The complete primary structure of toxin CM-1b from Hemachatus haemachatus (Ringhals) snake venom

Taziaw~. VoL 18, DD. 191-198 . O Perpmon Pmw Ltd. 1980. Printed in

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0041-0101/80/0301-0191502 .00/0

THE COMPLETE PRIMARY STRUCTURE OF TOXIN CM-lb FROM HEIMACHATUS HAEMACHAT US (RINGHALS) SNAKE VENOM FRANCOIS

J. Joue~r and Nico TALiAARn

Molecular Hiochemiatry Division, National Chemical Research Laboratory, Coundl for Sdentlfic and Industrial Research, P.O. Box 395, Pretoria 0001, Republic of South Africa (.lcoeptedjor pubHaotion 91W1y 1979)

F. J. Jov>1>~r and N. Tu rMan . The complete primary structure of ttnân CM-lb finm Heneocltatua levdnachatus (Ringhals) snakevenom. Toxicon 18,191-198 .1980.-Toxin CM-1b was purified fi+om Hanadiatns haanachatus venom. The purified toxin contains 57 amino acids including 8 half~ystine residues in a single polypeptide chain. The complete primary structure of CM-lb has been established. The sequence and the invariant amino acid residues of CM-lb resemble those of the Napa-type toxins . In toxin CM-lb one of the mncturnUy invariant amino add residues pmline 48, of the short nwrotoxina has bees roplaoed by a arginine, while it contains none of the juncttonally invariant amino add residues. INTRODUCTION

the toxic polypeptides, viz. long neurotoxins, short neurotoxins and cytotoxins (Srltxnoi~ and Bores, 197, elapid snake venoms contain also polypeptidea of lowtoxicity. The latter include the short neurotoxin homologues (Jous~tr, 1975), cytotoxin homologues (C~ru ssoN, 1974), trypsin inhibitors (TAxAxesHI et al., 1974) and trypsin inhibitor homologues (STRYDOM, 197 . In comparing the primary structure of the neurotoxins and cytotoxins, it was obvious that certain invariant amino acid positions in the structural chain are important for the general folding of the molecule, and others are important for the toxic activity (RxD>~N et al., 1973 and KAItr.ssoN,1979). Jovsllitr (1977a) purified and sequenced the low-toxic polypeptides CM-2a and CM-3 from Naja haje aratalifera venom. The sequences of CM-2a and CM-3 and some of the invariant amino acids do not show a high degree of similarity with those of a short neurotoxin, a long neurotoxin, a cytotoxin or an Angusticeps-type protein. Furthermore, the t,D6o values of toxins CM-2a and CM-3 are about 10- and 150-fold of those, respectively, of the cytotoxin and neurotoxin groups . They are also immunochemically distinct from the cytotoxin and neurotoxin groups. Toxins CM-2a and CM-3 probably represent a new type of elapid venom toxins, the so-called Naja-type toxins (SraYnoas, 1977, personal communication) . Concerning the basic polypeptides of Ringhals venom, $TRYDObi and BOTBS (1971) sequenced two short neurotoxins from this venom. Fkxr urm and BASt~t (1973) fractionated the venom into 14 major fractions and sequenced one of them (12B). Fraction 12B is a non-neurotoxic, hemolytic, basic protein and is structurally related to the cytotoxins. Recently Jorrs~r (1977b) described the sequences of toxins 9B, 11 and 12A also from lZinghals venom. Tho sequences of toxin 11 and 12A resemble those of the cytotoxin group. Toxin 9B is less toxic than the cytotoxins and is structurally homologous to the cytotoxina. BBSII)B3

192

FRANCOIS J. JOUHERT and NICO TALJAARD

In continuation of the study on polypeptides from Hemachatus haemachatus venom, this communication presents the primary structure of a relative innocuous toxin (CM-lb) also from this venom . MATERIALS AND METHODS Desiccated ilarwckatus baemachatlra (Ringhals) venom was supplied by D. Mailer, Professional Snake Catcher (Pty.) Ltd., 215 Barkston Drive, Blairgowne, Johannesburg, 2001. The souris of the trypsin, a-chymohypsln and chemical reagents have been described previously (Jousanr, 1975, 197 . The phyaioochemical methods ; the toxicity determination by i.v. lnjxtlon ; the sequence elucidation ofthe toxin and the peptides ; and the nomenclature ofthe peptides have also bean detailed in previousoommunicatiom (JoueBRT, 1975, 197 . RESULTS Purification and some properties of toxin CM-Ib The fractionation of crude Ringhals venom on Amberlite CG-50 has been previously described (see Fig . 1 of SrRVDOM and BOTB4, 1971) . Eleven major and a number of minor peaks were obtained . Peak I was further fractionated on CM-cellulose using a linear ammonium acetate/acetic acid buffer (pH 5) gradient and the elution pattern is depicted in Fig. 1 .

