http://www.abbs.info e-mail:[email protected]

ISSN 0582-9879                     ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(8): 683–688 CN 31-1300/Q

α-Neurotoxins of Naja atra and Naja kaouthia Snakes in Different Regions

WEI Ji-Fu^1,2, LÜ Qiu-Min¹, JIN Yang¹, Li Dong-Sheng¹, XIONG Yu-Liang¹, WANG Wan-Yu¹*

( ¹Department of Animal Toxicology, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China;² the Graduate School of the Chinese Academy of Sciences, Beijing 100039, China )

Abstract Recent studies have shown that there are geographic variation of α-neurotoxins in Naja kaouthia, but the cause is not clear yet. In this work, venoms were collected from adult Naja atra in Zhejiang Province and Naja kaouthia in Yunnan Province, well identified by morphological characters and cytochrome b gene analysis in summer season to avoid age and seasonal variation in the venom composition. Then α-neurotoxins were purified and cloned from these two kinds of snakes. Three α-neurotoxins from Naja kaouthia (Yunnan) and two from Naja atra (Zhejiang) were identified. Together with previously reportedα-neurotoxins in Naja kaouthia (Thailand) and Naja atra (Taiwan Province), it was found that the α-neurotoxins of Naja kaouthia in Yunnan Province were similar to those of Naja atra in Zhejiang and Taiwan Provinces, but different from those of Naja kaouthia in Thailand. This result can hardly be explained by population phylogeny or geographic distance. It might be due to the different climate, habitat and prey in Thailand in comparison with those in Yunnan, Zhejiang and Taiwan Provinces.

Key words Naja kaouthia; Naja atra; α-neurotoxins; prey

Cobra snakebite is a serious problem in China. According to Wüster[1], there are two species of cobras in China, Naja atra and Naja kaouthia. The former is present in most areas of China including Taiwan Province and the latter is only found in Southwest China including Yunnan, Sichuan and Guangxi Provinces. Naja kaouthia is also present in Bihar, Assam, Bengal, Nepal, Indo-China and the Triangle in Upper Burma.

The major toxic components in cobra venoms are postsynaptic neurotoxins orα-neurotoxins which block the nerve transmission by binding specifically to the nicotinic acetylcholine receptor, leading to flaccid paralysis and even death by respiratory failure. Based on the amino acid sequences, α-neurotoxins can be divided into two major groups, the long (65－72 residues with 5 disulfide bridges) and short neurotoxins (60－62 residues with 4 disulfide bridges). In spite of the diversity in their primary structure, these two kind of α-neurotoxins share a common three-loop structure[2,3]. Phylogenetic analysis showed that they were probably derived from a common ancestor[4]. Cobrotoxin is the main α-neurotoxin found in Taiwan Naja atra venom. It contains 62 amino acid residues in a single polypeptide chain[5]. In 1997, another short neurotoxin called cobrotoxin b was purified from the same source[6]. However, the major α-neurotoxin in Naja kaouthia (Thailand) venom is a long neurotoxin, α-cobratoxin, the minor α-neurotoxin is different from cobrotoxin in one residue[7]. Recently, we have purified and sequenced three short neurotoxins from the venom of Yunnan Naja kaouthia, one being a novel short neurotoxin, and the other two the same as cobrotoxin and cobrotoxin-b[8,9]. So, it seemed that there existed geographic variation of α-neurotoxins in Naja kaouthia. However, the geographic variation of α-neurotoxins in Naja atra was not reported till now.

The systematics of Asian Naja is in a confusion. Many problems are due to the fact that these snakes are often extremely variable even within populations, especially in their coloration and pattern. This variation has often made the identification extremely difficult, as snakes in one population may look more different from one another than from those of another population thousands of miles away[1,10].

To avoid species misidentification, we collected Zhejiang Naja atra and Yunnan Naja kaouthia, well identified by morphological characters and partial cytochrome b gene analysis. Venoms were collected from adult snakes of these two species of cobra in summer season to avoid age and seasonal variation in the venom composition. Then α-neurotoxins were purified from these two venoms. On the other hand, α-neurotoxins were also cloned from the same snake. The similarity and variation of α-neurotoxins between these venoms were discussed.

