Biochimica et Biophysica Acta, 537 (1978) 1--8 © Elsevier/North-Holland Biomedical Press

BBA 38018

PURIFICATION, SOME PROPERTIES AND THE PRIMARY STRUCTURES OF THREE REDUCED AND ~CARBOXYMETHYLATED TOXINS (CM-5, CM-6 AND CM-10a) FROM NAJE HAJE HAJE (EGYPTIAN COBRA) VENOM *

FRANCOIS JOUBERT and NICO TALJAARD

National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001 (Republic of South Africa)

(Received April 17th, 1978)

Summary

Three reduced and S-carboxymethylated toxins (CM-5, CM-6 and CM-10a) were purified from Naja haje haje {Egyptian cobra) venom. Whereas toxin CM-5 comprises 71 amino acid residues and five intrachain disulphide bridges, toxins CM-6 and CM-10a contain each 61 residues and four disulphide bridges. The complete primary structures of the three toxins have been established. The toxicity, the immunochemical properties, the sequence and the invariant amino acid residues of toxin CM-5 resemble the properties of the long neurotoxin group, while those of toxin CM-6 and CM-10a are related to the short neuro- toxin group. Further, the sequences of the three toxins from Naja haje haje venom reveal a high degree of homology with those of the corresponding neurotoxins isolated from Naja haje annulifera or Naja nivea venoms.

Introduction

The sub-species of the Naja haje complex, viz. Naja haje haje, Naja haje annulifera, Naja haje anchietae and Naie haje arabica, is distributed throughout Africa and Arabia [1]. In a number of papers [2--8], the amino acid sequences of two short neurotoxins and 14 cytotoxins of the venom from Naja haje annulifera have been elucidated. A group of workers [9--11] purified several neurotoxins, determined the partial sequences of some of them and

* S u p p l e m e n t a r y d a t a t o this article are d e p o s i t e d w i t h , a n d c a n b e o b t a i n e d f r o m : Elsevier Sc ient i f i c P u b l i s h i n g C o m p a n y , B B A D a t a D e p o s i t i o n , P .O . B o x 1 3 4 5 , 1 0 0 0 B H A m s t e r d a m , T h e N e t h e r l a n d s . R e f e r e n c e shou ld be m a d e t o N o . B B A / D D / 0 8 6 / 3 8 0 1 8 / 5 3 7 ( 1 9 7 8 ) 1. T h e s u p p l e m e n t a r y i n f o r m a t i o n i n c l u d e s : c h r o m a t o g r a m s o f the t r y p t i c a n d c h y m o t r Y p t i c d iges t s , a m i n o acid c o m p o s i t i o n o f the p e p - t ides , N - t e r m i n a l s e q u e n c e s o f i n t a c t t o x i n s a n d a d d i t i o n a l s e q u e n c e s tudies o n s o m e o f the pept ides , o f the r e d u c e d a n d S - c a r b o x y m e t h y l a t e d t o x i n s CM-5, CM-S a n d CM-10a .

established the complete primary structure of one long neurotoxin from the venom of Naja haje, obtained from Miami Serpentarium, Florida, U.S.A. or Ethiopia.

Concerning the toxins from Naje haje haje venom, Joubert and Taljaard [12] sequenced four cytotoxins from this venom. The primary structures of the cytotoxins resemble those of the cytotoxins from Naje hale annulifera and Naja nivea venoms. In continuation of this comparison, the present communi- cation describes the properties and complete amino acid sequences of three neurotoxins from Naja haje haje venom.

Experimental procedure

The sources of the Naja haje haje venom, trypsin, ~-chymotrypsin, subtilisin and the chemical reagents have been described previously [3,6]. The physico- chemical methods; the toxicity determinations by intravenous injection; the immunochemical examination by the Ouchterlony technique; the reduction and S-carboxymethylation; the digestion with trypsin, ~-chymotrypsin and subtilisin; the fractionation of enzyme digests by chromatography on DEAE- cellulose and paper chromatography and/or high-voltage paper electrophoresis; the amino acid analyses of the toxins and of the peptides; the sequence deter- mination of reduced and S-carboxymethylated toxins and peptides by Edman degradation with the Beckman sequencer or manually and the nomenclature of the peptides have all been detailed in previous communications [3,6].