o.s

CM-Ib

N

4

CM-le 0.3 i-

1

CM-la

600 Eluats volume (ml)

1200

Fm. 1. Cmzo~roa~nrxsr of mina I ox CM-c~.r ur~ . Toxin I (0~2 B) was loaded on the column (0~9 x 1S cm) and elution affected by a 2L linear gradient of0"OS to 0~6 M ammonium acetate~cetic acid buffea~ofpH S at aflow of SO ml/hr. The column temperature was 20°C and the eluate was monitored at 280 nm. The chromatogram revealed three major fractions . One of the major &actions contained toxin CM-lb . The toxin appeared to be homogeneous by chromatography, amino acid analysis and N-terminal end group determination . The lethality oftoxin CM-lb determined by i.v. injections (LDsu value = 11"7 ~ Iß ~g/g mouse), is indicative of a low toxicity relative to that normally encountered in the neurotoxin group, ca. 0~1 pg/g mouse, S~rxxDOM, 1973 (Dissertation, Univ. of South Africa, Pretoria). It is, however, comparable to the Lnsu values found for toxin CM-2a and CM-3 from Naja haje annulifera venom (JOVH~tT, 1977a) . The amino acid composition of toxin CM-lb is given in Table 1. Examination of the toxin with Elhnan's reagent (ELLMAN, 1959), both in the presence and absence of guanidinium chloride, revealed that the toxin was devoid of free sulphydryl groups.

193

Àmino-Add Sequence of a Snake Toxin TAHa.H

1. A~o ACm COl~OerrION OF H~UC~ AI~ S 1'8U TOXW CM-lb AND rrs "ra~rrnC PBp1~He (OIVHN Ae mo10 OF ABeII)UH PHR mOlO 1'07~i OR F~lmH)

Anvno add Cya (Cm)" Aep T'hr 3er ß1u Pm (31y Ala Val Met Ib Leu Tyr Phe Lye Iüa Arg Trp Total Yield Purlßcation

(n

Toxin CM-lb

T-1

T-2

7"6 (8) S-0 (~ 6"7 (7) 3"4 (4) 7"0 (7) 2"S (3) 2"2 (2) 0 2"6 (3) 1"0 (1) 0 2"0 (2) 2"9 (3) 3"8 (4) 4"0 (4) 1 "1 (1) 3"1 (3) 0 37

1"2 (1)

1"0 (1)

2"0 (2)

1"0 (1) 0"9 (i) 3"1 (3) 0"7 (1)

T-2a

T-2b 1-0 (1)

1 "0 (1)

1 "6 (2)

1"0 (1) 3-0 (3) 0"7 (1) 1 "6 (2)

1 "0 (1) 1"0 (1) 1"0 (1) 6 38-0

2"0 (2) 11 6"4

BPAWt

1"0 (1) 2 17"2

HPAWt

1"0 (1) 9 13 "4

BPAWt

T-3

3"2 3"U 3"9 2"8 1"9 2-0 1"9

(3) (3) (4) (3) (2) (2) (2)

T-4

3"3 (3) 1"9 Cj) 2-0 (2)

1-0 (1) 1"0 (1) 1"0 (1) 2"0 (2) 4-0 (4) 1"0 (1) 1"9 (2) 31 14"8

HPAWt

1-0 (1) 1"1 (1) 9 64"0

~S~arboxymet>~ylcyeteine. tPaper ohromotog~aPhY with butanol-PYridino-acetic add-watea~ .