1 Materials and Methods

1.1 Materials

Yunnan Naja kaouthia venom was collected from Wenshan County, South Yunnan Province, China. Zhejiang Naja atra venom was collected from Jinghua City, Zhejiang Province, China. SP-Sephadex C-25, Superdex-75, endoproteinase Glu-C and low-molecular-weight markers were from Pharmacia Fine Chemical, Uppsala, Sweden. Rats (180－200 g) were from Animal Center, Kunming Institute of Zoology, CAS, China. Other chemicals and reagents were of analytical grade.

1.2 Isolation of α-neurotoxins

α-neurotoxins from Zhejiang Naja atra and Yunnan Naja kaouthia venoms were isolated according to the procedure of Lu et al.[8]. The lyophilized venom (5 g) was dissolved in 20 mL 0.05 mol/L ammonium acetate buffer (pH 5.8) and applied to a SP-Sephadex C-25 column (5 cm×60 cm) pre-equilibrated with the same buffer. The adsorbed proteins were eluted with a linear gradient of 0－1 mol/L NaCl. Separation on Superdex-75 column (1.6 cm×40 cm) was performed with the same buffer containing 0.15 mol/L NaCl. The sample was then loaded on a Resource S ion-exchange column (Pharmacia Fine Chemical, Uppsala, Sweden). Finally, the toxins were purified on HPLC Nava-Pak C18 column (Dalian Elite Scientific Instruments Co., Ltd., Dalian, China)

1.3 Mass spectrometry

The mass spectra of the derivatives of isolated components above, their derivatives were recorded on a Bruker reflex III (Bruker) spectrometer, using α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxy-benzoic acid as matrices.

1.4 Protein sequence analysis

Amino acid sequencing was carried out with an Applied Biosystem 476A protein sequencer. The reduced and S-carboxymethylated (RCM-) protein was subjected to automated Edman degradation to determine the N-terminal sequence. The RCM-protein was hydrolyzed with Glu-C protease. The hydrolysates were separated by HPLC on a Nava-Pak C18 column (3.9 mm×30 mm). The amino acid sequence of each peptide fraction was determined.

1.5 Assay of neurotoxicity

Neurotoxicity was assayed according to the method of Cai et al.[11] Briefly, rats of either sex, weighing between 180－200 g were used. The diaphragm and nervus phrenicus preparation was quickly excised. The preparation was mounted in a 50 mL organ bath with the diaphragm and nervus phrenicus connected to two separate electrodes. After the preparation was equilibrated for 60 min in aerated (95% O2 and 5% CO₂) Krebs’ solution, components of different concentrations were added to the bath to inhibit contractions induced by electric stimulation.

1.6 Molecular cloning of short neurotoxins

Isolation of mRNA and reverse transcription were conducted using PolyATract system 1000-kit and Reverse transcription system kit (Promega Biotech) respectively, according to the manufacturer’s protocols. Two oligonucleotide primers, designed according to the signal peptide and 3'-noncoding regions of cobrotoxin gene with the forward sequence, 5'-ATGAAAACTCTGCTGACCTTGGTG-3' and the reverse one, 5'-GGATGGTCCTTGATGGATGAGAG-C-3', were synthesized[12].

PCR was carried out in 100 μL reaction buffer using total RT-PCR products as templates. The amplification was processed on a thermocycler 94 ℃/55 ℃/72 ℃ 1 min each. The recovered PCR products were cloned into pMD18-T vector (TaKaRa, Dalian, China) according to the TA-cloning procedures, and then transformed into E. coli strain JM 109.

The white transformants were screened by PCR using the above primers, and the positive clones were sequenced in TaKaRa Biotechnology Co. Ltd. (Dalian, China).

