Biochimica et Biophysica A cta, 1159 (1992) 255-261 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

255

BBAPRO 34320

Chemical modification of arginine residues in a-bungarotoxin Shinne-Ren Lin

a

and Chun-Chang Chang b

a Department of Chemistry, Kaohsiung Medical College, Kaohsiung (Taiwan, ROC) and b Department of Biochemistry, Kaohsiung Medical College, Kaohsiung (Taiwan, ROC) (Received 6 May 1992)

Key words: a-Bungarotoxin; Chemical modification; Arginine residue; Nicotinic acetylcholine receptor

The reaction of a-bungarotoxin (a-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of a-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of a-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of a-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in a-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.

Introduction

Up to date, more than 80 highly homologous a-neurotoxins from the venoms of families Elapidae and Hydrophiidae have been sequened. They are structurally grouped into 2 classes, short-chain neurotoxins containing 60-62 amino-acid residues with four disulfide bonds and long-chain neurotoxins of 70-74 residues with five disulfides [1-3]. ot-Bungarotoxin (o~BuTX), a long-chain neurotoxin isolated from the venom of Bungarus multicinctus has been widely used to study the structure-function of nicotinic acetylcholine receptor (AChR) owing to its specific and irreversible binding to the receptor. We have recently demonstrated that the amino-groups in a-BuTX may participate in the multipoint contact between the toxin and AChR, but none of the individual amino groups are essential for the binding [4]. ~-BuTX contains three arginine residues located on loop 2 (Arg-25 and Arg-36) and in the C-terminal tail (Arg-72). It has been assumed that the invariant Arg-36 appears to play an important electrostatic role in the toxin binding to AChR, while the experimental evidence is relatively poor in comparison with that from other functional groups in a-neurotoxins [1-3,5]. In this work, the role

Correspondence to: C.-C. Chang, Department of Biochemistry, Kaohsiung Medical College, Kaohsiung, Taiwan, ROC 80708.

of arginine residues in lethality, affinity to AChR and immunological activity of a-BuTX was studied by selective modification. Materials and Methods

Bungarus multicinctus venom was collected in our laboratory and a-BuTX was isolated as described by Chen and Chang [6] and further purified according to the method used by Kosen et al. [7]. Torpedo californica was purchased from Pacific Biomarine (Venice, CA, USA) and the electric organs were removed and stored in - 70°C. [125I]a-BuTX (291 C i / m m o l ) was purchased from Amersham, UK. TPCK-Trypsin, Streptococcus aureus V8 proteinase and trifluoroacetic acid (TFA) were from Sigma (St. Louis, USA). 1,2-Cyclohexanedione (CHD) was obtained from Aldrich and p-hydroxyphenylglyoxal (HPG) from Pierce (Rockford, IL, USA). The reverse-phase high-performance liquid chromatography (RP-HPLC) column (TSK-gel, ODS-120T, 0.46 × 25 cm) was from Toyo Soda, Japan. All other reagents were of analytical grade. Modification with CHD. Modification was performed essentially according to the method described by Patthy and Smith [8]. a-BuTX (1 ~mol) in 2 ml of 0.2 M sodium borate buffer (pH 9.0) was incubated with 100-fold molar excess of CHD. The reaction was allowed to proceed at 37°C for 1 h and the mixture was

256 desalted by gel filtration on a Sephadex G-50 column (1.8 x 90 cm) equilibrated with 10% acetic acid. In order to modify more arginine residues, the reaction was carried out in 0.2 M sodium borate buffer (pH 9.0) containing 6 M urea with 1000-fold molar excess of CHD according to the procedure described by Wolfenstein-Todel and Santom6 [9]. Modification with p-hydroxyphenylglyoxal. This was performed according to the procedure described by Yamasaki et al. [10] and Kharrat et al. [11]. a-BuTX (1.5/zmol) in 2 ml 0.1 M NaHCO 3 buffer (pH 9.0) was incubated with 100-fold molar excess of HPG. The reaction was allowed to proceed at room temperature for 1 h and the mixture was desalted by passage through a Sephadex G-25 column (2.0 x 45 cm) equilibrated with 0.05 M sodium acetate (pH 5.0). The modified derivatives were separated on a CM-52 column (1.5 X 28 cm) equilibrated with 0.05 M sodium acetate buffer (pH 5.0) and eluted with a linear gradient of 0-0.15 M NaC1 in the same buffer. The three main fractions (Fig. 3) were further purified on a SP-Sephadex C-25 column (1.0 x 28 cm) with a linear gradient from 0.03 M (pH 5.0) to 0.15 M (pH 6.8) of ammonium acetate buffer. Localization of the incorporated groups. The derivatives were reduced and carboxymethylated (RCM) by the procedure described by Crestfield et al. [12], followed by proteolytic digestion. RCM-proteins (1 mg) in 1 ml of 0.1 M ammonium bicarbonate (pH 8.0) were digested with trypsin for 6 h or with S. aureus V8 proteinase for 8 h in 0.05 M sodium phosphate buffer (pH 7.8) at 37°C, substrate/enzyme ratio of 50:1 (w/w). The hydrolysates were separated by RP-HPLC on Toyo Soda ODS-120T column (0.46 x 25 cm) equilibrated with 0.1% TFA and eluted with gradients of acetonitrile in 0.1% TFA as shown in the figure legends. The peptides were lyophilized for amino-acid analysis. Assay for lethal toxicity. Mice weighing 16-18 g were injected intraperitoneally with 0.2-ml samples of serial 2-fold dilutions. Four mice of both sexes were used for each dilution and the LDs0 values were calculated according to the 50% end-point method of Reed and Muench [13].

