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The Role of the N-Terminal Leucine Residue in Snake Venom Cardiotoxin II (Naja naja atra)

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS233, 713 – 716 (1997)

ARTICLE NO.RC976503

The Role of the N-Terminal Leucine Residue in Snake

Venom Cardiotoxin II (Naja naja atra)

Chi-Yue Wu,*

,1

Wan-Chen Chen,* Chewn-Lang Ho,* Shui-Tein Chen,* and Kung-Tsung Wang*

,

*Institute of Biological Chemistry, Academia Sinica, 128, Yan-Chiu-Yuan Road, Sec II, Nankang, Taipei, Taiwan 11529, Republic of China; and †Department of Chemistry, National Taiwan University, 1, Roosevelt Road, Sec IV, Taipei, Taiwan 11529, Republic of China

effects, including lethal toxicity, hemolysis, cytolysis,

The N-terminal leucine residue of snake venom muscle contractures, membrane depolarization, and cardiotoxin II (CTX II) (Naja naja atra) was systemati- activation of tissue phospholipase C (4-7).

cally replaced with D-leucine (CTXII-L1-D-L), glycine Among all CTXs, their N-terminal residue is highly (CTXII-L1G) or deleted [CTXII-(2-60)] to study the role

conserved and is either leucine (90.3%) or isoleucine

of leucine residue in CTX II molecule. CTX II,

CTXL1-(7.3%) except for CTX IV (Naja naja atra) (5, 8, 9).

D-L, CTXL1G and CTX(2-60) were produced by

chemi-Therefore, it is very interesting to explore the reason

cal synthesis method and purified by high

perfor-why the N-terminal residue of CTX is so highly

con-mance liquid chromatography. Owing to folding

prob-served. In the present report, we choose CTX II (Naja

lem in CTXII-(2-60), only CTX II, CTXII-L1-D-L and

naja atra) as a model molecule and systematically

engi-CTXII-L1G were produced in a pure form and

charac-neer its N-terminal leucine residue by D-leucine

terized by amino acid analysis, mass spectrometry and

(CTXII-L1-D-L), glycine (CTXII-L1G) or deleted [CTXII-peptide mapping. In the structural aspect, changing the

(2-60)] using chemical synthesis method as developed

Leu-1 byD-Leu or Gly causes a drastic alteration in the

in our laboratory (10) to study the effects of N-terminal

whole CTX II structure as detected by circular

dichro-substitutions on the structure and biological function

ism, 1-anilino-naphthalene-8-sulfonate (ANS)

fluores-cence assay. In the functional aspect, both CTXII-L1-D-L of CTX II.

and CTXII-L1G are still retained substantial biological activity of CTX II. Therefore, the results indicate that

MATERIALS AND METHODS

both the chirality and the side-chain of the N-terminal leucine residue of CTX II are important elements in

Chemical synthesis. Samples were chemically synthesized us-maintaining the whole CTX II structure. In addition,

ing a Fmoc amino acid strategy with an Applied Biosystems 431A this study is the first report in elucidating the reason peptide synthesizer using the protocols and reagents provided by why the first N-terminal residue of most CTXs (90.3%) the manufacturers. The previously described procedures (10) were is leucine residue. q 1997 Academic Press used to air-oxidize and purify each sample. The purity of each sample was determined by analytical RP-HPLC (Rainin, C18, 5

mm, 4.6 1 250 mm).

Determination of amino acid composition and molecular mass.

Amino acid composition and molecular mass of each sample were Snake venom cardiotoxins (CTXs) (also called

cyto-determined as previously described (10). The extinction coefficients toxins), a group of basic proteins containing 60-63 of 1% toxin solutions in water at 280 nm were 6.64 for CTX II and amino acids and 4 disulfide bonds, are major lethal 6.60 for each CTX II analog.

components of elapid snake venom (1, 2). In contrast

Peptide mapping. Sample (0.2 mg) was digested by thermolysin to cobra neurotoxins, which interact with the postsyn- (5 units) in Tris-HCl buffer (0.05M, pH 7.5) at 37

7C for 24 hr. Di-aptic acetylcholine receptor (3), CTXs show no defined gested sample was analyzed by analytical RP-HPLC (Rainin, C

18, 5

cellular targets and have very diverse pharmacological mm, 4.6 1 250 mm).