dmlno acid sequence of reduced and S-carboxymethylated toxin CM-lb The tryptic and chymotryptic digests of the reduced and S-carboxymethylated toxin CM-lb were fractionated on columns of DEAF-cellulose as illustrated in Figa. 2 and 3. If necessary, the peptides were further purified by the methods indicated in Tables 1 and 2, which also list the amino acid composition of the peptides. The amino-terminal sequence of the reduced and S~arboxymethylated toxin was determined by using the Beckman sequencer (Table 3), and additional sequence studies were performed on some of the tryptic and chymotryptic peptides (Table 4). Figure 4 reveals the complete primary structure of reduced and S~arboxymethylated toxin CM-lb. The amino-terminal sequence of the toxin established directly the alignment of peptides T-1 to T-3 and C-1 to C-3. The remaining tryptic peptide T-4 must be derived from the carboxy-terminus of toxin CM-lb. DISCUSSION

The complete primary structures of two Naja-type toxins are currently known. In Fig. 5 the amino acid sequence of toxin CM-lb from Hemachatus haemachatus venom is compared to the known sequences of the raja-type toxins . The high degree of homology within the Naja-type toxins, consisting of CM-2a and CM-3 from Naja haje annulifera venom, is quite apparent. Furthermore, the sequence of CM-lb is homologous to those of the Najatype toxins . ICARLSaON (1979) noticed for the primary structures ofneurotoxins and non-neurotoxins, isolated from elapid or hydrophid snakes, that 10 positions are identical in all sequences. These amino acids will be called the structurally invariant residues. Further, IülerssoN (1979) established that three additional positions are invariant in all neurotoxins but not in non-neurotoxins. These will be called the functionally invariant residues, and they are assumed to be important for neurotoxicity. Figure Sd reveals the invariant amino acids of the short neurotoxin group and indicates the structurally andfunctionally invariant residues .

194

FRANCOIS J. JOUBERT and NICO TALTAARD

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Amino-Acid Sequence of a Snake Tautin

195

T-I + T-4

0.4 0 N t 0.2

T-2a

T-2Lti T-2 s~~~-~~ ~ ~ ~ i ~--~ ~~ sao i2oo L7uah vdurrr (ml)

FIO. 2. ~IROMATOORAPHY OF THB TAYPTIC DIOBST OF RI~UCFD AID 1+wmv CM-lb ox D1?AF-rrui vl,o~,

T®

The reduced and S-carboxymethylated toxin (10 pmole) was digested with trypsin (1 ~ w/w) for 2 hr at 30°C. The digest was applied on the column (0~9 x 130 cm) and eluted with a 2L linear gradient of 0"023 to 0~6 M NH,HCO, solution at a flow rate of 30 ml/hr. The column teanperature was 20°C and the eluate was monitored at 230 nm.

0.4

0.2

C-aa

c-a c-s C-4

soc

C-4a

~2b

Eluab volurrw (ml)

FIO.

3.

c-sb C-8al C-3o

i2oo

CSAOIdATOOBAPHY OF THS QYlg1AYFTIC DIOHSI' OF AZ~UCBD AID z~YUZZ~ toms CM-lb ox Dl~-c~.TuLaea.

S-c++naoxr-

The reduced and S~arboxymethylated toxin (10 Amok) was digested with chymotrypsin . The experimental conditions were the same as in Fig. 2.

The sequences of the Naja-type toxins (Figs. Sa and Sb) contain nine of the ten structurally invariant amino acid residues, viz. Cysteine 3, 17, 24, 43, 47, 59, 60, 65 and glycine 42, while the proline 48 is exchanged for a arginine . The sequences apparently contain only one of the functionally invariant amino acids, viZ. arginine 34 while the tryptophan 29 and glycine 35 are, respectively, replaced in the Naja-type toxins by phenylalanine and asparagine. It was quite obvious that in toxin CM-lb, from Hemachatus baentachattrs venom (Fig. Sc), all the structurally invariant amino acids are conserved with exception of proline 48 which has also been exchanged for an arginine. Furthermore, the sequence of toxin CM-lb contains none of the threefunctionally invariant amino acids. The L.n6o value of toxin CM-lb is 117-fold higher, relative to the neurotoxin group. The structural feature which lowers the toxicity of CM-lb by several orders of magnitude, is presumably the replacement of three functionally invariant amino acids usually found in the neurotoxins. .