Preparation of AChR-rich membrane fragments. These were prepared from T. californica electric tissues according to the published procedure of ContiTronconi et al. [14] with a slight modification as previously described [4]. The specific activity of the preparation (expressed as nmol of a-BuTX binding/mg protein), measured by the [125I]a-BuTX binding assay, was 0.6 nmol/mg protein. Receptor binding assay. The binding activity of aBuTX and its modified derivatives was determined by modifying the centrifugation assay of Conti-Tronconi et al. [14] as previously described [4]. A constant amount

of AChR-rich membrane fragments (80 /zg) and [125I]a-BuTX (3.44 nM) were incubated with increasing concentrations of a-BuTX or its derivatives at room temperature for 2 h, followed by continuous incubation at 4°C for 16 h. The amount of bound toxin was calculated from the different radioactivity between the total radioactivity input and supernatant. The mean values of duplicate determinations were plotted and the molar concentration of the native or modified toxin able to inhibit 50% of the specific binding of the labeled a-BuTX was defined as ICs0 [15,16]. Both ICs0 values from duplicate determinations fell within 10% of the mean value. Immunological methods. Rabbit anti-a-BuTX sera were prepared according to the procedure previously described [6]. Antigenicity was determined by competitive ELISA as previously described [4]. Results

Reaction of a-BuTX with CHD The reaction product of a-BuTX with 100-fold molar excess of CHD in 0.2 M borate buffer (pH 9.0) was purified by CM-52 chromatography. The major fraction proved to be homogeneous by polyacrylamide gel electrophoresis was subject to amino-acid analysis. As presented in Table I, only one of the three arginine residues in a-BuTX was modified. No more arginine reacted, even with 1000-fold molar excess of CHD, in TABLE I

Amino-acid compositions of a-bungarotoxin and its derivatives modified with CHD or HPG All values are expressed as molar ratios based on leucine = 2.0. C H D and H I through H 3 were prepared by reaction with C H D and HPG, respectively. A m i n o acid

Native

Asp Thr Ser Glu Pro Gly Ala 1/2Cys-Cys Val Met Ile leu Tyr Phe His Lys Arg Trp

4.1 6.7 5.7 5.1 7.7 4.0 5.2 9.8 5.1 0.8 1.8 2.0 1.8 0.9 1.8 5.9 2.9 1.0

n.d., not determined.

(4) (7) (6) (5) (8) (4) (5) (10) (5) (1) (2) (2) (2) (1) (2) (6) (3) (1)

Modified derivative CHD

HI

H2

H3

4.2 6.8 5.7 5.1 8.0 4.1 5.1 9.8 5.1 1.1 1.9 2.0 1.8 0.9 1.9 6.1 1.9 n.d.

3.8 6.9 5.8 5.0 7.8 4.1 5.1 9.7 4.8 0.8 1.8 2.0 1.9 1.1 1.8 6.1 1.1 n.d.

3.9 6.9 5.7 4.9 7.9 4.2 5.1 9.6 4.8 0.9 1.9 2.0 1.9 1.2 1.8 6.0 2.2 n.d.

4.0 7.1 5.8 5.0 7.9 4.2 5.1 9.7 4.9 1.0 1.9 2.0 1.8 0.9 1.8 5.9 2.2 n.d.

