Amino acid sequence of toxin F, a snake venom toxin that blocks neuronal nicotinic receptors

30

Brain Re.s-catch, 385 (i98(~)30-57 Elsevier

BRE 12062

Amino Acid Sequence of Toxin F, a Snake Venom Toxin That Blocks Neuronal Nicotinic Receptors RALPH H. LORING 1, DAVID ANDREWS ~, WILLIAM LANE 2 and RICHARD E. ZIGMOND I 1Department of Pharrnacology, Harvard Medical School. Boston. MA 02115, and :Microchemistr~ Laboratory, Harvard Universio,, Cambridge. MA 02138 (U. S. A. ) (Accepted 11 March 1986) Key words: Toxin F

Bungarotoxin 3.1 x-Bungarotoxin Neuronal nicoumc receptor Bungarusmultteinctus venom -(~-bungarotoxin Amino acid sequence Nicotinic antagonis~

The amino acid sequence was determined for toxin F, a component of Bungarus rnulticinctus venom that blocks niconmc ~ransm~ssion in the chick ciliary ganglion and the rat superior cervical ganglion. Toxin F was purified by a procedure that includes preparative isoelectric focusing and ion exchange chromatography. Seventy nanomolar toxin F blocks nicotimc transmission in the chick ciliarv ganglion; however, the toxin only weakly blocks the binding of 125I-a-bungarotoxinto membranes derived from Torpedo caliJbrnica electroplax (ICs0 = 1,uM). These data raise the possibility that toxin F may preferentially recognize neuronal nicotinic receptors, Toxin F focused identically on an isoelectric focusing gel with samples of two similar toxins, bungarotoxin 3.1 and x-bungarotoxin. The sequence of toxin F is identical with that recently reported for x-bungarotoxin. When the N-terminal portion of bungarotoxin 3.1 was sequenced, it was found to be identical to the other two toxins. These and other data suggest that the 3 toxins are. in fact. the same.

INTRODUCTION The study of n e u r o n a l nicotonic receptors has been hampered by the lack of a ligand that binds to these receptors with a high affinity and can be used to quantify and localize them. In contrast, the study of nicotinic receptors from skeletal muscle and fish electric organs has been greatly facilitated by the use of a-bungarotoxin and other a - n e u r o t o x i n s isolated from the venoms of various snakes 6. While a-bungarotoxin does bind to specific sites in autonomic ganglia and in the central nervous system, in most instances this a - n e u r o t o x i n does not block nicotinic transmission in n e u r o n a l tissues (e.g. Brown and Fumagallil). Thus, the use of these toxins in characterizing neuronal nicotinic receptors is controversial (see Oswald and F r e e m a n e° for a review). We have previously purified a m i n o r c o m p o n e n t from the venom of Bungarus multicinctus which we refer to as toxin F. This toxin blocks nicotinic transmission in the chick ciliary ganglion Is and in the rat

superior cervical ganglion 14. Toxin F can be distinguished from a-bungarotoxin by its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) and on isoelectric focusing gels 15. U n d e r conditions in which the toxin completely blocks synaptic nicotinic transmission, carbachol-induced depolarizations in the chick ciliary ganglion are also blocked is. Together. these results suggest that toxin F will be useful in characterizing ganglionic ntcotinlc receptors. Over 130 different snake venom toxins have been sequenced v. These toxins are classified both by their site of action (e. g. pre- or postsynaptic) and their sequence homology 7"12. Since toxin F is a new neurotoxin, comparison of its sequence to other toxins is an important step in understanding the basis of its pharmacology In addition, toxin F resembles two neurotoxins purified by others from the venom of B. multicmctus based on their ability to block neuronal nicotinic receptors. These toxins, referred to as bungarotoxin 3.1 I ref. 24) and x-bungarotoxin2 are similar

Correspondence." R.E. Zigmond, Department of Pharmacology. ttarvard Medical School. 250 Longwood Avenue. Boston. MA (J2115, U .S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

31 to toxin F both in molecular weight and pharmacological action. Sequencing these toxins should help establish the relationships among them.

