- Email: [email protected]
Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity
PII: !soo41-0101(~5-7
Toxicon, Vol. 34, No. 9, pp. 987-1001, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Gnat Britain. All rights reserved cul41~101/96 $15.00 + 0.00
TWO NEUROTOXINS (BMK I AND BMK II) FROM THE VENOM OF THE SCORPION BUTHUS MARTENSI KARSCH: PURIFICATION, AMINO ACID SEQUENCES AND ASSESSMENT OF SPECIFIC ACTIVITY YONG HUA JI,’ P. MANSUELLE; S. TERAKAWA,3 C. KOPEYAN,’ N. YANAIHARA,4 KE HSU’ and H. ROCHAT’* ‘Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai, 200031, P.R. China, ZLaboratoire de Biochimie, CNRS URA 1455, IFR Jean Roche, Faculte de Medecine Secteur Nord, Bd. Pierre Dramard, 13916, Marseille, Cedex 20, France, ‘Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan and 4Laboratory of Bioorganic Chemistry, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422, Japan (Received
12 January 1996; accepted 2 April 1996)
Y. H. Ji, P. Mansuelle, S. Terakawa, C. Kopeyan, N. Yanaihara, K. Hsu and H. Rochat. Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity. Toxicon 34,987-1002, 1996.-Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthu,s martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V, protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus,
Abbreviations: BmK I and BmK II, toxins I and II from the scorpion Buthusmartensi Karsch; AaH I, II, III and IV, toxins I, II, III and IV from the scorpion Androctonus astral Hector (North Africa); Lqq III and V, toxins III and V from the scorpion Leiurus quinquestriatw quinquestriatus (Sudan); Bot I, II and XI, toxins I, II and XI from the scorpion Ruth occitanus tunetanus (Algeria); Be M9 and M14, toxins M9 and Ml4 from the scorpion Euthus epeus (Asia); CMC, carboxymethyl-cysteine; DABITC, 4-N,N-dimethylaminoazobenzene-4’isothiocyanate; MLD, minimum lethal dose; RCM, reduced and S-carboxymethylated, TTX, tetrodotoxin; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone. 987
988
Y. H. Jl et al.
functional and structural similarities suggest that BmK I and BmK II belong to group 3 of scorpion a-type toxins. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
To date, more than 100 toxins have been isolated and identified in venoms of scorpions belonging to the Buthidae family (Rochat et al., 1979; Keegan, 1980). Most constitute a family of basic, low mol. wt, single-chain polypeptides, cross-linked by four intramolecular disulfide bridges (Kopeyan et al., 1974; Fontecilla-Camps et al., 1980; Gregoire and Rochat, 1983). They have a high affinity for the voltage-dependent sodium channel with different species specificity and thus are categorized as anti-mammal and/or anti-insect toxins (Rochat et al., 1979; Loret et al., 1992). Anti-mammal toxins have been further divided into two groups, a-toxins and B-toxins, according to their mode of action: a-toxins, the binding of which on the membrane is potential dependent, prolong the Na+ inactivation phase of the action potential, while p-toxins affect the Na+ activation phase (Jover et al., 1980a, 1980b; Couraud et al., 1978, 1982). Both types of toxin bind to distinct sites on the sodium channel. Scorpion neurotoxins have thus been extensively promoted as tools for investigating the components and molecular mechanism of sodium channels (Ovchinikov et al., 1982; Catterall, 1988). The Asian scorpion Buthus martensi Karsch (BmK) is a species belonging to the Buthidae family. It is widely distributed from northern China to Mongolia and Korea. In Chinese traditional medicine, even at the present time, the scorpion is still used as a drug to treat neurological symptoms, such as incomplete paralysis and mimetic paralysis. However, little knowledge is available of the neurotoxic components of this species. In the present study, the purification, amino acid sequences and electrophysiological properties of two anti-mammal neurotoxins (BmK I and BmK II) from the Chinese scorpion B. martensi Karsch are described. MATERIALS
AND
METHODS
Extraction of venom gland About 5000 adult (length approx. 7 cm) B. martensi Karsch (BmK) were purchased from local culture farms in Henan and Shandong Provinces, China. Telsons (the last caudal segments containing the venom gland) were cut with scissors and ground in a glass bowl. A small quantity of water (20 ml) was added to the ground material, and the mixture was suspended in water and centrifuged at room temperature. The precipitate was re-extracted with the same volume of water and the centrifuge supernatant was collected and lyophilized. Lyophilized materials were stored at - 40°C until use.
Toxicity tests The toxicity of the fractions of the extract was tested using mice (20 + 2 g body weight), Calliphora fly larvae ( c 100 mg bodv weight) and cricket Gryllus emma Ohmachi ( z 500 mg body weight) as described by Loret et al. (1992) and_Zlotkin et al. (1987). The lyophilized sample was weighed and dissolved in a modified Harreveld or Krebs solution and injected i.p. into each mouse (100 pl), fly larva (5 ~1) and cricket (2.5 pl). Quantitative estimation of the toxicity of the crude venom and purified toxins in mammals and insects was based on the determination of the dose killing 50% of mice (LD~) by i.p. injection, the minimum lethal dose (MLD) on mice by intracerebroventricular (i.c.v.) injection, or the contraction paralysis unit (CPU) that induces 50% of spastic paralysis in fly larvae or crickets.
Isolation and puriJication of toxins About 550 mg of the BmK venom extract was dissolved in 10 ml of 0.01 M NH,HCO, solution and centrifuged at 1000 g for 10 min. The supernatant was then loaded on a CM-Sephadex (Pharmacia) ion-exchange column
Buthus martensi Neurotoxins
989
was based on the determination of the dose killing 50% of mice (LDm) by i.p. injection, the minimum lethal dose (MLD) on mice by intracerebroventricular (i.c.v.) injection, or the contraction paralysis unit (CPU) that induces 50% of spastic paralysis in fly larvae or crickets.
Isolation and purification of toxins
About 550 mg of the BmK venom extract was dissolved in 10 ml of 0.01 M NH,HCO, solution and centrifuged at 1000 g for 10 min. The supernatant was then loaded on a CM-Sephadex (Pharmacia) ion-exchange column (2 x 65 cm) and eluted stepwise with 0.01, 0.05, 0.10, 0.25 and 0.50 M of NH,HCO, solutions at 20°C at a flow rate of 36 ml/h. Fractions were collected (6 ml each). Eluates were monitored at 280 run with a UV monitor (Uvicord II, L,KB, Broma, Sweden). The neurotoxic fractions were further purified by high-performance liquid chromatography (HPLC) using a Kratos system composed of Spectroflow elements: two 400 solvent delivery systems, a 757 absorbence detector, a 480 injector/valve module and a 450 solvent programmer on a Lichrosorb RP-18 column (7 urn, 0.6 x 25 cm). The eluents employed were 0.15 M ammonium formate (PH 2.7) as solvent A, and 70% acetonitrile in solvent A as solvent B. A linear gradient from 23.8% to 26.6% acetonitrile in solvent A over 40 min was used. The last step of purification of each toxin was an isocratic chromatography with 27.3% acetonitrile in solvent. Elution was monitored at 280 nm and performed at a flow rate of 1.5 ml/min.
Polyacrylamide gel electrophoresis
Assessment of purity of the isolated toxins was carried out on 13% polyacrylamide gel slabs (pH 4.5), according to the method of Reisfeld et al. (1962).
Reduction and S-carboxymethylation
The purified toxins were reduced with a 240-fold molar excess of dithioerythritol in 5 M guanidine HCI, 0.25 M Tri-acetate buffer and 14 mM EDTA (PH 8.6). Reduction was carried out in the dark at 40°C under nitrogen for 20 hr. S-alkylation was then performed at room temperature in the presence of a l.Zfold molar excess of iodoacetic acid (sodium salt) over SH groups under nitrogen for 30 min. The S-alkylated derivatives were dialysed against water at 4°C for 72 hr in a 1 ml Eppendorf tube sealed with Spectra/Par Molecular-porous dialysis membrane (3500 mol. wt cut-off) and then dried with a stream of nitrogen.
Enzymatic cleavages
The reduced and S-carboxymethylated (RCM) toxins were subjected to enzymatic cleavages. For each digestion. 12 or 25 nmoles of sample were used. Digestion with TPCK-treated trvnsin (Sigma) was in 0.2 M NIethylmorpholine buffer, pH 8.1, at 37°C for 4hr using 7.5% or 10% (wii) en&e. ‘Digestion with Staphylococcus aureus V, protease (Miles Laboratories) was done in the above conditions for 40 hr with 5 or 13% (w/w) enzyme. Digestion with carboxypeptidase A (Worthington, Freehold, NJ, U.S.A.) was performed in N-ethylmdrphdline buffer at 37°C with 20% (w/w) em&e and with .stirring. Aliquots were removed at 5, 15, 30, 60, 120 and 240 min after the onset of incubation, acidified with 10% acetic acid, and freeze-dried. The free amino acid content in the digest was assessed in an Amino Acid Analyser (Beckman 6300).
Isolation of peptides
Thin-layer chromatography (TLC) plastic sheets of silica gel 60 (90 x 90 x 0.2 mm, Merck, Germany) were used to isolate the peptides from each enzymatic cleavage. Enzymatic digest (l-2 nmol) were slowly dropped on the left corner of a-TLC-plastic sheet. T&o-dimensional chromatography was performed, the first -dimension in 25% ammonia/ethanol (40160. v/v) and the second in acetic acid/ethanol/water (9/51/40. v/v/v). The dried sheet was then prefixed by spraying with 1% (v/v) ethyl-diisopropylamine in acetone, before’ staining with 0.01% (w/v) fluorescamine in acetone (Hoffman La Roche Laboratories, Switzerland) as described by Fishbein et al. (1980). Stained peptides were scraped from the sheet while monitoring with a UV lamp at 366 nm and carefully collected into a 1 ml Eppendorf tube. Volumes of 5 x 250 ul of 50% acetic acid and 3 x 250 ul of the solvent used in the first dimension were used to extract the collected peptides. The extracts were centrifuged at 3000 g for 5 min, supematants were filtered through a Pasteur pipette (150 x 7 mm) plugged with glass wool and finally dried with nitrogen. One-fifth or one-tenth of the extracts was subjected to amino acid analysis and the remainder was used for automatic Edman degradation in a Beckman 890 M sequencer.
Amino acid composition
Native and RCM-toxin (about 1 nmole) and peptides were hydrolysed with a Pica Tag Station (Waters) in 6.0 N HCl with 1% (w/v) phenol at 110°C for 24 or 70 hr in a tube sealed in vacua. Amino acid analyses were performed in duplicate in a Beckman Amino Acid Analyser.