Results

Purification and some properties of toxin CM-5, CM-6 and CM-10a The fractionation of crude Naja haje haje venom by gel filtration and ion-

exchange chromatography has previously been described (See Figs. 1 and 2 of ref. 12). Fractions CM-5, CM-6 and CM-10a were further purified by rechro- matography on CM-cellulose at pH 5. The fractions (0.2 g) were each loaded on columns (0.9 × 15 cm) of CM-cellulose and eluted by a linear gradient of 0.05-- 0.6 M ammonium acetate/acetic acid buffer of pH 5 over 2 L. This afforded major peaks, viz. toxins CM-5, CM-6 and CM-10a. The toxins appeared to be homogeneous by chromatography, amino acid analysis, N-terminal end group determination and immunodiffusion.

Some of the properties of the three toxins are summarised in Table I and the amino acid composition of the toxins are given in Table II. Examination of the

T A B L E I

SOME OF T H E P R O P E R T I E S OF T O X I N S CM-5, CM-6 A N D CM-10a

Molecular weights were ca lcu la ted f r o m the a m i n o acid s e q u e n c e s

T o x i n Molecular weight . Tox ic i t y L D s 0 ~g/g m o u s e Serological group

CM-5 7751 0.11 ± 0.2 I l l CM-6 6 7 9 6 0 .10 -+ 0.2 I CM-10a 6 8 4 0 0 .08 + 0.1 I

T A B L E II

A M I N O ACID C O M P O S I T I O N O F T O X I N CM-5, CM-6 A N D CM-10a

Samples were h y d r o l y s e d for 24 h . Values are given as too l o f residue per tool of t o x i n

A m i n o acid CM-5 CM-6 CM-10a

Analys is Sequence Analys i s Sequence Analys i s Sequence

Aspartic acid 9.0 9 6.9 7 5,1 5 Threon ine 6.8 7 6 .4 7 5.0 5 Serine 2.9 3 3.8 4 2,4 3 Glutamic acid 1.2 1 6.7 7 5,8 6 Prol ine 5.6 6 3.9 4 4 ,0 4 Glycine 4.8 5 4.8 5 6.0 6 Alanine 3.0 3 0 0 0 0 Half-cystine * 9.6 10 7.7 8 7.6 8 Valine 4.3 5 1.1 1 2.1 2 Methionine 1.8 2 0 0 1.0 1 I so leuc ine 2.7 3 2.8 3 4.3 5 Leucine 1 . I 1 1.2 1 0 0 Tyros ine 1.0 1 1.0 1 2.0 2 Phenylalanine 2.9 3 0 0 0 0 Lysine 4.7 5 5.8 6 6.7 7 Histidine 0.9 1 1.8 2 1.8 2 Arginine 5.0 5 3.6 4 3.6 4 TryptoPhan 0.7 1 0.7 1 0 .8 1 Free su lphydry l 0 0 0 0 0 0

Tota l 71 61 61

* Determined as S - carboxymethy l cys t e ine .

toxins with EUman's reagent both in the presence and absence of 6 M guanidi- nium chloride, revealed that the toxins were devoid of free sulphydryl groups.

Amino acid sequence of reduced and 8-carboxymethylated toxins CM-5, CM-6 and CM-10a

The details of the amino acid sequence elucidation of the reduced and S~arboxymethylated toxins are given in the supplementary data. This includes: chromatograms of the fractionation of the tryptic and chymotryptic digests of the toxins on DEAE-ceUulose; tables of the further purification of the peptides and the amino acid composition of the peptides; tables of the amino-terminal sequences of the intact toxins; and additional sequence studies on some of the tryptic and subtilisin peptides.

The complete amino acid sequence of reduced and S~arboxymethylated toxin CM-5 is shown in Fig. 1. The known amino-terminal sequence of the toxin established directly the alignment of peptides T-1 and T-2 and C-1 to C-4. Peptide T-6a contained no C-terminal arginine or lysine and must be derived from the carboxy-terminal peptide of toxin CM-5. The known amino acid com- position of chymotryptic peptides C-5a and C-5b provided, respectively, the necessary overlaps between peptides To2 and T-3, T-3 and T-4 and T-4 and T-5.

