228

Biochimica et Biophysica Actal 5 7 9 ( 1 9 7 9 ) © Elsevier/North-Holland

Biomedical

228--233

Press

BBA 38220

SOME PROPERTIES AND THE COMPLETE PRIMARY STRUCTURES OF TWO REDUCED AND S-CARBOXYMETHYLATED POLYPEPTIDES (SsC1 AND SsCl0) FROM DENDROASPIS JAMESONI KAIMOSAE (JAMESON'S MAMBA) VENOM *

FRANCOIS

J. JOUBERT

and NICO

TALJAARD

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

December

13th,

1978)

Key words: Snake venom; Toxin; Protein sequence; (Jameson's mamba)

Summary Two polypeptides (protein SsCI and toxin SsC10) were purified from

Dendroaspis ]amesoni kaimosae venom. Whereas protein SsC1 comprises 61 amino acid residues, toxin SsC10 contains 58 and they each comprise four disulphide bridges. The complete primary structures of the two polypeptides have been elucidated. The sequences of protein SsC1 and toxin SsC~0 are structurally homologous to the short neurotoxins Type I, but they are much less toxic. In toxin SsC10 one of the functionally invariant amino acid residues, lysine 26, of the Type I neurotoxin has been replaced by a serine. In contrast protein SsC~ has the feature that it contains ten or eleven structurally invariant amino acids and apparently only one of the five functionally invariant residues.

Introduction Several basic low-molecular-weight polypeptides devoid of enzymatic activity have been isolated from venoms of elapid and hydrophid snakes. One group of highly poisonous polypeptides which act on the postsynatic sites; is know as the neurotoxins. Various workers [1--3] comparing the primary structures of * S u p p l e m e n t a r y d a t a t o t h i s article is 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 : E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press, 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 , 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 s h o u l d b e m a d e t o N o . BBA/DD[104/38220/579 ( 1 9 7 9 ) 2 2 8 - - 2 3 3 . The supplementary 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 tryptic and c h y m o t r y p t i c digests, a m i n o a c i d c o m p o s i t i o n o f t h e p e p tides, N - t e r m i n a l s e q u e n c e s o f intact polypeptides a n d a d d i t i o n a l s e q u e n c e s t u d i e s o n s o m e o f t h e pepfides, o f t h e r e d u c e d S - c a r b o x y m e t h y l a t e d p r o t e i n $ 5 C 1 a n d t o x i n $ 5 C 10.

229 short neurotoxins (Type I) and long neurotoxins (Type II) established that a minimum number of amino acids in certain chain positions are necessary for neurotoxic activity. A number of low-toxicity polypeptides have been purified and have been f o u n d to be structurally homologous to the Type I neurotoxin group. These polypeptides have one or more of the so-called essential amino acids substituted by other amino acids. Viljoen and Botes [4,5] purified and sequenced the low-toxic polypeptides F7 and F8 (the so-called angusticeps type proteins [6]) f r o m Dendroaspis angusticeps venom. F7 and F8 are structurally homologous to the Type I neurotoxins but contain only one of the functionally invariant amino acids [1]. This is also the case for angusticeps type protein SsC4 [7] from D. jamesoni kaimosae venom b u t the sequence reveals two of the essential amino acids for neurotoxic activity. J o u b e r t [8] sequenced t w o proteins, viz. CM-10 and CM-12 from Naja haje annulifera venom. These proteins are still toxic, although less so than normal for type I neurotoxins, and have the structural feature that one constant aspartic residue of the Type I neurotoxins has been replaced b y a glycyl residue. Concerning the components of D. jamesoni kaimosae venom, Strydon [9] sequenced a Type I ~eurotoxin and T y p e II neurotoxin from this venom, and J o u b e r t and Taljaard [7] reported the sequence of an angusticeps type protein of low toxicity. In continuation of the study on the polypeptides of low toxicity, the present communication describes the purification, some properties and the primary structure of protein SsC~ and toxin SsC~0 also from this venom. Experimental procedure Desiccated Dendroaspis jamesoni kaimosae venom was supplied by D, Muller, Professional Snake Center (Pty) Ltd., 215 Brakston Drive, Blairgowrie, Johannesburg, 2001. The source of the trypsin, a-chymotrypsin and chemical reagents have been described previously [8,10]. The physicochemical methods; the toxicity determination by intravenous injection and the sequence elucidation o f the polypeptides and the peptides have also been detailed in previous communications [8,10]. Results

