Biochimica et Biophysica Acta, 491 (1977) 53-66 © Elsevier/North-Holland Biomedical Press BBA 37602 P R I M A R Y S T R U C T U R E S OF C A R D I O T O X I N A N A L O G U E S II A N D IV F R O M THE V E N O M OF N A J A N.4JA A T R A *
NORIO KANEDA a, TOYOSAKU SASAKIb and KYOZO HAYASHI a aDepartment of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, and bHimeji Institute of Technology, Himeji (Japan) (Received September 16th, 1976)
SUMMARY Cardiotoxin analogues II and IV were isolated from the venom of Naja naja atra by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The venom contains at least four cardiotoxin analogues that account for about 54 of the weight of the lyophilized crude venom. These four cardiotoxin analogues, named cardiotoxin analogues I, II, III, and IV, show strong cytotoxicity to Yoshida sarcoma cells but the lethal toxicity is one-order less. These toxins contain 60 amino acid residues in a single peptide chain. Cardiotoxin analogue IV differs from cardiotoxin analogue II only by the presence of arginine in place of a leucine residue at position 1. A comparison of the amino acid sequences of these toxins with that of cobrotoxin, a neurotoxin containing 62 amino acid residues obtained from the same snake venom, shows that about 20 amino acid residues, including 8 half cystine residues, are identical, assuming 3 residues deletion and 2 residues insertion in the cardiotoxin molecule.
INTRODUCTION A number of biologically active components have been isolated from venoms of snakes belonging to the family Elapidae [1]. One group of basic polypeptides, which act on the pre- or post-synaptic sites, are known as venom neurotoxins [2]. Another group having relatively low toxicity to animals induce a variety of effects which have been separately recognized and studied in various laboratories. As a consequence confusion in their nomenclature arose and only recently they were found * Supplementary data to this article, giving details of amino acid compositions, chromatography and electrophoresis patterns and amino acid sequences, are deposited with, and can be obtained from: Elsevier Scientific Publishing Company, BBA Data Deposition, P.O. Box 1527, Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/055/37602/491(1977)53. Abbreviations and symbols: The following abbreviations are used: CB- cyanogen bromide fragments, T- tryptic peptides derived from CB-I obtained by cyanogen bromide cleavage of reduced and S-carboxymethylated-cardiotoxinanalogues II and IV. T'-, tryptic peptides derived from each CB-II1 obtained by cyanogen bromide cleavage of reduced and S-carboxymethylated-cardiotoxin analogues II and IV. -
-
54 to be closely related if not identical. These strongly basic polypeptides isolated from a specific venom or from venoms of different species or subspecies have been designated as cardiotoxin [3], skeletal muscle depolarizing factor [4], cobramines A and B [5], cytotoxin [6], toxin y [7], toxin direct lytic factor [8], protein 12B [9] and others. In this paper, we use the name "cardiotoxins" because of their effect on the heart and because of the precedent established by Lee et al. [10]. Within the last few years, these strongly basic cardiotoxic polypeptides have been isolated in pure form from the venoms of several species of cobra [9, 11-25]. Cardiotoxins are about one-order less toxic than many neurotoxins from Elapidae snake venoms [2]. By gel filtration on Sephadex G-50 followed by chromatography on CMcellulose, we obtained four cardiotoxin analogues which exhibited cytotoxicity to Yoshida sarcoma cells. Because the amino acid composition data suggested a high degree of structural homology among these toxins, we felt that the primary structure determination of these cardiotoxins might help to reveal the significance of the relationship between cardiotoxins and neurotoxins and also aid in the identification of functionally important amino acid residues. Previously, we reported on the amino acid sequences of cardiotoxin analogues I and III (---- cardiotoxin) isolated from the venom of Naja naja atra [15, 44]. The present paper deals with the complete amino acid sequences of cardiotoxin-analogues II and IV from the same venom. MATERIALS AND METHODS Lyophilized Naja naja atra venom, a-chymotrypsinogen A, cytochrome c, and insulin were obtained from Sigma Chemical Co., Ltd., U.S.A. Bovine trypsin (recrystallized three times) (EC 3.4.21.4) was obtained from Worthington Biochemical Corp., U.S.A. Sephadex G-25 and G-50 were the products of Pharmacia Fine Chemicals, Sweden. Carboxymethyl (CM)-cellulose was purchased from Seikagaku Kogyo Co., Ltd., Japan. All other chemicals were commercial preparations of the highest quality available.
Disc gel electrophoresis Polyacrylamide disc gel electrophoresis was performed by the method of Williams [26], using disc gel electrophoresis apparatus of Mitsumi Kagaku Sangyo, Co., Ltd., Japan, and 15 ~ gels at pH 4.0. Electrophoresis was performed with an initial current of 3 mA per gel. After concentration of the sample, the current was increased to 5 mA per gel. Gels were stained with Amido Black 10B and rinsed several times with 10 ~ acetic acid solution.
Determination of molecular weight by gel filtration The molecular weight of the purified cardiotoxin-analogues II and IV was determined by the gel filtration method described by Andrews [27]. A column (1.4 x 144 cm) of Sephadex G-50 equilibrated with 0.05 M phosphate buffer containing 0.1 M KCI, pH 7.5, was used. Elution was carried out with the same buffer at a flow rate of 30 ml per h at room temperature. The calibration curve was made by using blue dextran, a-chymotrypsinogen A, cytochrome c and insulin as markers.
55
Toxicity The toxicity was measured by subcutaneous or intravenous injection (mice, 16-18 g) of progressively diluted venom solutions as described previously [15]. Five male mice were used for each dilution and the LDs0 was calculated according to the 50 ~ end peint method of Reed and Muench [28]. Five controls were used under the same conditions but without venom.