Fig. 2 reveals the complete amino acid sequence of reduced and S-carboxy- methylated toxin CM-6. Sequencing the amino-terminal segment of the toxin provided directly the position of peptides T-1 and T°2. Since peptide T-7 was

4

I0 20 H2N-I le-Ar g-Cys-Phe- I le-Thr-Pro-Asp-Val-Thr-Se r-Gln-Al a-Cys-Pro-Asp-Gly-His-Va l-Cys-

-T-'I

~--T-la ~ T-ib

T-Id

( - c - i X . . , c-2-

( Sequencer

30 40 Ty r-Th r-Lys-Me t -Trp-Cys -Asp-Asn-Phe -Cys-Gly-Me t -At g-Gly-Lys -At g-Val-Asp-Leu-Gly-

- T-I ).( T-2 ~ T-3 ~ . T-4

T.- 1 I ~ . - T - 1 c-..~ ~ T-3a ~.~ T-4a

T- 1 d-.....~) ~ T-4a-- Sa

--C-.~" c-3 ~. c-4 X c -5

C-4a )( C-4~ ~ C-5a

.Sequencer

XO-Sb-

50 60 Cys-A la-Al a-Thr-Cys-Pro-Thr-Val-Lys-Pro-Gly-Val-Asp-I le-Lys-Cys-Cys-Se r-Thr-Asp-

-" T-/: X T - 5 ~

T-4~ ~ T-Sa

- - T-4a- S a - - . ~ T-4a-Sb ~ T-4a-Sc. , ~ T- 4 a- Sd--..-.~

C-5

-C-5b

70 Asn-Cys-As n-Pro-Phe-Pro-Thr-Arg-Lys-Arg-Se r-OH

T-5 V~ T-S---)

T-5a "M T-6a )

~--T- 6 b.~-6 c~

- c - 5 ~ c - e

C-Sb

Fig. 1. The complete amino acid sequence of reduced and S-carboxymethylated toxin CM-5 from Naja haje haje venom.

the only tryptic peptide not terminating in either lysine or arginine, this pep- tide was assigned as the carboxyterminal peptide of toxin CM-6. By comparing the tryptic peptides T-3, T-4, T-5 and T-6 of toxin CM-6 to the corresponding tryptic peptide of the toxin ~ ofNa]a haje annulifera venom [2], it was obvious that the amino acid compositions and the sequences of the peptides are identi- cal. Consequently, peptides T-3, T-4, T-5 and T-6 of toxin CM-6 were aligned in the same order as those had been in toxin ~.

The complete amino .acid sequence of reduced and S-carboxymethylated toxin CM.10a is illustrated in Fig. 3. The known amino-terminal sequence of the toxin allowed peptides T-1 and T-2 and C-1 and C-2 to be placed in that order from the NH2-terminus, and established an overlap for T-2 and T-3. The

iO 20 H2N-Leu-Glu-Cys-His-Asn-Gln-Gln-Ser-Ser-Gln-Pr o-Pro-Thr-Thr-Lys-Thr-Cys-Pro-Gly-Glu-

• T-I X T-2

( Sequencer"

3O 40 Thr -Asn-Cy s -Tyr -Ly s-Ly s -Ar g-T rp-Ar g-A sp-Hi s-Ar g-G ly-S er - I i e-Thr -G lu-Arg-Gly-Cy s-

- - T - 2 ~" T-3 ~ T-4

( T-3a, )

4 T-4a---~

Sequencer

T-4b

%( T-5 ~ T-6---

; 4--T-6a--

Gly-Cys-Pro-Ser-Val-Lys-Lys-Gly-I le-Glu-I le-Asn-Cys-Cys-Thr-Thr-Asp-Lys-Cy s-Asn-Asn-OH

T-6 X T-7 l,

T - 6 a X T - 7 a ')

F i g . 2. T h e c o m p l e t e a m i n o a c i d s e q u e n c e o f r e d u c e d a n d S - c a r b o x y m e t h y l a t e d t o x i n C M - 6 f r o m Naja haje haje v e n o m .

IO 20 H2N-Me t - I l e - C y s - H i s - A s n - G l n - G l n - S e r - S e r - G l n - P r o - P r o - T h r - I l e - L y s - T h r - C y s - P r o - G l y - G i u -

T-I M T-2

T-2a

( T-2b

C-I

C-lb

,i

• C-l~ ~,

C-ic

• C-l~

C-ld-

Sequencer

30 40 Thr -Asn-Cys-Tyr-Lys-Lys-Gln-Trp-Arg-Asp-His-Ar 8-G ly-Thr -I le-I le-Glu-Arg~G1y-Cys-

--T-2 ~( T-3 .~. T-4 ~.. T-5 ~ T-6---

T-2a ~- eT-3&~ T-3b ";