Purification and some properties of protein SsC1 and toxin SsClo The fractionation of crude D. ]amesoni kaimosae venom by gel filtration and ion exchange chromatography has been previously described (see Figs. 1 and 2 of Ref. 7). Protein SsC1 was obtained by using fraction SsC, as such. Fraction SsCl0 was, however, further fractionated on DEAE-cellulose at pH 7.8. The fraction (0.2 g) was loaded on the column (0.9 X 15 cm) of DEAE-cellulose and eluted by a linear gradient of 0.05 to 0.60 M ammonium hydrogen carbonate solution over 2 1. The chromatogram showed a major peak and several minor peaks. The major peak provided toxin SsCl0. The polypeptides appeared to be homogenous by chromatography, amino acid analysis, and N-terminal end group determination. The toxicities of the

230

TABLE I A M I N O A C I D C O M P O S I T I O N O F P R O T E I N $5C1 A N D T O X I N $ 5 C 1 0 S a m p l e s w e r e h y d r o l y s e d f o r 24 h V a l u e s are given as tool of r e s i d u e p e r tool of p r o t e i n .

A m i n o acid

S 5C 1

A s p a r t i c acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Half-cystine * Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Free s u l p h y d r y l

S 5 C I0

Analysis

Sequence

Analysis

Sequence

8.3 5.7 1.8 4.0 5.3 4.9 0.2 7.6 0.1 1.0 3.2 2.1 2.9 2.0 3.2 1.1 4.6 0.8 0

9 6 2 4 5 5 0 8 0 1 4 2 3 2 3 1 5 1 0

7.0 3.4 6.2 2.0 2.3 3.0 2.0 7.8 1.9 0.1 5.0 1.0 1.6 0 6.8 1.9 3.8 0.7 0

7 4 6 2 2 3 2 8 2 0 5 1 2 0 7 2 4 1 0

Total

61

58

* D e t e r m i n e d as S - c a r b o x y m e t h y l c y s t e i n e .

10 20 H 2 N - A r g - • • e - C y s - T y r - A s n - H i s - L e u - G • y - T h r - L y s - P r • - P r • - T h r - T h r - G • u - C y s - T h r - G • n - G • u - A s pT-l T-la C-I

-

"

C-2

~

Sequencer

30 40 Ser-Cys-Tyr-Lys-Asn-••e-Trp-Arg-Asn-••e-Thr-Phe-Asp-Asn-••e-Arg-Arg-G•y-Cys-G•y~=

T-I

T-2 ,

~

T-3

~ ~

T-4

--T-la C-2

~.

C-3

~

C-4 •

~

C-5 ~ - - - - - -

C-6 ~

~Sequencer~

Cys-Phe-Thr-Pro-Arg-Gl

T-4 ~C-6

~-~--

50 y-Asp-Me t-Pro-Gly-Pro-Tyr-Cys-C

~-~

60 ys.-Glu-S er-Asp-Lys-Cys~Asn-Le~

T-5 C-7

~-

C-8

Fig. 1. T h e c o m p l e t e p r i m a r y s t r u c t u r 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 p r o t e i n S 5 C 1.

231 polypeptides was determined by intravenous injections. Protein SsC~ is not toxic, failing to kill mice at a dose of 130 tzg/g body weight. In contrast, the LDs0 value determined for toxin SsC10 (5.5 + 0.6 ttg/g mouse), is indicative of a low toxicity relative to that normally encountered in the neurotoxin group (approx. 0.1 tzg/g mouse) [11]. The amino acid composition of two polypeptides is given in Table I. Examination of protein SsC1 and toxin SsC~0 with Ellman's reagent [12], both in the presence and absence of 6 M guanidinium chloride, revealed that the polypeptides were devoid of free sulphydryl groups.