MATERIALS AND METHODS The original procedure for isolating toxin F ~5 was modified to incorporate a preparative isoelectric focusing step. Fractions derived from the venom of B. multicinctus were purchased from Biotoxins (St. Cloud, FL), lyophilized and dialyzed against 2 liters of doubly distilled water using Spectrapor membrane tubing No. 3 (Spectrum Medical Industries, Los Angeles, CA). Twenty-one milliliters of dialysate conraining 21){_1mg of protein were added to 1 ml of pH 9-11 ampholytes, 0.3 ml of pH 8-9.5 ampholytes (LKB, Rockville, MD), and 2.1 g of dry Sephadex G50 superfine (Pharmacia, Piscataway, N J). The slurry was degassed for 5 min and poured into a neoprene rubber mold (inside measurements 11 cm × 11 cm × 2 ram), which was sealed with vaseline to a glass plate 1 mm thick. After drying the slurry on a slide warmer until it had 'hardened" into a self-supporting block, isoeleclric focusing was performed on the block using 3 W continuous power for 4.25 h at 4 °C. The anode buffer was 2 N N a O H and the cathode buffer was 0.4~4 (v/v) pH 7.0-9.0 ampholyte. Two 50-ul samples containing 1 mg/ml cytochrome c and horse heart myoglobin (Sigma, St. Louis, MO) were added near one edge of the block to serve as visual indicators of the progress of electrofocusing. After subjecting the block to an additional 7.2 W for 10 rain, it was sliced into 2.5-mm strips, 1.5 ml H 2 0 was added to each strip and the pH was measured. A sample of the supernatant from each strip was diluted 1:10 in distilled water, and the optical density was determined at 280 nm. Each strip was then eluted with an additional 13.5 ml 0.1 M ammonium acetate, pH 5.0. Pooled material from the preparative isoelectric focusing step was dialyzed overnight against 2 liters 0.1 M ammonium acetate, pH 5.1) (4 °C) and then chromatographed on a CM cellulose column.(1.5 x 22 cm) at 4 °C using a gradient of(). 1-0,3 M ammonium acetate, pH 5./)-7.4 (600 ml) and a flow-rate of 15 ml/h. Fractions were assayed for pharmacological potency by determining their ability to block nicotinic transmission in chick ciliary ganglia s . Analytical

isoelectric focusing and S D S - P A G E were performed as reported previously 15. Binding of l-'Sl-a-bungarotoxin to membranes isolated from electric tissue of Torpedo californica was assayed essentially as previously described 4't~ using Torpedo physiological saline as the buffer (raM): NaC1 250, KCI 5, CaCI 2 3. MgCI: 2, NaPO4 5. pH 7.0. Polypeptides (0.1-1 nmol) were sequenced on an Applied Biosystems 470A gas-liquid-solid phase sequencer tl. The resulting amino acid phenylthiohydantoins were identified on a Hewlett-Packard 1090 HPLC using a Dupont Zorbax ODS column. Amino acid composition was determined by dissolving peptides in 6 N HCI, sealing in vacuo, and heating at 110 °C for 24 h. The resulting hydrolysates were analyzed on a Beckman 121M amino acid analyzer interfaced to an IBM Instruments CS9000 computer svstern. All enzymatic digestions were performed on completely reduced and alkylated toxin >. Reduced and alkvlated toxin (1-10 nM) was dissolved in 10(t ul of 1% N H a H C O ~ in a 1.5 ml Eppendorf tube. To this solution was added TPCK-trypsin (Millipore, Freehold, N J) or staphylococcal V8 protease (SV8) (Pierce Chemical, Rockford, IL). Tryptic digestions were carried out at 1:100 enzyme to substrate ratio (w/w) for 4 h fit 37 °C. SV8 digestions were carried out at 1:21) enzyme to substrate ratio (w:w) for 18 h at 37 °C. Digestions were terminated by freezing and lyophilizing the samples. Peptide mixtures were resolved using Vydac columns (The Separations Group, Hesperia, CA} on a Hewlett-Packard 1084B HPLC system. Tryptic peptides were separated on a C-18 column. The column was run at room temperature with a flow rate of 1 ml/min, starting with a concentration of 90% solvent A (t).1% trifluoroacetic acid (TFA)), 10% solvent B 10.1% T F A in acetonitrile). The concentration of solvent B was increased to 1005; over 60 rain, after injection of the peptide solution in _?0,ul of solvent A. SV8 peptides were resolved in a similar way, using a phenyl column and the same gradient conditions. Carboxyl-terminal sequence analysis w a s carried out with carboxypeptidase y10,13 (Pierce Chemical, Rockford, IL). Reduced and alkylated toxin was dissolved in 50 mM N a O A c (pH 5.5) find carboxypeptidase was added (1" I()(L enzyme:substrate). Aliquots were withdrawn at timed intervals and lyophilized. The samples were derivatized with phenylisothio-

32

2.13

r

I

1,5 I0

L-f- .....