990
Y. H. JI et al.
Amino acid sequencing The partial amino acid sequences of native toxins were obtained with the DABITCjPITC double-coupling manual method (Chang, 1983). RCM-toxins and peptides were then sequenced in a Beckman 890M()M sequencer using a 0.1 M quadrol program in the presence of Polybrene (Pierce). PTH-derivatives of amino acids were identified with a HPLC system composed of two Beckman 114 M solvent delivery module pumps, a Waters WISP 710 A automatic injector, a Beckman 165 variable wavelength detector and a Kontron Anacomp 220. The elution system was operated according to Hawke et al. (1982).
Electrical recordings The effect of toxins on nerve excitability was examined in a preparation of the lateral giant axon of the crayfish abdominal cord by intracellular recording and under voltage-clamp conditions as described by Terakawa et al. (1989) and Moore (1971). For intracellular recording, a glass microelectrode of 20 Mil tip resistance was inserted into the axon at the circumoesophageal connective.The potential was measured through Ag-AgCl wire placed in the microelectrode and connected to a high-input impedance amplifier (M-707, WP Instruments, New Haven, Connecticut, U.S.A.), and recorded on to videotape after PCM encoding with a DC-modified digital audio processor (PCM-SOIES, Sony, Tokyo, Japan). A modified Harreveld solution containing (mM) NaCl 195, KC1 5, CaClz 2, MgCl* 1, HEPES 1 (pH adjusted to 7.4 by HCl) was used as a physiological saline. For voltage clamping, the lateral giant axon was cleaned of associated small nerve fibers at the circumoesophageal connective for a length of 4 mm. The axon was mounted in an acrylic chamber 5 mm long and 2 mm wide. The chamber was sectioned with two gaps of petroleum jelly (VaselineTM) placed perpendicularly to the axon at an interval of I mm. The central section was filled with the physiological saline and the side sections were filled with 205 mM KCI solution. Two microelectrodes of 15 M.Q tip resistance were inserted into the middle portion of axon in the central section. Two pieces of Ag-AgCl wire placed in respective microelectrodes were both connected to a single input of a potential recorder. Voltage-clamping currents were supplied from the KCI solution in the side sections to the axonal membrane in the central section using the axoplasm as a conductor. The current was measured through a virtual ground made of Ag-AgCl wire placed in the central section, and recorded into a computer using a 16-bit A/D converter. The leak current through the axonal membrane and the gaps was subtracted using a multiple of an inward current induced by a stepwise hyperpolarization of 5 mV.
RESULTS
Purijication of two anti-mammal toxins (BmK I and BmK II) The BmK venom gland extract was tested for toxicity in mice by i.p and i.c.v. injections (LD~~ and MLD: 2.4 and 0.074 mg/kg body weight, respectively). The specific toxicity (i.c.v.) was 676, and blowfly larvae CPU was about 1 ug (Table 1). The extract was separated into 13 fractions on a CM-Sephadex C-50 ion-exchange column. Step gradient chromatography was monitored at 280 nm (Fig. 1A). By qualitative bioassays, fractions XI and XII were found to be the most toxic to mice, as confirmed by the LD~,,values of the two fractions, which were 0.48 and 0.63 mg/kg body weight; the MLD were 0.013 and 0.015 mg/kg body weight, and the specific toxicities (i.c.v.) 3846 and 3333. The CPU of both fractions was determined to be lower than 0.5 ug per blowfly larvae. Fraction XI was submitted to reverse-phase HPLC on a Lichrosorb RP-18 column (0.6 x 25 cm). Two major components, identified as MT I and MT II, were eluted (Fig. 1Ba) and both fractions were further purified by isocratic HPLC, eluting as single peaks (Fig. 1Bb and c). The purity of both polypeptides was assessed by examining a single band in the polyacrylamide gel electrophoresis (data not shown). Finally, the two polypeptides in fractions MT I and MT II were called BmK I and BmK II. Toxicity tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II: the MLD of BmK I and BmK II were 36 ng and 222 ng per mouse of 20 + 2 g, respectively. The value of specific toxicity (i.c.v.) of BmK I was 27,778, 41 times higher than that of venom extract, while that of BmK II was 4505, 6.7 times higher than that of venom extract. Moreover, both toxic polypeptides were also potent in insects. The CPU of BmK I and II was 0.75 and 1.25 ug per cricket, respectively. Table 1 summarizes the quantitative data of the purification of BmK I and II.
IA 1A 1B
1B
XI XII MTI(Bmk I)
MTII(Bmk II) 2.09
409 179 62 3.94
8521 18,646 4921
-
2.4 0.48 0.63
-
‘. * +:;a1 toxicity (Lb
21 104 79 -
/ODZSO)
i.p.* Specific toxicity
injection; Scontraction paralysis unit.
Protein (0~~~ units)
*After intraperitoneal injection; t after intracerebroventricular
HPLC RI’ C-18
Step Venom extract CM-Sephadex C-50
Ref. to Fig.
Toxin fraction
i.p.+ LDB (mg/kg mouse)
0.0111
i.c.v.t MLD (mg/kg mouse) 0.074 0.013 0.015 0.0018 9414
276,351 688,461 206,667 109,444
i.c.v.t Total toxicity
4505
676 3846 3333 27,77X
0.88
0.29
1 0.5
1.25
0.75
2 1.5
i.c.v.7 CPU1 (pg) Specific blowfly CPI-J$ (pg) toxicity larvae cricket (MLD/oD~~ ) (110 + 10 mg) (500 f 20 mg)
Table 1. Quantitative data of the purification of toxins BmK I and II of Buthus martensi Karsch
E3 2 i: G a a 8 ?I. R
L E
Y. H. JI et al.
992
Amino acid compositions of‘ BmK I and II The amino acid compositions of native and RCM-BmK I and II are listed in Table 2. Both toxins are composed of 64 amino acid residues. There are no Met or Phe residues. The differences between BmK I and II appear at positions 59 (Val, Ile) and 62 (Lys, Asn).
Amino acid sequence of BmK I Native BmK I (about 10 nmoles) was first sequenced with the manual DABITCjPITC about 8 nmoles of RCM-BmK I were double-coupling method. Alternatively,
__
0.01
__tc__
w
1
I
111
-
0.05
0.10
--I--0.25
+
0.50
Xf
IV 3 XII
Q VI X
300
600
900
EffIuent
(b)
I ‘ig. 1. (A) Ion-exchange
1200
(ml)
(4
MT1
”
0.006
_____~_ _____........-. __-__..
J
XIII
MT II
0-
_
5 I
x
.. . J 20
\
chromatography of the venom of the scorpion B. martensi Karsch on a CM-Sephadex C-50 column. The venom extract containing about 550 mg of protein was loaded on the column (2 x 65 cm). The concentration of NH4HC03 (elution buffer) is indicated on the top. Flow rate: 36 ml/hr, 6 ml/tube. (B) HPLC patterns of (a) fraction XI, (b) MT 1, and (c) MT II. (a) A 0.2 mg sample of fraction XI, (b) 20 pg of MT I (BmK I), and (c) 15 ug of MT II (BmK II) dissolved in 20 u1 of water were injected. Column: Lichrosorb RP-18 (7 urn, 0.6 cm ID x 25 cm). Elution buffer: (a) solvent A:O. 15 M ammonium formate, pH 2.7; (b,c) 0.1% TFA-water. Solvent B: 70% acetonitrile. AUFS: (a) 0.12; (b,c) 0.015 at 280 nm. Flow rate: 1.5 ml/mm The ordinate to the right indicates the concentration of acetonitrile in the elution buffer (broken line).
e
*
Buthus martensi
Neurotoxins
993
Table 2. Amino acid compositions of toxins BmK I and II Amino acid
BmK I Native protein
CYS
BmK II RCM-protein
RCM-protein
6.78 (8)
7.16 (8)
CMC Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His LYS Arg Trp Total
Native protein
(8) (9) (1) (1) (4) (4) (6) (4) (5)
9.16 (10) 0.96 (1) 1.00 (1) 3.62 (4) 3.88 (4) 6.27 (6) 4.12 (4) 4.15 (4)
6.80 (8) 9.59 (10) 0.96 (1) 0.99 (1) 4.02 (4) 4.14 (4) 6.18 (6) 4.13 (4) 3.91 (4)
2.88 (3) 2.03 (2) 4.25 (5)
2.98 (3) 2.05 (2) 4.52 (5)
3.80(4) 2.02 (2) 3.94 (5)
3.69 (4) 2.06 (2) 4.33 (5)
1.97(2) 5.00 (5) 3.17 (3) -_(2) 64
1.98(2) 5.04 (5) 3.48 (3) -_(2) 64
2.00(2) 4.32 (4) 3.10 (3) -_(2) 64
1.89 (2) 4.15 (4) 3.38 (3) -(2) 64
8.75 0.98 0.94 4.01 3.97 6.21 4.10 4.85
(9) (1) (1) (4) (4) (6) (4) (5)
6.83 8.72 0.95 0.93 3.89 4.19 6.28 4.14 4.92
Experimental values are deduced from two duplicate analyses after 20 and 70 hr hydrolyses. Numbers in parentheses indicate the number of residues deduced from the sequence analysis.
automatically degraded in a Beckman 890 MM protein sequencer. Sequence analysis occurred from the N-terminal position up to position 31, with ambiguous identification of positions 29 and 30 (Fig. 2a). For further sequencing, RCM-BmK I (25 nmoles) was digested by S. aureus V, and subjected to TLC on 12 TLC-silica gel 60 sheets (90 x 90 x 0.2 mm). As shown in Fig. 3A, the chromatography profile contains eight fluorescing peptide traces of which six, marked V,-1 to V,-6, were collected. Taking into
(A)
BmK
I
(a) RCM-BmK
I VRDAYIAKPHNCVYECARNEYCNDLCTK
LPDNVPIRVPGKCH
(b) vg-1 (c) vg-5 (d) T-11
BntK II (B)
(e)RCM-BmK (r')x78-5 (c) T-9 (h) VS-3 (i.) T-4
____
??A_____----____-----_______----
LCTKDGAKSGYCQWVGKYG------GAKSGYCQWVGKYG??CWC-YGNGCWCIEL-------
VRDAYIAKPHNCVYECARNEYCNDLCTKDGAKSGYCQWGKYGNGCWCIELPDNVPIRIPGNCH II VRDAYIAKPHNCVYECARNEYCNDLCTKDGAK--------------------------------LCTKDGAKSGYCQWVGKYG------YGNGCWCIELPD----LPDNVPIRIPGNCH IPGNCH
Fig. 2. Determination of the amino acid sequences of BmK I and BmK II. (A) (a) Automatic degradation of RCM-BmK I (8 nmoles) in a Beckman 890 M sequencer: average yield =: 86%; (b) degradation of peptide 5164 (V,-1, 7.5 nmoles): average yield = 85%; (c) degradation of peptide 2550 (V,-5, 7 mnoles): average yield = 82%; (d) degradation of peptide 42-58 (T-11, 4.5 nmoles): average yield = 92%. (B) (e) Automatic degradation of RCM-BmK II (10 mnoles): average yield = 88%; (t) degradation of peptide 25-50 (V,-5, 4 nmoles): average yield =: 81%; (g) degradation of peptide 42-58 (T-9, 4 nmoles): average yield = 79%; (h) degradation of peptide 5164 (J,-3, 1.5 nmoles): average yield = 80%; (i) degradation of peptide 59-64 (T-4, 3 nmoles): average Yield = 78%.
