Protein Identification of Venoms of the African Spitting Cobras, Naja mossambica and Naja nigricincta nigricincta.
ABSTRACT: Cobra snakes, including Naja mossambica and Naja nigricincta nigricincta, are one of the major groups of snakes responsible for snakebites in southern Africa, producing significant cytotoxicity and tissue damage. The venom of N. mossambica has been briefly characterised, but that of N. n. nigricincta is not reported. The current study identifies the venom proteins of N. mossambica and N. n. nigricincta. This is achieved using sodium dodecyl sulphate (SDS)-polyacrylamide gel eletrophroresis (PAGE), followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Most of the proteins were less than 17 kDa in both snakes. N. mossambica was found to have 75 proteins in total (from 16 protein families), whereas N.n. nigricincta had 73 (from 16 protein families). Of these identified proteins, 57 were common in both snakes. The proteins identified belonged to various families, including the three-finger toxins (3FTx), Cysteine-rich secretory proteins (CRiSP), Phospholipase A2 (PLA2) and Venom metalloproteinase M12B (SVMP). The current study contributes to the profile knowledge of snake venom compositions, which is of fundamental value in understanding the proteins that play a major role in envenomation.
Project description:The equatorial spitting cobra, Naja sumatrana, is a distinct species of medically important venomous snakes, listed as WHO Category 1 in Southeast Asia. The diversity of its venom genes has not been comprehensively examined, although a few toxin sequences annotated to Naja sputatrix were reported previously through cloning studies. To investigate this species venom genes' diversity, de novo venom-gland transcriptomics of N. sumatrana from West Malaysia was conducted using next-generation sequencing technology. Genes encoding toxins represented only 60 of the 55,396 transcripts, but were highly expressed, contributing to 79.22% of total gene expression (by total FPKM) in the venom-glands. The toxin transcripts belong to 21 families, and 29 transcripts were further identified as full-length. Three-finger toxins (3FTx) composed of long, short, and non-conventional groups, constituted the majority of toxin transcripts (91.11% of total toxin FPKM), followed by phospholipase A? (PLA?, 7.42%)-which are putatively pro-inflammatory and cytotoxic. The remaining transcripts in the 19 families were expressed at extremely low levels. Presumably, these toxins were associated with ancillary functions. Our findings unveil the diverse toxin genes unique to N. sumatrana, and provide insights into the pathophysiology of N. sumatrana envenoming.
Project description:BACKGROUND: Kraits (genus Bungarus) and cobras (genus Naja) are two representative toxic genera of elapids in the old world. Although they are closely related genera and both of their venoms are very toxic, the compositions of their venoms are very different. To unveil their detailed venoms and their evolutionary patterns, we constructed venom gland cDNA libraries and genomic bacterial artificial chromosome (BAC) libraries for Bungarus multicinctus and Naja atra, respectively. We sequenced about 1500 cDNA clones for each of the venom cDNA libraries and screened BAC libraries of the two snakes by blot analysis using four kinds of toxin probes; i.e., three-finger toxin (3FTx), phospholipase A2 (PLA2), kunitz-type protease inhibitor (Kunitz), and natriuretic peptide (NP). RESULTS: In total, 1092 valid expressed sequences tags (ESTs) for B. multicinctus and 1166 ESTs for N. atra were generated. About 70% of these ESTs can be annotated as snake toxin transcripts. 3FTx (64.5%) and ? bungarotoxin (25.1%) comprise the main toxin classes in B. multicinctus, while 3FTx (95.8%) is the dominant toxin in N. atra. We also observed several less abundant venom families in B. multicinctus and N. atra, such as PLA2, C-type lectins, and Kunitz. Peculiarly a cluster of NP precursors with tandem NPs was detected in B. multicinctus. A total of 71 positive toxin BAC clones in B. multicinctus and N. atra were identified using four kinds of toxin probes (3FTx, PLA2, Kunitz, and NP), among which 39 3FTx-positive BACs were sequenced to reveal gene structures of 3FTx toxin genes. CONCLUSIONS: Based on the toxin ESTs and 3FTx gene sequences, the major components of B. multicinctus venom transcriptome are neurotoxins, including long chain alpha neurotoxins (?-ntx) and the recently originated ? bungarotoxin, whereas the N. atra venom transcriptome mainly contains 3FTxs with cytotoxicity and neurotoxicity (short chain ?-ntx). The data also revealed that tandem duplications contributed the most to the expansion of toxin multigene families. Analysis of nonsynonymous to synonymous nucleotide substitution rate ratios (dN/dS) indicates that not only multigene toxin families but also other less abundant toxins might have been under rapid diversifying evolution.
Project description:Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms-that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.
Project description: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.