1.7 Amplification and sequencing of partial sequence of cytochrome b

DNA extractions were performed by first digesting snake liver tissues for overnight at 55 ℃ in 2 mL lyses buffer (100 mmol/L Tris-HCl, pH 8.0; 50 mmol/L EDTA; 10 mmol/L NaCl; 0.5% SDS) containing proteinase K at a concentration of 0.06 g/L. Further digesting was carried out in the same lyses buffer for approximately 3 h at 65 ℃ with constant motion, followed by two times of extraction in chloroform. Then DNA was precipitated with ethanol and washed with 80% ethanol. The precipitated DNA was dissolved with TE buffer and diluted to an appropriate concentration (100－400 mg/ L) prior to PCR. A 767 bp fragment of the cytochrome b gene was amplified by PCR reaction according to Slowinski et al.[13].

2 Results

2.1 Purification of α-neurotoxins

As shown in Fig.1(A), the Yunnan Naja kaouthia venom was separated into several protein peaks in SP-Sephadex C-25 chromatography. The peak VI, peak VII and peak VIII were with neurotoxin activity. Three neurotoxins named as NT-I, NT-II, NT-III were purified from these peaks according to Lu et al.[8]. The Zhejiang Naja atra venom was separated with the same procedure, and the peak 6 and 8 were with neurotoxin activity as shown in Fig.1(B).

Fig.1 Separation of Naja kaouthia and Naja atra venom on SP-Sephadex C-25 column

(A) Separation of Yunnan Naja kaouthia venom. Peaks VI, VII and VIII contained neurotoxin activity. (B) Separation of Zhejiang Naja atra venom. Peaks 6 and 8 contained neurotoxin activity.

The peak 6 of Zhejiang Naja atra was further loaded on a Superdex-75 column (1.6 cm×40 cm), and peak 6-3 with neurotoxicity was pooled (Fig.2).

Fig.2 Further separation of the peak 6 in Fig.1(B) on a Superdex-75 column (1.6 cm×40 cm)

The peak containing neurotoxin activity was indicated by arrow.

The peak 8 and the peak 6-3 were loaded on FPLC Resource S column respectively (Fig.3). The two neurotoxins were further purified by HPLC Nava-Pak C18 column (Fig.4) and named as NT-I' and NT-III'. Their molecular weight were 6952.84 and 6829.07 respectively by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS.

Fig.3Purification of the neurotoxins of Zhejiang Naja atra on FPLC Resource S column

(A) Separation of NT-I'. (B) Separation of NT-III′. The peaks containing neurotoxin activity were indicated by arrows.

Fig.4 Purification of the NT-I′ and NT-III′ of Zhejiang Naja atra on HPLC Nava-Pak C18 column

(A) Separation of NT-I′. (B) Separation of NT-III′. Retention times of the neurotoxins were marked on the top of the peaks.

2.2 Protein sequence analysis

The two neurotoxins NT-I' and NT-III' from Zhejiang Naja atra were subjected to amino acid sequencer to determine the N-terminal sequence. The N-terminal sequences up to 40 amino acids were determined. Furthermore, the two neurotoxins were subjected to reduction and S-carboxymethylation. The RCM-neurotoxins were then digested with Glu-C protease. The hydrolysates of NT-I' and NT-III' were separated into four and three peptide fractions on Nava-Pak C18 column, respectively. The sequences of hydrolysates of NT-I' were: INCCTTDRCNN, RGCGCPSVKNGIE, TNCYKKRWRDHRGYRTE and LECHNQQSSQTPTTTGCSGGE. The sequences of hydrolysates of NT-III' were: TNCYKKWWSDHRG-TIIE, LECHNQQSSQTPTTKTCSGE and RGCGCPK-VKPGVNLNCCTTDRCNN.

2.3 cDNA cloning of short neurotoxins of Yunnan Naja kaouthia and Zhejiang Naja atra

PCR amplification using venom cDNA mixtures as templates with the designed primers resulted in a PCR fragment estimated to be about 300 bp (data not shown). The PCR fragments were then subcloned by TA-cloning kit. More than 30 clones were selected for sequencing. The cDNAs of NT-I, NT-II and NT-III from Naja kaouthia and NT-I', NT-III' from Naja atra were identified, and it was found that the cDNA sequences of NT-I and NT-III were the same as NT-I' and NT-III' respectively. Combined with the sequences of hydrolysates of NT-I' and NT-III', the whole amino acid sequences of NT-I' and NT-III' were determined (Fig.5). Sequence analysis also showed that the nucleosides and amino acid sequences of NT-I, NT-I' and NT-III, NT-III' were the same as those of cobrotoxin and cobrotoxin-b, respectively.