Assay of cytotoxicity Assay of cytotoxicity to Yoshida sarcoma cells was performed by modification of the method described by Braganca et al. [6]. The tumors were maintained in Donryu rats by transplantation of 0.2 ml of fluid derived from the intraperitoneal cavity. In this study, the ascitic fluid containing Yoshida sarcoma cells was withdrawn from the intraperitoneal cavity 3-4 days after transplantation with this tumor. The tumor cells were freed from blood cells according to the method of Mckee, Lonberg-Holm and Jehl [29]. 0.1 ml of serial dilutions of the active material in phosphate buffered saline solution was added to 0.1 ml of cell suspension (4.106 cells/ml). The mixture was incubated with gentle shaking at 37 °C and at the end of 30 min 0.2 ml of trypan blue (0.4 ~ in saline) was added to the incubation mixture. Part of this mixture was placed on a haemocytometer slide glass and the proportion of damaged cells was calculated by dividing the number of cells permeable to trypan blue by the total number of cells. Cells incubated without the active fraction were permeable to trypan blue to the extent of 5-7 ~ . Cytotoxicity was defined as the concentration (expressed as fig protein/ml) of active material which caused 50 ~ of the cells to become permeable to trypan blue.
Purification of cardiotoxin analogues The cardiotoxins were purified as follows: 4 g of lyophilized venom was dissolved in about 20 ml of 1 ~ acetic acid and applied to a column of Sephadex G-50 (4 x 193 cm) at room temperature. The Sephadex column was eluted with 1 ~o acetic acid at a flow rate of 42 ml/h. Fractions of 15 ml each were collected and assayed for absorbance at 280 nm. After pooling, each pool was lyophilized. The pool containing compounds with a molecular weight of about 6000-7500 yielded 3.118 g (or 78.0 of the crude venom). The entire sample was dissolved in 10 ml of 0.005 M sodium acetate buffer, pH 5.8 and applied to a CM-cellulose column (2.8 x 140 cm) equilibrated with the same buffer. The column was washed with the starting buffer and eluted with a gradient formed from 10 liters of equilibration buffer in the mixing vessel and 10 liters of 0.5 M sodium acetate buffer, pH 6.5, in the reservoir. Fractions of 15 ml each were collected, lyophilized, redissolved in a minimum amount of 1 acetic acid, and gel-filtered to remove sodium acetate. Four cardiotoxin-like components, cardiotoxin analogue I, cardiotoxin analogue II, cardiotoxin analogue III, and cardiotoxin analogue IV, were isolated by this method. Cardiotoxin analogues II and IV were further purified on a column of CMcellulose and eluted with a gradient of increasing pH and salt concentration. The gradient was established by means of a mixing vessel containing 1000 ml of 0.2 M sodium acetate buffer, pH 6.0, connected to a reservoir containing 1000 ml o f 0.8 M sodium acetate buffer, pH 6.8. Fractions of 4 ml each were collected at a flow rate of 46 ml/h at room temperature. The pooled protein fraction forming the major peak
56 was lyophilized, the residue redissolved in 1 ~o acetic acid, sodium acetate removed by Sephadex G-25 gel filtration and the resulting solution lyophilized.
Amino acid composition Samples (0.05 to 0.1 ffmol) were hydrolyzed in an evacuated, sealed tube with 0.5-1.0 ml of 5.7 M HC1 for 24 h at 110 °C, the acid removed by evaporation under reduced pressure, and the residue dissolved in 1.1 ml of 0.2 N sodium citrate buffer, pH 2.20. Analysis was performed with an Hitachi KLA 3B automatic amino acid analyzer by the method of Spackman et al. [30].
Reduction and alkylation The carboxymethyl derivatives of cardiotoxin-analogues II and IV were prepared by the method of Crestfield et al. [31]. The samples were dissolved in 0.2 M Tris.HC1 buffer, pH 8.2, containing 5 M guanidine-HC1 and fl-mercaptoethanol was added. The glass-stoppered tube was flushed with nitrogen gas and left standing at 37 °C for 4 h. For alkylation, a freshly prepared solution of iodoacetic acid in 2 N NaOH was added with stirring and the pH of the solution maintained at pH 8.6 with 2 N NaOH. After 30 rain the solution was desalted by passing through a column of Sephadex G-25. The reduced and S-carboxymethylated-cardiotoxin analogues II and IV were each pooled and lyophilized.
Cyanogen bromide cleavage Fragmentation of reduced and S-carboxymethylated-cardiotoxin analogues (about 10 ffmol) was performed in 70 ~ formic acid with a 300-fold molar excess of cyanogen bromide for 24 h at room temperature [32]. After dilution with water, the reaction mixture was lyophilized to remove excess reagent, the residues dissolved in 0.2~ acetic acid and applied to a column (1.8 × 111 cm) of Sephadex G-50 equilibrated with 0.2 ~ acetic acid. The chromatographic profile was established by following the absorbance at 280 nm. Fractions with absorbance were pooled and the pool lyophilized.
Trypsin digestion of CB-I and CB-III generated by cyanogen bromide cleavage Trypsin digestion was carried out at 37 °C for 8 h in 0.1 M ammonium bicarbonate buffer, pH 8.0, with a weight ratio of enzyme to substrate of 1:50. After digestion, the digest was lyophilized.