~

1,0 :r 3

~5

0.5

50

)0

L NALTIOh

10

2O

30

4O

3

Fraction Number

Fig. I. Preparative isoclectric focusing of B. muhicinclus venom components on a granulated gel bed. Isoelectric focusing of fraction IV (from Biotoxins Incorporated) was carried out as indicated in the text. The circles indicate the optical density of a 1: 10 dilution of the supernatant taken from the gel strips. The squares indicate the pH of each supernatant. The bar represents those fractions that were pooled for further purification. cyanate and resolved using H P L C on an Altex-ODS column. RESULTS

NLJMBEi.

Fig. 2. Material isolated from Bungarus multicincms xenom side-fraction IV by preparative lsoelccmc focusing (pl range 8.5-8.91 was cluted from a CM-cellulose column as shown• The solid line represents the ammonium acetate concentranon in the gradient {600 roll which went from 0. t M. pH 5.0 to ll.3 M. pH 7.4. The numbered lines at the bottom of the figure indicate the pooled fractions.

toxin F (pH 8.5-8.9) was pooled and chromatographed on a CM-cellulose column yielding 7 sharply defined peaks (Fig. 2). Only two of these peaks displayed any blocking activity on chick ciliary ganglia when tested at a concentration of 2 ug/ml. Peak l partially blocked nicotinic transmission after a 1-h incubation and the effect reversed quickly upon washout ( < 1 h). Analysis is isoelectric focusing and SDS-

The subfractions of B. m u l t i c i n c t u s venom that

P A G E indicated that peak 1 was heterogeneous.

were used as the starting material for our purification of toxin F were derived from a CM-Sephadex column

Peak 3 blocked transmission within 30 rain at a concentration of 2 ~lg/ml, and the blockade was only par-

during the purification of a-bungarotoxin. A n elution profile (supplied by Biotoxins) indicated that the venom components had eluted from the CM-Sephadex column virtually identically to the profile published as Fig. 1 in Loring et al.l~. Two subfractions were supplied, corresponding to fractions Ili and IV in our previous purification. We were unable to detect any material in fraction III with an isoelectric point similar to toxin F, and only a weak blockade of nicotinic transmission was observed with this fraction. Fraction 1V blocked nicotinic transmission in chick ciliary ganglia at a concentration of 5 t~g/ml. Preparative isoelectric focusing of this fraction revealed a major peak with a mean pl of about 9.1, a slightly more acidic trailing shoulder and minor acidic components (Fig. 1). The material nearest the pl of

tially reversed ( - 40%) over the course of a 2-h washout. When peak 3 was rechromatographed on CMcellulose, only a single peak was observed, and this material blocked transmission in 1 h at a concentration of 0.5~g/ml ( ~ 70 nM). M a t e r i a l f r o m peak 3 behaved identically to samples of toxin F derived from our previous purification 15 on analytical isoelectric focusing gels, on S D S - P A G E (data not s h o w n ) a n d on amino acid analysis: The final yield of toxin F (peak 3) was 2.3 mg. The a m i n o acid composition of toxin F is listed in Table I, column 1. Two h u n d r e d micrograms of toxin F were reduced and carboxymethylated, and then a portion of this material was subjected to SV8 protease digestion i The two peptide fragments were separated as described in Materials and Methods, and

33 SEQUENCE DETERMINATION

TABLE I N-TERMINAL END

Amino acid composition o/lo):in F and SV8 protease fragments oJ to.~in F

Values in parentheses are the numbers of residues derived from the amino acid sequence. The letters in parentheses arc the single letter code equivalents for the amino acids. Tovin F'

Asp*Asn (D+N) Thr (T) Ser(S) GIu+GIn(E+Q) GIv(O) Ala (A) Val (Vi lie(I) Leu (L) Tyr (Y) Phc(F) Lys(K) His(H) Arg (R) Pro(P) Cys (C)

7.2_+0.4(7) 5.9_+0.3(6) 5.6_+(3.3(6) 6.2_+0.3(6) 4.2_+ 1.2(3) 1.9_+0.1(2) 1.9+0.3(21 3.1 +0.2(4) 3.9_+0.1(41 1.2+0.1(11 3.1 +0.4(3) 1.9_+0.1(2) 1.2,+0.1(1) 3.7_+0.4(4) 4.8+0.1(5) N.D. (10)

SVSprotease SV~protea~e fragment A ,/?agment B fN-terminal) (C-terminal)

3.9 (3) 3.0 (31 3.8 (4) 4.7 (4) 3.0(2) 1.5(1) 0.5(1) 2.8(4) 2.0(2) 0.1 (0) 2.2(2) 2.(1(2) 0.1 (0) 1.8(2) 4.2(4) N.D. (5)

4.8 (4) 3.1 (3) 2.2(2) 2.6 (2) 1.6(1) 1.3(1) 0.7(11 0(0) 2.(1(21 1.0(I) 1.2(1) 0.1(0) 0.9(1) 1,9(2) 1.1 (1) N,D. (5)

TOTAL TOXIN ] ~ RT L I XPXXTPQT PNGQDI F[ KAQXXKF TOTAL TOXIN 2 R T C L I S P S S T P O T ~ P N G QO I(CIF t K A O{C)D K FCr)S SV8 N-FRAGMENT+ RT C -- T C 0 I CF TRYPTIC FRAGMENT I fA)O [ D K F C S BUNGAROTOXIM 3.!*

RT

POSITION: FIN&SEQUENCE

I RTC[

R... P...