Fig.5 Complete cDNA sequences and deduced amino acid sequences of NT-I(NT-I′), NT-II, NT-III(NT-III′)

The nucleotide residues of coding region were numbered in the 5′ to 3′ direction. Beneath the nucleotide sequence is the deduced amino acid sequence (in bold).

2.4 Analysis of partial sequence of cytochrome b

A 767 bp fragment of the cytochrome b gene was amplified by PCR reaction and sequenced (data not shown). According to Slowinski et al.[13], Naja atra and Naja kaouthia had the closest evolutional relationship compared with other cobras. The partial sequence of cytochrome b of Thailand Naja kaouthia had 93% similarity with that of Taiwan Naja atra. However, the similarity of partial sequence of cytochrome b between Thailand and Yunnan Naja kaouthia, Zhejiang and Taiwan Naja atra, were 97% and 99%, respectively. It indicated there was no possibility of species misidentify.

3 Discussion

Variation in snake venom composition is a ubiquitous phenomenon at all taxonomic levels. Many factors including phylogeny, geographic origin, season, age and prey preference may influence venom composition. Recently, Mukherjee et al.[14] found that there existed high variations in two closely related snakes, Naja kaouthia and Naja naja collected in the same place and in the venom composition of Naja naja from three neighboring districts of India excluding age, sex and seasonal variation[14,15]. In this work, we first collected the male snakes with almost the same size. Morphological characters and cytochrome b gene analysis of Zhejiang Naja atra and Yunnan Naja kaouthia were undertaken to avoid species misidentification. Snake venoms were collected in summer to eliminate the influence of season. Results showed that the two venoms from Yunnan Naja kaouthia and Zhejiang Naja atra differed in chromatographic elution profile through the identical SP-Sephadex C-25 column. Moreover, NT-II in the former venom was not found in the latter. Both venoms contained two short neurotoxins which are identical to cobrotoxin and cobrotoxin-b respectively. In both venoms, no components with molecular weight similar to that of α-cobratoxin were detected in the peaks with neurotoxicity by mass spectrometry (data not shown). However, Yunnan Naja kaouthia venom was distinct from that of Thailand. α-cobratoxin, the main α-neurotoxin of Thailand Naja kaouthia, was not found in Yunnan Naja kaouthia. It seemed that intraspecies variation of α-neurotoxins in Naja kaouthia venom was larger than interspecies variation between Naja atra and Naja kaouthia. It can hardly be explained by population phylogeny or geographic distance (Yunnan is nearer to Thailand than to Zhejing or Taiwan).

In recent years, intraspecies variation of certain components in snake venoms has received considerable attention[16－18]. On the other hand, there are also reports that different snake species venoms contained same or similar components. Tsai et al.[17] found two PLA2s designated as CRV-R6a and CRV-R6b, cloned from the venom of Calloselasma rhodostoma, had a structure identical to that of TmPL-III, a PLA2 from Trimeresurus mucro-squamatus, although Trimeresurus mucrosquamatus and Calloselasma rhodostoma were only loosely related in evolution. The Lys-49 PLA2 from Trimeresurus mucrosquamatus venoms was also identical to that from Deinagkistrodon acutus[19]. Cytotoxin 3, cytotoxin 10 and CX1-NAJSP of Naja atra[20], Thailand Naja kaouthia[21] and Naja sputatrix[22] had the same sequence. So the variation of purified components in the same snake venom or similarity of purified components in different snake venoms is not an unusual phenomenon.

