Isolation and characterization of a potent curarizing polypeptide from Tityus discrepans scorpion venom

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PII: !30041-0101(97)ooo41-x

Toxicon, Vol. 35, No. 12, pp. 168H689, 1997 1997 Elsevier Science Ltd. All rights reserved Printed in C%eat Britain 00414101/97 %17.00 + 0.00

ISOLATION AND CHARACTERIZATION OF A POTENT CURARIZING POLYPEPTIDE FROM TITYUS DISCREPANS SCORPION VENOM G. D’SUZE,’ C. SEVCIK,’ J. F. PEREZ* and J. W. FOX3 ILaboratory of Cellular Neuropharmacology, Instituto Venezolano de Investigaciones Cientificas, CBB, Apartado 21827, Caracas, 1020-A, Venezuela, *Laboratory of Gastrointestinal Physiology, Instituto Venezolano de Investigaciones Cientificas, CBB, Apartado 21827, Caracas, 1020-A, Venezuela and ‘Biomolecular Research Facility, University of Virginia, Health Sciences Center, Box 441, Charlottesville, VA 22908, U.S.A. (Received 16 December

1996; accepted 24 February 1997)

G. D’Suze, C. Sevcik, J. F. Perez and J. W. Fox. Isolation and characterization of a potent curarizing polypeptide from Tityus discrepuns scorpion venom. Toxicon 35, 1683-1689, 1997.-The pure TdI-1 polypeptide that blocks miniature endplate potentials (MEPPs) and abolishes or reduces endplate potentials (EPPs) below the action potential threshold was identified from the crude fraction of Tityus discrepans venom. The toxin is a potent reversible non-depolarizing muscle relaxant that blocks more than 95% of the EPP at a 2 PM (0.1 mg/ml) concentration. On a molar basis, TdI-1 is as potent as or more potent than many muscle relaxants since, at the concentration used, the toxin suppressed more than 95% of the EPP. Using matrix-assisted laser desorption time of flight (MALD-TOF) ionization mass spectrometry, TdI-1 was found to have an unsually large mol. wt for a scorpion toxin, close to 48,000. The N-terminal sequence of the first 23 residues of TdI-1 was also determined. The fragment differs from the N-terminal sequences of all 140 peptidic scorpion toxins found in the SWISSPROT and PIR databases using the search engine of the felix.EMBL-Heidelberg.de computer (European Molecular Biology Laboratory, Heidelberg, Germany). 0 1997 Elsevier Science Ltd

INTRODUCTION

Tityus discrepans is a scorpion that lives in the north-central area of Venezuela and is the most common source of human envenomation in this country. In a previous study (D’Suze et al., 1995) four fractions, called TdF-I, -11, -111 and -IV, were isolated from T. discrepuns venom. Fraction I has curarizing effects on the frog neuromuscular junction (D’Suze et al., 1995). The present communication describes the isolation and N-terminal sequence of a polypeptide that suppresses completely and reversibly the miniature endplate potentials (MEPPs) and blocks neuromuscular transmission. Using matrix-assisted laser desorption time of flight (MALD-TOF) mass spectrometry the polypeptide was shown to 1683

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have a mol. wt of approx. 48,000. This is unusually large for a scorpion known scorpion toxins range from 3000 to 7000 in mol. wt.

MATERIALS

AND

toxin,

since all

METHODS

The venom resuspended in water was chromatographed on Sephadex’ G-50 medium as previously described (D’Suze et al., 1995). The methods to bioassay toxin activity on frog (Hyla crepitans) neuromuscular junctions and to purify the peptides using high-performance liquid chromatography (HPLC) were essentially the same as those described by D’Suze et al. (1995, 1996), except that an additional step of reverse-phase HPLC on an analytical C4 column (VydacTM) was added. The HPLC-purified peptide was subjected to MALD-TOF mass spectrometry (Skoog and Leary, 1992) with a Lasermat 1000 mass spectrometer, using a-cyano-4-hydroxyconamic acid as the matrix. Samples of toxins were isolated by HPLC. The isolated toxin was reduced, carboxymethylated and sequenced as described by Shannon et al. (1989). The purity of the HPLC peak was checked using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12% acrylamide. The data were subjected to non-parametric statistical analysis. Results are presented as medians and their 95% confidence interval, calculated by the procedure of Hodges and Lehmann (in Hollander and Wolfe, 1973). The significance of differences between pairs of experimental conditions was calculated with the Mann-Whitney (Wilcoxon) test. (See Hollander and Wolfe, 1973, for details on all non-parametric statistical methods mentioned above.) Differences between treatments were considered significant if the probability that they occur by chance was < 5% (P 5 0.05).