L I SP S ST P OT- P N 5 S10 r 15 ?(l I SRS POTCPNGQOIC~L

P5 K30 • K AQCD C%

C-TERMINAL END TOTAL TOXIN ] (CONT.)* TOTAL TOXIN 2 (CONT.1 SV8 C-FRAGMENT i SV8 C-FRAGMENT 2 TRYPTIC FRAGMENT 2 TRYPTIC FRAGMENT 3 TRYPTIC FRAGMENT 4 CARBOXVPEPT!DASF

,

VAT

XQ

OGCVATCPQFXSNY(R!XLL IT)(NI(N) GCVATCPOFRSNYQSL E GCVATCPO C SNY SLLCCTTDNCN NH

~

GP V

3~

ROSITION: FINAL SEQUENCE:

E~G

GPV GPV

40

Pv

45

50

55

60

65

EQGCVATCROFRSNV#StLSfTTDNCNH

*Not reduced or carbox~nnethylated. +Only the indicated residues were identified.

Fig. 3. Determination of the sequences <11toxin F and the partial sequence of bungarotoxin 3.1. A kev to the single letter amino acid code is found in parentheses next to the 3-letter code in Table I. Also, the following additional symbols were used: parentheses indicate a tentative identification of an amino acid residue, an X indicates an inconclusive identification of a residue and an underlined blank indieatcs either that no residue was observed at that cycle or, in the case of the SV8 N-fragment, that no attempt was made to identify the residue.

' Values axeraged from 3 determinations + S.D, N,D., not determinecl. Approximately 3 ug of bungarotoxin 3.1 (supplied the amino acid compositions of these two peptides are given in Table I, columns 2 and 3. A n o t h e r two hundred micrograms of toxin F were reduced and carboxymethylated. A portion of this material was digested by trypsin, and the fragments were sepa-

by D.K. Berg), 10ug of x-bungarotoxin (supplied by V.A. Chiappinelli) and 1(I J~g of toxin F were subjected to analytical isoelectric focusing on a slab gel. All 3 samples focused identically with a major band

rated by HPLC. Sixty micrograms of unreduced toxin (total toxin 1), lI/0 ug of reduced and carboxymethylated toxin (total toxin 2) and the various proteolytic fragments were sequenced. The results are pre-

-11,4

sented in Fig. 3. The various fragments overlap to produce an unambiguous sequence up to residue 65. Since histidine is present in the amino acid composition of the intact molecule (Table I, column 11 and the C-terminal peptide (Table I, column 3), but is not present in the first 65 residues, histidine was tentatively assigned to the 66th residue. Upon digestion of toxin F with carboxypeptidase, first histidine and later asparagine were released, confirming histidine and asparagine as the respective ultimate and penultimate C-terminal residues (Fig, 3). In addition, 25 ug of native bungarotoxin 3.1 (supplied by D.K. Berg) was sequenced up to residue 16. The resulting sequence was identical to what one would expect for a sample of native toxin F (Fig. 3).

- 8.7

9,2 -8.5

-8.1

-7,8 I

2

3

4

5

Fig. 4. Comparison of bungarotoxin 3.1, x-bungarotoxin and toxin F by isoclectric focusing. From left to right arc: (1) IU/~g of ~-bungarotoxin, (2)ca. 3,ug of bungarotoxin 3.1, (3) 10Hgot toxin F, (4) 10 ,ug of x-bungarotoxin, and (5) 10/~g of cytochrome c.

