of Androctonus australis Hector

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1 Eur. J. Biochem. 7 (7) - The Amino Acid Sequence of Neurotoxin I of Androctonus australis Hector Her& ROCHAT, Catherine ROCHAT, Franpois MIRANDA, Serge LISSITZKY, and Pehr EDMAN Laboratoire de Biochimie MBdicale, FacultB de MBdecine, Marseille, and St. Vincent's School of Medical Research, Melbourne (Received July, 7) The complete amino acid sequence of neurotoxin I of the North African scorpion Androctonus australis Hector collected in Algeria (area of Chellala) is described. X-Methylation of cysteine residues formed by reduction of disulphide bonds was found useful for their identification in sequence determination using the phenylisothiocyanate method. From the N-terminal sequence (38 residues) established in the protein sequenator, and from the sequence of chymotryptic peptides the complete sequence of the protein has been deduced. The molecule consists of a single polypeptide chain cross-linked by four disulphide bridges. An isoneurotoxin (') has been found in the venom of A. australis collected in Tunisia (area of Tozeur). It differs from neurotoxin I in the replacement of the valine residue in position 7 by an isoleucine residue. Two neurotoxins (I and ) have been purified from the venom of the North African scorpion Androctonus australis Hector [l]. Both neurotoxins are small molecular weight basic proteins consisting of a single peptide chain cross-linked by four disulphide bridges. Neurotoxins I and I contain 3 and 4 amino acid residues, respectively. Lysine and threonine were found as N- and C-terminal amino acids in toxin I. This paper describes the complete amino acid sequence of toxin I. EXPERIMENTAL PROCEDURE MATERIALS The venom of Androctonus australis Hector was obtained from animals collected either in Algeria (area of Chellala or Mecheria) or in Tunisia (area of Tozeur). Alpha-chymotrypsin (lyophilized) and diisopropyl phosphofluoridate-treated carboxypeptidase A were purchased from Worthington (Freehold, N.J., U.S.A.). Methyl iodide (laboratory reagent grade, B. D. H., Poole, England) was distilled and stored as previously described []. Guanidine hydrochloride (B. D. H.) was purified according to Spackman et al. [3]. Reagents and solvents used for sequence determination by phenylisothiocyanate degradation were purified according to Edman and Begg [4]. Sephadex G-5 and G-5 (beads, fine grade) were obtained from Pharmacia (Uppsala, Sweden). All other chemicals were reagent grade products. Deionized quartz-redistilled water was used throughout. METHODS Neurotoxin I was purified as previously described [I]. Preparation of Reduced and S-Methylated Toxin (RM-Toxin) The toxin was reduced with -mercaptoethanol in 5 M guanidine. A -fold excess ofmercaptoethanol over half-cystine was used. The cysteine residues were then methylated with methyl iodide. The techniques and conditions for reduction and methylation were those described by Rochat et al. []. The RM-toxin was recovered by gel filtration on a column of Sephadex G-5 equilibrated with.m acetic acid. Amino Acid Analysis Samples of the native or RM-toxin, and of chymotryptic peptides were digested with. N HC in sealed evacuated tubes at " for h. The hydrolysates were analyzed either in a Technicon Amino Acid Auto-Analyzer according to Piez and Morris [5] or in a Spinco automatic Amino Acid Analyzer, model B, according to Spackman et al. []. Using the former method separation of proline and X-methylcysteine (MeCys) was sufficient to allow the quantitative estimation of both amino acids. Digestion with a-chymotrypsin Digestion of RM-toxin by a-chymotrypsin was performed under ph-stat control. The protein