Amino acid sequences of reduced and S-carboxymethylated protein SsC1 and toxin SsClo The details of the amino acid sequence elucidation of the reduced and Scarboxymethylated protein SsC~ and toxin SsC10 are given in the supplementary data. Figs. 1 and 2 reveal, respectively, the complete primary structures of reduced and S-carboxymethylated protein SsCI and toxin SsC~0"

I0 20 H2N-Arg- II e-Cys-Tyr-Asn-His-Gln-Ser-A sn-Thr-Pro-Ala-Thr-Thr-Lys-Ser-Cys-Va l-Glu-Asn-

T-2a

~.

C-J -

-=

T-2b

~-~

C-2

~

C-3

Sequencer

--~

30 40 Ser-C ys-Tyr-Lys-Ser- II e-Tr p-Al a - A s p - H i s - A r g-Gl y-Thr- II e- II e - L y s - A r g - G ly-Cys-GlyT-3

~-

T-4

~.

T-5

.~=

T-4a ~ C-3

~=

C-4

~

Sequencer

4

C-5

~=

-

T-6 ~ T - 7 - ~ , ~ - - T - 8

~

-

-~,~--T-9 -D.~------ T - I 0

T-6a~

C-6

~

50 Cys-Pro-Arg-Val-Lys-Ser-Lys-Ile-Lys-Cys-Cys-Lys-S ~

T-6

C-6a

er-Asp-Asn-Cys-Asn-Leu-OH ~

T-! I

T-6a

~ C - 6

~

C-7

~

C-8

C-6a Fig. 2. The complete primary structure of reduced and S-ca~boxyrnethylated toxin $5C I0.

232

Discussion In Fig. 3 the primary structures of protein SsC~ and toxin SsC~0 from D. jamesoni kaimosae are compared to known sequences of the Type I neurotoxins from the various mamba venoms, viz. D. jamesoni kaimosae, D. polylepis polylepis and D. viridis. The high degree of homology of the sequences is quite apparent in Fig. 3. In comparing the primary structures of 27 homologous proteins with either neurotoxic, cytotoxic or lytic activity, for elapid or hydrophid snakes, Ryd6n et al. [1] observed eleven structurally invariant amino acids in all the sequences. These amino acids are believed to be important for determining the general folding of the polypeptide chain. In the sequences of the Type I neurotoxins (Fig. 3 a--c) this is found to be so and these residue positions corresponds to cysteine 3, 17, 23, 40, 42, 54, 55, 60, tyrosine 24, glycine 39 and proline 43. Interestingly enough all these residues are conversed in toxin SsC10, while protein SsC 1 contains only ten of the eleven structurally invariant amino acids; the proline 43 being replaced by a phenylalanine residue. 20

I0

(a)

R [ C Y N H Q S T TQA

(b)

R ]CYNHQ

(c)

R [C

(d)

R [ C Y N~i Q S ( ~ T

T T K S C - E E N S C Y K KIY W'-R[D H R G T I""

STTPATT

YNHQSTTPATT

KS

C - G E N S C

KSC

-

T Q

xx

x@x

xDxx

40

x[q

I E R G C G C P K V K P~V

(c)

[ E R G C G C P K V K QIG]I H

R G C G CF

SCY

I

K K T W S D H R G T

I

xxx

S C Y K

×

50

(a)

(e)

GEN

Y K K T W S D H R G T

P A T T K S C - V E N S C Y K S I WIAID H R G T I

(e) (f)

30

G-

IIW__R N I T F D N

xNx

60 IIH C C Q S D K C N Y

L[H C C Q S D K C N N

T P R G D M P G P YIC CIE[S D K C N L

x x xIZTl

x x

F-6x

Fig. 3. Comparison of the amino acid sequences of (a) Type I neurotoxin ~ from O. p o l y l e p i s p o l y l e p i s venom [13]; (b) Type I neurotoxin 4.11.3 from D. viridis venom [14]; (c) Type I neurotoxin V t from D . . i a m e s o n i kaimosae venom [9]; (d) toxin S 5 C 10 from D. j a m e s o n i kaimosae venom; (e)protein $5 C 1 from D. j a m e s o n i kaimosae venom and (f) the Type I neurotoxin group [8], where X shows variant amino acid residues. The positions of the invariant amino acids arc boxed. The circles in a boxed region indicate variant amino acids. The IUPAC oneqetter notation for amino acids is used [15].