34

Torpedo membranes. Since the binding

at a pH of ~ 8.8 and with more acidic secondary

does bind to

bands appearing at pH ~ 8.7 and ~ 8.5 (Fig. 4). Toxin F was compared with (x-bungarotoxin in

of a-bungarotoxin to Torpedo m e m b r a n e s is essentially irreversible (e.g. Raftery el al.-~?), we deter-

terms of its ability to displace the binding of 1251-cz-

mined whether the binding of our sample of Ioxin could be reversed. Membranes diluted to 1 u M r.~-

bungarotoxin to m e m b r a n e s isolated from electric

Torpedo californica which are rich in acetyl-

bungarotoxin binding sites were incubated overnight

choline receptors. Various concentrations of either

in l0 y M toxin F. When these m e m b r a n e s wcrc then

c~-bungarotoxin or toxin F were incubated overnight in the presence of m e m b r a n e s diluted to contain 5

incubated with 4(1 nM 1251-(z-bungavotoxin for 5 rain,

tissue of

nM c~-bungarotoxin binding sites. 125I-a-bungarotox-

the binding of the radioactive toxin was onl}. t0cJ of that seen in control m e m b r a n e s !data not shown).

in (10 nM) was then allowed to bind to the membranes for 20 min before filtering. Preincubation with 2 nM a-bungarotoxin reduced the ~25I-ct-bungarotox-

However, if the mixture of membrmles and toxin F was diluted l//0-fold (to 1(I nM m e m b r a n e s , 1()11nM

in to about 50% as expected (Fig. 5). However, ap-

toxin binding, the binding was 63'~ of control (data not shown). Seventy-three percenl of control binding was observed when a similar assa~ was performed on 10 nM m e m b r a n e s that had been incubated overnight

proximately 1/xM toxin F was required to reduce the binding by an equivalent amount. These data suggest that the affinity of toxin F for the Torpedo ct-bungarotoxin binding site is about 500-fold lower than the affinity of a - b u n g a r o t o x i n for the same site. However, the presence of a small a m o u n t of cx-bungarotoxin contaminating our sample of toxin F would also lead to a blockade of the binding of ~251-(z-bungarotoxin. Thus, the question arises as to whether toxin F itself

toxin F) 10 rain before assaying for 1251
in 100 nM toxin F. Thus. within 1(] rain after the concentration of toxin F had been reduced [0(l-fold by dilution, most of the available ~J.-bungarotoxm binding sites had reappeared. ['hcsc data suggest that toxin F itself is binding to nicotinic receptors m l)~r-

pedo m e m b r a n e s and that this binding is highly reversible. The data also provide a limit for the extent to which our sample of toxin F could be contaminated

TOXIN BINDING TO TORPEDOMEMBRANES

with ct-bungarotoxin or other toxins which brad with high affinity to

I00!

Torpedo acetvlchohne receptors

DISCUSSION

z5 rn

i

H

Ravdin and Berg 24 were the first to report the purifi-

5c I i

25 [

e -9

-

i

__o . . . .

J

-8

Log [Toxin]

Fig. 5. Displacement of J2~I-a-bungarotoxinbinding to Torpedo membranes by unlabeled a-bungarotoxin and toxin F. Membranes prepared from Torpedo californica electroplax were diluted to 5 nM a-bungarotoxin binding sites in 50 #1 of Torpedo physiological saline containing 1 mg/ml bovine serum albumin. After incubating overnight in the indicated concentration of unlabeled toxin, 10 nM 125I-a-bungarotoxin was added for 20 rain at room temperature before the mixture was filtered through a Millipore cellulose acetate filter (EGWP) and washed with 3 ml of Torpedophysiological saline containing 0.1 mg/ml bovine serum albumin. 100% Binding was determined in the absence of any unlabeled toxin. The circles show the displacement by a-bungarotoxin; the triangles are data for toxin F. Each point represents the average of duplicate samples.

cation of a c o m p o n e n t of Bungarus mtdticinctus venom that blocked neuronal nicotinic receptors on dissociated chick ciliary neurons. This material appeared to be smaller than <~-bungarotoxin on SDSP A G E and was termed Bgt 3.1 based on its elution from an ion-exchange column. When toxin F was purified, it showed a slightly different elution profile. and thus it was unclear whether or not the two toxins were the same 14lS. Following the isolation of toxin 1~. Chiappinelli2 reported the isolation of a toxin from Bungarus multicinctus venom that he designated as xbungarotoxin. This material was claimed to differ from toxin F based on its isoelectric point 23. However, in our hands not only does x-bungarotoxin focus identically with toxin F. but the sequences of the two materials are identical ~see G r a n t and Chiappinellf' for the sequence of ~-bungarotoxml. Bungarotoxin