Isolation of tryptie peptides The peptides generated by trypsin were isolated by a combination of high voltage paper electrophoresis and paper chromatography. High voltage paper electrophoresis was performed using a solvent system of pyridine/acetic acid/water (1:10:289, by volume), pH 3.6, at 30 to 35 V per cm in a tank (Toyo Kagaku Sangyo Co., Ltd., Japan) containing Esso naphtha No. 5 as a coolant. Guide strips (5 mm) were cut and stained with ninhydrin reagent. Peptides were eluted from the paper with 0.1 M pyridine adjusted to pH 5.0 with acetic acid. The purities of extracted peptides were examined by descending paper chromatography on Toyo paper No. 51 using a solvent system of 1-butanol/acetic acid/pyridine/water (15:3:10:l 2, by volume) [solvent (a)] in a glass tank for 14 to 16 h. The chromatograms were stained with ninhydrin reagent.
57 When the peptides were chromatographically impure, further purification was carried out by paper chromatography using solvent (a), 1-butanol/acetic acid/water (4:1:5, by volume) [solvent (b)], or water-saturated phenol [solvent (c)].
Sequence analyses The amino-terminal sequences of reduced and S-carboxymethylated-cardiotoxin analogues II and IV, and carboxy-terminal fragments generated by cyanogen bromide cleavage were determined using a modified Edman's phenylthiohydantoin procedure [33]. The amounts of the phenylthiohydantoin derivatives were determined semiquantitatively by measuring their absorbance at 269 nm before chromatography. The derivatives were then identified by thin layer chromatography on Kieselgel F254 plates (E. Merck, Germany) using solvent systems IV and V described by Jeppsson and SjSquist [34] and on polyamide sheets (Carle Place, New York, U.S.A.) using a solvent system of toluene/n-pentane/acetic acid (60:30:35, by volume); 250mg [2-(4'-butylphenyl)-5-(4'-biphenyl)-l,3,4-oxdiazole]/liter [35]. The aqueous phase, containing phenylthiohydantoin arginine was evaporated to dryness, the residue dissolved in 0.1 ml of 70 ~ ethanol, an aliquot spotted on filter paper and tested for arginine with Sakaguchi's reagent. Phenylthiohydantoin leucine and phenylthiohydantoin isoleucine were determined, when they occurred, by amino acid analyses of the tryptic peptides. RESULTS
Purification of cardiotoxin analogues H and IV Lyophilized crude venom dissolved in 1 ~ acetic acid was applied to a column of Sephadex G-50 and eluted with 1 ~ acetic acid. A typical elution pattern is shown in Fig. 1. Four peaks (I to IV) with absorption at 280 nm were obtained. The pool from peak IV containing the cardiotoxin analogue, was further purified on a column of CM-cellulose. As shown in Fig. 2, many peaks appeared. Among these, four frac2.000
¢q
1.000
o .
Fig. 4. Amino acid sequence of cardiotoxin analogue II. Horizontal arrows below the amino acid residues denote the sequence of cyanogen bromide fragments derived from reduced and S-carboxymethylated-cardiotoxin analogue II and tryptic peptides. ~ , sequence elucidated by Edman degradation. T and T' represent the peptides produced by trypsin digestion of fragments CB-I and CB-III, respectively. Numbers below amino acid residues show the yields ( ~ ) of phenylthiohydantoin amino acids estimated from the molar absorbance coefficient at 269 nm and from the amounts of samples used in the Edman degradation.
63 ed f r o m the a m i n o - t e r m i n a l p o r t i o n . The f o u r t h peak, CB-1I, c o r r e s p o n d e d to phenylalanylhomoserine. Stepwise E d m a n d e g r a d a t i o n o f the c a r b o x y - t e r m i n a l f r a g m e n t C B - I I I (11.6 mg) revealed the following a m i n o acid sequence: -Va•-Ser-Asn-Leu-Thr-Va•-Pr•-Va•-Lys-Arg-G•y-Cys-••e•Asp-Va•-Cys-Pr•-Lys-Asn-Ser-A•a-LeuVal-Lys-Tyr-Val-Cys-Cys-Asn-Thr-Asp-Arg-Cys-Asn-OH O n the basis o f the a b o v e results, the a m i n o acid sequence o f c a r d i o t o x i n a n a l o g u e I V was f o u n d to be C B - I - I I - I I I . F o r conclusive evidence, fragments CB-I a n d C B - I I I were digested by trypsin, the resulting peptides s e p a r a t e d b y a c o m b i n a t i o n o f high voltage p a p e r electrophoresis a n d p a p e r c h r o m a t o g r a p h y as described for cardiotoxin a n a l o g u e II, a n d analyzed for a m i n o acid c o m p o s i t i o n (see D a t a Deposition). The overall p r i m a r y structure o f c a r d i o t o x i n a n a l o g u e IV was ascertained as shown in Fig. 5,
TABLE III AMINO ACID COMPOSITIONS OF THE FRACTIONS GENERATED BY CYANOGEN BROMIDE CLEAVAGE OF REDUCED AND S-CARBOXYMETHYLATED-CARDIOTOXIN ANALOGUE IV Numbers in parentheses represent the values from the amino acid sequence. In peak 1, the value of alanine was taken as 2.0. In CB-I and CB-III, the value of alanine was taken as 1.0, and in peak 4, the value of phenylalanine was taken as 1.0. Tryptophan was determined spectrophotometrically. Amino acid
S-Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Pbenylalanine Tryptophan Lysine Histidine Homoserine Arginine
Peak 1 (unfragmented cardiotoxin analogue IV)
Cyanogen bromide fragments Peak 2 (CB-III)
Peak 3 (CB-I)
8.48 (8) 8.67 (8) 2.97 (3) 2.36 (2) 0.33 (0) 4.58 (4) 2.33 (2) 2.00 (2) 0 6.70 (7) 1.88 (2) 1.12 (1) 5.58 (5) 3.06 (3) 1.79 (2) 0 7.73 (8) 0 0 3.00 (3)
5.25 (5) 5.77 (6) 1.89 (2) 1.77 (2) 0 1.98 (2) 1.05 (1) 1.00 (1) 0 5.28 (6) 0 1.21 (1) 1.95 (2) 0.96 (1) 0 0 2.86 (3) 0 0 1.95 (2)
3.38 (3) 2.24 (2) 1.00 (1) 0 0 1.85 (2) 1.04 (1) 1.00 (1) 0 1.15 (1) 0 0 2.91 (3) 1.87 (2) 1.27 (1) 0 5.95 (5) 0 0.80 (1) 0.96 (1)
Peak 4 (CB-II)
1.00 (1)
1.01 (1)
64 H-Ar g-Lys-Cys-Asn-Lys-Leu-Val-Pr o-Leu- Phe-Tyr-Lys-Thr-Cys-Pr o47 76 74 60 60 67 64 66 50 54 58 32 37 34 31 +--T-l-+ ~ T-2 ~ ~" T-3 ~~ T-4 ~ #------- T-6 T-5 > ( ._..~
. ~
._._~
_._..,
2(i
___.,
CB-I ._....._, _._..., ._..., ._.._~ .__..~ .._...~.._._~
.....~.
