The omega-loop of cobra cytotoxins tolerates multiple amino acid substitutions.

Cobra cytotoxins (CTs), the three-fingered proteins, feature high amino acid sequence homology in the beta-strands and variations in the loop regions. We selected a pair of cytotoxins from Naja kaouthia crude venom to clarify the sequence-structure relationships. Using chromatography and mass spectroscopy, we separated and identified the mixture of cytotoxins 2 and 3, differentiated by the only Val 41/Ala 41 substitution. Here, using natural abundance 13C, 15N NMR-spectroscopy we performed chemical shift assignments of the signals of the both toxins in aqueous solution in the major and minor forms. Combining NOE and chemical shift data, the toxins' spatial structure was determined. Finally, we proved that the tip of the "finger"-2, or the loop-2 of cytotoxins adopts the shape of an omega-loop with a tightly-bound water molecule in its cavity. Comparison with other NMR and X-ray structures of cytotoxins possessing different amino acid sequences reveals spatial similarity in this family of proteins, including the loop-2 region, previously considered to be flexible.

Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana.

Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small ß-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of 13C,15N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.

Consensus sequence L/PKSSLL mimics crucial epitope on Loop III of Taiwan cobra cardiotoxin.

Phage display is effective in screening peptides that mimic venom's neutralizing epitopes. A phage display cyclized heptapeptide library (C7C library) was panned with purified divalent antivenin IgG, which neutralizes Naja naja atra venom (NAV) and Bungarus multicinctus venom (BMV). The selected heptapeptide sequences were aligned with known protein sequences of NAV and BMV in GenBank. One of the four consensus sequences, L/PKSSLL, mimicked the crucial epitope on Loop III of Taiwan cobra cardiotoxin that is associated with the venom's lethal potency. In dot blot analysis, several clones showed varying reactivities for NAV monovalent antivenin and lesser cross-reactions with BMV monovalent antivenin. The KSSLLRN-carrying phage occurred four times in selected clones and showed the strongest reactivity to NAV monovalent antivenin. Furthermore, the QDSLLPS-carrying phage also presented significant dot blot signal, indicating that the SLL sequence shared by these two clones may be a crucial antibody-binding site.

Molecular cloning and evolution of the genes encoding the precursors of taiwan cobra cardiotoxin and cardiotoxin-like basic protein.

Genomic DNAs encoding the precursors of eight cardiotoxins and two cardiotoxin-like basic proteins (CLBP) were isolated from the liver of Naja naja atra (Taiwan cobra). The cardiotoxin and CLBP genes have three exons like alpha-neurotoxin precursors. The promoter regions of these genes are highly conserved and contain the consensus transcriptional factor-binding sites for TBP, NF-1, CACCC-binding site, Spl and EFII, suggesting that these genes are regulated using similar transcriptional mechanisms. The introns and flanking regions of these genes share a high degree of nucleotide sequence identity, but except for the signal peptide domain the protein-coding regions are much more diversified than introns. The ratio of nonsynonymous to synonymous substitution is higher than one, reflecting that adaptive selection occurred during the evolution of cardiotoxin and CLBP proteins. Phylogenetic trees separate CLBPs and cardiotoxins into two clusters, suggesting that the CLBP gene and the cardiotoxin gene diverged earlier before the appearance of numerous cardiotoxins and CLBP.

Modification of substrate inhibition of synaptosomal acetylcholinesterase by cardiotoxins.

Different types of cardiotoxin (I-V and n) were isolated and purified from the venom of the Taiwan cobra (Naja naja atra). The effects of these cardiotoxins were studied on membrane-bound acetylcholinesterase, which was isolated from a sheep's brain cortex. The results showed that cardiotoxins I-III, V, and n activated the enzyme by modification of substrate inhibition, but cardiotoxin IV's reaction was different. The inhibition and activation of acetylcholinesterase were linked to the functions of the hydrophobicity index, presence of a cationic cluster, and the accessible arginine residue. Our results indicate that Cardiotoxins have neither a cationic cluster nor an arginine residue in their surface area of loop I; therefore, in contrast to fasciculin, cardiotoxins are attached by loop II to the peripheral site of the enzyme. As a result, fasciculin seems to stabilize nonfunctional conformation, but cardiotoxins seem to stabilize the functional conformation of the enzyme. Based on our experimental and theoretical findings, similar secondary and tertiary structures of cardiotoxins and fasciculin seem to have an opposite function once they interact with acetylcholinesterase.

Alpha-neurotoxin gene expression in Naja sputatrix: identification of a silencer element in the promoter region.

Alpha-neurotoxin (alpha-NTX) from the venom of cobra, Naja sputatrix, is a highly lethal post-synaptic toxin that is responsible for the lethality caused by the venom. However, this toxin is found at low levels (3%) in the crude venom. The expression of its gene is determined by a promoter which is 90% similar to the promoter of another three-fingered toxin, cardiotoxin (CTX), which is produced in large amounts (60%) in the same venom. Functional analysis of the NTX-2 gene promoter demonstrated the presence of a silencer element of 24 nucleotides (nt -678 to -655) at its 5(') flanking region. This element has been found to play a major role in the down-regulation of NTX-2 gene expression. A point mutation on this silencer appears to attenuate its repressive property in CTX-2 gene.