2 Voi. 7, No., 7 H. ROCHAT, C. ROCHAT, F. MIRANDA, S. LISSITZKY, and P. EDMAN 3 (. pmoles) was dissolved in water to a concent,ration of.o/, (w/v) and the ph was adjusted to 8. by addition of.5 N NaOH. Nitrogen was passed over the surface of the solution and temperature was maintained at 37". a-chymotrypsin dissolved in water was added in an enzyme to substrate ratio of :IOO (w/w). A second addition of chymotrypsin (one half the amount added first) at the end of the digestion did not induce additional NaOH consumption. The reaction was stopped after 75 min by addition of.o N HCI to ph 3. and the solution was lyophilized. Determination of N-Terminal Sequences These determinations were made by the phenylisothiocyanate method [I I]. Automatic sequencing with the protein sequenator [4] was used for the RM-toxin. Manual sequencing of chymotryptic peptides was carried out according to Blomback et al. [. Phenylthiohydantion amino acids were identified by thin layer chromatography using solvents D and E [4]. An additional solvent system (ethylene chloride-glacial acetic acid, 3 : 7, v/v) [3], was also used. Purification of Chymotryptic Peptides Purification of chymotryptic peptides was mainly achieved by gel filtration on Sephadex. The digest was dissolved in 3 /, acetic acid ( ml) and filtered at a flow-rate of ml/h through a x cm column of Sephadex G-5 equilibrated in the same solvent. Absorbance of the effluent at 8 nm was monitored with a Uvicord I analyzer (LKB, Stockholm, Sweden) and fractions of 4ml were collected. A.5 ml sample of each fraction was removed, dried under vacuum, digested with alkali (.5 N NaOH for h at lloo) and analyzed by the ninhydrin reaction [7]. The fractions were suitably pooled and lyophilized. Reversible adsorption on Sephadex G-5 was carried out according to Miranda et al. [S]. Fraction I (see Results) was dissolved in water and filtered on a x 5 cm column of Sephadex G-5 equilibrated in water at a flow-rate of 3 ml/h. Twenty-two hours later water was replaced by. N acetic acid and the column effluent was monitored at 8 and nm with a Beckman DU spectrophotometer. Paper chromatography in n-butanol-acetic acid-water (4: : 5, by vol.) and high voltage paper electrophoresis at ph 3. in pyridine-acetic acidwater (: :8, by vol.) on Whatman 3 MM filter paper were used according to Katz et al. [] to purify or to control the purity of the chymotryptic peptides. When necessary, peptides were eluted with 3 /, acetic acid and the solvent removed by freezedrying. Digestion with Carboxypeptidase A The RM-toxin or its chymotryptic peptides were submitted to carboxypeptidase A hydrolysis at ph 8. in the ph-stat [lo]. Nitrogen was passed over the surface of the solution and temperature was maintained at 5". Carboxypeptidase A was used in an enzyme to substrate rat~io of : (w/w). Aliquots of the digest were removed at various intervals (,, 4, 8 and min), acidified with.n HC and dried under vacuum for amino acid analysis. RESULTS N-Terminal Sequence of RM-Toxin Recovery of the reduced and S-methylated toxin was 7O/,. Table shows the amino acid composition of toxin I before and after reduction and S-methylation. The yield of S-methylcysteine calculated on the cystine originally present amounted to 5O/,. Thirty eight cycles in the sequenator using.7 pmole of the RM-toxin established the N-terminal sequence. When the toxin was purified from the venom of animals collected in Tunisia (Tozeur) a mixture of phenylthiohydantoin-valine and phenylthiohydantoin-isoleucine was recovered at step 7. However, when the toxin was obtained from animals collected in Algeria (Chellala or Mecheria) only the valine derivative was found at step 7. Table. Amino acid composition of native, and reduced and S-methylated toxin I Figures in brackets are the theoretical number of residues. n. d. = not determined Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidiue Arginine Tryptophan Half-cystine S-Methylcysteine Native ComDosition of residuesirnoie Reduced and S-methylated 8. ().. (). 5.8 () 5.. ().. () 5.7. () 5.. (). 4. (5). () () (4) 4..7 (3).8. (). 5.8 () 5.. (). ().. n. d. n. d.. (8). - 7.

