis tigrinus tigrinus. Ten Duvernoy’s glands were proteins were separated from the reagents by C18 broken into small pieces after freezing in liquid nitrogen, reverse-phase HPLC, and the amino-acid sequence was and then extracted in 30 mL of 50 mM Tris/HCl pH 8.0 for determined by sequencing the peptides obtained by diges- 4hat4°C. After ultracentrifugation, the supernatant was tion with endoprotease Lys-C. All the samples were applied onto Q-Sepharose Fast Flow column (1.6 · 10 cm) analyzed on Applied Biosystems protein sequencers (mod- in the same buffer, and eluted with a linear gradient from 0 els 473 A and 477). to 0.5 M NaCl. A major peak eluted at 0.05 M NaCl, which was subsequently purified by chromatography on Superdex cDNA cloning of proteins 200 pg (2.6 · 60 cm column). Ablomin was purified by three successive chromato- The cDNAs encoding tigrin, ablomin, and latisemin were graphic steps. Five hundred milligrams of lyophilized obtained using the RT-PCR method. Typically, venom A. blomhoffi venom was dissolved in 3 mL of 20 mM gland total RNA was isolated from the venom gland with TM imidazole/HCl pH 6.8 containing 0.2 M NaCl, and insol- ISOGEN (Wako Pure Chemical Industries, Japan) uble materials were removed by centrifugation and according to the manufacturer’s protocol. 5¢ and 3¢ RACE filtration (0.22 lm). The filtrate was loaded onto a were carried out to determine the nucleotide sequence of the Superdex 75 pg column (2.6 · 60 cm)and eluted with 5¢ and 3¢ end cDNAs with the SMARTTM RACE cDNA the same buffer. The ablomin fractions from two gel amplification kit (Clontech). The amino-acid sequences of filtration runs (a total of 1000 mg of snake venom)were peptides derived from purified proteins were used to design pooled and dialyzed against 50 mM Tris/HCl, pH 8.0, and degenerate primers. For the first amplification of tigrin and applied to the Q-Sepahrose Fast Flow column latisemin cDNA, degenerate primers were used for both (1.6 · 15 cm). The column was eluted with a linear sense and antisense primers. For ablomin cDNA, PCR was gradient of NaCl from 0 to 0.4 M at a flow rate of performed with single degenerate primer (sense or antisense) 2mLÆmin)1. Chromatographic fractions containing and an primer recognizing an adaptor sequence that had ablomin were then dialyzed against 20 mM imidazole- been attached to the 5¢ or 3¢ endofcDNAs.Inthecaseof HCl, pH 6.0, and fractionated on a SP–Sepharose High triflin, PCR was carried out using habu cDNA library as a Performance column (1.6 · 11 cm). This column was template [40] with a degenerate primer and an adaptor developed with a linear gradient of NaCl in the imidazole primer. The PCR products were subcloned into the pGEM )1 buffer (0–0.4 M,2mLÆmin ). T-easy vector (tigrin and triflin)or pUC19 vector (ablomin For the purification of triflin, 500 mg of the venom of and latisemin)and sequenced with the DSQ 2000 L DNA Trimeresurus flavoviridis was applied to the SP–Sepharose sequencer (Shimadzu, Japan). Primers used this study are Fast Flow column (1.6 · 30 cm)with 10 m M phosphate described as follows: tigrin, sense 5¢-AA(C,T)GT(A,C,G,T) buffer, pH 6.8, and eluted with a linear gradient from 0 to GA(C,T)TT(C,T)AA(C,T)TC(A,C,G,T)GA(A,G)TC-3¢ 0.15 M NaCl, as described previously [39]. Fractions con- (corresponding to amino acids 1–8 in tigrin)and antisense taining triflin were detected by Western blotting using anti- 5¢-(A,G)TT(A,G)CA(A,G)TT(A,G)TT(A,G)TA(A,G)TC tigrin serum. These fractions were pooled and fractionated (A,G)TC-3¢ (corresponding to amino acids 187–193 in on Superdex 75 pg (2.6 · 60 cm)in a 50-m M Tris/HCl, tigrin); ablomin, sense 5¢-GGCCATTA(C,T)ACTCAG(A,G) pH 8.0, containing 0.2 M NaCl. Finally, triflin was purified T(A,G)G-3¢ (corresponding to amino acids 114–120 in by chromatography on a Blue-Sepharose Fast Flow column ablomin)and antisense 5 ¢-C(C,T)A(C,T)CTGAGT(A,G) (1.6 · 15.5 cm)in 50 m M Tris/HCl, pH 8.0, which was TAATGGCC-3¢ (corresponding to amino acids 114–120 eluted with a linear gradient from 0 to 0.5 M NaCl.