3~
AI a-Gly-Lys-Asn-Leu-Cys-Tyr-Lys-Met-Ph e-Me t-Val- S er-Asn-Leu31 26 16 17 22 18 18 i0 15 15 13 __~ _~'7 _~6 80 (
T-7
) +-T-8 -~
--
~ +CB-II--> (
Thr-Val- Pro-Val-Lys-Ar g-Gly-Cys- Ii e-As p-Val-Cys-Pro-Lys-Asn42 56 52 54 39 22 49 44 47 45 38 36 34 20 24 T'-I ) ~-T'-2+~ T'-3 ) t CB-III .... 60 Ser-Ala-Leu-Val-Lys-Tyr-Val-Cys-Cys-Asn-Thr-As p-Ar g-Cys -Asn-OH 21 22 21 21 13 19 17 16 16 ii i0 ll 3 7 5 T'-4
) ~-
T'-5
) ~-T'-6---~
Fig. 5. Amino acid sequence of cardiotoxin analogue IV. Symbols and numbers expressed are the same as in Fig. 4. DISCUSSION During our studies on the isolation and characterization of biologically active principles from the venom of Elapidae snakes, we observed that Naja naja atra venom differed strikingly from Naja naja venom with regard to the ion exchange chromatographic profile and the structural characteristics of the principle neurotoxic components [36]. However, we observed a remarkable similarity in the chromatographic profile of cardiotoxins from the two venoms. The present amino acid sequence studies indicate that cardiotoxin analogues isolated from the venom of Naja naja atra contain 60 amino acid residues in single peptide chains which are similar to those of cytotoxins isolated from the venom of Naja naja [12, 13, 14, 37]. Sequencing was carried out by manual Edman degradation and about half the total sequences of the parent reduced and S-carboxymethylated toxins were elucidated. As a result, two methionine residues were found in the central part of the toxin molecule. Cyanogen bromide cleavage of reduced and S-carboxymethylated toxins was carried out to obtain the carboxy-terminal fragment, which was used for determining the remaining amino acid sequence. Using the carboxy-terminal fragment, the amino acid sequences of residues 27 to 60 were determined. Thus, the sequences of the toxins were established by direct Edman degradation as shown in Figs. 4 and 5. The proposed amino acid sequences were further confirmed by amino acid analyses of the tryptic peptides of the CB-fragments obtained by cyanogen bromide cleavage of reduced and S-carboxymethylated toxins. In the tryptic digestion of the CBfragments, it was observed that the peptide bond between Leu(9)-Phe(10) in CB-I was hydrolyzed considerably but other leucyl peptide bonds were not hydrolyzed. In comparing neurotoxic polypeptides isolated from the venom of Elapidae and Hydrophiidae, Tu [38] classified the toxins into two types, Type I consisting of
65 61-62 amino acid residues and Type II consisting o f 71-74 amino acid residues.
Naja naja v e n o m contained Type II as major neurotoxins and Type I as minor neurotoxins, and Naja naja atra v e n o m contained only Type I neurotoxins [36]. However, all cardiotoxins isolated from a variety o f species representing the genera Naja [11-25] and Haemachatus [9] consisted of 60--61 amino acid residues. Cardiotoxins and neurotoxins were observed to be highly h o m o l o g o u s in their primary structures (see D a t a Deposition). In addition to the invariant distribution o f the eight half-cystine residues which are important for maintaining the protein in its active conformation, 24 amino acid residues are found in c o m m o n a m o n g these cardiotoxins, and two additional groups have similar side chain functions: Ile/Leu/Val and Arg/Lys. Even greater similarity can be seen in the amino- and carboxy-terminal sequences. These regions are considered to be essential to the formation o f active sites in cardiotoxins. The amino acid sequence o f cardiotoxin analogue IV differs from that o f cardiotoxin analogue II only by the presence o f the arginine residue in position 1. The replacement can be explained by a single base replacement in the D N A triplet codes ( C U U to C G U , C U C to C G C , C U A to C G A , or C U G to C G G ) . Cardiotoxin analogue IV is the first cardiotoxin found with an arginine residue at the amino terminus, although some neurotoxins have an arginine residue at that position [39-43]. ACKNOWLEDGMENTS The authors are greatly indebted to Professor I. Yamashina o f our laboratory for the valuable discussions in the course of this study. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
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NORIO KANEDA a, TOYOSAKU SASAKIb and KYOZO HAYASHI a aDepartment of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, and bHimeji Institute of Technology, Himeji (Japan) (Received September 16th, 1976)
SUMMARY Cardiotoxin analogues II and IV were isolated from the venom of Naja naja atra by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The venom contains at least four cardiotoxin analogues that account for about 54 of the weight of the lyophilized crude venom. These four cardiotoxin analogues, named cardiotoxin analogues I, II, III, and IV, show strong cytotoxicity to Yoshida sarcoma cells but the lethal toxicity is one-order less. These toxins contain 60 amino acid residues in a single peptide chain. Cardiotoxin analogue IV differs from cardiotoxin analogue II only by the presence of arginine in place of a leucine residue at position 1. A comparison of the amino acid sequences of these toxins with that of cobrotoxin, a neurotoxin containing 62 amino acid residues obtained from the same snake venom, shows that about 20 amino acid residues, including 8 half cystine residues, are identical, assuming 3 residues deletion and 2 residues insertion in the cardiotoxin molecule.