Y. H. JI et al.
994
(W
(4 Vs-5 e ,O Vs-6 + v*-4
+‘I--11
T_9
h T-7 l ) T-8
A
T-10 T-6 t
T;2
a T-5
1
l T-4
T-l
T!3 *.
W
T-9,
‘;” I T-l
1 T-4 t T-3
First Dimension 25% NH3/Ethanol (40/60, Fig. 3. Separation of peptides of RCM-BmK I and RCM-BmK II on TLC sheets. Enzymatic digest (l-2 nmoles) was slowly dropped on the left corner. (A) Cleavage of RCM-BmK I with S. aureu~ V,. (B) Cleavage of RCM-BmK I with trypsin. (C) Cleavage of RCM-BmK II with S. ~UY~USV,. (D) Cleavage of RCM-BmK II with trypsin.
account the amino acid compositions of peptides (Table 3) only V,-1 and V,-5 were subjected to sequencing. The sequence of V,-1 (7.5 nmoles) was determined and assumed to be located at the C-terminal region, from position 51 to the end of the protein (Fig. 2b). The V,-5 peptide (7 nmoles) accounted for positions 25-43 (Fig. 2~). This sample contained about 0.9 nmole of a peptide, the sequence of which corresponded to position 3048. To RCM-BmK I was subjected to tryptic digestion. establish the complete sequence, Figure 3B shows the chromatography of tryptic peptides of RCM-BmK I (25 nmoles) on a TLC sheet. There were at least 11 peptides, marked T-l to T-l 1 (amino acid compositions in Table 4). Of these tryptic peptides, T-l 1 was sequenced (ten residues) and found to overlap with the sequence of the fragments V,-1 and V,-5. Finally, the complete amino acid sequence of BmK I was established by the link of T-11 fragment (Fig. 2d).
Buthus mnrtensi Neurotoxins
995
Carboxypeptidase A digestion of RCM-BmK 1 was carried out to confirm the C-terminal residue. The kinetic of release is in favour of the C-terminal sequence Cys-His. The complete sequence of BmK I is shown in Fig. 2A. Amino acid sequence of BmK II The automatic degradation of RCM-BmK II (10 nmoles) gave the sequence l-32, which was identical to that of BmK I (Fig. 2e). The TLC separation of the RCM-BmK II digests obtained with trypsin and S. aureus V, is shown in Fig. 3 (C and D). The amino acid compositions of enzymatic fragments are listed in Tables 5 and 6. Of these enzymatic peptides, V,-5 and VB-3, T-9 and T-4 corresponded to the sequence of positions 25-50 (Fig. 2f), 5.1-64 (Fig. 2h), 42-58 (Fig. 2g), and 59-64 (Fig. 2i), respectively. The sequencing of these four enzymatic peptides led to the determination of the complete amino acid sequence of BmK II (Fig. 2B). Furthermore, the C-terminal residue, e.g. His-64 in BmK II, was directly defined by Edman degradation of enzymatic peptides V,-3 and T-4. Electrophysiological e#ect of BmK I The effect of BmK I on nerve excitability was examined in the crayfish axon model (Fig. 4). Intracellular recording showed that the toxin prolonged the descending phase of the action potential without affecting the rising phase (Fig. 4A). Under voltage clamp (Fig. 4B), BmK I greatly prolonged the inward current. No outward current was observed during the clamping pulse of 5 msec. Addition of tetrodotoxin (0.1 PM) to the external medium in the presence of BmK I eliminated this inward current completely and left the outward current almost the same as that observed before the application of scorpion toxin.
Table 3. Amino acid compositions of S. aureus Ve peptides obtained from RCM-BmK I Amino acid CMC Asp Thr Ser GlU Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Trp Total Yield (%)
S. aureus Va Pevtides V,-3 l&20* -V,-4 21-24*
v*-2 1-3*
Vs-6 415*
0.99 (1)
1.18 (1) 1.27 (1)
0.28 (1) 1.10 (1)
1.22 (1) 1.07 (1)
1.07 (1)
0.77 (1) 2.13 (2) -
0.62 (1)
1.72<2) 1.08 (1)
0.80 (1)
1.44 (2) -
2.70 2.16 0.83 0.91 2.21
(4) (2) (1) (1) (2)
4.85 (5) 1.10 (1) 1.06 (1)
0.87 (1)
0.91(l)
V,-5 25-50*
0.93 (1) 0.69 (1) 1.42 (2)
-
Traces (1)
0.87 (1) 1.04 (1) -
Traces (1)
3 80
12 80
5 90
2.57 (3) 4 80
-(2) 26 56
Experimental values are deduced from duplicate analyses after 20 hr hydrolyses. Numbers in parentheses indicate the number of residues deduced from the sequence analysis. *Sequence positions.
v,-1 51-64* Traces (1) 1.09 (2)
2.72 (3) 1.14 (1) 1.83 (2) 0.87 (1) 0.66 (1)
0.88 (1) 0.93 (1) Traces (1) 14 60
ti
B 3 :: >;
02 11 (I)(1) 86’0
08 c!,
-
(I) SI'I
88 SZ (z)i;; Sk::
PS 91
I;; g:; (1) VS.0 (2) LS’I (1) EO’I
I;; ;y:; (z) 28’1
(I) PE’I
(I) 7.8’0 7 (5) LE’Z
I;,’ :;:; (z) OI’Z (I) - 51’1
(I) Z6'0 (El SI'E (0 PO‘Z *8'?EE 6-L
*81-E 8-L
i;; g:;
(E) E8'Z
(I) zo.1 I;; ;;:;
01-L
(Z) 58'1
(2) LI’Z I
*IbEE
(I) LO'1 (I) 06'0
;;; ;;:I (I) LO'1
*8S-ZP II-L
BP 01
~
(I) ZL'O
I;; g:;
ecP9-E9P-l
(1) L8’0
(I) PZ’I
09 2
*81-6 E-l
(I) SO'1 (I) 28'0
(1) LO'1 (1) 86'0
(I) f8’0
(I) LI’I
(I) 68’0
8 01
+ZE-6Z Z-Z
(I) f6’0
i;; ii:;
(I) 86’0
PZ P
~s!s~IEu~ amanbas ay] u10g pasnpap sanp!sa.t 30 laqurnu ay] almpu! .sasblolp6q ‘q OZ lal3” sasKpzue alwydnp u.1013 paanpap PZ P
~
9f Z
(I) 98’0
(I) 6L’0
(I) 89’0
-
1
-
-
(I) 96'0
-
7
(1) 56’0
(Z) L8‘0
;:; ;;:;
*Z-I 9-L *Z9-65 S-J sapgdad 3gdi(lL
*8Zp61 L-J.
py
OUyIIV
3MI3
"10 Ias 1Y.L dsv
old
I"‘4 "IV 4t)
w
sh S!H ayd “6.L na7 alI
BJV
dlL
(%) Pl3!A WoL
woysod manbag, sasay]ualed u! s.raqumN am sanIv,t [Eluau+adxa
8P PI
(z) OO’Z
(I) ZL’O -_(I) L6‘0
(I) 62'1 (I) 69'0
(I) 18'0
(1) 01'1
*ZEp61 I-L
Buthus martensi Neurotoxins
997
These findings indicate that BmK I binds to the sodium channel and blocks its inactivation process. DISCUSSION
Although the Chinese scorpion B. martensi Karsch is generally not regarded as being dangerously venomous, the venom is considered to be a “rather active toxin” (Balozet, 1971), suggesting that it probably contains toxic components. The toxicity (LDSo) of this venom to mice was found to be 2.4 mg/kg body weight, which is moderate in comparison with venom of other species. From fraction XI, the major toxic component, two toxic anti-mammal polypeptides (BmK I and BmK II) were isolated. The toxicities of these toxins are lower than those of toxins purified from other dangerous venomous species. Miranda et al. (1970) were the first to report a general method of purification of scorpion toxins. The method involved (1) extraction by distilled water, (2) gel filtration with recycling on Sephadex G-SO, and (3) ion-exchange equilibrium chromatography. We have shortened this method in the present work by using only cation-exchange equilibrium chromatography on a CM-Sephadex C-50 column (Fig. lA), followed by HPLC, enabling us to obtain two anti-mammal toxins, BmK I and II (Fig. 1B). When BmK I was added to a crayfish axon preparation, there was incomplete inactivation of Na+ channels, an effect common to a-type toxins. When the amino acid sequences of BmK I and BmK II were compared to those of other scorpion toxins, it was found that the sequences of both toxins, as other a-toxins, differed significantly from those of P-type neurotoxins isolated from the venoms of South American and North American scorpions (group 5). These sequences showed a high degree of similarity with toxins of group 3 (Fig. 5) according to the classification of Rochat et al. (1979). Nevertheless, it is interesting to note that the C-terminal ends of BmK toxins are unmodified, whereas the ends of other toxins of group 3 are amidated. In addition, the two toxins are potent in
Table Amino
5. Amino
acid compositions
of S. aureus V8 peptides
acid VR-1 l-3*
CMC Asp Thr Ser Glu Pro Gly Ala Val Met Be Leu
1.02 (1)
v*-2 1620* 0.24 (1) 0.86 (1)
from RCM-BmK
0.48 (1) 2.90 (3)
0.94 (1) 2.02 (2)
0.93 (1) 2.67 (3) 1.04 (1) 0.13 (1)
V,-5 25-50* 2.94 2.20 0.71 1.07 2.15
(4) (2) (1) (1) (2)
5.09 (5) 1.07 (1) 0.90 (1)
0.95 (1) 0.48 (1) 1.41 (2) 0.48 (1) 0.61 (1)
Tyr Phe His Lys Arg Trp Total Yield (%)
obtained
S. aurew V, peptides V,-3 51-64* V,-4 21-24*
0.88 (1) 0.60 (1) 1.23 (2)
1.24 (1) 2.83 (3) 0.47 (1)
1.07 (1)
0.53 (1)
3 22
5 37
14 9
Experimental values are deduced Numbers in parentheses indicate *Sequence positions.