Circular dichroism spectra. Circular dichroism spectra were re-corded at 257C in quartz cells (path length 1 mm) using a JASCO 1To whom correspondence should be addressed: Chi-Yue Wu,

In-model J-720 circular dichroism spectrophotometer equipped with a stitute of Biological Chemistry, Academia Sinica, 128, Yan-Chiu- thermoelectric temperature controller and constantly flushed with Yuan Road, Sec II, Nankang, Taipei, Taiwan 11529. Fax: 886-2- nitrogen. The protein concentration was 0.23 mg/mL in 1 mM Tris-7883473. HCl buffer (pH 8.0). Five scans were averaged for each of CTX

sam-The abbreviations used are: ANS, 1-anilino-naphthalene-8-sul- ples and for the solvent. fonate; CTX, cardiotoxin; Fmoc, 9-fluorenylmethoxycarbonyl; HMP;

4 - hydroxymethylphenoxymethylcopolystyrene - 1% - divinylbenzene; ANS fluorescence spectra. Fluorescence spectra of ANS binding of samples were measured between 420 and 600 nm using the excita-RP-HPLC, reverse-phase high performance liquid chromatography;

TFA, trifluoroacetic acid. tion wavelength of 400 nm on a Hitachi fluorescence spectrophotome-0006-291X/97 $25.00

Copyrightq 1997 by Academic Press All rights of reproduction in any form reserved. 713

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Vol. 233, No. 3, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS

Synthesis of CTX II and its analoges. CTXII-(2-60)

peptide-resins were first produced by coupling each amino acid residue from C-terminal Asn-60 to N-termi-nal Lys-2 on 4-hydroxymethyl-phenoxymethylco-polystyrene-1%-divinylbenzene (HMP) resin. Con-sequently, CTX II, CTXII-L1-D-L and CTXII-L1G peptide-resins were produced by coupling the last N-terminal residue, L-Leu, D-Leu, or Gly, respectively, onto CTXII-(2-60) peptide-resin. After 95% trifluoro-acetic acid (TFA) cleavage of each peptide-resin, each crude reduced CTX sample (purity about 30%) was pu-rified by semi-preparative C18RP-HPLC, and then the

FIG. 1. HPLC profiles of purified CTX II and CTX analogs as

purified sample was directly air-oxidized in the phos-analyzed by analytical RP-HPLC. (1) CTX II, (2) CTXII-L1-D-L, and

phate buffer for 7 days (10). Each crude oxidized sam-(3) CTXII-L1G. Conditions: C18column; 5%-95% CH3CN containing

ple was purified by semi-preparative C18RP-HPLC and 0.1% TFA, 30 min; 1 ml/min, UV 280 nm.

further purified by analytic C18 RP-HPLC.

Unfortu-nately, owing to folding problem in CTXII-(2-60), only CTX II, CTXII-L1-D-L and CTXII-L1G were obtained ter, Model F-4010. The measurements were performed 10 min after

in a pure form (above 99%), as shown in Fig. 1. mixing the sample (10mM) and ANS (200mM).

Characterization of CTX II and its analogs. Amino

Biological activity assay. Lethal toxicity of CTX sample was

mea-acid compositions of CTX samples were determined by sured by intravenous injection of sample into the tail veins of

experi-amino acid analysis. The results show that values for mental mice (19-21g). Six mice were used at each dose, and the

toxicity was expressed as LD50(11). Six control mice were treated each CTX sample can be consistent with the corre-under the same conditions, but without CTX sample. sponding theoretical values of each amino acid residue

The ability of the CTX sample to stimulate muscle contractions

except for cystine, methionine and tyrosine, as shown was assayed using a chicken biventer cervices muscle preparation

in Table 1. Molecular mass of each CTX sample was (12). The muscle preparation was maintained in 20 ml Krebs solution

determined by electrospray mass spectroscopy. The re-and oxygenated with 95% O2and 5% CO2at 377C. The muscle was

stimulated indirectly with supramaximal rectangular electric pulses sults show that molecular masses of CTX II (6742.13), of 0.5 ms duration at a frequency of 0.2 Hz. The isometric contrac- CTXII-L1-D-L (6742.13) and CTXII-L1G (6686.02) are tions were recorded with a Grass FT 03 force displacement

trans-6742.03, 6742.69 and 6687.00, respectively, as shown ducer attached to a Grass 7D polygraph. Toxins (1.5mM) were

ap-in Table 1. The molecular mass data are consistent plied to the bathing medium to assess their ability to increase muscle

contractions. with their corresponding theoretical value,

respec-TABLE 1

Amino Acid Compositions and Molecular Masses of CTX Samples

CTX II CTXII-L1-D-L CTXII-L1G

Amino acid Theoretical Found Theoretical Found Theoretical Found

Asp 8 7.45 8 7.85 8 7.33 Thr 3 2.50 3 2.37 3 2.07 Ser 2 1.35 2 1.78 2 1.04 Pro 4 3.62 4 3.71 4 3.73 Gly 2 2.10 2 2.01 3 3.05 Ala 2 2.00 2 2.00 2 2.00 Val 7 6.10 7 6.40 7 6.25 Met 2 0.20 2 0.17 2 0.12 Ile 1 0.89 1 0.80 1 0.87 Leu 6 5.14 6 5.30 5 4.50 Tyr 3 1.56 3 1.95 3 1.86 Phe 2 1.65 2 1.90 2 1.67 Lys 8 7.22 8 7.20 8 7.03 Arg 2 2.05 2 2.00 2 1.63 1/2CYSSCY 8 2.54 8 2.81 8 2.56 Molecular weight 6742.13 6742.69 6742.13 6742.69 6686.02 6687.00 714 AID BBRC 6503 / 6929$$$282 04-11-97 08:56:03 bbrcg AP: BBRC