Real-time detection of gene promoter activity: quantitation of toxin gene transcription.

We have developed a new method for quantification of promoter activity in cell lines transfected with recombinant plasmids containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) by real-time PCR. As the efficiency of transfection has a direct influence on the total mRNA produced, we have used the neomycin-resistance gene present within the same vector DNA to normalize the measurement of mRNA levels. Three promoters from genes encoding toxins (pre-synaptic neurotoxin phospholipase A(2), post-synaptic alpha neurotoxin and cardiotoxin), believed to have evolved from the same ancestor but exhibiting different promoter activities, have been employed in this study to demonstrate the feasibility and accuracy of the method in CAT gene reporter analysis.

Expression of cardiotoxin-2 gene. Cloning, characterization and deletion analysis of the promoter.

This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.

Evidence for factors regulating transfer cell-specific expression in maize endosperm.

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (< 100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.

Postsynaptic alpha-neurotoxin gene of the spitting cobra, Naja naja sputatrix: structure, organization, and phylogenetic analysis.

The venom of the spitting cobra, Naja naja sputatrix contains highly potent alpha-neurotoxins (NTXs) in addition to phospholipase A2 (PLA2) and cardiotoxin (CTX). In this study, we report the complete characterization of three genes that are responsible for the synthesis of three isoforms of alpha-NTX in the venom of a single spitting cobra. DNA amplification by long-distance polymerase chain reaction (LD-PCR) and genome walking have provided information on the gene structure including their promoter and 5' and 3' UTRs. Each NTX isoform is approximately 4 kb in size and contains three exons and two introns. The sequence homology among these isoforms was found to be 99%. Two possible transcription sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and -45. The promoter also contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also present. The high percentage of similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with the alpha-NTX and kappa-NTX genes from other land snakes suggests that the NTX gene has probably evolved from a common ancestral gene.

In situ hybridization and immunohistochemical analysis of the expression of cardiotoxin and neurotoxin genes in Naja naja sputatrix.

Secretory processes and their regulation have been extensively studied in mammalian salivary parotid glands. However, little is known regarding the secretory mechanism in the venom glands of snakes, which invariably produce one of the most complex of all animal secretions. The pharmacologically important and toxic components of the Malayan spitting cobra (Naja naja sputatrix) venom include postsynaptic neurotoxins (NTX), presynaptic neurotoxins (phospholipase A2, PLA2), and cardiotoxins (CTX) which, for convenience, have been collectively referred to as "toxins." We report here for the first time the mechanism of toxin gene expression by studying the accumulated mRNA level and protein synthesis rates for the three toxins over a period of 8 days after stimulation of venom synthesis by manual "milking" of the venom gland. Immunofluorescence and in situ hybridization were used to localize the toxins and their mRNAs in venom gland sections. The rate of protein synthesis, as determined by immunofluorescence and liquid chromatography-mass spectrometry (LC-MS) techniques, increased in parallel with the increase in the toxin mRNA content in the secretory epithelial cells, suggesting that transcriptional regulation of the toxin genes is involved. (J Histochem Cytochem 47:551-560, 1999)

Effect of D57N mutation on membrane activity and molecular unfolding of cobra cardiotoxin.

Cobra cardiotoxins (CTXs) are able to adopt a three-fingered beta-strand structure with continuous hydrophobic patch that is capable of interacting with zwitterionic phospholipid bilayer. In addition to the four disulfide bonds that form the rigid core of CTXs, Asp57 near the C-terminus interacts electrostatically with Lys2 near the N-terminus (Chiang et al. 1996. Biochemistry. 35:9177-9186). We indicate herein, using circular dichroism and the time-resolved polarized tryptophan fluorescence measurement, that Asp57 to Asn57 (D57N) mutation perturbs the structure of CTX molecules at neutral pH. The structural stability of the D57N mutant was found to be lower, as evidenced by the reduced effective concentration of the 2,2,2-trifluoethanol (TFE)-induced beta-sheet to alpha-helix transition. Interestingly, the single mutation also allows a greater degree of molecular unfolding, because the rotational correlation time of the TFE-induced unfolding intermediate is larger for the D57N mutant. It is suggested that the electrostatic interaction between N- and C-termini also contributes to the formation of the functionally important continuous hydrophobic stretch on the distant end of CTX molecules, because both the binding to anilinonaphthalene fluorescent probe and the interaction with phospholipid bilayer were also reduced for D57N mutant. The result emphasizes the importance of the hydrophobic amino acid residues near the tip of loop 3 as a continuous part of the three-fingered beta-strand CTX molecule and indicates how a distant electrostatic interaction might be involved. It is also implicated that electrostatic interaction plays a role in expanding the radius of gyration of the folding/unfolding intermediate of proteins.