4 45
from duplicate analyses after 20 hr hydrolyses. the number of residues deduced from the sequence
-(2) 26 99 analysis
II V,-6 4-15* 1.33 (1) 1.49 (1)
1.38 (1) 0.99 (1) 1.79 (2) 0.93 (1) 0.97 (1) 1.08 (2) 0.84 (1) 1.02 (1)
12 45
998
Y. H. JI et d.
1 llllIll;lllllIl~l”~
6
,’
Buthus martensi Neurotoxins
999
both mammals and insects. Another toxin (LQQ III) from the scorpion Leiurus quinquestriatus quinquestriatus, which belongs to group 3, was recently found to have the same characteristics (Kopeyan et al., 1993). The structure-activity relationships of scorpion neurotoxins have been extensively studied by modifying particular residues involved in toxic and pharmacological activities. It has been demonstrated that the charged residues located in the N-terminal and C-terminal regions of scorpion toxins play a multiple role in the interaction with proteic components of the sodium channel. For example, the modification of Lys-58 in AaH II by N-hydroxysuccinimidyl biotin results in an almost complete loss of activity. The modification of the homologous lysine in Lqq V reduces activity to 20% (Darbon et al., 1983). An analogue of AaH II, Bot XI, in which Lys-58 is replaced by Val-58, is 250-fold less active (Sampieri et al., 1987). This loss of toxicity might be explained by this substitution. A single modification of Lys-60 in Lqq V and of Arg-60 in AaH I results in residual activities of 40 and 14%. When Arg-56 in AaH II is modified with phenylglyoxal, the residual activity of the toxin is 20% (Kharrat et al., 1990). In addition to the above demonstrations, this study found valuable evidence
(A:) (a)
Control
0 -50 -100 mV IL 0
5
10 1s msec
(B:)
I
A------
a. Control b. BmK 1 c. BmK I + TTX d. Membrane potential
1 ms
40 mV
Fig. 4. Effects of BmK I on a crayfish lateral giant axon. (A) Eilect of BmK I on intracellularly recorded action potentials: (a) control record of the crayfish action potential; (b, c) records obtained 3 and 15 min after application of 0.3 pm01 BmK I, respeci.ively. (B) Effect of BmK I under voltage clamp: (a) control record of ionic currents; (b) record after application of BmK I; (c) record after application of 0.1 pmol TTX; (d) record of the membrane potential during the voltage clamp.
Y. H. JI
1000
et al.
BOf II
G______~_E_______~._~________~_________~_~~__~_~__~___~____~~_
___,z
Born III Be M9 BeMI4
G--G---Q-E----H-FPGSSG-DT--KEK--T-H.-GFLPGS-”A---DN--NK---V-G-E--_ ~_______________~~K~~___~_._EN..E_----IL-____~____Q________~__ ~______,,,,~_.._T..L -P--DSE-K-N--D~~____L-RF__~___K~___~____K,_~~_~
_..
of the amino acid sequence of BmK I and II with that of group 3 according to the classification by Rochat et al. (1979). BmK I and II: toxins I and II from the scorpion Buthus martensi Karsch (Asia). Lqq III: toxin III from the scorpion Leiurus quinquestriatus quinquestriatus (Sudan). Bot I and II: toxins I and II from the scorpion &thus occitanus tunetanus (Algeria). Born III: toxin II from the scorpion Buthus occitanus Mardochei (Morocco). Be M9 and M14: toxins M9 and Ml4 from the scorpion Buthus epeus (Asia). Fig. 5. Comparison
for the important role in the bioactivity of toxins of a basic residue in the C-terminal region (here, position 62). It is interesting to note that BmK I was six times more potent than BmK II. The conservative mutation in position 59 seems to be unimportant in terms of activity. When comparing the sequences of the two toxins (Fig. 5), the only significant difference is in position 62, where a basic residue (Lys) is found in BmK I, while a neutral residue (Asn) is found in BmK II. The presence of a positive charge in C-terminal position 62 may be important for the binding of BmK I to the receptor on sodium channels. This conversion could explain the lower toxicity of BmK II. Another illustration of the importance of the basic residue in this part of the molecule is given by the toxicity of AaH IV (group l), which was recently found to be at least four times lower than that of others (Mansuelle et al., 1992). The low toxicity of AaH IV may result from two substitutions in the C-terminal positions 60 and 61, where two basic residues (Arg-60 and Lys-61), responsible for strong toxicity in other neurotoxins, were replaced by two acidic residues (Asp-60 and Asp-61) in AaH IV. These observations suggest that positively charged residues (Lys or Arg) at position 62, and generally in the C-terminal region, play an important role in maintaining the potency of scorpion neurotoxins.
Acknowledgements-We are greatly indebted to Drs C. Granier and M. Hoshino for their fruitful discussions, and to Mrs T. Brando for her kind help in operating the amino acid analyser. The present study was supported by a fellowship from the CNRS of France, granted to Y. H. Ji and in part by the Grant-in-Aid for the International Scientific Research Program from the Ministry of Education, Science and Culture of Japan.
REFERENCES Balozet, L. (1971) Scorpionism in the old world. In: Venomous Animals and Their Venoms, Vol. III, Venomous Invertebrates, Ch. 56, pp. 3499371 (Biicherl, W. and Buckley, E. E., Eds). New York: Academic Press. Catterall, W. A. (1988) Structure and function of voltage-sensitive ion channels. Science 242, 50-61. Chang, J. Y, (1983) Manual micro-sequence analysis of polypeptides using dimethylaminoazobenzene isothiocyanate. Meth. Enzymol. 91, 455466. Couraud, F., Rochat, H. and Lissitsky, S. (1978) Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells. Biochem. biophys. Res. Commun. 83, 1525-1530. Couraud, F., Jover, E., Dubois, J. M. and Rochat, H. (1982) Two types of scorpion toxin receptor sites, one related to the activation, the other to the inactivation of the action potential sodium channel. Toxicon 20,9-16. Darbon, H., Jover, E., Couraud, F. and Rochat, H. (1983) Scorpion neurotoxin derivatives suitable as potential markers of sodium channels. Znt. J. Pept. Prof. Res. 22, 179-186.
Buthus martensi Neurotoxins
1001
Fishbein, J. C., Place, A. R., Roposon, I. J., Powers, D. A. and Sofer, W. (1980) Thin-layer peptide mapping: quantitative analysis and sequencing at the nanomole level. Analyt. Biochem. 108, 193-201. Fontecilla-Camps, J. C., Almassy, R. J., Suddath, F. L., Watt, D. D. and Bugg, C. E. (1980) Three-dimensional structure of a protein from scorpion venom: a new structural class of neurotoxins. Proc. natn. Acad. Sci. U.S.A.
77, 64966500.
Gregoire, J. and Rochat, H. (1983) Covalent structures of toxin I and II from the scorpion Buthus occitanus tunetanus. Toxicon 21, 153-162. Hawke, D., Yuan, P. M. and Shively, J. E. (1982) Separation of PTH amino acid by reverse phase HPLC. Analyt. Biochem. 120, 302-3 11.
Jover, E., Couraud, F. and Rochat, H. (1980) Two types of scorpion neurotoxins characterized by their binding to two separate receptor sites on rat brain synaptosomes. Biochem. biophys. Res. Commun. 95, 1607-1614. Jover, R., Martin-Moutot, N., Couraud, F. and Rochat, H. (1980) Binding of scorpion toxins to rat brain synaptosomal fraction, effects of membrane potential, ions and other neurotoxins. Biochemistry 19, 463467. Keegan, H. L. (1980) Scorpions of Medical Importance, p. 41. University Press of Mississippi, U.S.A. Kharrat, R., Darbon, R., Granier, C. and Rochat, H. (1990) Structure-activity relationships of scorpion a-neurotoxins: contribution of arginine residues. Toxicon 28, 5099523. Kopeyan, C., Martinez, C., Lissitzky, S., Miranda, F. and Rochat, H. (1974) Disulfide bonds of toxin II of the scorpion Androctonus austrlais Hector. Eur. J. Biochem. 47, 483489. Kopeyan, C., Mansuelle, P., Martin-Eauclaire, M. F., Rochat, H. and Miranda, F. (1993) Characterization of Toxin III of the scorpion Leiurus quinquestriatus quinquestriatus: a new type of alpha-toxin highly toxic both to mammalian and insects. Nat. Toxins 1, 308-312. Loret, E. P., Sampieri, R., Granier, C., Miranda, F. and Rochat, H. (1992) Scorpion toxins affecting insects. Meth. Neurosci. 8, 381-395.
Mansuelle, P., Martin, M.-R., Rochat, H. and Granier, C. (1992) The amino acid sequence of toxin IV from the Androctonus australis scorpion: differing effects of natural mutations in scorpion a-toxin on their antigenic and toxic properties. Nat. Toxins 1, 6169. Miranda, F., Kopeyan, C., Rochat, H., Rochat, C. and Lissitzky, S. (1970) Purification of animal neurotoxins. Eur. J. Biochem. 16, 514523.
Moore, J. W. (1971) Voltage clamp methods. In: Biophysics and Physiology of Excitable Membranes, pp. 143-167 (Adelman, W. J., Jr, Ed.). New York: Van Nostrand Reinhold. Ovchinikov, Yu. A. and Grishin, E. V. (1982) Scorpion neurotoxins as tools for studying fast sodium channels. Trends biochem. Sci. 7, 2628.
Reisfeld, R. .4., Lewis, U. J. and Williams, D. E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195, 281-283. Rochat, H., Bernard, P. and Couraud, F. (1979) Scorpion toxins: chemistry and mode of action. Adv. Cytopharmac. 3, 325-334.
Sampieri, F., Rochat, C., Martin, M.-R., Kopeyan, C. and Rochat, H. (1987) Amino acid sequence of toxin XI of the scorpion Buthus occitanus tunetanus. Int. J. Pept. Prot. Res. 29, 231-237. Terakawa, S., Kimura, Y., Hsu, K. and Ji, Y. H. (1989) Lack of effect of a neurotoxin from the scorpion Buthus martensi K.arsch on nerve fibers of this scorpion. Toxicon 27, 569-578. Zlotkin, E., Miranda, F. and Rochat, H. (1987) Chemistry and pharmacology of Buthinae scorpion venom. Arthropod Venoms 317, 369.