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Vol. 233, No. 3, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 2. HPLC profiles of digested (1) CTX II, (2) CTXII-L1-D-L and (3) CTXL1G by protease thermolysin. Conditions: C18column; 5%-95% CH3CN containing 0.1% TFA, 30 min; 1ml/min, UV 280 nm.

tively. In addition, peptide mapping was used to com-pare the disulfide-linkage patterns of these CTX

sam-FIG. 4. Fluorescence spectra of ANS in the presence of various ples. The results show that the peak patterns in HPLC

CTX samples. Curves: (1) CTX II, (2)CTXII-L1-D-L and (3) CTXL1G. profiles of thermolysin-digested CTX II, CTXII-L1-D-L The dotted line shows the fluorescence of ANS in the absence of and CTXII-L1G are similar to each other except for a sample.

few minor peaks which are resulted from the first N-terminal residue differences among these CTX analogs, as shown in Fig. 2. This result suggests that the

disul-II are markedly decreased for CTXdisul-II-L1-D-L and fide-linkage patterns of these CTX analogs are similar

CTXII-L1G spectra. In the near ultraviolet region, CTX to each other.

II displays a broad positive peak in the 256-277 nm

Structural comparison of CTX II and its analogs. wavelength range. This region is much decreased for

Circular dichroism and ANS fluorescence assays were CTXII-L1G and is completely reverse to a negative used to compare the tertiary structures of CTX II, trough for

CTXII-L1-D-L. CTXII-L1-D-L and CTXII-L1G.

The fluorescence emission of ANS is known to in-In the far ultraviolet region of circular dichroism

crease when it binds to hydrophobic regions of a protein spectrum, CTX II displays a negative trough at 211.0

(13). Therefore, ANS is a suitable hydrophobic probe nm (01100 deg cm2

dmol01

), a smaller positive band at

for comparison of the accessibility of the hydrophobic 224.1 nm (600 deg cm2

dmol01

), and a larger positive

core in CTX II and its analogs. Figure 4 shows the peak at 195.0 nm (400 deg cm2

dmol01

), indicating

pre-fluorescence spectra of ANS in the presence of various dominantb-sheet organization, as shown in Fig. 3. The

CTX samples. Only a limited increase in fluorescence negative trough presented in CTX II is shifted from

is observed in the presence of CTX II, whereas a sub-211 nm to 206 nm (02400 deg cm2

dmol01

) for

CTXII-stantial fluorescence increase is observed in the pres-L1-D-L and to 204 nm (04400 deg cm2 dmol01) for

ence of CTXII-L1-D-L or CTXII-L1G. In addition, the CTXII-L1G. The two positive peaks presented in CTX

wavelength of maximum emission is shifted from 520 nm in CTX II to 510 nm in both CTX II analogs. This result indicates that the ANS molecule has become en-closed in a more hydrophobic environment in CTXII-L1-D-L or CTXII-L1G than CTX II (14). Thus, the con-sequence of ANS fluorescence analysis suggests that there are more accessible hydrophobic regions of both CTXL1-D-L and CTXL1G for ANS than CTX II.

Biological activities of CTX II and its analogs.

Le-thal toxicity and muscle contracture assays were used to evaluate the biological activity of each CTX sample. Lethal toxicity (expressed as LD50) of CTX II,

CTXII-L1-D-L and CTXII-L1G are 2.47, 4.32 and 6.77 mg/g, respectively, as shown in Table 2. In muscle con-tracture assay, 1.5mM CTXII-L1-D-L and CTXII-L1G stimulated muscle contraction with a force of 200 and 170 mg, respectively, approximately 26% and 22%, re-FIG. 3. Circular dichroism spectra of (1) CTX II, (2)CTXII-L1-D

-L and (3) CTX-L1G. spectively, of force elicited by 1.5mM CTX II (780 mg). 715

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Vol. 233, No. 3, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2 important structural element. In addition, this report

also provides a possible reason to interpret why the Biological Activities of CTX II and Its Analogs

first N-terminal residue of most CTXs (90.3%) is leu-LD50 Muscle contracturea cine residue. On the other hand, the present report is Toxin (mg/g body wt) (mg) the first example of engineering CTX mutants using chemical synthesis, and this method may provide a fea-CTX II 2.47 (2.34-2.62) 780 { 130 (3)

sible and efficient route to study the structure/function CTXII-L1-D-L 4.32 (4.11-4.53)* 200 { 10 (3)*

CTXII-L1G 6.77 (6.54-7.01)* 170 { 40 (3)* relationship of other biologically active polypeptides consisting of approximately 60 residues.

aValues given are means { S.E.M. with the numbers of experi-ments in parentheses.