233 In all sequences of short neurotoxins as well as long neurotoxins R y d e n et al. [1] also noticed five additional functionally invariant amino acids. These residues are probably important for the specific interaction of the neurotoxins with the nicotinic acetylcholine receptor protein. In the sequences of the Type I neurotoxins from various m a m b a venoms [Fig. 3a--3c] these invariant residues correspond to lysine 26, tryptophan 28, aspartic acid 30, arginine 32 and glycine 33. All these residues are conserved in toxin SsCl0, with the exception of lysine 26 which has been exchanged by a serine. In contrast, protein SsC1 contains only one of the functionally invariant amino acids, viz. tryptophan 28, while the lysine 26, aspartic acid 30, arginine 32 and glycine 33 are, respectively, replaced b y t w o asparagines, threonine and phenylalanine. Concerning the structure vs. function relationship of the neurotoxins from snake venom, it is apparent 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 neurotoxic activity [1]. The LDs0 value of toxin $5C10 is 55-fold higher, relative to the neurotoxin group. The structural feature which lowers the toxicity of SsC10 by several orders of magnitude, is presumably the replacement of one of the five functionally invariant amino acids usually found in the neurotoxins. In Fig. 2(f), the invariant amino acids of the T y p e I neurotoxin group [8] are indicated. Interesting enough all these residues are conversed in toxin SsCl0 with the exception of the one functionally invariant residue, lysine 26 which has been substituted by a serine and glutamic acid 37 which has been replaced by as lysine. Protein SsCl is n o t toxic, failing to kill mice at a dose of 130/~g/g b o d y weight. A comparison of the primary structure of protein SsCI with those of the Type I neurotoxins from mamba venoms and also the short neurotoxin group, reveals some differences. All of the structurally invariant amino acids, are conserved in protein $5C~ with the exception of the proline 43. In contrast, it contains only one of the five functionally invariant residues and five of the ten additional invariant amino acid residues of the Type I neurotoxin group. Acknowledgement The authors are indebted to Mrs. A. Ruelle for her assistance with the Beckman sequencer. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Ryd~n, L., Gabel, D. and Eaker, D. (1973) Int. J. Peptide Protein Res. 5, 261--273 S t r y d o m , D.J. (1973) Comp. Biochem. Physiol. 44B, 269--281 Karlsson, E. (1973) E x p e r i e n t i a 29, 1 3 1 9 - - 1 3 2 7 Viljoen, C.C. and Botes, D.P. (1973) J. Biol. Chem. 248, 4915---4919 Viljoen, C.C. and Bores, D.P. (1974) J. Biol. Chem. 2 4 9 , 3 6 6 - - 3 7 2 S t r y d o m , D.J. (1976) Eur. J. Biochem. 69, 169--176 Joubert, F.J. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 741--749 Joubert, F.J. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 53--72 S t r y d o m , A.J.C. (1973) Biochim. Biophys. Acta 328, 49 1--509 Jo ubert, F.J. (1976) Eur. J. Biochem. 6 4 , 2 1 9 - - 2 3 2 S t r y d o m , D.J. (1973) Dissertation, University of South Africa, Pretoria Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70--77 S t r y d o m , D.J. (1972) J. Biol. Chem. 247, 4 0 2 9 - - 4 0 4 2 Banks, B.E.C., Miledi, R. and Shipolini, R.A. (1974) Eur. J. Biochem. 45, 4 5 7 - - 4 6 8 IUPAC (1968) Eur. J. Biochem. 5, 151--153