Toxicon, Vol. 34, No. 9, pp. 987-1001, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Gnat Britain. All rights reserved cul41~101/96 $15.00 + 0.00
TWO NEUROTOXINS (BMK I AND BMK II) FROM THE VENOM OF THE SCORPION BUTHUS MARTENSI KARSCH: PURIFICATION, AMINO ACID SEQUENCES AND ASSESSMENT OF SPECIFIC ACTIVITY YONG HUA JI,’ P. MANSUELLE; S. TERAKAWA,3 C. KOPEYAN,’ N. YANAIHARA,4 KE HSU’ and H. ROCHAT’* ‘Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai, 200031, P.R. China, ZLaboratoire de Biochimie, CNRS URA 1455, IFR Jean Roche, Faculte de Medecine Secteur Nord, Bd. Pierre Dramard, 13916, Marseille, Cedex 20, France, ‘Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan and 4Laboratory of Bioorganic Chemistry, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422, Japan (Received
12 January 1996; accepted 2 April 1996)
Y. H. Ji, P. Mansuelle, S. Terakawa, C. Kopeyan, N. Yanaihara, K. Hsu and H. Rochat. Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity. Toxicon 34,987-1002, 1996.-Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthu,s martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V, protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus,
Abbreviations: BmK I and BmK II, toxins I and II from the scorpion Buthusmartensi Karsch; AaH I, II, III and IV, toxins I, II, III and IV from the scorpion Androctonus astral Hector (North Africa); Lqq III and V, toxins III and V from the scorpion Leiurus quinquestriatw quinquestriatus (Sudan); Bot I, II and XI, toxins I, II and XI from the scorpion Ruth occitanus tunetanus (Algeria); Be M9 and M14, toxins M9 and Ml4 from the scorpion Euthus epeus (Asia); CMC, carboxymethyl-cysteine; DABITC, 4-N,N-dimethylaminoazobenzene-4’isothiocyanate; MLD, minimum lethal dose; RCM, reduced and S-carboxymethylated, TTX, tetrodotoxin; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone. 987
988
Y. H. Jl et al.
functional and structural similarities suggest that BmK I and BmK II belong to group 3 of scorpion a-type toxins. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
To date, more than 100 toxins have been isolated and identified in venoms of scorpions belonging to the Buthidae family (Rochat et al., 1979; Keegan, 1980). Most constitute a family of basic, low mol. wt, single-chain polypeptides, cross-linked by four intramolecular disulfide bridges (Kopeyan et al., 1974; Fontecilla-Camps et al., 1980; Gregoire and Rochat, 1983). They have a high affinity for the voltage-dependent sodium channel with different species specificity and thus are categorized as anti-mammal and/or anti-insect toxins (Rochat et al., 1979; Loret et al., 1992). Anti-mammal toxins have been further divided into two groups, a-toxins and B-toxins, according to their mode of action: a-toxins, the binding of which on the membrane is potential dependent, prolong the Na+ inactivation phase of the action potential, while p-toxins affect the Na+ activation phase (Jover et al., 1980a, 1980b; Couraud et al., 1978, 1982). Both types of toxin bind to distinct sites on the sodium channel. Scorpion neurotoxins have thus been extensively promoted as tools for investigating the components and molecular mechanism of sodium channels (Ovchinikov et al., 1982; Catterall, 1988). The Asian scorpion Buthus martensi Karsch (BmK) is a species belonging to the Buthidae family. It is widely distributed from northern China to Mongolia and Korea. In Chinese traditional medicine, even at the present time, the scorpion is still used as a drug to treat neurological symptoms, such as incomplete paralysis and mimetic paralysis. However, little knowledge is available of the neurotoxic components of this species. In the present study, the purification, amino acid sequences and electrophysiological properties of two anti-mammal neurotoxins (BmK I and BmK II) from the Chinese scorpion B. martensi Karsch are described. MATERIALS
AND
METHODS
Extraction of venom gland About 5000 adult (length approx. 7 cm) B. martensi Karsch (BmK) were purchased from local culture farms in Henan and Shandong Provinces, China. Telsons (the last caudal segments containing the venom gland) were cut with scissors and ground in a glass bowl. A small quantity of water (20 ml) was added to the ground material, and the mixture was suspended in water and centrifuged at room temperature. The precipitate was re-extracted with the same volume of water and the centrifuge supernatant was collected and lyophilized. Lyophilized materials were stored at - 40°C until use.
Toxicity tests The toxicity of the fractions of the extract was tested using mice (20 + 2 g body weight), Calliphora fly larvae ( c 100 mg bodv weight) and cricket Gryllus emma Ohmachi ( z 500 mg body weight) as described by Loret et al. (1992) and_Zlotkin et al. (1987). The lyophilized sample was weighed and dissolved in a modified Harreveld or Krebs solution and injected i.p. into each mouse (100 pl), fly larva (5 ~1) and cricket (2.5 pl). Quantitative estimation of the toxicity of the crude venom and purified toxins in mammals and insects was based on the determination of the dose killing 50% of mice (LD~) by i.p. injection, the minimum lethal dose (MLD) on mice by intracerebroventricular (i.c.v.) injection, or the contraction paralysis unit (CPU) that induces 50% of spastic paralysis in fly larvae or crickets.
Isolation and puriJication of toxins About 550 mg of the BmK venom extract was dissolved in 10 ml of 0.01 M NH,HCO, solution and centrifuged at 1000 g for 10 min. The supernatant was then loaded on a CM-Sephadex (Pharmacia) ion-exchange column
Buthus martensi Neurotoxins
989
was based on the determination of the dose killing 50% of mice (LDm) by i.p. injection, the minimum lethal dose (MLD) on mice by intracerebroventricular (i.c.v.) injection, or the contraction paralysis unit (CPU) that induces 50% of spastic paralysis in fly larvae or crickets.
Isolation and purification of toxins
About 550 mg of the BmK venom extract was dissolved in 10 ml of 0.01 M NH,HCO, solution and centrifuged at 1000 g for 10 min. The supernatant was then loaded on a CM-Sephadex (Pharmacia) ion-exchange column (2 x 65 cm) and eluted stepwise with 0.01, 0.05, 0.10, 0.25 and 0.50 M of NH,HCO, solutions at 20°C at a flow rate of 36 ml/h. Fractions were collected (6 ml each). Eluates were monitored at 280 run with a UV monitor (Uvicord II, L,KB, Broma, Sweden). The neurotoxic fractions were further purified by high-performance liquid chromatography (HPLC) using a Kratos system composed of Spectroflow elements: two 400 solvent delivery systems, a 757 absorbence detector, a 480 injector/valve module and a 450 solvent programmer on a Lichrosorb RP-18 column (7 urn, 0.6 x 25 cm). The eluents employed were 0.15 M ammonium formate (PH 2.7) as solvent A, and 70% acetonitrile in solvent A as solvent B. A linear gradient from 23.8% to 26.6% acetonitrile in solvent A over 40 min was used. The last step of purification of each toxin was an isocratic chromatography with 27.3% acetonitrile in solvent. Elution was monitored at 280 nm and performed at a flow rate of 1.5 ml/min.
Polyacrylamide gel electrophoresis
Assessment of purity of the isolated toxins was carried out on 13% polyacrylamide gel slabs (pH 4.5), according to the method of Reisfeld et al. (1962).
Reduction and S-carboxymethylation
The purified toxins were reduced with a 240-fold molar excess of dithioerythritol in 5 M guanidine HCI, 0.25 M Tri-acetate buffer and 14 mM EDTA (PH 8.6). Reduction was carried out in the dark at 40°C under nitrogen for 20 hr. S-alkylation was then performed at room temperature in the presence of a l.Zfold molar excess of iodoacetic acid (sodium salt) over SH groups under nitrogen for 30 min. The S-alkylated derivatives were dialysed against water at 4°C for 72 hr in a 1 ml Eppendorf tube sealed with Spectra/Par Molecular-porous dialysis membrane (3500 mol. wt cut-off) and then dried with a stream of nitrogen.
Enzymatic cleavages
The reduced and S-carboxymethylated (RCM) toxins were subjected to enzymatic cleavages. For each digestion. 12 or 25 nmoles of sample were used. Digestion with TPCK-treated trvnsin (Sigma) was in 0.2 M NIethylmorpholine buffer, pH 8.1, at 37°C for 4hr using 7.5% or 10% (wii) en&e. ‘Digestion with Staphylococcus aureus V, protease (Miles Laboratories) was done in the above conditions for 40 hr with 5 or 13% (w/w) enzyme. Digestion with carboxypeptidase A (Worthington, Freehold, NJ, U.S.A.) was performed in N-ethylmdrphdline buffer at 37°C with 20% (w/w) em&e and with .stirring. Aliquots were removed at 5, 15, 30, 60, 120 and 240 min after the onset of incubation, acidified with 10% acetic acid, and freeze-dried. The free amino acid content in the digest was assessed in an Amino Acid Analyser (Beckman 6300).
Isolation of peptides
Thin-layer chromatography (TLC) plastic sheets of silica gel 60 (90 x 90 x 0.2 mm, Merck, Germany) were used to isolate the peptides from each enzymatic cleavage. Enzymatic digest (l-2 nmol) were slowly dropped on the left corner of a-TLC-plastic sheet. T&o-dimensional chromatography was performed, the first -dimension in 25% ammonia/ethanol (40160. v/v) and the second in acetic acid/ethanol/water (9/51/40. v/v/v). The dried sheet was then prefixed by spraying with 1% (v/v) ethyl-diisopropylamine in acetone, before’ staining with 0.01% (w/v) fluorescamine in acetone (Hoffman La Roche Laboratories, Switzerland) as described by Fishbein et al. (1980). Stained peptides were scraped from the sheet while monitoring with a UV lamp at 366 nm and carefully collected into a 1 ml Eppendorf tube. Volumes of 5 x 250 ul of 50% acetic acid and 3 x 250 ul of the solvent used in the first dimension were used to extract the collected peptides. The extracts were centrifuged at 3000 g for 5 min, supematants were filtered through a Pasteur pipette (150 x 7 mm) plugged with glass wool and finally dried with nitrogen. One-fifth or one-tenth of the extracts was subjected to amino acid analysis and the remainder was used for automatic Edman degradation in a Beckman 890 M sequencer.
Amino acid composition
Native and RCM-toxin (about 1 nmole) and peptides were hydrolysed with a Pica Tag Station (Waters) in 6.0 N HCl with 1% (w/v) phenol at 110°C for 24 or 70 hr in a tube sealed in vacua. Amino acid analyses were performed in duplicate in a Beckman Amino Acid Analyser.