50.5 min (Tdl-1)

B

I

i0 Nonaliied

Elutii

Volume

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3 s

3 0

Elution Time (min)

60

Fig. 1. HPLC of TdF-I. The fraction was chromatographed on a 10 pm, 10 x 250 mm, VydacTM Protein C4 (Vydac, Hesperia, CA, U.S.A.) column, with a linear gradient that changed from water with 0.12% TFA at a rate of l%/min to 60% acetonitrile in water with 0.1% TFA. The flow rate was 2 ml/min; 4.7 mg of TdF-I was injected into the column. The entire separation was carried out at room temperature. Absorbance was determined at 230 nm; the peak containing TdI-1 is marked with an arrow. Inserted is the elution pattern of Tityus discrepans venom using a Sephadex G-50 column at 25°C. The abscissa is the UV absorbance at 280 nm, and the ordinate is the volume of 20 mM ammonium acetate, expressed as normalized column (0.9 x 200 cm) volume. Roman numerals identify the four fractions containing different biological activities. The flow rate was 18.9 ml/hr per cm2.

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RESULTS

Toxin purQ?cation The scorpion venom was chromatographed on Sephadex G50@ columns as previously described (D’Suze et al., 1995); the elution profile is shown as an insert in Fig l(A). Fraction I from the G-50 chromatography step was lyophilized, resuspended in water and subjected to HPLC on a semipreparative Vydac TMProtein C4 column (250 x 10 mm, 10 pm). As can be seen in Fig. l(B), 20 different peaks were eluted from the HPLC column. The fraction eluting at 50.5 min was then rechromatographed twice on an analytical VydacTM Protein C4 column (250 x 4.6 mm, 5 pm). Following this chromatography step, a single peak (TdI-1) was observed eluting at 47.5 min [Fig 2(A)]. Since this HPLC fraction showed a single band (mol. wt 5 1,500) during SDS-PAGE, it was considered homogeneous [Fig. 2(B)]. Efects of the toxins on neuromuscular transmission The toxin with a HPLC retention time of 50.5 min, called TdI-1, modified endplate potentials (E:PPs). TdI-1 suppressed completely and reversibly the MEPPs and blocked neuromuscular transmission. A representative experiment of the effect of TdJ-1 on the

45.7 min I

kD

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2

& m

0

II

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..5 kD

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0 Elution

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‘.5 kD TdFI

TdI-1

MW

Fig. 2. HPLC and SDS-PAGE of TDI-1. The toxin fraction eluting at 50.5 min from the Vydac C4 (Vydac, Hesperia, CA, U.S.A.) semipreparative column (lo pm, 10 x 250 mm) was further purifiedthrough aVydac C4 analytical column (5 pm, 4.6 x 250 mm), using the same gradient described in Fig. 1. The peptide (TdI-1) and TdF I were subject to SDS-PAGE with 12% acrylamide to verify the purity of the HPLC process.

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A

r

e_h---“___~-_..__---

L

C _q---------_-___--

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__ -. -..~---J-

D

z 9

I

20.48

ms

Fig. 3. Reversible block of neuromuscular transmission in frog H. crepitans sartorius muscle. (A) Action potential recorded from one fibre at the neuromuscular junction under control conditions, resting potential (RP) - 73 mV. (B) Evoked endplate potential recorded from a neuromuscular junction exposed to 2 PM TdI-1 for 29 min, RP - 89 mV. (C) Evoked endplate potential recorded from a neuromuscular junction exposed to 2 PM TdI-1 and washed with normal saline solution for 22 min, RP - 90 mV. (D) Extrajunctional action potential recorded from a fibre exposed to 2 PM TdI-1 and washed with normal saline solution for 27 min, RP - 87 mV. The calibration is valid for all traces: a vertical bar indicates 40 mV, a horizontal bar indicates 20.48 msec.

Curarizing

Toxin

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evoked EPPs is shown in Fig. 3. An action potential elicited by nerve stimulation and recorded from an endplate region is presented at the top of Fig. 3(A). Since the contraction was not ablolished, the microelectrode was dislodged from the fibre after the spike. A record from an endplate region after 29 min of exposure to 2 PM (0.1 mg/ml) of TdI-1 is shown in Fig. 3(B); the EPPs are reduced to below 4 mV. Another two traces recorded after 22 min [Fig. 3(C)] and 27 min [Fig. 3(D)] washing with normal saline are presented in the lower part of the figure. An evoked EPP that recovered just below the threshold of action potential is shown in Fig. 3(C), while Fig. 3(D) presents a large action potential recorded in a fibre away from the neuromuscular junction; the weak mechanical artefact that followed the spike suggested that most of the endplates were still blocked by the toxin and that the muscle twitch was still very weak. Molecular weight determination

Two analyses of the HPLC pure toxin using MALD-TOF mass spectrometry produced three peaks in each analysis. These results are summarized in Table 1. The data in the table suggest that the peptide produces three ions with relative valencies (z) equal to 4, 2 and 1, and mass/charge ratios (m/z) close to 12,000, 24,000 and 48,000, respectively. We estimated t:he mol. wt of the peptide as 48,078 (47,792, 48,253) (median and 95% confidence interval of six values), by multiplying the m/z ratios by their corresponding charge. N-Terminal sequence analysis

TdI-I was reduced and carboxymethylated and its N-terminal amino acid sequence elucidated as described by Shannon et al. (1989). Only one PTH amino acid was observed from the protein at each cycle of the Edman reaction, providing some evidence as to the purity of the toxins separated by HPLC. The amino-terminus of the peptide was: DFKVFCEVPL

FLCQXXFNDX

YXT - ,

where X indicates unidentified PTH amino acids.