T-2~

C-lb ~ C~2 )~ C-3

C-Id l, ( C-3a ~ C-3b

Cequence~

50 60 G l y - C y s - P r o - S e r - V a l - L y s - L y s - G l y - V a l - O l y - ~ I e -Ty r -Cys -Cye -Lys - T h r - A sp - L ys - Cy s-Asn-Arg-OH

¢ T-7a ' )

T-7~ ~_. T-7c----~

• T-7d ')

¢-3 ~ - C-'; ~ C-5

F i g . 3. T h e c o m p l e t e a m i n o a c i d s e q u e n c e o f r e d u c e d a n d S - c a r b o x y m e t h y l a t e d t o x i n C M - 1 0 a f r o m Naja

known amino acid composition of chymotryptic peptides C-3a and C-3b furnished, respectively, overlaps between T-3 and T-4, T-4 and T-5, and T-6 and T-7.

Discussion

The properties of three toxins, viz. CM-5, CM-6 and CM-10a, isohted from Naja haje hale venom have been studied. Whereas toxin CM-5 comprises 71 amino acid residues, toxins CM-6 and CM-10a both contain 61. Toxin CM-5 contains 10 half-cystine residues and toxin CM-6 and CM-10a both 8, and since no free sulphydryl groups could be demonstrated in the intact toxins they are, respectively, cross-linked by five and four intramolecular disulphide bridges. The amino acid composition of all three toxins are characterised by high con- tents of aspartic acid and its corresponding amide, threonine, proline, glycine and lysine and a low content of methionine, leucine, tyrosine, histidine and tryptophan. Further toxin CM-6 and CM-10a are devoid of alanine and phenyl- alanine, toxin CM-6 also of methionine and toxin CM-10a of leucine. The amino acid composition of toxins CM-5 and CM-10a resemble, respectively, that of toxin ~ from Naja nivea [13] and toxin CM-14 from Naja haje annuli- fera venoms [3], and that of toxin CM-6 is identical to that of toxin a from Naie hale annulifera venom [2].

The complete amino acid sequences of toxin CM-5, CM-6 and CM-10a were readily elucidated (Figs. 1--3) by using, trypsin, chymotrypsin and subtilisin digestions. Apart from the cleavage of the Tyr2~-Thr 22 bond (toxin CM-5) and the Tyr24-Lys 2s and also TyrS:-Cys(CM Cys) ~3 bonds (toxin CM-10a) by trypsin, the proteolytic enzymes (trypsin and chymotrypsin) in general exhibited their anticipitated specificities. Tryptic peptide T-4a (18 residues) of toxin CM-5 was redigested with subtilisin (See Fig. 1). As previously observed [14] subtilisin caused extensive cleavage of the peptide and major splits occurred at Ala 43- Thr 44, Thr47-Val 4s and GlySl-Val s2 bonds.

Regarding the biological properties of the toxins, their toxicities, LDs0 values (Table I), are of the same order as values which were found for the short neurotoxins and long neurotoxins from elapid and hydrophid snake venoms [15]. The immunochemical properties Of the toxins were investi- gated by the Ouchterlony technique. A single distinct precipitin band for each toxin was found. Further, the toxins were compared with a long neuro- toxin (a), a short neurotoxin (fl) and a cytotoxin (VII1) from Naja nivea venom [13,16]. For toxin CM-5 complete crossing of the precipitin bands was ob- served for the short neurotoxin and the cytotoxin but the long neurotoxin revealed complete coalescence. However, for toxins CM-6 and CM-10a com- plete crossing of the bands was found for the long neurotoxin and cytotoxin whilst complete coalescence for the short neurotoxin. Consequently, toxin CM-5 is immunochemically related to the long neurotoxin group III [17] and toxin CM-6 as well as toxin CM-10a are related to the short neurotoxin group I [17].

In Fig. 4 the primary structures of toxin CM-5, CM-6 and CM-10a from the venom of Naja haje haje are compared to those of the corresponding neuro- toxins from Naja haje annulifera, Naja haje and Naja nivea venoms. The

(a)

(b) (c)

(a)

(b)

(c)

(d)

(e)

(f)