990
Y. H. JI et al.
Amino acid sequencing The partial amino acid sequences of native toxins were obtained with the DABITCjPITC double-coupling manual method (Chang, 1983). RCM-toxins and peptides were then sequenced in a Beckman 890M()M sequencer using a 0.1 M quadrol program in the presence of Polybrene (Pierce). PTH-derivatives of amino acids were identified with a HPLC system composed of two Beckman 114 M solvent delivery module pumps, a Waters WISP 710 A automatic injector, a Beckman 165 variable wavelength detector and a Kontron Anacomp 220. The elution system was operated according to Hawke et al. (1982).
Electrical recordings The effect of toxins on nerve excitability was examined in a preparation of the lateral giant axon of the crayfish abdominal cord by intracellular recording and under voltage-clamp conditions as described by Terakawa et al. (1989) and Moore (1971). For intracellular recording, a glass microelectrode of 20 Mil tip resistance was inserted into the axon at the circumoesophageal connective.The potential was measured through Ag-AgCl wire placed in the microelectrode and connected to a high-input impedance amplifier (M-707, WP Instruments, New Haven, Connecticut, U.S.A.), and recorded on to videotape after PCM encoding with a DC-modified digital audio processor (PCM-SOIES, Sony, Tokyo, Japan). A modified Harreveld solution containing (mM) NaCl 195, KC1 5, CaClz 2, MgCl* 1, HEPES 1 (pH adjusted to 7.4 by HCl) was used as a physiological saline. For voltage clamping, the lateral giant axon was cleaned of associated small nerve fibers at the circumoesophageal connective for a length of 4 mm. The axon was mounted in an acrylic chamber 5 mm long and 2 mm wide. The chamber was sectioned with two gaps of petroleum jelly (VaselineTM) placed perpendicularly to the axon at an interval of I mm. The central section was filled with the physiological saline and the side sections were filled with 205 mM KCI solution. Two microelectrodes of 15 M.Q tip resistance were inserted into the middle portion of axon in the central section. Two pieces of Ag-AgCl wire placed in respective microelectrodes were both connected to a single input of a potential recorder. Voltage-clamping currents were supplied from the KCI solution in the side sections to the axonal membrane in the central section using the axoplasm as a conductor. The current was measured through a virtual ground made of Ag-AgCl wire placed in the central section, and recorded into a computer using a 16-bit A/D converter. The leak current through the axonal membrane and the gaps was subtracted using a multiple of an inward current induced by a stepwise hyperpolarization of 5 mV.
RESULTS
Purijication of two anti-mammal toxins (BmK I and BmK II) The BmK venom gland extract was tested for toxicity in mice by i.p and i.c.v. injections (LD~~ and MLD: 2.4 and 0.074 mg/kg body weight, respectively). The specific toxicity (i.c.v.) was 676, and blowfly larvae CPU was about 1 ug (Table 1). The extract was separated into 13 fractions on a CM-Sephadex C-50 ion-exchange column. Step gradient chromatography was monitored at 280 nm (Fig. 1A). By qualitative bioassays, fractions XI and XII were found to be the most toxic to mice, as confirmed by the LD~,,values of the two fractions, which were 0.48 and 0.63 mg/kg body weight; the MLD were 0.013 and 0.015 mg/kg body weight, and the specific toxicities (i.c.v.) 3846 and 3333. The CPU of both fractions was determined to be lower than 0.5 ug per blowfly larvae. Fraction XI was submitted to reverse-phase HPLC on a Lichrosorb RP-18 column (0.6 x 25 cm). Two major components, identified as MT I and MT II, were eluted (Fig. 1Ba) and both fractions were further purified by isocratic HPLC, eluting as single peaks (Fig. 1Bb and c). The purity of both polypeptides was assessed by examining a single band in the polyacrylamide gel electrophoresis (data not shown). Finally, the two polypeptides in fractions MT I and MT II were called BmK I and BmK II. Toxicity tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II: the MLD of BmK I and BmK II were 36 ng and 222 ng per mouse of 20 + 2 g, respectively. The value of specific toxicity (i.c.v.) of BmK I was 27,778, 41 times higher than that of venom extract, while that of BmK II was 4505, 6.7 times higher than that of venom extract. Moreover, both toxic polypeptides were also potent in insects. The CPU of BmK I and II was 0.75 and 1.25 ug per cricket, respectively. Table 1 summarizes the quantitative data of the purification of BmK I and II.
IA 1A 1B
1B
XI XII MTI(Bmk I)
MTII(Bmk II) 2.09
409 179 62 3.94
8521 18,646 4921
-
2.4 0.48 0.63
-
‘. * +:;a1 toxicity (Lb
21 104 79 -
/ODZSO)
i.p.* Specific toxicity
injection; Scontraction paralysis unit.
Protein (0~~~ units)
*After intraperitoneal injection; t after intracerebroventricular
HPLC RI’ C-18
Step Venom extract CM-Sephadex C-50
Ref. to Fig.
Toxin fraction
i.p.+ LDB (mg/kg mouse)
0.0111
i.c.v.t MLD (mg/kg mouse) 0.074 0.013 0.015 0.0018 9414
276,351 688,461 206,667 109,444
i.c.v.t Total toxicity
4505
676 3846 3333 27,77X
0.88
0.29
1 0.5
1.25
0.75
2 1.5
i.c.v.7 CPU1 (pg) Specific blowfly CPI-J$ (pg) toxicity larvae cricket (MLD/oD~~ ) (110 + 10 mg) (500 f 20 mg)
Table 1. Quantitative data of the purification of toxins BmK I and II of Buthus martensi Karsch
E3 2 i: G a a 8 ?I. R
L E
Y. H. JI et al.
992
Amino acid compositions of‘ BmK I and II The amino acid compositions of native and RCM-BmK I and II are listed in Table 2. Both toxins are composed of 64 amino acid residues. There are no Met or Phe residues. The differences between BmK I and II appear at positions 59 (Val, Ile) and 62 (Lys, Asn).
Amino acid sequence of BmK I Native BmK I (about 10 nmoles) was first sequenced with the manual DABITCjPITC about 8 nmoles of RCM-BmK I were double-coupling method. Alternatively,
__
0.01
__tc__
w
1
I
111
-
0.05
0.10
--I--0.25
+
0.50
Xf
IV 3 XII
Q VI X
300
600
900
EffIuent
(b)
I ‘ig. 1. (A) Ion-exchange
1200
(ml)
(4
MT1
”
0.006
_____~_ _____........-. __-__..
J
XIII
MT II
0-
_
5 I
x
.. . J 20
\
chromatography of the venom of the scorpion B. martensi Karsch on a CM-Sephadex C-50 column. The venom extract containing about 550 mg of protein was loaded on the column (2 x 65 cm). The concentration of NH4HC03 (elution buffer) is indicated on the top. Flow rate: 36 ml/hr, 6 ml/tube. (B) HPLC patterns of (a) fraction XI, (b) MT 1, and (c) MT II. (a) A 0.2 mg sample of fraction XI, (b) 20 pg of MT I (BmK I), and (c) 15 ug of MT II (BmK II) dissolved in 20 u1 of water were injected. Column: Lichrosorb RP-18 (7 urn, 0.6 cm ID x 25 cm). Elution buffer: (a) solvent A:O. 15 M ammonium formate, pH 2.7; (b,c) 0.1% TFA-water. Solvent B: 70% acetonitrile. AUFS: (a) 0.12; (b,c) 0.015 at 280 nm. Flow rate: 1.5 ml/mm The ordinate to the right indicates the concentration of acetonitrile in the elution buffer (broken line).
e
*
Buthus martensi
Neurotoxins
993
Table 2. Amino acid compositions of toxins BmK I and II Amino acid
BmK I Native protein
CYS
BmK II RCM-protein
RCM-protein
6.78 (8)
7.16 (8)
CMC Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His LYS Arg Trp Total
Native protein
(8) (9) (1) (1) (4) (4) (6) (4) (5)
9.16 (10) 0.96 (1) 1.00 (1) 3.62 (4) 3.88 (4) 6.27 (6) 4.12 (4) 4.15 (4)
6.80 (8) 9.59 (10) 0.96 (1) 0.99 (1) 4.02 (4) 4.14 (4) 6.18 (6) 4.13 (4) 3.91 (4)
2.88 (3) 2.03 (2) 4.25 (5)
2.98 (3) 2.05 (2) 4.52 (5)
3.80(4) 2.02 (2) 3.94 (5)
3.69 (4) 2.06 (2) 4.33 (5)
1.97(2) 5.00 (5) 3.17 (3) -_(2) 64
1.98(2) 5.04 (5) 3.48 (3) -_(2) 64
2.00(2) 4.32 (4) 3.10 (3) -_(2) 64
1.89 (2) 4.15 (4) 3.38 (3) -(2) 64
8.75 0.98 0.94 4.01 3.97 6.21 4.10 4.85
(9) (1) (1) (4) (4) (6) (4) (5)
6.83 8.72 0.95 0.93 3.89 4.19 6.28 4.14 4.92
Experimental values are deduced from two duplicate analyses after 20 and 70 hr hydrolyses. Numbers in parentheses indicate the number of residues deduced from the sequence analysis.
automatically degraded in a Beckman 890 MM protein sequencer. Sequence analysis occurred from the N-terminal position up to position 31, with ambiguous identification of positions 29 and 30 (Fig. 2a). For further sequencing, RCM-BmK I (25 nmoles) was digested by S. aureus V, and subjected to TLC on 12 TLC-silica gel 60 sheets (90 x 90 x 0.2 mm). As shown in Fig. 3A, the chromatography profile contains eight fluorescing peptide traces of which six, marked V,-1 to V,-6, were collected. Taking into
(A)
BmK
I
(a) RCM-BmK
I VRDAYIAKPHNCVYECARNEYCNDLCTK
LPDNVPIRVPGKCH
(b) vg-1 (c) vg-5 (d) T-11
BntK II (B)
(e)RCM-BmK (r')x78-5 (c) T-9 (h) VS-3 (i.) T-4
____
??A_____----____-----_______----
LCTKDGAKSGYCQWVGKYG------GAKSGYCQWVGKYG??CWC-YGNGCWCIEL-------
VRDAYIAKPHNCVYECARNEYCNDLCTKDGAKSGYCQWGKYGNGCWCIELPDNVPIRIPGNCH II VRDAYIAKPHNCVYECARNEYCNDLCTKDGAK--------------------------------LCTKDGAKSGYCQWVGKYG------YGNGCWCIELPD----LPDNVPIRIPGNCH IPGNCH
Fig. 2. Determination of the amino acid sequences of BmK I and BmK II. (A) (a) Automatic degradation of RCM-BmK I (8 nmoles) in a Beckman 890 M sequencer: average yield =: 86%; (b) degradation of peptide 5164 (V,-1, 7.5 nmoles): average yield = 85%; (c) degradation of peptide 2550 (V,-5, 7 mnoles): average yield = 82%; (d) degradation of peptide 42-58 (T-11, 4.5 nmoles): average yield = 92%. (B) (e) Automatic degradation of RCM-BmK II (10 mnoles): average yield = 88%; (t) degradation of peptide 25-50 (V,-5, 4 nmoles): average yield =: 81%; (g) degradation of peptide 42-58 (T-9, 4 nmoles): average yield = 79%; (h) degradation of peptide 5164 (J,-3, 1.5 nmoles): average yield = 80%; (i) degradation of peptide 59-64 (T-4, 3 nmoles): average Yield = 78%.