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FRANCOIS J. JOLrBF.RT and NICO TALJAAitD 10

p0

HZ N-Leu-Glu- CyS-Tyr - Gh -Lys-Ser-Lys-Val-Val-Thr - Cys-Gln- Pro-Glu-Gln-Lys-Phe--Cys-Tyr T-I ---~ ~

T~2

~T-2a+ ~

C-2 C-2a-- -" Sequencer

T3~-

T2b

lC-3 ~C-3a-

C-2b

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C-4

~- C-S

C- 4a-

Sequ~encer

~C-6 ~C-6o

C-5o

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C-6o -~~

Fx~. 4.

TäE

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OOI~LÛrfi PRI!(ARY 311tUCTURB OF ßF~UC~ A2m S-C~aHOxr~u~rsn Toamv CM-lb mioie Henwchatus haemadwras vsi~ors .

TASt$

Step 1 2 3 4 S 6 7 8 9

lo

11 12 13 14 15 16 17 18 19 20 21 22

3. N-x~eolvAi, a~uavca os x~uc~n Alm S vwm~r CM-lb van~a a~ Av~rAric BECKMAN s~u~rc~e" Gas chromatography Normal Silylated Leu/Ile > 400 L~ > 400 Glu 400 CysJSer 76 Cys % Tyr > 400 Tyr > 400 Gln 76 Gln 240 Lya 20 Lys 36 Cys/Ser 80 Ser 92 Lys 16 Lya 15 Va1248 Va1308 val22s va1228 Thr sa Thr 6s Cys/Ser 38 Cys 18 Gln 90 Gln ~ Pro 76 Pro 32 Glu 68 Gln 24 Lys 12 Lys 36 Phe SO Phe 66 Cys/Ser 12 Cys 14 Tyr 30 Tyr 60 Cys/Ser 14 Ser 16 Asp 15

TLC Leu Glu Cys Tyr Gln Lys Ser Lys Val val Thr Cys Gln Pro Glu Gln Lys Pho -

Ideatiflcation Leu Glu

Cyaf

Tyr Gln Lys Ser Lys Val val Thr Cysf Gln Pto Glu Gln Lya Phe Cysf Tri' Ser Asp

'840 nmolo was loaded . The quadrol programme ~eclcman No.122974 Mod.) wasused . Tho numbeas signifyylolds of phertylthiohydantoin-amino acids in nmole. TLC = Thin layer chra®atography" fIdentlfled as S~arbo~grmethylcyst~ne.

Amino-Acid Sequence of a Snake Toxin

197

TAaIB ~l. AMnVO ACm SBQU8NCF8 OF 801i8 OF TH8 TRYPTIC AND CHY1gTRYP1'IC PEPTII)ES OF RHDUCBD AND S-CAAHOXYIIBrS7ffiA1'8D TOXRd CM-Ib"

Peptide Residues

Sequences

C-3

19-26

Cys-Tyr-S~Asp-Târ-Met-Thr-Phe

C~4

27-34

Phe-Pro-Asn-His-Pro-Val-Tyr-Leu

C-5

35-39

SeQ-0ly~,j~s-Thr-Phe

C-6

40-57

T~

49-57

Cys-Arg-Thr-Asp-Cilu~er-Gly-Glu-Ar~-L~s-Gj~s > > ~ ~ > (Thr, Thr, Asp, Arg, ~. Asn, Lys) G~s-C~Thr-ThrAsp-Arg-(~s-Asn-Lys

sequences of peptideT-1(reeidue 1~, T-2 (residues 7-17) and thepartial sequence of T-3 (residues 188) follow from the sequence of the amino-teaminal segm~t of toxin CM-1 b (Table 3) . The upper half-arrows show the residues identified by using the automatic Beckman sequenncr . The lower half-arrows indicate the residues identified by the manual F.dman procedure. I

3

.

(0 )

LECY

(b)

LECY --- --OMSKVVTCKPEEKFCYSDVFMP~

(C)

L E C Y - -- - -OIKIS K V V T CIOI P EIOIK F C~Y S DIT ~AAIT F

(d)

XX®XNXXSXXXXTXXX®XXXXXX

--- --OMSKVVTCKPEETFCYSDVFMP

40

50

f0

TO

(0)

I

(b)

-VYTSGCSSYCR-DGTGEK - --CCTTDRCNGARGG

(C)

P

(d)