INTRODUCTION A number of biologically active components have been isolated from venoms of snakes belonging to the family Elapidae [1]. One group of basic polypeptides, which act on the pre- or post-synaptic sites, are known as venom neurotoxins [2]. Another group having relatively low toxicity to animals induce a variety of effects which have been separately recognized and studied in various laboratories. As a consequence confusion in their nomenclature arose and only recently they were found * Supplementary data to this article, giving details of amino acid compositions, chromatography and electrophoresis patterns and amino acid sequences, are deposited with, and can be obtained from: Elsevier Scientific Publishing Company, BBA Data Deposition, P.O. Box 1527, Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/055/37602/491(1977)53. Abbreviations and symbols: The following abbreviations are used: CB- cyanogen bromide fragments, T- tryptic peptides derived from CB-I obtained by cyanogen bromide cleavage of reduced and S-carboxymethylated-cardiotoxinanalogues II and IV. T'-, tryptic peptides derived from each CB-II1 obtained by cyanogen bromide cleavage of reduced and S-carboxymethylated-cardiotoxin analogues II and IV. -
-
54 to be closely related if not identical. These strongly basic polypeptides isolated from a specific venom or from venoms of different species or subspecies have been designated as cardiotoxin [3], skeletal muscle depolarizing factor [4], cobramines A and B [5], cytotoxin [6], toxin y [7], toxin direct lytic factor [8], protein 12B [9] and others. In this paper, we use the name "cardiotoxins" because of their effect on the heart and because of the precedent established by Lee et al. [10]. Within the last few years, these strongly basic cardiotoxic polypeptides have been isolated in pure form from the venoms of several species of cobra [9, 11-25]. Cardiotoxins are about one-order less toxic than many neurotoxins from Elapidae snake venoms [2]. By gel filtration on Sephadex G-50 followed by chromatography on CMcellulose, we obtained four cardiotoxin analogues which exhibited cytotoxicity to Yoshida sarcoma cells. Because the amino acid composition data suggested a high degree of structural homology among these toxins, we felt that the primary structure determination of these cardiotoxins might help to reveal the significance of the relationship between cardiotoxins and neurotoxins and also aid in the identification of functionally important amino acid residues. Previously, we reported on the amino acid sequences of cardiotoxin analogues I and III (---- cardiotoxin) isolated from the venom of Naja naja atra [15, 44]. The present paper deals with the complete amino acid sequences of cardiotoxin-analogues II and IV from the same venom. MATERIALS AND METHODS Lyophilized Naja naja atra venom, a-chymotrypsinogen A, cytochrome c, and insulin were obtained from Sigma Chemical Co., Ltd., U.S.A. Bovine trypsin (recrystallized three times) (EC 3.4.21.4) was obtained from Worthington Biochemical Corp., U.S.A. Sephadex G-25 and G-50 were the products of Pharmacia Fine Chemicals, Sweden. Carboxymethyl (CM)-cellulose was purchased from Seikagaku Kogyo Co., Ltd., Japan. All other chemicals were commercial preparations of the highest quality available.
Disc gel electrophoresis Polyacrylamide disc gel electrophoresis was performed by the method of Williams [26], using disc gel electrophoresis apparatus of Mitsumi Kagaku Sangyo, Co., Ltd., Japan, and 15 ~ gels at pH 4.0. Electrophoresis was performed with an initial current of 3 mA per gel. After concentration of the sample, the current was increased to 5 mA per gel. Gels were stained with Amido Black 10B and rinsed several times with 10 ~ acetic acid solution.
Determination of molecular weight by gel filtration The molecular weight of the purified cardiotoxin-analogues II and IV was determined by the gel filtration method described by Andrews [27]. A column (1.4 x 144 cm) of Sephadex G-50 equilibrated with 0.05 M phosphate buffer containing 0.1 M KCI, pH 7.5, was used. Elution was carried out with the same buffer at a flow rate of 30 ml per h at room temperature. The calibration curve was made by using blue dextran, a-chymotrypsinogen A, cytochrome c and insulin as markers.
55
Toxicity The toxicity was measured by subcutaneous or intravenous injection (mice, 16-18 g) of progressively diluted venom solutions as described previously [15]. Five male mice were used for each dilution and the LDs0 was calculated according to the 50 ~ end peint method of Reed and Muench [28]. Five controls were used under the same conditions but without venom.