3 4 Primary Structure of A. australis Toxin Eur. J. Biochem. Effluent (liter) Fig.. Gel filtration of RM-toxin chymotryptic digest. The digest (. poles) was filtered through a X cm column of Sephadex G-5 in 3 /, acetic acid. Flow-rate, ml/h. Full line, absorbance at 8 nm; dotted line, absorbance at 57 nm after alkaline hydrolysis of the fractions and ninhydrin reaction. Double-headed arrows indicate pooling of fractions.5 - a. b.a Purification of Chymotryptic Peptides Chymotryptic digestion was performed on. pmoles of RM-toxin prepared from toxin I (Chellala). From the amount of NaOH consumed, it was calculated that 5.75 peptides bonds had been split. Four fractions were obtained by gel filtration of the chymotryptic digest on Sephadex G-5 (Fig.). Only two of them (I and ) required further purification. Submitted to high voltage paper electrophoresis, fraction I gave two peptides (IIa and IIb). Resolution of fraction I was carried out by reversible adsorption on Sephadex G-I5 (Fig.). The three peptides (IIIa, IIIb and IIIc) obtained could be used for sequence studies without additional purification. Table shows the amino acid composition of the seven chymotryptic peptides. The composition of the peptides accounts for the entire composition of the reduced and S-methylated toxin. Amino Acid Sequence of Chymotryptic Peptides Peptide I (Residues 4-3) : H-MeCys-Lys- Asp-Leu-Pro- Asp- Asn-Val-Pro- Ile-Lys- Asp-Thr-Ser- Arg-Lys-MeCys-Thr-OH Seventeen degradation steps performed on 5 pmoles established the N-terminal sequence. As peptide I contained two threonine residues, it was inferred that the last amino acid was threonine. a, z. e w I 4.5 I I Effluent (litef) Fig.. Reversible adsorption of fraction I on Sephadex. Fraction, obtained by gel filtration of the chymotryptic digest (Fig.l), was dissolved in water and filtered through a x 4 cm column of Sephadex G-5 in water. The vertical arrow indicates change from water to.m acetic acid. Flow-rate, 3 ml/h. Full line, absorbance at nm; dotted line, absorbance at 8 nm. The large increase in absorbance at nm just before the elution of peptide IIIc is due to the strong absorbance of acetic acid at this wavelength. Double headed arrows indicate pooling of fractions C-Terminal Sequence of RM-Toxin Digestion of RM-toxin with carboxypeptidase A gave the C-terminal sequence -Lys-MeCys-Thr-OH. Peptide I a (Residues 37-4) : H-Leu-Val-Pro-Ser-Gly-Leu- OH Three degradation steps established the N-terminal sequence H-Leu-Val-Pro-. Carboxypeptidase A digestion released.5 mole of leucine per mole of peptide. Partial hydrolysis by. PIT HC at " for 48 h produced three peptides and free serine which were separated by fingerprinting. The amino acid composition of the peptides was Gly, Leu; Ser, Gly, Leu; and Leu, Val, Pro. These results established the sequence. Peptide IV (Residues 43-45) : H- Ala-MeCys-Trp-OH Two degradation cycles established the N-terminal sequence H-Ala-MeCys-. Carboxypeptidase digestion released tryptophan (.75 mole of tryptophan per mole of peptide), thereby establishing the sequence of this tripeptide. Alignment of Peptides From the results of (a) the automatic degradation of RM-toxin, (b) the digestion of RM-toxin with carboxypeptidase A and (c) the amino acid sequences of chymotryptic peptides I, IIIa and IV, it was possible to derive the complete sequence of RM-toxin (Fig. 3).