35 3.1 also focuses identically and, assuming the presence of 1/2 cysteine residues at positions 3 and 14, the first 16 residues of bungarotoxin 3.1 are identical with the corresponding residues of the other two toxins. Given these data, the similar pharmacological specificity of bungarotoxin 3.1, and the fact that the amino acid composition of bungarotoxin 3.1 is virtually indistinguishable from that of toxin F (D.K. Berg, personal communication), we believe that bungarotoxin 3.1 is also identical to toxin F, or, at most, differs by a few amino acid residues. Other toxins present in B. multicinctus venom have been described that block neuronal nicotinic receptors. Bungarotoxin 3.3 (ref. 24) has a molecular weight of ~ 8000 on SDS-PAGE and blocks depolarization in cultured chick ciliary neurons induced by iontophoretic application of acetylcholine. We have found a similar toxin in this venom and shown that it has a pl of greater than 9.2 and an initial N-terminal sequence of methionine-glutamine (Loring, Andrews, Lane and Zigmond, unpublished observation). However, this toxin requires a concentration of 10 ag/ml ( - 1.25/~M) to block nicotinic transmission in the chick ciliary ganglion, and the effect is reversible within 45 min. Another toxin or family of toxins has been described in 3 different laboratories and has been termed bungarotoxin II-S1 (found to block transmission in the rat superior cervical ganglion21), bungarotoxin P4 (found to block acetylcholine-evoked catecholamine release from bovine chromaffin cells 26) and bungarotoxin P15 (found to block transmission in the guinea pig hypogastric nerve-vas deferens preparationS). All 3 toxins have a molecular weight of 15,000 on SDS-PAGE and are potent phospholipases s'2226. In contrast, toxin F has no detectable phospholipase activity 15. Bungarotoxin P4 has been partially sequenced -'{' and shows greater homology with presynaptically acting toxins than with postsynaptic toxins. Snake venom toxins are classified both by their mode of action and by sequence homology 7,12. Postsynaptic (i.e., "a') neurotoxins, which block muscle acetylcholine receptors, are subdivided into "short' and "long" neurotoxins, but share a common 3-dimensional structure consisting of 3 major loops held together by highly conserved disulfide bonds. Short neurotoxins have 60-64 amino acid residues and 4

disulfide bonds. Long neurotoxins generally have 70-74 residues, with an additional disulfide bond inserted into the second major loop. One exception is the toxin LS-III from Laticauda semifasciata which has only 66 residues but does have the 5 disulfide bonds and is considered a long neurotoxin. Toxin F also has 66 amino acid residues, and, after adjusting for a single amino acid insertion in the third loop, all 10 half cystine residues are in alignment with the long neurotoxin pattern. Toxin F has other features of a long neurotoxin including a characteristic deletion (compared to short neurotoxins) in the first loop and the presence of an invariant alanine-threonine pair at positions 44 and 45. As pointed out by Grant and Chiappinelli 9, toxin F shares almost equal homology (ca. 50%) with members of the long neurotoxin family and with a peptide referred to as Naja haja haja CM2 (Sequence No. MT01 in Dufton and Hider7), which has low toxicity and no known pharmacological action. Toxin F has less homology with short neurotoxins and least homology with other types of snake venom toxins. It is not known what parts of the sequences of postsynaptic neurotoxins are responsible for their ability to block muscle acetylcholine receptors, but from X-ray crystallographic and other data, it is believed that critical amino acid residues that are present on a common concave surface of the toxins are involved in the binding 1727. Both long and short neurotoxins have an invariant tryptophan that is present in the second loop (position 26) and is functionally important 12'17. Recently, however, the absolute necessity of this tryptophan has been questioned. As reviewed by Karlsson 12. the tryptophan can be chemically modified under certain conditions with onlv 20-50% losses in toxicity. X-ray crystallographic data for the short neurotoxin erabutoxin g 1s29 and the long neurotoxin ct-cobratoxin 3° place the tryptophan as a necessary hydrophobic binding site on the concave surfaces of those toxins. However, the 3-dimensional structure of the long neurotoxin. (x-bungarotoxin 27.2s places its tryptophan on the opposite face of the toxin. In its place, a tyrosine on the third loop ((z-bungarotoxin position 54) has been hypothesized to substitute for the tryptophan as the hydrophobic binding site 7. The tryptophan which normally occurs in (zneurotoxins at position 26 is substituted by a glutamine in toxin F. However, toxin F also has a tyrosine in the