Y. H. JI et al.
994
(W
(4 Vs-5 e ,O Vs-6 + v*-4
+‘I--11
T_9
h T-7 l ) T-8
A
T-10 T-6 t
T;2
a T-5
1
l T-4
T-l
T!3 *.
W
T-9,
‘;” I T-l
1 T-4 t T-3
First Dimension 25% NH3/Ethanol (40/60, Fig. 3. Separation of peptides of RCM-BmK I and RCM-BmK II on TLC sheets. Enzymatic digest (l-2 nmoles) was slowly dropped on the left corner. (A) Cleavage of RCM-BmK I with S. aureu~ V,. (B) Cleavage of RCM-BmK I with trypsin. (C) Cleavage of RCM-BmK II with S. ~UY~USV,. (D) Cleavage of RCM-BmK II with trypsin.
account the amino acid compositions of peptides (Table 3) only V,-1 and V,-5 were subjected to sequencing. The sequence of V,-1 (7.5 nmoles) was determined and assumed to be located at the C-terminal region, from position 51 to the end of the protein (Fig. 2b). The V,-5 peptide (7 nmoles) accounted for positions 25-43 (Fig. 2~). This sample contained about 0.9 nmole of a peptide, the sequence of which corresponded to position 3048. To RCM-BmK I was subjected to tryptic digestion. establish the complete sequence, Figure 3B shows the chromatography of tryptic peptides of RCM-BmK I (25 nmoles) on a TLC sheet. There were at least 11 peptides, marked T-l to T-l 1 (amino acid compositions in Table 4). Of these tryptic peptides, T-l 1 was sequenced (ten residues) and found to overlap with the sequence of the fragments V,-1 and V,-5. Finally, the complete amino acid sequence of BmK I was established by the link of T-11 fragment (Fig. 2d).
Buthus mnrtensi Neurotoxins
995
Carboxypeptidase A digestion of RCM-BmK 1 was carried out to confirm the C-terminal residue. The kinetic of release is in favour of the C-terminal sequence Cys-His. The complete sequence of BmK I is shown in Fig. 2A. Amino acid sequence of BmK II The automatic degradation of RCM-BmK II (10 nmoles) gave the sequence l-32, which was identical to that of BmK I (Fig. 2e). The TLC separation of the RCM-BmK II digests obtained with trypsin and S. aureus V, is shown in Fig. 3 (C and D). The amino acid compositions of enzymatic fragments are listed in Tables 5 and 6. Of these enzymatic peptides, V,-5 and VB-3, T-9 and T-4 corresponded to the sequence of positions 25-50 (Fig. 2f), 5.1-64 (Fig. 2h), 42-58 (Fig. 2g), and 59-64 (Fig. 2i), respectively. The sequencing of these four enzymatic peptides led to the determination of the complete amino acid sequence of BmK II (Fig. 2B). Furthermore, the C-terminal residue, e.g. His-64 in BmK II, was directly defined by Edman degradation of enzymatic peptides V,-3 and T-4. Electrophysiological e#ect of BmK I The effect of BmK I on nerve excitability was examined in the crayfish axon model (Fig. 4). Intracellular recording showed that the toxin prolonged the descending phase of the action potential without affecting the rising phase (Fig. 4A). Under voltage clamp (Fig. 4B), BmK I greatly prolonged the inward current. No outward current was observed during the clamping pulse of 5 msec. Addition of tetrodotoxin (0.1 PM) to the external medium in the presence of BmK I eliminated this inward current completely and left the outward current almost the same as that observed before the application of scorpion toxin.
Table 3. Amino acid compositions of S. aureus Ve peptides obtained from RCM-BmK I Amino acid CMC Asp Thr Ser GlU Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Trp Total Yield (%)
S. aureus Va Pevtides V,-3 l&20* -V,-4 21-24*
v*-2 1-3*
Vs-6 415*
0.99 (1)
1.18 (1) 1.27 (1)
0.28 (1) 1.10 (1)
1.22 (1) 1.07 (1)
1.07 (1)
0.77 (1) 2.13 (2) -
0.62 (1)
1.72<2) 1.08 (1)
0.80 (1)
1.44 (2) -
2.70 2.16 0.83 0.91 2.21
(4) (2) (1) (1) (2)
4.85 (5) 1.10 (1) 1.06 (1)
0.87 (1)
0.91(l)
V,-5 25-50*
0.93 (1) 0.69 (1) 1.42 (2)
-
Traces (1)
0.87 (1) 1.04 (1) -
Traces (1)
3 80
12 80
5 90
2.57 (3) 4 80
-(2) 26 56
Experimental values are deduced from duplicate analyses after 20 hr hydrolyses. Numbers in parentheses indicate the number of residues deduced from the sequence analysis. *Sequence positions.
v,-1 51-64* Traces (1) 1.09 (2)
2.72 (3) 1.14 (1) 1.83 (2) 0.87 (1) 0.66 (1)
0.88 (1) 0.93 (1) Traces (1) 14 60
ti
B 3 :: >;
02 11 (I)(1) 86’0
08 c!,
-
(I) SI'I
88 SZ (z)i;; Sk::
PS 91
I;; g:; (1) VS.0 (2) LS’I (1) EO’I
I;; ;y:; (z) 28’1
(I) PE’I
(I) 7.8’0 7 (5) LE’Z
I;,’ :;:; (z) OI’Z (I) - 51’1
(I) Z6'0 (El SI'E (0 PO‘Z *8'?EE 6-L
*81-E 8-L
i;; g:;
(E) E8'Z
(I) zo.1 I;; ;;:;
01-L
(Z) 58'1
(2) LI’Z I
*IbEE
(I) LO'1 (I) 06'0
;;; ;;:I (I) LO'1
*8S-ZP II-L
BP 01
~
(I) ZL'O
I;; g:;
ecP9-E9P-l
(1) L8’0
(I) PZ’I
09 2
*81-6 E-l
(I) SO'1 (I) 28'0
(1) LO'1 (1) 86'0
(I) f8’0
(I) LI’I
(I) 68’0
8 01
+ZE-6Z Z-Z
(I) f6’0
i;; ii:;
(I) 86’0
PZ P
~s!s~IEu~ amanbas ay] u10g pasnpap sanp!sa.t 30 laqurnu ay] almpu! .sasblolp6q ‘q OZ lal3” sasKpzue alwydnp u.1013 paanpap PZ P
~
9f Z
(I) 98’0
(I) 6L’0
(I) 89’0
-
1
-
-
(I) 96'0
-
7
(1) 56’0
(Z) L8‘0
;:; ;;:;
*Z-I 9-L *Z9-65 S-J sapgdad 3gdi(lL
*8Zp61 L-J.
py
OUyIIV
3MI3
"10 Ias 1Y.L dsv
old
I"‘4 "IV 4t)
w
sh S!H ayd “6.L na7 alI
BJV
dlL
(%) Pl3!A WoL
woysod manbag, sasay]ualed u! s.raqumN am sanIv,t [Eluau+adxa
8P PI
(z) OO’Z
(I) ZL’O -_(I) L6‘0
(I) 62'1 (I) 69'0
(I) 18'0
(1) 01'1
*ZEp61 I-L
Buthus martensi Neurotoxins
997
These findings indicate that BmK I binds to the sodium channel and blocks its inactivation process. DISCUSSION
Although the Chinese scorpion B. martensi Karsch is generally not regarded as being dangerously venomous, the venom is considered to be a “rather active toxin” (Balozet, 1971), suggesting that it probably contains toxic components. The toxicity (LDSo) of this venom to mice was found to be 2.4 mg/kg body weight, which is moderate in comparison with venom of other species. From fraction XI, the major toxic component, two toxic anti-mammal polypeptides (BmK I and BmK II) were isolated. The toxicities of these toxins are lower than those of toxins purified from other dangerous venomous species. Miranda et al. (1970) were the first to report a general method of purification of scorpion toxins. The method involved (1) extraction by distilled water, (2) gel filtration with recycling on Sephadex G-SO, and (3) ion-exchange equilibrium chromatography. We have shortened this method in the present work by using only cation-exchange equilibrium chromatography on a CM-Sephadex C-50 column (Fig. lA), followed by HPLC, enabling us to obtain two anti-mammal toxins, BmK I and II (Fig. 1B). When BmK I was added to a crayfish axon preparation, there was incomplete inactivation of Na+ channels, an effect common to a-type toxins. When the amino acid sequences of BmK I and BmK II were compared to those of other scorpion toxins, it was found that the sequences of both toxins, as other a-toxins, differed significantly from those of P-type neurotoxins isolated from the venoms of South American and North American scorpions (group 5). These sequences showed a high degree of similarity with toxins of group 3 (Fig. 5) according to the classification of Rochat et al. (1979). Nevertheless, it is interesting to note that the C-terminal ends of BmK toxins are unmodified, whereas the ends of other toxins of group 3 are amidated. In addition, the two toxins are potent in
Table Amino
5. Amino
acid compositions
of S. aureus V8 peptides
acid VR-1 l-3*
CMC Asp Thr Ser Glu Pro Gly Ala Val Met Be Leu
1.02 (1)
v*-2 1620* 0.24 (1) 0.86 (1)
from RCM-BmK
0.48 (1) 2.90 (3)
0.94 (1) 2.02 (2)
0.93 (1) 2.67 (3) 1.04 (1) 0.13 (1)
V,-5 25-50* 2.94 2.20 0.71 1.07 2.15
(4) (2) (1) (1) (2)
5.09 (5) 1.07 (1) 0.90 (1)
0.95 (1) 0.48 (1) 1.41 (2) 0.48 (1) 0.61 (1)
Tyr Phe His Lys Arg Trp Total Yield (%)
obtained
S. aurew V, peptides V,-3 51-64* V,-4 21-24*
0.88 (1) 0.60 (1) 1.23 (2)
1.24 (1) 2.83 (3) 0.47 (1)
1.07 (1)
0.53 (1)
3 22
5 37
14 9
Experimental values are deduced Numbers in parentheses indicate *Sequence positions.