4 Vol. 7, No., 7 H. ROCHAT, C. ROCHAT, F. MIRANDA, S. LISSITZKY, and P. EDMAN 5 Table. Amino acid composition of chymotryptic peptides from RN-toxin I Amino acid I I a IIb IIIa I b IIIc IV Total Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Half-cystine Methionine Isoleucine Leucine Tyrosine Phenvlalanine LysiGe Histidine Arginine Tryptophan8 8-Methylcysteine Total 3.8 (4).5 (). ().3 (). ().77 (). ().7 (3).4 (). ().7 (). ().3 ().87 ().8 (). () residues per mole 3.5 (4).8 ().7 (). (3).8 ().8 (). (). ().8 ().5 ().7 ().4 ().5 ().55 ().7 ().5 ().8 (). ().3 (). (). () Yield.3 () () 7.77 () () () 3 83 Spectrophotometric determination I c J I b. I a H-Lys-Arg-Asp-G)ly-Tyr-Ile-Val-Tyr-Pro-Asn-Asn-Cys-V~l~Tyr-His-~s-Val-Pro-Pro-Cys- J. IIb. a Asp-Gly-Leu-Cys-Lys-Lys-Asn-Gly-Gly-Ser-Ser-Gly-Ser-Ser-Cys-Phe-Leu-Val-Pro-Ser IV 4 I Gly-Leu-Ala-Cys-Trp-Cys-Lys-Asp-Leu-Pro~Asp-Asn-Val-Pro-Ile-Lys-Asp-Thr-Ser-Arg- 5 Lys-Cys-Tkr-OH Fig.3. Amino acid sequence of A. australis neurotoxin I. Arrows show peptide bonds split by or-chymotrypsin. Roman numerals refer to chymotryptic peptides Prom their amino acid composition, peptides IIIc, IIIb, IIa and IIb have to be placed in this order from the N-terminus. The N-terminal sequence of peptide IIIa starts with H-Leu-Val- and it must therefore follow peptide I b according to the results of automatic degradation. As peptide I contains the two threonine residues of the toxin, it must be placed at the C-terminus. Peptide IV, which accounts for the remaining amino acids of the RM-toxin, must be inserted between peptides IIIa and I. DISCUSSION Gel filtration on Sephadex -5 equilibrated with 3 /, acetic acid was found very efficient for the purification of the chymotryptic peptides of RMtoxin. Although peptides are generally eluted from Sephadex columns according to their molecular volume, some are more or less strongly adsorbed to the gel depending on their content of aromatic amino acids. For example, the nonapeptide IIIb, which contains two tyrosine residues, emerges from the column with the same elution volume as peptides IIIa ( residues) and IIIc (5 residues) and after peptide IIa ( residues). The tripeptide IV, which contains one tryptophan residue, is eluted in a volume greater than the total volume of the column. Reversible adsorption on Sephadex was previously used for scorpion neurotoxins purification [S]. The method gave excellent results in the separation of the three peptides contained in fraction. Dextran gels contain small amounts of carboxyl groups which can exchange basic molecules if the gel is equilibrated in water and the exchangeable material dissolved in the same solvent. Sephadex -5 was selected in preference to Sephadex G-35 because of its lower water regain. Peptide IIIa, which is neutral and does not contain aromatic residues, is eluted first. Peptide I b, which is also neutral but contains two tyrosine residues, is eluted later as a broad peak. Eventually, acetic acid is necessary to elute the strongly adsorbed basic peptide I c.

5 "I H. ROCHAT et al. : Primary Structure of A. australis Toxin I Eur. J. Biochem. Recoveries were almost quantitative. We believe that the use of reversible adsorption on Sephadex might solve many problems of peptide separation. Due to the easy characterization of the phenylthiohydantoin derivative of 8-methylcysteine by thin layer chromatography [], the conversion of cysteine into X-methylcysteine residues appears to be very useful for amino acid sequence determination. The amount of 8-methylcysteine estimated after hydrolysis of the reduced and X-methylated toxin I (Table ) is in good agreement with our previous results on reduced and S-methylated toxin I of A. australis Hector []. It may be ascertained that the four disulphide bridges have been reduced and the eight cysteine residues thus formed alkylated. The X-methylcysteine residue in position was rapidly released by carboxypeptidase A from the RM-toxin. Ten minutes at 5" were enough to recover mole of both threonine and 8-methylsysteine per mole of RM-toxin. Peptide IIb behaved similarly and its C-terminal sequence could be established as -Ser-MeCys-Phe-OH. The easy release by carboxypeptidase A of X-methylcysteine was not unexpected [4] since it differs from the easily released