Daltry et al.[23] demonstrated a significant relationship between geographical variation and venom composition, and hypothesized that geographical variation in venom composition may reflect natural selection for higher efficiency in killing and /or digesting different preys in different regions. The following evidences implied that the composition of snake venom was strongly influenced by environmental factors including habitat, climate and preys[16,24]. The habitat, climate of Wenshan (South Yunnan), Jinhua (South Zhejiang) and Taiwan are similar in summer, but different from those in Thailand.

Cobras were reported to have a broad diet spectrum including small mammals, frogs, birds and lizards. So, its diet analysis in different regions is laborious, expensive and hazardous, and it’s very hard to get the true all-round diet. Small mammals especially Mus and Rattus genus are the main prey of Naja atra and Naja kaouthia[25]. So statistic analysis of Mus and Rattus genus suitable for cobras’ feeding in different regions could reflect their rough diets composing. We found that four Mus and Rattus genus in Zhejiang and Yunnan, five in Taiwan, and eleven in Thailand were the possible prey of cobras[26] , which showed that Thailand was greatly different from Zhejiang, Yunnan and Taiwan Provinces in diet of cobras.

Long neurotoxins and short neurotoxins might coexist in the common ancestor of Naja atra and Naja kaouthia since there existed both long neurotoxins and short neurotoxins in modern Naja kaouthia and other cobra venoms[4,7], but then evolved independently since there was no intercourse between their genes. Even in the same species, if two populations in the different area with different habitats have been separated from each other for long time in the geological history, namely there was a vicariance between them, it was certain that the two different populations would vary from each other. Due to the isolation of Trans-Himalayas Mountains and four big rivers (Irrowardi River, Saween River, Meigong River and Jinsha River), Naja kaouthia snakes can be divided to two populations. One is distributed in Bengal and Northern Berma, and the other in Southern Yunnan, Southwestern Sichuan and Southeastern Guangxi in China. These two populations had different habitat, climate and prey, so that it was very possible that they varied from each other, which could explain the variation of α-neurotoxins in Thailand and Yunnan Naja kaouthia venom. Though there was also a geographical obstacle (Taiwan Strait) between Zhejiang and Taiwan Naja atra snakes, they shared similar habitat, climate and prey, so they contained same α-neurotoxins. This hypothesis could further explain the similarity of α-neurotoxins between Yunnan Naja kaouthia and Zhejiang Naja atra. Such cobras populations as those of South Yunnan and Zhejiang that shared similar habitat, climate and prey might contain similar α-neurotoxins after the divergence of Naja kaouthia and Naja atra from the common ancestor.

References

1 Wüster W. Taxonomic changes and toxicology: Systematic revisions of the Asiatic cobras (Naja naja species complex). Toxicon, 1996, 34: 399－406

2 Yang CC. Structure and function of cobra neurotoxin. Adv Exp Med Biol, 1996, 391: 85－96

3 Tsetlin V. Snake venom alpha-neurotoxins and other ‘three-finger’ proteins. Eur J Biochem, 1999, 264: 281－286

4 Chang L, Lin S, Wang J, Hu WP, Wu B, Huang H. Structure-function studies on Taiwan cobra long neurotoxin homolog. Biochim Biophys Acta, 2000, 1480: 293－301

5 Yang CC. Cobrotoxin: Structure and function. J Nat Toxins, 1999, 8: 221－332

6 Chang LS, Chou YC, Lin SR, Wu BN, Lin J, Hong E, Sun YJ et al. A novel neurotoxin, cobrotoxin b, from Naja naja atra (Taiwan cobra) venom: Purification, characterization, and gene organization. J Biochem (Tokyo), 1997, 122: 1252－1259

7 Chiou SH, Lin WW, Chang WP. Sequence characterization of venom toxins from Thailand cobra. Int J Pept Protein Res, 1989, 34: 148－152

8 Lu QM, Meng QX, Li DS, Zhu SW, Jia YH, Xiong YL, Wang WY. Comparative study of three short-chain neurotoxins from the venom of Naja kaouthia (Yunnan, China). J Nat Toxins, 2002, 11: 221－229

9 Meng QX, Wang WY, Lu QM, Jin Y, Wei JF, Zhu SW, Xiong YL. A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: Isolation and purification, primary and secondary structure determination, and tertiary structure modeling. Comp Biochem Physiol C Toxicol Pharmacol, 2002, 132: 113－121