36 third loop (position 53) which might substitute for the

Although we find that toxin F blocks neuronal nic-

tryptophan in the m a n n e r proposed for a-bungaro-

otinic transmission in the chick ciliary ganglion at a

toxin. Alternatively, the lack of tryptophan in toxin F

concentration of ~ 7(1 nM applied for l h, 400 nM toxin F had no effect on neuromuscuhtr transmission

may be partially responsible for the apparent specificity of this toxin for neuronal nicotinic receptors• Various models have been proposed in which cer-

in co-cultured chick ciliary neurons and muscle fibers (H, Gottlieb and R. Loring, unpublished obserwt-

tain essential residues of postsynaptic toxins mimic acetylcholine or d-tubocurarme ' . In all of these

tion). Similar unpublished results indicating that the

models the arginine-glycine residues at positions 34

acetylcholine receptors have been mentioned for

and 35 play a central role. This arginine-glycine pair is invariant in a-neurotoxins, and chemical modifica-

bungarotoxin 3.1 (ref. 25) and z - b u n g a r o t o x m . This low potency is not surprising m light of the fact that

tion studies demonstrate that the arginine is functio-

toxin F binds weakly to the simihtr acetylcholine receptor found in T o r p e d o electroplax tissue, as shown

•

71":

nally important k'. Toxin F is unique as a postsynaptic

toxin has a low potency for blockmg skeletal muscle •

9

toxin in that the arginine-glycine pair is followed by a

by competition for a - b u n g a r o t o x i n binding sites.

proline residue (position 36). A proline at this posi-

These data suggest that the toxin preferentially rec-

tion might alter the orientation of the arginine-glycine pair relative to other a-neurotoxins, which, in turn, might alter the binding properties of the toxin. Toxin F has another unusual substitution at position 29 which lies in the small disulfide loop unique to

ognizes neuronal nicotinic receptors such as those found in chick ciliary ganglia.

ACKNOWLEDGEMENTS

the long neurotoxins. Usually, this position is filled by one of the neutral amino acids such as alanine, glycine or glutamine. Toxin F has a basic lysine at position 29. Since this region would also be expected to interact with the receptor, this substitution might also alter the toxin's specificity. Other unusual features of •

"

"0

the toxin are discussed by G r a n t and Chtappmelh . including a shortened "tail" region, a phenyhflanine/ tvrosine substitution at position 22 and a threonine/ serine substitution at position 60.

REFERENCES 1 Brown. D.A. and Fumagalti, L.. Dissociation of ct-bungarotoxin binding and receptor block in the rat superior cervical ganglion, Brain Research, 129 (1977) 165-168. 2 Chiappinelli, V.A., x-Bungarotoxin: a probe for the neuronal nicotinic acetylcholine receptor in the avian ciliary ganglion. Brain Research. 277 (1983) 9-22. 3 Chiappinelli, V.A., x-Bungarotoxin: a probe for the neuronal nicotinic acetylcholine receptor, TIPS 5 (1984) 425-428. 4 Chiappinelli, V.A., Cohen, J.B. and Zigmond, R.E., The effects of a- and fl-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia, Brain Research, 211 (1981) 107-126. 5 Chiappinelli, V.A. and Zigmond, R.E., a-Bungarotoxin blocks nicotinic transmission in the avian ciliary ganglion, Proc. Natl. A cad, Sci. U.S.A.. 75 (1978) 2999- 3003. 6 Conti-Tronconi. B.M. and Raftery, M.A., The nicotinic cholinergic receptor: correlation of molecular structure

This 12651, Milton cipient

research was supported by NIH Grants NS NS 22472 and an award from the William F. Fund at Harvard University. R , E ; Z . is a reof an N I M H Research Scientist D e v e l o p m e n t

Award (MH 00162). We thank Dr. D.J. Strydom for many helpful discussions and J.B. Cohen for purified T o r p e d o electroplax m e m b r a n e s . We also thank Drs. D.K. Berg and V.A. Chiappinetli for samples of bungarotoxin 3.1 and x-bungarotoxin, respectively•

with functional properties, Annu. Roy, Biochem., 51 (1982) 491-530 7 Dufton, M.J and Hider• R.C., Conformational propemcs of the neurotoxms and cvtotoxms tsolated from elapid snake venom. CRC Crit. Rev. Biochem. 14 ~1983~ 113-17t S Gotti. C.. Omni. C.. Berti. F. and Ctementi, F., Isolation ol a polypeptide from the venom of Bungarus muhicinctus that bind~ to ganglia and blocks the ganglionic transmission in mammals. Neuroscience. 15 (1985) 563-575. 9 Grant. G.A. and Chiappinelli, V.A.. Kappa-bungarotoxin: complete amino acid sequence of a neuronal nicotinic receptor probe, Biochemistry, 2411985) 1532-1537. 10 Hendrickson. R.L. and Meredith. S.('.. Amino acid analysis bv reverse phase high-performance liquid chromatography: precotumn derivatization with phenylisothyocyanate. Anal. Biochem.. 136 (1984) 65-74. 11 Hewick. R.M.. Hunkapillar, M.W. Hood. L.E. and Dryer, W..I,. A gas-liquid solid phase peptide and protein sequenator, J. Biol Chem. 256 [198l ~700n-7997.