methionine by only a methylene group. Chymotrypsin showed the usual specificity. Carboxyl peptide bond cleavage at two of the three tyrosine residues, at the single phenylalanine and tryptophan residues and at two of the leucine residues was observed. The Tyr-Pro (residues 8-) and Leu-Pro (residues 4-5) bonds were resistant. The phenylisothiocyanate degradation of the toxin preparation originating from Tozeur showed that it was, in fact, a mixture of two isotoxins differing only in that one had a valine residue (toxin I) and the other an isoleucine residue (toxin ') at position 7 [5]. This substitution can be explained by a single nucleotide change in the coding triplet. The conservative character of the change of a single nonpolar residue explains that separation of toxins I and I' has been so far unsuccessful []. Six amino acid residues in the sequence of toxin I appear in duplicate. The homodipeptides are concentrated in the N-terminal section of the chain and especially in the positions 8 to 34. The amino acid sequence of some snake neurotoxins are now known [i7-]. These are also basic proteins of small molecular weight (approx. 7) consisting of a single chain cross-linked by four disulphide bridges and lacking methionine. However, their amino acid sequences, which exhibit a remarkable degree of homology within themselves, are completely different from A. australis toxins I and '. The only similarities in the primary structure are the high frequency of pairs of the same residue (nine in Naja haje toxin alpha [ls] and six in A. australis toxins I and ') and the sequence Asn-Gln-Gln-Ser- Ser-Gln-Pro-Pro (residues 5 to ) of N. haje t,oxin alpha which may be compared to the sequence Asn- Gly-Gly-Ser-Ser-Gly-Ser-Ser (residues 7 to 34) of A. australis toxins I and '. Partial N-terminal amino acid sequences of an additional number of scorpion neurotoxins [] have shown a high degree of homology between these proteins although less pronounced than that observed for snake neurotoxins. Molecular phylogeny, structure-function relationships and the structural basis for the difference in mode of action of snake and scorpion neurotoxins are at present being studied. This work was supported by the Centre National de la Recherche Scientifique (RCP ) and the Direction des Recherche5 et Moyens d'essais. REFERENCES. Rochat, C., Rochat, H., Miranda, F., and Lissitzky, S., Biochemistry, (7) 57.. Rochat, C., Rochat, H., and Edman, P., Anal. Biochem. in the press. 3. Spackman, D. H., Stein, W. H., and Moore, S., J. Biol. Chem. 35 () Edman, P., and Begg, G., Eur. J. Biochem. (7) Piez, K. A., and Morris, L., Anal. Biochem. () 87.. Spackman, D. H., Stein, W. H., and Moore, S., Anal. Chem. 3 (58). 7. Moore,S., and Stein,W.H., J. Biol. Chem. (54) Miranda. F.. Rochat., H.., and Lissitzkv. S.. J. Chromatoo. 7 () 4.. Katz. A. M., Drever, W. J.. and Anfinsen. C. B.. J. Biol. Chem. 34 (5) Slobin, L. I., and Carpenter, F. H., Biochemistry, (3).. Edman, P., Acta Chem. Scand. 4 (5) 83.. Blomback, B., Blomback, M., Edman, P., and Hessel, B., Biochim. Biophys. Acta, 5 () Kluh, J., and Edman, P., personal communication. 4. Ambler, R. P., In Methods in Enzymology (edited by C. H. W. Hirs), Academic Press, New York and London 7, Vol. XI, p Rochat, H., These Doctorat s-sciences, Marseille.. Miranda, F., Kupeyan, C., Rochat, H., Rochat, C., and Lissitzky, S., Eur. J. Biochem. (7) Eaker, D. L., and Porath, J., 7th Innt. Congr. Biochem. (edited by the Science Council of Japan), Tokyo 7, Abstracts, p Botes, D.P., and Strydom,D. J., J. Biol. Chem. 44 () Yang, C. C., Yang, H. J., and Huang, J. S., Biochim. Biophys. Acta, 88 () 5.. Botes, D. P., communication at the nd International Symposium on Animal and Plant toxins, Tel-Aviv, Israel, Feb. 7.. Tamiya, N., Sato, S., Endo, Y., Seto, A., and Yoshida, H., communication at the nd InternationalSymposium on Animal and Plant toxins, Tel-Aviv, Israel, Feb. 7.. Rochat, H., Rochat, C., Kupeyan, C., Miranda, F., Lissitzky, S., and Edman, p., unpublished results. H. Rochat, C. Rochat, F. Miranda, and S. Lissitzky Laboratoire de Biochimie MBdicale Facultk de MBdecine et de Pharmacie 7 Boulevard Jean-Moulin, F-3 Marseille, France P. Edman St. Vincent's School of Medical Research Victoria Parade, Melbourne, Victoria 35, Australia