Assay of cytotoxicity Assay of cytotoxicity to Yoshida sarcoma cells was performed by modification of the method described by Braganca et al. [6]. The tumors were maintained in Donryu rats by transplantation of 0.2 ml of fluid derived from the intraperitoneal cavity. In this study, the ascitic fluid containing Yoshida sarcoma cells was withdrawn from the intraperitoneal cavity 3-4 days after transplantation with this tumor. The tumor cells were freed from blood cells according to the method of Mckee, Lonberg-Holm and Jehl [29]. 0.1 ml of serial dilutions of the active material in phosphate buffered saline solution was added to 0.1 ml of cell suspension (4.106 cells/ml). The mixture was incubated with gentle shaking at 37 °C and at the end of 30 min 0.2 ml of trypan blue (0.4 ~ in saline) was added to the incubation mixture. Part of this mixture was placed on a haemocytometer slide glass and the proportion of damaged cells was calculated by dividing the number of cells permeable to trypan blue by the total number of cells. Cells incubated without the active fraction were permeable to trypan blue to the extent of 5-7 ~ . Cytotoxicity was defined as the concentration (expressed as fig protein/ml) of active material which caused 50 ~ of the cells to become permeable to trypan blue.
Purification of cardiotoxin analogues The cardiotoxins were purified as follows: 4 g of lyophilized venom was dissolved in about 20 ml of 1 ~ acetic acid and applied to a column of Sephadex G-50 (4 x 193 cm) at room temperature. The Sephadex column was eluted with 1 ~o acetic acid at a flow rate of 42 ml/h. Fractions of 15 ml each were collected and assayed for absorbance at 280 nm. After pooling, each pool was lyophilized. The pool containing compounds with a molecular weight of about 6000-7500 yielded 3.118 g (or 78.0 of the crude venom). The entire sample was dissolved in 10 ml of 0.005 M sodium acetate buffer, pH 5.8 and applied to a CM-cellulose column (2.8 x 140 cm) equilibrated with the same buffer. The column was washed with the starting buffer and eluted with a gradient formed from 10 liters of equilibration buffer in the mixing vessel and 10 liters of 0.5 M sodium acetate buffer, pH 6.5, in the reservoir. Fractions of 15 ml each were collected, lyophilized, redissolved in a minimum amount of 1 acetic acid, and gel-filtered to remove sodium acetate. Four cardiotoxin-like components, cardiotoxin analogue I, cardiotoxin analogue II, cardiotoxin analogue III, and cardiotoxin analogue IV, were isolated by this method. Cardiotoxin analogues II and IV were further purified on a column of CMcellulose and eluted with a gradient of increasing pH and salt concentration. The gradient was established by means of a mixing vessel containing 1000 ml of 0.2 M sodium acetate buffer, pH 6.0, connected to a reservoir containing 1000 ml o f 0.8 M sodium acetate buffer, pH 6.8. Fractions of 4 ml each were collected at a flow rate of 46 ml/h at room temperature. The pooled protein fraction forming the major peak
56 was lyophilized, the residue redissolved in 1 ~o acetic acid, sodium acetate removed by Sephadex G-25 gel filtration and the resulting solution lyophilized.
Amino acid composition Samples (0.05 to 0.1 ffmol) were hydrolyzed in an evacuated, sealed tube with 0.5-1.0 ml of 5.7 M HC1 for 24 h at 110 °C, the acid removed by evaporation under reduced pressure, and the residue dissolved in 1.1 ml of 0.2 N sodium citrate buffer, pH 2.20. Analysis was performed with an Hitachi KLA 3B automatic amino acid analyzer by the method of Spackman et al. [30].
Reduction and alkylation The carboxymethyl derivatives of cardiotoxin-analogues II and IV were prepared by the method of Crestfield et al. [31]. The samples were dissolved in 0.2 M Tris.HC1 buffer, pH 8.2, containing 5 M guanidine-HC1 and fl-mercaptoethanol was added. The glass-stoppered tube was flushed with nitrogen gas and left standing at 37 °C for 4 h. For alkylation, a freshly prepared solution of iodoacetic acid in 2 N NaOH was added with stirring and the pH of the solution maintained at pH 8.6 with 2 N NaOH. After 30 rain the solution was desalted by passing through a column of Sephadex G-25. The reduced and S-carboxymethylated-cardiotoxin analogues II and IV were each pooled and lyophilized.
Cyanogen bromide cleavage Fragmentation of reduced and S-carboxymethylated-cardiotoxin analogues (about 10 ffmol) was performed in 70 ~ formic acid with a 300-fold molar excess of cyanogen bromide for 24 h at room temperature [32]. After dilution with water, the reaction mixture was lyophilized to remove excess reagent, the residues dissolved in 0.2~ acetic acid and applied to a column (1.8 × 111 cm) of Sephadex G-50 equilibrated with 0.2 ~ acetic acid. The chromatographic profile was established by following the absorbance at 280 nm. Fractions with absorbance were pooled and the pool lyophilized.
Trypsin digestion of CB-I and CB-III generated by cyanogen bromide cleavage Trypsin digestion was carried out at 37 °C for 8 h in 0.1 M ammonium bicarbonate buffer, pH 8.0, with a weight ratio of enzyme to substrate of 1:50. After digestion, the digest was lyophilized.