Structure and organization of the cardiotoxin genes in Naja naja sputatrix.

We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix. The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-kappaB and PuF have been identified in the 5'-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.

Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs.

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Differentially expressed snoRNAs in Bungarus multicinctus (Taiwan banded krait).

Twenty novel snoRNAs forming extensive sequence complementarities to mature 5S rRNA were identified from Bungarus multicinctus by reverse transcription-polymerase chain reaction. It was found that the snoRNA species were differentially transcribed in different tissues as evidenced by single stranded conformational polymorphism analysis and direct nucleotide sequence analysis. Although the diversity in the sequences of snoRNAs is observed, comparison of these snoRNA genes reveals that the regions involved in binding to 5S rRNA are highly conserved and form two 12-nt-15-nt tracts of complementarity to phylogenetically invariant sequences in eukaryotic 5S rRNAs. Nevertheless, the lower conservation of box C/D or box H/ACA in these snoRNAs was observed. Likewise, the sequences in several fish and human genes forming perfect duplexes with 5S rRNA also did not highly retain these box elements. These results may infer that the box elements are dispensable for the function of snoRNA species identified in the present study. Moreover, the novel finding of the differentially expressed snoRNA variants in B. multicinctus suggests that the snoRNA genes are selectively processed in different tissues and are likely associated with tissue-specific regulation of their host gene transcripts.

Genomic structures of cardiotoxin 4 and cobrotoxin from Naja naja atra (Taiwan cobra).

Two genomic DNAs with the size of 2.3 kb and 2.4 kb, which were isolated from the liver of Naja naja atra (Taiwan cobra), encoded the precursors of cardiotoxin 4 and cobrotoxin, respectively. Both genes shared virtually identical overall organization with three exons separated by two introns, which were inserted in the similar positions of the gene's coding regions. Moreover, their nucleotide sequences shared approximately 84.2% identity. This result reveals the evolutionary relationship between cardiotoxin and cobrotoxin. The exon/intron structures of cardiotoxin 4 and cobrotoxin genes were similar to that reported for erabutoxin c gene, a neurotoxin genomic DNA from a sea snake (Laticauda semifasciata). However, in contrast to the finding that the intron 2 of these genes had a similar size, a notable variation with the size of intron 1 was observed (1233 bp, 1269 bp and 197 bp for cardiotoxin 4, cobrotoxin and erabutoxin c genes, respectively). The different size with intron 1 is due to the middle region at the first intron of cardiotoxin 4 and cobrotoxin genes, which encoded small nucleolar RNA (snoRNA), being absent in that of erabutoxin c gene. These results, together with the finding of the potential mobility of snoRNA genes during evolution, suggest that intron insertions or deletions of snoRNA genes occur with the evolutionary divergence of snake neurotoxins and cardiotoxins.

Novel SnoRNAs from Naja naja atra (Taiwan cobra) and Bungarus multicinctus (Taiwan banded krait), form extended sequence complementarity to 5S rRNA.

During the mapping and sequencing of Naja naja atra cobrotoxin and cardiotoxin 4 genes, we have found that novel small nucleolar RNAs (snoRNAs) are encoded in the first intron of the two genes. The snoRNAs in Naja naja atra were amplified from the venom glands cDNA mixtures of Naja naja atra by reverse transcription-polymerase chain reaction using the primers designed from the first intronic sequences of cobrotoxin and cardiotoxin 4 genes. Likewise, the snoRNAs in Bungarus multicinctus were also amplified by the same primers. Comparison of these snoRNA genes reveals that the regions involved in binding to 5S rRNA are highly conserved among these genes, and form 12-nt and 15-nt tracts of complementarity to phylogenetically invariant sequences in eukaryotic 5S rRNAs. The box C sequence in these snoRNAs is consensus, however, variations with the sequence of box D motif are observed. The present study is the first case of intron-encoded snoRNAs contain extended regions of perfect complementarity to mature 5S rRNA.

cDNA sequence analysis of cardiotoxin variants from Taiwan cobra.

Five cDNAs encoding cardiotoxin variants were constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription-polymerase chain reaction. A high degree of nucleotide sequence homology was observed between these variants and other determined ones. Among them, a novel cardiotoxin 6 had 61 amino acid residues rather than 60 ones that usually observed with Naja naja atra cardiotoxins. The other cardiotoxin variants were the homologues of cardiotoxins 1, V or N with one or two amino acid substitutions, respectively. These results probably reflect the involvement of RNA editing in the production of cardiotoxin variants in the venom of Taiwan cobra.

cDNA sequence analysis of a novel cardiotoxin-like protein from Taiwan banded krait.

The cDNAs encoding a novel protein was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus by reverse transcription-polymerase chain reaction. The deduced amino acid sequence of this novel protein contained 65 amino acid residues with 8 cysteine residues. Comparative sequence analysis showed that it was structurally related to cardiotoxin rather than neurotoxins. These results suggest that the venom of Bungarus multicinctus may contain cardiotoxin(s) which was not noticed before.