ACKNOWLEDGMENTS * p õ 0.05 as compared with the value of CTX II.

We are grateful to Prof. Wen-Chang Chang of the Institute of Biological Chemistry, Academia Sinica, and Dr. Ming-Fai Tam of the Institute of Molecular Biology, Academia Sinica, for performing the DISCUSSION

mass spectrometry; and Prof. Shyh-Horhg Chiou and Ms. Hui-Ming Yu of the Institute of Biological Chemistry, Academia Sinica, for In our study, as comparison with the conformations assisting with the measurement of fluorescence spectrum and the peptide synthesis, respectively. Support for this research provided by of CTX II and CTXII-L1-D-L, a significant structural

Academia Sinica and the National Science Council, Taipei, Taiwan is difference is presented which is resulted from changing

gratefully acknowledged. the chirality of Leu-1 in CTX II from the L- to the D

-form. In the same way, as comparison of CTX II and

REFERENCES CTXII-L1G, a substantial structural difference is also

presented which is resulted from the deletion of the 1. Fryklund, L., and Eacker, D. (1975) Biochemistry 14, 2865 – 2871.

side-chain of Leu-1 in CTX II. These results clearly

2. Bougis, P. E., Marchot, P., and Rochat, H. (1986) Biochemistry indicate that both the chirality and the side-chain of

25, 7235 – 7243. the first N-terminal Leu residue of CTX II are

im-3. Changeux, J. P. (1981) Haevey Lect. 75, 85 – 254. portant elements in the maintenance of CTX II

confor-4. Lee, C. Y. (1972) Ann. Rev. Pharmacol. 12, 265 – 28confor-4. mation. Although the three-dimensional structure of

5. Dufton, M. J., and Hider, R. C. (1991) in Snake Venom (Harvey, CTX II (Naja naja atra) had been determined (15), only

A. L., Ed.), pp. 259 – 302, Pergamon Press, New York. limited information about N-terminal residue could be

6. Harvey, A. L. (1991) Handbook of Natural Toxins, 5th ed., pp. obtained which is not enough for interpreting our ex- 85 – 106, Dekker, New York.

perimental results. Therefore, it is necessary to further 7. Fletcher, J. E., and Jiang, M. S. (1993) Toxicon 31, 669 – 695. determine the three-dimensional structures of both 8. Chiou, S. H., Chuang, M. H., Hung, C. C., Huang, H. C., Chen, CTXII-L1G and CTXII-L1-D-L to find out more data to S. T., Wang, K. T., and Ho, C. L. (1995) Biochem. Mol. Biol. Int.

35, 1103 – 1112. elucidate why changing the first residue causes the

9. Chen, K. Y., Huang, W. N., Jean, J. H., and Wu, W. G. (1991) J. drastic alteration in the conformation of CTX II

mole-Biol. Chem. 266, 3252 – 3259. cule. This work is in progress in our laboratory.

10. Wu, C. Y., Chen, S. T., Ho, C. L., and Wang, K. T. (1996) J. Chin. Owing to drastic tertiary structure differences in

Chem. Soc. 43, 67 – 71. CTX II and it analogs, the functional role of N-terminal

11. Litchfield, J. T., and Wilcoxon, F. (1949) J. Pharmacol. Exp. leucine residue in CTX II can not been elucidated

un-Ther. 96, 99 – 113.

equivocally. However, according to these functional 12. Ginsborg, B. L., and Warriner, J. (1960) Br. J. Pharmacol. 15, data, we can infer that the native conformation and 410 – 411.

the Leu-1 of CTX II are probably not essential for its 13. Stryer, L. (1965) J. Mol. Biol. 13, 482 – 495.

biological activity. 14. Turner, D. C., and Brand, L. (1968) Biochemistry 7, 3381 – 3390. In conclusion, the structural and functional analyses 15. Bhaskaran, R., Huang, C. C., Tsai, Y. C., Jayaraman, G., Chang,

D. K., and Yu, C. (1994) J. Biol. Chem. 269, 23,500 – 23,508. suggest that the N-terminal leucine residue plays an

716

數據

FIG. 1. HPLC profiles of purified CTX II and CTX analogs as
FIG. 4. Fluorescence spectra of ANS in the presence of various
TABLE 2 important structural element. In addition, this report

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