Isolation of tryptie peptides The peptides generated by trypsin were isolated by a combination of high voltage paper electrophoresis and paper chromatography. High voltage paper electrophoresis was performed using a solvent system of pyridine/acetic acid/water (1:10:289, by volume), pH 3.6, at 30 to 35 V per cm in a tank (Toyo Kagaku Sangyo Co., Ltd., Japan) containing Esso naphtha No. 5 as a coolant. Guide strips (5 mm) were cut and stained with ninhydrin reagent. Peptides were eluted from the paper with 0.1 M pyridine adjusted to pH 5.0 with acetic acid. The purities of extracted peptides were examined by descending paper chromatography on Toyo paper No. 51 using a solvent system of 1-butanol/acetic acid/pyridine/water (15:3:10:l 2, by volume) [solvent (a)] in a glass tank for 14 to 16 h. The chromatograms were stained with ninhydrin reagent.
57 When the peptides were chromatographically impure, further purification was carried out by paper chromatography using solvent (a), 1-butanol/acetic acid/water (4:1:5, by volume) [solvent (b)], or water-saturated phenol [solvent (c)].
Sequence analyses The amino-terminal sequences of reduced and S-carboxymethylated-cardiotoxin analogues II and IV, and carboxy-terminal fragments generated by cyanogen bromide cleavage were determined using a modified Edman's phenylthiohydantoin procedure [33]. The amounts of the phenylthiohydantoin derivatives were determined semiquantitatively by measuring their absorbance at 269 nm before chromatography. The derivatives were then identified by thin layer chromatography on Kieselgel F254 plates (E. Merck, Germany) using solvent systems IV and V described by Jeppsson and SjSquist [34] and on polyamide sheets (Carle Place, New York, U.S.A.) using a solvent system of toluene/n-pentane/acetic acid (60:30:35, by volume); 250mg [2-(4'-butylphenyl)-5-(4'-biphenyl)-l,3,4-oxdiazole]/liter [35]. The aqueous phase, containing phenylthiohydantoin arginine was evaporated to dryness, the residue dissolved in 0.1 ml of 70 ~ ethanol, an aliquot spotted on filter paper and tested for arginine with Sakaguchi's reagent. Phenylthiohydantoin leucine and phenylthiohydantoin isoleucine were determined, when they occurred, by amino acid analyses of the tryptic peptides. RESULTS
Purification of cardiotoxin analogues H and IV Lyophilized crude venom dissolved in 1 ~ acetic acid was applied to a column of Sephadex G-50 and eluted with 1 ~ acetic acid. A typical elution pattern is shown in Fig. 1. Four peaks (I to IV) with absorption at 280 nm were obtained. The pool from peak IV containing the cardiotoxin analogue, was further purified on a column of CM-cellulose. As shown in Fig. 2, many peaks appeared. Among these, four frac2.000
¢q
1.000
o .
Fig. 4. Amino acid sequence of cardiotoxin analogue II. Horizontal arrows below the amino acid residues denote the sequence of cyanogen bromide fragments derived from reduced and S-carboxymethylated-cardiotoxin analogue II and tryptic peptides. ~ , sequence elucidated by Edman degradation. T and T' represent the peptides produced by trypsin digestion of fragments CB-I and CB-III, respectively. Numbers below amino acid residues show the yields ( ~ ) of phenylthiohydantoin amino acids estimated from the molar absorbance coefficient at 269 nm and from the amounts of samples used in the Edman degradation.
63 ed f r o m the a m i n o - t e r m i n a l p o r t i o n . The f o u r t h peak, CB-1I, c o r r e s p o n d e d to phenylalanylhomoserine. Stepwise E d m a n d e g r a d a t i o n o f the c a r b o x y - t e r m i n a l f r a g m e n t C B - I I I (11.6 mg) revealed the following a m i n o acid sequence: -Va•-Ser-Asn-Leu-Thr-Va•-Pr•-Va•-Lys-Arg-G•y-Cys-••e•Asp-Va•-Cys-Pr•-Lys-Asn-Ser-A•a-LeuVal-Lys-Tyr-Val-Cys-Cys-Asn-Thr-Asp-Arg-Cys-Asn-OH O n the basis o f the a b o v e results, the a m i n o acid sequence o f c a r d i o t o x i n a n a l o g u e I V was f o u n d to be C B - I - I I - I I I . F o r conclusive evidence, fragments CB-I a n d C B - I I I were digested by trypsin, the resulting peptides s e p a r a t e d b y a c o m b i n a t i o n o f high voltage p a p e r electrophoresis a n d p a p e r c h r o m a t o g r a p h y as described for cardiotoxin a n a l o g u e II, a n d analyzed for a m i n o acid c o m p o s i t i o n (see D a t a Deposition). The overall p r i m a r y structure o f c a r d i o t o x i n a n a l o g u e IV was ascertained as shown in Fig. 5,
TABLE III AMINO ACID COMPOSITIONS OF THE FRACTIONS GENERATED BY CYANOGEN BROMIDE CLEAVAGE OF REDUCED AND S-CARBOXYMETHYLATED-CARDIOTOXIN ANALOGUE IV Numbers in parentheses represent the values from the amino acid sequence. In peak 1, the value of alanine was taken as 2.0. In CB-I and CB-III, the value of alanine was taken as 1.0, and in peak 4, the value of phenylalanine was taken as 1.0. Tryptophan was determined spectrophotometrically. Amino acid
S-Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Pbenylalanine Tryptophan Lysine Histidine Homoserine Arginine
Peak 1 (unfragmented cardiotoxin analogue IV)
Cyanogen bromide fragments Peak 2 (CB-III)
Peak 3 (CB-I)
8.48 (8) 8.67 (8) 2.97 (3) 2.36 (2) 0.33 (0) 4.58 (4) 2.33 (2) 2.00 (2) 0 6.70 (7) 1.88 (2) 1.12 (1) 5.58 (5) 3.06 (3) 1.79 (2) 0 7.73 (8) 0 0 3.00 (3)
5.25 (5) 5.77 (6) 1.89 (2) 1.77 (2) 0 1.98 (2) 1.05 (1) 1.00 (1) 0 5.28 (6) 0 1.21 (1) 1.95 (2) 0.96 (1) 0 0 2.86 (3) 0 0 1.95 (2)
3.38 (3) 2.24 (2) 1.00 (1) 0 0 1.85 (2) 1.04 (1) 1.00 (1) 0 1.15 (1) 0 0 2.91 (3) 1.87 (2) 1.27 (1) 0 5.95 (5) 0 0.80 (1) 0.96 (1)
Peak 4 (CB-II)
1.00 (1)
1.01 (1)
64 H-Ar g-Lys-Cys-Asn-Lys-Leu-Val-Pr o-Leu- Phe-Tyr-Lys-Thr-Cys-Pr o47 76 74 60 60 67 64 66 50 54 58 32 37 34 31 +--T-l-+ ~ T-2 ~ ~" T-3 ~~ T-4 ~ #------- T-6 T-5 > ( ._..~
. ~
._._~
_._..,
2(i
___.,
CB-I ._....._, _._..., ._..., ._.._~ .__..~ .._...~.._._~
.....~.