37 12 Karlsson, E., Chemistry oI protein toxins in snake venoms. In C.Y. Lee tEd.), Snake Venoms, Handbook of Experimental Pharmacolow, Vol. 52. Springer-Verlag, Berlin, 1979. pp. 159-212. 13 Koop, D.R., Morgan, E.T., Tart, G.E. and Coon, M.J,, Purification and characteristics of a unique isozyme of cvtochrome P-450 from liver microsomes of ethanol-treated rabbits, J. Biol. Chem.. 257 (1982) 8472-8480. 14 Loring, R H , . Chiappinelli. V.A,, Zigmond. R.E. and Cohen, J.B., Characterization of a snake venom neurotoxin v~hich blocks nicotinic transmission in autonomic ganglia, Soc. Neurosci. Abstr., 9(1983) 1143. 15 Loring, R,H., Chiappinelli. V.A,, Zigmond. R,E. and Cohen, J.B., Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion. Neuroscience, 11 (1984) 989-999. 16 koring, R.H., Jones, S.W., Matthews-Bellinger, J. and Salpeter, M.M., IZSl-a-bungarotoxin: effects of radiodecomposition on specific activity. J. Biol. Chem., 257 11982) 1418--1423. 17 Low. B.W., The three-dimensional structure of postsynaptic snake neurotoxins: considerations of structure and function. In C.Y. Lee, tEd.), Snake Venoms, Handbook of Experimental Pharmacology, Vol. 52, Springer, Berlin, 1979, pp. 213-257. 18 Low, B.W., Preston, H.S., Sato, A., Rosen. L.S., Searl, ,I.E.. Rudko, A.D. and Richardson, J.S., Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acctvlcholine receptor, Proe. Natl. Acad. Sci. U.S,A.. 73 (1976) 2991-2994. 19 Moss, B.A.. Peptide mapping at the nanomole level, in I. Lefkovits and B. Pernis (Eds.), Immunological Methods, Academic Press, New York, 1979. pp. 69-80. 20 Oswald, R.E. and Freeman. J.A., a-Bungarotoxin binding and central nervous system nicotinic receptors, Neuroscience, 611981) 1-14.

21 Quik, M. and Lamarca, V., Blockade of transmission in rat sympathetic ganglia by a toxin which copurifies with abungarotoxin, Brain Research, 238 (1982) 385-399. 22 Quik, M., Inhibition of nicotinic receptor mediated ion fluxes in rat symphathetic ganglia by BGT lI-Sl, a potent phospholipase, Brain Research, 325 (19851 79-88. 23 Raftery, M.A., Schmidt, J. and Clark, D G . , Specificity ol (z-bungarotoxin binding to Torpedo cal(fbrnica electroplax. Arch. Biochem. Biophvs., 152 ( 19721 882-886. 24 Ravdin, P.M. and Berg, D.K., Inhibition of neuronal acctylcholine sensitivity bv c~-toxins lrom Bungaru,s multici~w~ tus venom, Proc. Natl. Acad. Sci. U..~,A., 76 (1979) 2072 2/)76. 25 Ravdim P.M., Nitken, R.M. and Berg, D.K.. Internalization of a-bungarotoxin on neurons induced hv a neurotoxm that blocks neuronal acetvlcholine sensitivity, J. Neuros'ci.. 1 (1981) 849-861. 26 Saiani, k., Kageyama, H., Conti-Tronconi, B.M. and Guidotti, A.. Purification and characterization of an (z-bungarotoxin peptide which blocks nicotinic receptor function in primary culture of adrenal chromaffin cells, Mol. Pharmaco/.. 25 (1984) 327-334. 27 Stroud, R.M,. Structure of an acetvlcholine receptor, a hypothesis for a dynamic mechanism oI its action. In R.H. Sarna tEd.), Proceedings ()! the Second SUN YA ('(m verration in the Di~cipline Biomolecular Stereodvm*mics. Vol. 2, Adenine Press, New York, 1982. pp. 55-73. 28 Stroud, R.M., Acetvlcholmc receptor structure, Neztrosci. Comment., l (1983) 124-138. 29 Tsernglou, P. and Petsko. G.. Thc crystal structure of a postsynaptic neurotoxin from a sea-snake at 2.2. A resolution. FEBS Lett., 68 (1976) 1-4. 30 Walkinshaw, M.D., Sacnger, W, and Maelickc, A., Threedimensional structure of the 'long ncurotoxin from cobra venom, Pro('. Natl. Acad. S c i . U.,S.A.. 77 ( 198111 24(10-2404.