DISCUSSION

In a prevj.ous communication D’Suze et al. (1995) found a curarizing fraction (TdI) in T. discrepans that had not been described in any other species of scorpion. It was not known whether the curarizing activity existed in other species. From the crude Td-I fraction of :r. discrepans venom a pure TdI-1 protein has now been identified that blocks

Table

1. MALD-TOF

Analysis 1 2

mass

spectrometry of the curarizing from T. discrepans

polypeptide

Mass (m/z) 12,091 (48,364) 12,040 (48,160)

24,071 (48,142) 24,029 (48,058)

The median and 95% confidence interval shown in parentheses are 48,058 (47,877.5,

47,792 (47,792) 47,697 (47,697) of the six values of mol. wt (m) 48,253).

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MEPPs, reduces EPPs below action potential threshold and abolishes them at higher concentrations. The N-terminal sequence of TdI-1 was also determined, and indicated that the protein is a unique scorpion toxin. The sequence differs from the N-terminal sequence of all 140 peptidic scorpion toxins that we could find in the SWISSPROT and PIR databases using the search engine of the felix.EMBL-Heidelberg.de computer (European Molecular Biology Laboratory, Heidelberg, Germany). Likewise, attempts to match the TdI-1 N-terminal sequence to all the sequences in the named databases did not produce any significant matches. Using MALD-TOF mass spectrometry, TdI-1 was found to have an unusually large mol. wt for a scorpion toxin, close to 48,000. All previously known scorpion toxins have mol. wts less than 6000. Irreversible neuromuscular transmission-blocking polypeptides are known to occur in elapid venoms, e.g. CY-and K-bungarotoxin and the Naja a-toxins (reviewed by Chiappinelli, 1993). These toxins, however, have a mol. wt close to 7000, which is approximately six times less than that of TdI-1. The a-conotoxins are well-known reversible curarizing polypeptides isolated from marine snails belonging to the Conus genus. These toxins are very small polypeptides of 13-25 amino acids (Gray et al., 1981, 1988; Zafaralla et al., 1988; Pardi et al., 1989; Fainzilber et al., 1991; Myers et al., 1991; Ramilo et al., 1992). Likewise, huwentoxin-I is a reversible neuromuscular transmissionblocking polypetide that occurs in the venom of the spider Selenocosmia huwena (Liang et al., 1993; Zhang and Liang, 1993; Zhou et al., 1997). Huwentoxin-I is a 33 amino acid polypeptide and has a mol. wt of 3750. The action of TdI-1 seems similar to the action of clinically useful non-depolarizing muscle relaxants (Agoston and Bowman, 1990). On a molar basis, TdI-1 is as potent as many of them, since at a concentration of 2 PM the toxin reduced the EPP amplitude by more than 95%. Since we were unable to find proteins or peptides with significant homologies with Tdl-1 in the SWISSPROT and PIR databases, we conclude that this toxin represents a new class of curarizing peptidic toxins.

Acknowledgements-The authors are indebted to the people of San Antonio de Los Altos and their Fire Department for supplying the scorpions, to Mr Nehemias Mujica for the supply and maintenance of the frogs and to Mr Moises Sandoval for the maintenance of the scorpions. This research was supported by grants Sl-2086 (CS) and Sl-95000722 (GD) from CONICIT, Venezuela. Mrs Dhuwya Otero-Palma drew Fig. I.

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Skoog, D. A. and Leary, J. J. (1992) Principles of tnstrumental Analysis, 4th edn, pp. 446448. Saunders, Fort Worth. Zafaralla, G. C., Ramilo, C., Gray, W. R., Karlstrom, R., Olivera, B. M. and Crux, L. J. (1988) Phylogenetic specificity of cholinergic ligands: a-conotoxin SI. Biochemistry 27, 7102-7105. Zhang, D. Y. and Liang, S. P. (1993) Assignment of the three disulfide bridges of huwentoxin-I, a neurotoxin from the Selenocosmia huwena. Journal of Protein Chemistry 12, 735-740. Zhou, P. A., Xie, X. J., Li, M., Yang, D. M., Xie, Z. P., Zong, X. and Liang, S. P. (1997) Blockade of neuromuscular transmission by huwentoxin-I, purified from the venom of the Chinese bird spider Selenocosmiu huwena. Toxlzon 35, 3945.