I R C F - -

R C F

R C F

40

I R V D L G

R V D L G

R V D L G

L E C H N Q

L E C H N Q

L E C H N Q

I0 - I T P D V

I T P D V

I T P D V

50 C A A T C P

C A A T C P

C A A T C P

10 Q s s Q P P

Q s s Q P P

Q s s Q P P

~O 30 T S q A C P D

T S Q A C P D G - H V C ¥ T K M W C D N F C G M R

T S Q A C P D G - H V C Y T K M W C D N F C G M R

T V K P G V D

V K P G V ~

V K P G V

T T K T C P -

T T K T C P -

T T K T C P -

6O l0 I K C C S T D N C N P F P T R ~

I K C C S ~ D N C N P F P T R K R S

I K C C S T D N C N P F P T R

20 G E T N C Y K K R W R D H . . . . R G

G E T N C Y K K R W R D H . . . . RG

G E T N C Y K K R W R D H . . . . RG

4O (d) IS I T Z R C

(e) ISS I T E g G

(f) I T E R G

(g) It( I C H S Q

(h) I~ I C H N Q

( i ) I c :a N q

4o

(h) I I Z S C

(1) ,, i i Z R G

50 C G - - C P S V K K G I E

C G C P S V K K G I E

C G C P S V K K C I E

IO Q S S Q[R~p T I Z T C P -

Q s s Q P P T I K T C P

q S S q P P T I K T C P

~0 C G - - C P S V K K G V G c C C P S V K K C v C

C G C P S V K K G V G

6o I N C C T T D K C N i l I N C C T T D K C N

I N C C T T D K C N

2O G E T N C Y K K R W R D H . . . . R ! /

G E T N C Y K K R W R D H R

G E T N C Y K K ~ W R D H R

60 I Y C C K T D K C N i l I Y C C K T ~ K C N

I Y C C K T K C N

Fig. 4. Comparison of the pr imary s tructures o f n e u r o t o x i n s f r o m various snake v e n o m s . (a) Nala hale, III [11]; (b) Nala nivea, c~ [18]; (c) Nala hale hale CM-5; (d) Nala nivea, 8 [13]; (e) Nala hale annulifera, a [2]; (f) Nala hale hale, CM-6; (g) Nala nivea, [3 [18]; (h) Naia hale annulifera, CM-14 [3] and (i) Nala hale hale, CM-IOa. T he s e q u e n c e s w e r e a l igned w i t h r e sp e c t to the p o s i t i o n o f the c y s t e i n e res idues and the p o s i t i o n s o f the invariant a m i n o acids are b o x e d . The I U P A C one- la t t er n o t a t i o n for a m i n o acids is used ( ( 1 9 6 8 ) Eur. J. B i o c h e m . 5, 1 5 1 - - 1 5 3 ) .

sequences of CM-5 and long neurotoxin III from Naja haje [11] venom reveal six differences. However, the higher degree of homology of toxin CM-5 com- pared to long neurotoxin a from Naja nivea venom [18], is quite obvious; their sequences differ only in three positions. The venom Naja haje annulifera does not appear to contain a long neurotoxin. The primary structure of toxin CM-6 is identical to those of short neurotoxin ~ from Naja nivea venom [13] and also short neurotoxin a from Naja haje annulifera venom [2]. Comparison of the sequences of toxin CM-10a from Naja haje hale venom to those of short neuro- toxin ~ from Naja nivea venom [18] and short neurotoxin CM-14 from Naja haje annulifera venom [3] reveals only two differences for each toxin which indicate a high degree of similarity between their sequences.

Concerning the structure vs. function relationship, in comparing the primary structures of 27 different homologous proteins with either neurotoxic, cyto- toxic or lytic activity from venoms of elapid or hydrophid snakes, Ryd6n et al. [19] noticed that 11 positions are identical in all the sequences. In the sequences of Fig. 4 this is found to be so and these invariant residue positions correspond to cysteine 3, 17, 24, 45, 49, 60, 61, 66, tyrosine 25, glycine 44 and proline 50. These invarient amino acid residues are believed to be impor- tant for determining the general folding of the peptide chain and are called the

'structurally invariant' residues. Ryd~n et al. [19] established also five addi- tional 'functionally invariant' residues in all sequences of the long neurotoxins and short neurotoxins. These residues are expected to be important for the specific interaction of the neurotoxins with the nicotinic acetylcholine recep- tor protein. In the sequences of Fig. 4 these invariant residues correspond to lysine 27, tryptophan 29, aspartic acid 31, arginine 37, and glycine 38.

In conclusion, the toxicity, the immunochemical properties, the primary structure and the invariant amino acid residues of toxin CM-5 from Naja haje haje, indicate it belongs to the long neurotoxin group, and those of toxin CM-6 and CM-10a from the same venom to the short neurotoxin group.

Acknowledgements

The authors are indebted to Mrs. A. Ruelle for her assistance with the Beck- man sequencer.

References

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