10 Wüster W, Golay P, Warrell DA. Synopsis of recent developments in venomous snake systematics. Toxicon, 1997, 35: 319－340

11Cai JX, Sun X, Yang CJ, Chen XL, Li CD, Zhao QK, Tian YF et al. Column chromatography fractionation of ophiophagus hannah venom and study of neurotoxic peaks. Zoological Reasearch, 1980, 1: 319－325

12 Chang LS, Lin J, Chou YC, Hong E. Genomic structures of cardiotoxin 4 and cobrotoxin from Naja naja atra (Taiwan cobra). Biochem Biophys Res Commun, 1997, 239: 756－762

13 Slowinski JB, Keogh JS. Phylogenetic relationships of elapid snakes based on cytochrome b mtDNA sequences. Mol Phylogenet Evol, 2000, 15: 157－164

14 Mukherjee AK, Maity CR. Biochemical composition, lethality and pathophysiology of venom from two cobras——Naja naja and N. kaouthia. Comp Biochem Physiol B Biochem Mol Biol, 2002, 131: 125－132

15 Mukherjee AK, Maity CR. The composition of Naja naja venom samples from three districts of West Bengal, India. Comp Biochem Physiol A Mol Integr Physiol, 1998, 119: 621－627

16 Chijiwa T, Deshimaru M, Nobuhisa I, Nakai M, Ogawa T, Oda N, Nakashima K et al. Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan. Biochem J, 2000, 347: 491－499

17 Tsai IH, Chen YH, Wang YM, Liau MY, Lu PJ. Differential expression and geographic variation of the venom phospholipases A2 of Calloselasma rhodostoma and Trimeresurus mucrosquamatus. Arch Biochem Biophys, 2001, 387: 257－264

18 Wei JF, Wei Q, Lu QM, Tai H, Jin Y, Wang WY, Xiong YL. Purification, characterization and biological activity of an L-amino acid oxidase from Trimeresurus mucrosquamatus venom. Acta Biochim Biophys Sin, 2003, 35: 219－224

19 Wang YM, Wang JH, Pan FM, Tsai IH. LYS-49 phospholipase A2 homologs from venoms of Deinagkistrodon acutus and Trimeresurus mucrosquamatus have identical protein sequence. Toxicon, 1996, 34: 485－489

20 Chang LS, Huang HB, Lin SR. The multiplicity of cardiotoxins from Naja naja atra (Taiwan cobra) venom. Toxicon, 2000, 38: 1065－1076

21 Joubert FJ, Taljaard N. The complete primary structures of three cytotoxins (CM-6, CM-7 and CM-7A) from Naja naja kaouthia (Siamese cobra) snake venom. Toxicon, 1980, 18: 455－467

22 Jeyaseelan K, Armugam A, Lachumanan R, Tan CH, Tan NH. Six isoforms of cardiotoxin in Malayan spitting cobra (Naja naja sputatrix) venom: Cloning and characterization of cDNAs. Biochim Biophys Acta, 1998, 1380: 209－222

23 Daltry JC, Wuster W, Thorpe RS. Diet and snake venom evolution. Nature, 1996, 379: 537－540

24 Jorge da Silva N Jr, Aird SD. Prey specificity, comparative lethality and compositional differences of coral snake venoms. Comp Biochem Physiol C Toxicol Pharmacol, 2001, 128: 425－456

25 Zhao EM. Amphibia and Reptilia. In: Wang S ed. China Red Data Book of Endangered Animals, Beijing: Science Press, 1998, 274－279

26 Corbet GB, Hill JE ed. The Mammals of the Indomalayan Region: A Systematic Review, New York: Oxford University Press, 1992, 320－413

_______________________________________

Received: March 31, 2003 Accepted: May 18, 2003

This work was supported by a grant from the National Natural Science Foundation of China (No. 39670165)

*Corresponding author: Tel, 86-871-5192476; Fax, 86-871-5191823; e-mail, [email protected]