4 45
from duplicate analyses after 20 hr hydrolyses. the number of residues deduced from the sequence
-(2) 26 99 analysis
II V,-6 4-15* 1.33 (1) 1.49 (1)
1.38 (1) 0.99 (1) 1.79 (2) 0.93 (1) 0.97 (1) 1.08 (2) 0.84 (1) 1.02 (1)
12 45
998
Y. H. JI et d.
1 llllIll;lllllIl~l”~
6
,’
Buthus martensi Neurotoxins
999
both mammals and insects. Another toxin (LQQ III) from the scorpion Leiurus quinquestriatus quinquestriatus, which belongs to group 3, was recently found to have the same characteristics (Kopeyan et al., 1993). The structure-activity relationships of scorpion neurotoxins have been extensively studied by modifying particular residues involved in toxic and pharmacological activities. It has been demonstrated that the charged residues located in the N-terminal and C-terminal regions of scorpion toxins play a multiple role in the interaction with proteic components of the sodium channel. For example, the modification of Lys-58 in AaH II by N-hydroxysuccinimidyl biotin results in an almost complete loss of activity. The modification of the homologous lysine in Lqq V reduces activity to 20% (Darbon et al., 1983). An analogue of AaH II, Bot XI, in which Lys-58 is replaced by Val-58, is 250-fold less active (Sampieri et al., 1987). This loss of toxicity might be explained by this substitution. A single modification of Lys-60 in Lqq V and of Arg-60 in AaH I results in residual activities of 40 and 14%. When Arg-56 in AaH II is modified with phenylglyoxal, the residual activity of the toxin is 20% (Kharrat et al., 1990). In addition to the above demonstrations, this study found valuable evidence
(A:) (a)
Control
0 -50 -100 mV IL 0
5
10 1s msec
(B:)
I
A------
a. Control b. BmK 1 c. BmK I + TTX d. Membrane potential
1 ms
40 mV
Fig. 4. Effects of BmK I on a crayfish lateral giant axon. (A) Eilect of BmK I on intracellularly recorded action potentials: (a) control record of the crayfish action potential; (b, c) records obtained 3 and 15 min after application of 0.3 pm01 BmK I, respeci.ively. (B) Effect of BmK I under voltage clamp: (a) control record of ionic currents; (b) record after application of BmK I; (c) record after application of 0.1 pmol TTX; (d) record of the membrane potential during the voltage clamp.
Y. H. JI
1000
et al.
BOf II
G______~_E_______~._~________~_________~_~~__~_~__~___~____~~_
___,z
Born III Be M9 BeMI4
G--G---Q-E----H-FPGSSG-DT--KEK--T-H.-GFLPGS-”A---DN--NK---V-G-E--_ ~_______________~~K~~___~_._EN..E_----IL-____~____Q________~__ ~______,,,,~_.._T..L -P--DSE-K-N--D~~____L-RF__~___K~___~____K,_~~_~
_..
of the amino acid sequence of BmK I and II with that of group 3 according to the classification by Rochat et al. (1979). BmK I and II: toxins I and II from the scorpion Buthus martensi Karsch (Asia). Lqq III: toxin III from the scorpion Leiurus quinquestriatus quinquestriatus (Sudan). Bot I and II: toxins I and II from the scorpion &thus occitanus tunetanus (Algeria). Born III: toxin II from the scorpion Buthus occitanus Mardochei (Morocco). Be M9 and M14: toxins M9 and Ml4 from the scorpion Buthus epeus (Asia). Fig. 5. Comparison
for the important role in the bioactivity of toxins of a basic residue in the C-terminal region (here, position 62). It is interesting to note that BmK I was six times more potent than BmK II. The conservative mutation in position 59 seems to be unimportant in terms of activity. When comparing the sequences of the two toxins (Fig. 5), the only significant difference is in position 62, where a basic residue (Lys) is found in BmK I, while a neutral residue (Asn) is found in BmK II. The presence of a positive charge in C-terminal position 62 may be important for the binding of BmK I to the receptor on sodium channels. This conversion could explain the lower toxicity of BmK II. Another illustration of the importance of the basic residue in this part of the molecule is given by the toxicity of AaH IV (group l), which was recently found to be at least four times lower than that of others (Mansuelle et al., 1992). The low toxicity of AaH IV may result from two substitutions in the C-terminal positions 60 and 61, where two basic residues (Arg-60 and Lys-61), responsible for strong toxicity in other neurotoxins, were replaced by two acidic residues (Asp-60 and Asp-61) in AaH IV. These observations suggest that positively charged residues (Lys or Arg) at position 62, and generally in the C-terminal region, play an important role in maintaining the potency of scorpion neurotoxins.
Acknowledgements-We are greatly indebted to Drs C. Granier and M. Hoshino for their fruitful discussions, and to Mrs T. Brando for her kind help in operating the amino acid analyser. The present study was supported by a fellowship from the CNRS of France, granted to Y. H. Ji and in part by the Grant-in-Aid for the International Scientific Research Program from the Ministry of Education, Science and Culture of Japan.
REFERENCES Balozet, L. (1971) Scorpionism in the old world. In: Venomous Animals and Their Venoms, Vol. III, Venomous Invertebrates, Ch. 56, pp. 3499371 (Biicherl, W. and Buckley, E. E., Eds). New York: Academic Press. Catterall, W. A. (1988) Structure and function of voltage-sensitive ion channels. Science 242, 50-61. Chang, J. Y, (1983) Manual micro-sequence analysis of polypeptides using dimethylaminoazobenzene isothiocyanate. Meth. Enzymol. 91, 455466. Couraud, F., Rochat, H. and Lissitsky, S. (1978) Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells. Biochem. biophys. Res. Commun. 83, 1525-1530. Couraud, F., Jover, E., Dubois, J. M. and Rochat, H. (1982) Two types of scorpion toxin receptor sites, one related to the activation, the other to the inactivation of the action potential sodium channel. Toxicon 20,9-16. Darbon, H., Jover, E., Couraud, F. and Rochat, H. (1983) Scorpion neurotoxin derivatives suitable as potential markers of sodium channels. Znt. J. Pept. Prof. Res. 22, 179-186.
Buthus martensi Neurotoxins
1001
Fishbein, J. C., Place, A. R., Roposon, I. J., Powers, D. A. and Sofer, W. (1980) Thin-layer peptide mapping: quantitative analysis and sequencing at the nanomole level. Analyt. Biochem. 108, 193-201. Fontecilla-Camps, J. C., Almassy, R. J., Suddath, F. L., Watt, D. D. and Bugg, C. E. (1980) Three-dimensional structure of a protein from scorpion venom: a new structural class of neurotoxins. Proc. natn. Acad. Sci. U.S.A.
77, 64966500.
Gregoire, J. and Rochat, H. (1983) Covalent structures of toxin I and II from the scorpion Buthus occitanus tunetanus. Toxicon 21, 153-162. Hawke, D., Yuan, P. M. and Shively, J. E. (1982) Separation of PTH amino acid by reverse phase HPLC. Analyt. Biochem. 120, 302-3 11.
Jover, E., Couraud, F. and Rochat, H. (1980) Two types of scorpion neurotoxins characterized by their binding to two separate receptor sites on rat brain synaptosomes. Biochem. biophys. Res. Commun. 95, 1607-1614. Jover, R., Martin-Moutot, N., Couraud, F. and Rochat, H. (1980) Binding of scorpion toxins to rat brain synaptosomal fraction, effects of membrane potential, ions and other neurotoxins. Biochemistry 19, 463467. Keegan, H. L. (1980) Scorpions of Medical Importance, p. 41. University Press of Mississippi, U.S.A. Kharrat, R., Darbon, R., Granier, C. and Rochat, H. (1990) Structure-activity relationships of scorpion a-neurotoxins: contribution of arginine residues. Toxicon 28, 5099523. Kopeyan, C., Martinez, C., Lissitzky, S., Miranda, F. and Rochat, H. (1974) Disulfide bonds of toxin II of the scorpion Androctonus austrlais Hector. Eur. J. Biochem. 47, 483489. Kopeyan, C., Mansuelle, P., Martin-Eauclaire, M. F., Rochat, H. and Miranda, F. (1993) Characterization of Toxin III of the scorpion Leiurus quinquestriatus quinquestriatus: a new type of alpha-toxin highly toxic both to mammalian and insects. Nat. Toxins 1, 308-312. Loret, E. P., Sampieri, R., Granier, C., Miranda, F. and Rochat, H. (1992) Scorpion toxins affecting insects. Meth. Neurosci. 8, 381-395.
Mansuelle, P., Martin, M.-R., Rochat, H. and Granier, C. (1992) The amino acid sequence of toxin IV from the Androctonus australis scorpion: differing effects of natural mutations in scorpion a-toxin on their antigenic and toxic properties. Nat. Toxins 1, 6169. Miranda, F., Kopeyan, C., Rochat, H., Rochat, C. and Lissitzky, S. (1970) Purification of animal neurotoxins. Eur. J. Biochem. 16, 514523.
Moore, J. W. (1971) Voltage clamp methods. In: Biophysics and Physiology of Excitable Membranes, pp. 143-167 (Adelman, W. J., Jr, Ed.). New York: Van Nostrand Reinhold. Ovchinikov, Yu. A. and Grishin, E. V. (1982) Scorpion neurotoxins as tools for studying fast sodium channels. Trends biochem. Sci. 7, 2628.
Reisfeld, R. .4., Lewis, U. J. and Williams, D. E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195, 281-283. Rochat, H., Bernard, P. and Couraud, F. (1979) Scorpion toxins: chemistry and mode of action. Adv. Cytopharmac. 3, 325-334.
Sampieri, F., Rochat, C., Martin, M.-R., Kopeyan, C. and Rochat, H. (1987) Amino acid sequence of toxin XI of the scorpion Buthus occitanus tunetanus. Int. J. Pept. Prot. Res. 29, 231-237. Terakawa, S., Kimura, Y., Hsu, K. and Ji, Y. H. (1989) Lack of effect of a neurotoxin from the scorpion Buthus martensi K.arsch on nerve fibers of this scorpion. Toxicon 27, 569-578. Zlotkin, E., Miranda, F. and Rochat, H. (1987) Chemistry and pharmacology of Buthinae scorpion venom. Arthropod Venoms 317, 369.