257 100

0.2

40

80

o o

v ~

tM

0

0.3

,

,

O '1" O

,

B

11 ~o

60

.m m

-= 4 0 .J

4

9

_ - -

-

"-

2J

20

20

3

0.1

20

0

40

60

I

0

I

30

Time (min)

Time

Fig. 1. HPLC chromatogram of the tryptic digests of native (A) and CHD-modified e-BuTX (B). The tryptic digests were separated on a TSK gel ODS-120 T column (0.46x25 cm, Toyo Soda) at a rate of 1.0 ml/min with a linear gradient of acetonitrile in 0.1% TFA as shown by a dotted line. The absorbance was monitored at 215 nm.

I

60

90

(rain)

Fig. 2. Change of lethal toxicity of a-BuTX by reaction with HPG at various pH values, tr-BuTX (1 mg) in 1 ml of 0.1 M NaHCO 3 buffer was allowed to react with 100-fold molar excess of HPG at 37°C at pH 8.0 (o e), pH 9.0 ( • • ), pH 9.5 ( o o ) and pH 10.0 (/x ,x ), respectively. After suitable intervals of time, aliquots were taken for the determination of lethality.

the presence of 6 M urea. The tryptic-digest peptides from RCM-a-BuTX and its CHD-derivative were separated by RP-HPLC. Peptide mapping (Fig. 1) and amino-acid analysis of the isolated peptides (Table II) indicated that Arg-72 was modified in the reaction.

mined at 340 nm based on a molar extinction coefficient of 1.83-104 M -~ cm -~ [10]. Changes of the lethal toxicity of a-BuTX by reaction with HPG at varying pH values are shown in Fig. 2. The toxicity decreased rapidly in the reactions performed at pH 9.0 or higher and was lost pronouncely after 60 min. For the preparation of arginine-modified derivatives, otBuTX was allowed to react with HPG in 0.1 M

Reaction with HPG HPG has been reported to be an arginine-specific reagent and the extent of modification can be deter-

TABLE II

Amino-acid compositions of tryptic fragments of RCM- a-bungarotoxin The peptides obtained from Fig. 1 were analyzed.

Amino acid

Peak 1

CM-Cys Asp Thr Ser Glu Pro Gly Ala Val Met

2

3

0.9 (1) 1.1 (1)

1.0(1) 0.9 (1) 1.0 (1)

0.8(1) 2.7 (3) 0.9(1)

4

0.8 (1) 1.1 (1)

1.8 (2)

5

1.6 (2) 1.0 (1) 1.8 (2) 1.1 (1) 1.9(2) 1.1 (1)

1.0 (1)

6

2.6 (3) 1.9 (2) 1.8 (2) 1.0 (1) 2.0(2) 2.9 (3)

1.0 (1)

7

8 *

1.7 (2)

1.5 (2)

0.8 (1) 0.9(1) 0.9(1) 0.9 (1) 1.0 (1) 1.8(2) 1.5 (2)

1.0 (1) 1.1 (1) 1.1 (1) 1.0 (1) 1.2(1) 2.0(2) 1.6 (2)

1.9 (2) 1.7 (2) 2.1 (2)

1.2(1) 1.2(1)

9

10 *

1.5 (2) 0.8 (1) 0.8 (1) 0.7 (l) 0.8(1) 0.9 (1) 0.9(1) 1.7 (2) 1.6 (2)

0.8 (1)

11 *

1.8 (2) 0.9 (1)

1.9 (2) 1.1 (1)

1.6(2)

2.0(2)

0.9(1)

1.1 (1)

0.8 (1)

0.8 (1)

1.0 (1)

1.0 (1)

2.0 (2)

2.9 (3) 0.9(1)

1.0 (1)

1.0 (1)

1.0 (1)

1.0 (1)

Phe

Lys His Arg 1.0 (1) Corresponding peptide 71-74

1.0 (3) 0.9(1) 0.9(1) 65-74

1.0 (1) 0.9(1)

2.0 (2)

1.0 (1)

1.0 (1) 65-70

* The peptides containing the Trp-28 residue.

52-64

52-70

39-51

39-51

2.6 (3) 1.0 (1) 3.6 (4) 1.9(2) 0.9(1) 2.8 (3) 1.0(1) 1.9(2) 1.6 (2) 1.7 (2) 0.9(1) 0.9 (1)

Ile

Leu Tyr

12

26-~38

39-51

0.9(1) 1.0 (1)

1.1 (1)

0.9(1)

1.0 (1)

26-36

27-36

0.9(1) 1.1 (1) 1-25

258

O.3

H1 H2

O

0D 0.2 N