3~
AI a-Gly-Lys-Asn-Leu-Cys-Tyr-Lys-Met-Ph e-Me t-Val- S er-Asn-Leu31 26 16 17 22 18 18 i0 15 15 13 __~ _~'7 _~6 80 (
T-7
) +-T-8 -~
--
~ +CB-II--> (
Thr-Val- Pro-Val-Lys-Ar g-Gly-Cys- Ii e-As p-Val-Cys-Pro-Lys-Asn42 56 52 54 39 22 49 44 47 45 38 36 34 20 24 T'-I ) ~-T'-2+~ T'-3 ) t CB-III .... 60 Ser-Ala-Leu-Val-Lys-Tyr-Val-Cys-Cys-Asn-Thr-As p-Ar g-Cys -Asn-OH 21 22 21 21 13 19 17 16 16 ii i0 ll 3 7 5 T'-4
) ~-
T'-5
) ~-T'-6---~
Fig. 5. Amino acid sequence of cardiotoxin analogue IV. Symbols and numbers expressed are the same as in Fig. 4. DISCUSSION During our studies on the isolation and characterization of biologically active principles from the venom of Elapidae snakes, we observed that Naja naja atra venom differed strikingly from Naja naja venom with regard to the ion exchange chromatographic profile and the structural characteristics of the principle neurotoxic components [36]. However, we observed a remarkable similarity in the chromatographic profile of cardiotoxins from the two venoms. The present amino acid sequence studies indicate that cardiotoxin analogues isolated from the venom of Naja naja atra contain 60 amino acid residues in single peptide chains which are similar to those of cytotoxins isolated from the venom of Naja naja [12, 13, 14, 37]. Sequencing was carried out by manual Edman degradation and about half the total sequences of the parent reduced and S-carboxymethylated toxins were elucidated. As a result, two methionine residues were found in the central part of the toxin molecule. Cyanogen bromide cleavage of reduced and S-carboxymethylated toxins was carried out to obtain the carboxy-terminal fragment, which was used for determining the remaining amino acid sequence. Using the carboxy-terminal fragment, the amino acid sequences of residues 27 to 60 were determined. Thus, the sequences of the toxins were established by direct Edman degradation as shown in Figs. 4 and 5. The proposed amino acid sequences were further confirmed by amino acid analyses of the tryptic peptides of the CB-fragments obtained by cyanogen bromide cleavage of reduced and S-carboxymethylated toxins. In the tryptic digestion of the CBfragments, it was observed that the peptide bond between Leu(9)-Phe(10) in CB-I was hydrolyzed considerably but other leucyl peptide bonds were not hydrolyzed. In comparing neurotoxic polypeptides isolated from the venom of Elapidae and Hydrophiidae, Tu [38] classified the toxins into two types, Type I consisting of
65 61-62 amino acid residues and Type II consisting o f 71-74 amino acid residues.
Naja naja v e n o m contained Type II as major neurotoxins and Type I as minor neurotoxins, and Naja naja atra v e n o m contained only Type I neurotoxins [36]. However, all cardiotoxins isolated from a variety o f species representing the genera Naja [11-25] and Haemachatus [9] consisted of 60--61 amino acid residues. Cardiotoxins and neurotoxins were observed to be highly h o m o l o g o u s in their primary structures (see D a t a Deposition). In addition to the invariant distribution o f the eight half-cystine residues which are important for maintaining the protein in its active conformation, 24 amino acid residues are found in c o m m o n a m o n g these cardiotoxins, and two additional groups have similar side chain functions: Ile/Leu/Val and Arg/Lys. Even greater similarity can be seen in the amino- and carboxy-terminal sequences. These regions are considered to be essential to the formation o f active sites in cardiotoxins. The amino acid sequence o f cardiotoxin analogue IV differs from that o f cardiotoxin analogue II only by the presence o f the arginine residue in position 1. The replacement can be explained by a single base replacement in the D N A triplet codes ( C U U to C G U , C U C to C G C , C U A to C G A , or C U G to C G G ) . Cardiotoxin analogue IV is the first cardiotoxin found with an arginine residue at the amino terminus, although some neurotoxins have an arginine residue at that position [39-43]. ACKNOWLEDGMENTS The authors are greatly indebted to Professor I. Yamashina o f our laboratory for the valuable discussions in the course of this study. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
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