YTS~L~SSY~R

XKX1(IXDX-~iG

x x

x

ww

~

DGTGEK - --CCTTDRCNGARGG

YLSGCT-FCRTDES

X-XX ER

P-XVKXGXXXX

xx

xx

x

x

TTDRCNK -- --

XXXX©NX -- --

FIO . S. COIIPARDON OP TFIS PRIMARY STRUCTURF3 OF TOXINS FROM VARIOUS SNALS VSNOlaB . (a) Naja haje annulijera . CM-2a (JoueeRT, 1977a) ; (b) NgJa haje annul(fera, CM-3 (JouamIT,

1977a) ; (c) Hemachatus haemachatus, CM-1b and (d) the short neurotoxin groups (JovsraeT, 1975), where X shows variant amino acid residues . The (") indicates structurally invariant amino acid residues and the . indicates Jiulctlonally invariant amino acid residues (ICARIS90N, 1979). The positions of the invariant amino acids are boxed. The IUPAC one-letter notation for amino acids is used (F,ur. J. Biochem. S, 151, 1968).

In a previous study (IOUBIiItT, 1975) two toxins from the cobra, Naja haje arvlultfera, CM-10 and CM-12 were sequenced. These toxins have the feature that they contain the three functionally invariant amino acids of the short neurotoxins. CM-10 and CM-12 are still toxic, but the Ln 6 u values are, respectively, 50-fold and 625-fold those oftheneurotoxin group. Since other mutations have also occurred, the cumulative effect of all these changes accounts presumably for the large decrease in lethality (KARLSSOrr, 1979). dcknowlcalgerrlents-The authors areindebted to Mrs . RUFa r a forher assistance with the Beckman sequenncr.

198

FRANCOI3 J. JOUHERT and MOO TALJAARD REFERENCES

C~ox, F. H. H. (1974) The primary structures of two novel cytotoxin homologues from tho vaaom of forest cobra Ngja rnelanoleuca. Biockem. biophys. Res. Common. 59, 269. Eiaaux, E. L. (1939) Tissue sulpbydryl groups. Archs Bfodum. Biophys. 82, 70. F~en uND, L. and Ewe, D. (1973) Complete amino acid sequence of e non-neurotoxic hemolytic protein from the venom of Henurchatus lurpnachatus (African Ringhals cobra). Biochendstry 1i1,, 661. Joua~T, F. J. (1975) The amino acid sequences of three toxins (CM-10, CM-12 and CM-14) from Naja hgje murullfera (Egyptian cobra) venom. Hoppe"Seyla's Z.pbysiol. Chum . 356, 53. JoueEar, F. J. (1976) The amino-acid sequence of three toxins (CM-8, CM-11 and CM-13a) from Naja hgje annulijera (Egyptian cobra) venom. Eur. l. Biochem. 64, 219. Joiner, F. J. (1977a) The amino acid sequences of two toxins (CM-2a and CM-3) from Ngja hgje annuljfera (Egyptian cobra) venom. Hoppe-Seyler's Z. physic/. Clam. 358, 377. Jouaaxr, F. J. (1977b) The amino-acid sequences of three toxins (9B, 11 and 12A) from Hemachatus haemachatus (Ringhals) venom. Farr. J. Biochtm. 74, 387. Iüxissox, E. (1979) Snaky vmoms. In : C'lKndstry ojProtein Toxins in Snake Vtnoms, 13S Ed .) . New York : Springer. Rvn~x, L., G~e~., D. and Ewe, D. (1973) A model of the three-dimensional structure of snake venom neurotoxins based on chemical evideaoe. Int. J. Peptide Protein Ru. 5, 261 . S~rxYnoM, D. J. (1976) Purification and properties of low-mokciilar-weight polypeptides of Dendroaspis polylepispolylepis (Black mamba) venom . E7ur.1. gym. 69, 169. $7RYDOIN, A. J. C. and B018e, D. F. (1971) Purification, propettiea and complete amino acid sequence of two toxins from Ringhals (Haemachatus lnaenradratus) venom. l. biol. C/rem. 246, 1341 . SIRYDOM, D. J. and Hm~, D. P. (1976) Snake venom. In : Handbook ojBiochenristryandMolecular Biology, 3rd Edition, Proteins, Vol. III, p. 360, (F~rr, G. D., Ed.). Cleveland : CRC Press. Twr~. g., Iwerrwa~, S. and Suzuu, T. (1974) Distribution of proteinase inhibitors in snake venoms . Toxicon 193.

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