Project description:In Southeast Asia, envenoming resulting from cobra snakebites is an important public health issue in many regions, and antivenom therapy is the standard treatment for the snakebite. Because these cobras share a close evolutionary history, the amino acid sequences of major venom components in different snakes are very similar. Therefore, either monovalent or polyvalent antivenoms may offer paraspecific protection against envenomation of humans by several different snakes. In Taiwan, a bivalent antivenom-freeze-dried neurotoxic antivenom (FNAV)-against Bungarus multicinctus and Naja atra is available. However, whether this antivenom is also capable of neutralizing the venom of other species of snakes is not known. Here, to expand the clinical application of Taiwanese FNAV, we used an animal model to evaluate the neutralizing ability of FNAV against the venoms of three common snakes in Southeast Asia, including two 'true' cobras Naja kaouthia (Thailand) and Naja siamensis (Thailand), and the king cobra Ophiophagus hannah (Indonesia). We further applied mass spectrometry (MS)-based proteomic techniques to characterize venom proteomes and identify FNAV-recognizable antigens in the venoms of these Asian snakes. Neutralization assays in a mouse model showed that FNAV effectively neutralized the lethality of N. kaouthia and N. siamensis venoms, but not O. hannah venom. MS-based venom protein identification results further revealed that FNAV strongly recognized three-finger toxin and phospholipase A2, the major protein components of N. kaouthia and N. siamensis venoms. The characterization of venom proteomes and identification of FNAV-recognizable venom antigens may help researchers to further develop more effective antivenom designed to block the toxicity of dominant toxic proteins, with the ultimate goal of achieving broadly therapeutic effects against these cobra snakebites.
Project description:Naja ashei is an African spitting cobra species closely related to N. mossambica and N. nigricollis. It is known that the venom of N. ashei, like that of other African spitting cobras, mainly has cytotoxic effects, however data about its specific protein composition are not yet available. Thus, an attempt was made to determine the venom proteome of N. ashei with the use of 2-D electrophoresis and MALDI ToF/ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry techniques. Our investigation revealed that the main components of analysed venom are 3FTxs (Three-Finger Toxins) and PLA?s (Phospholipases A?). Additionally the presence of cysteine-rich venom proteins, 5'-nucleotidase and metalloproteinases has also been confirmed. The most interesting fact derived from this study is that the venom of N. ashei includes proteins not described previously in other African spitting cobras-cobra venom factor and venom nerve growth factor. To our knowledge, there are currently no other reports concerning this venom composition and we believe that our results will significantly increase interest in research of this species.
Project description:Snake venom is a complex mixture of proteins and peptides, and a number of studies have described the biological properties of several venomous proteins. Nevertheless, a complete proteomic profile of venom from any of the many species of snake is not available. Proteomics now makes it possible to globally identify proteins from a complex mixture. To assess the venom proteomic profiles from Naja naja atra and Agkistrodon halys, snakes common to southern China, we used a combination strategy, which included the following four different approaches: (i) shotgun digestion plus HPLC with ion-trap tandem MS, (ii) one-dimensional SDS/PAGE plus HPLC with tandem MS, (iii) gel filtration plus HPLC with tandem MS and (iv) gel filtration and 2DE (two-dimensional gel electrophoresis) plus MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) MS. In the present paper, we report the novel identification of 124 and 74 proteins and peptides in cobra and viper venom respectively. Functional analysis based upon toxin categories reveals that, as expected, cobra venom has a high abundance of cardio- and neurotoxins, whereas viper venom contains a significant amount of haemotoxins and metalloproteinases. Although approx. 80% of gel spots from 2DE displayed high-quality MALDI-TOF-MS spectra, only 50% of these spots were confirmed to be venom proteins, which is more than likely to be a result of incomplete protein databases. Interestingly, these data suggest that post-translational modification may be a significant characteristic of venomous proteins.
Project description:The protein abundances of phospholipases A? in cobra venom proteomes appear to vary among cobra species. To determine the unique distribution of snake venom phospholipases A? (svPLA?) in the cobras, the svPLA? activities for 15 cobra species were examined with an acidimetric and a colorimetric assay, using egg yolk suspension and 4-nitro-3-octanoyloxy benzoic acid (NOBA) as the substrate. The colorimetric assay showed significant correlation between svPLA? enzymatic activities with the svPLA? protein abundances in venoms. High svPLA? activities were observed in the venoms of Asiatic spitting cobras (Naja sputatrix, Naja sumatrana) and moderate activities in Asiatic non-spitters (Naja naja, Naja atra, Naja kaouthia), African spitters (subgenus Afronaja), and forest cobra (subgenus Boulengerina). African non-spitting cobras of subgenus Uraeus (Naja haje, Naja annulifera, Naja nivea, Naja senegalensis) showed exceptionally low svPLA? enzymatic activities. The negligible PLA? activity in Uraeus cobra venoms implies that PLA? may not be ubiquitous in all snake venoms. The svPLA? in cobra envenoming varies depending on the cobra species. This may potentially influence the efficacy of cobra antivenom in specific use for venom neutralization.
Project description:In contrast to comprehensively investigated antibacterial activity of snake venoms, namely crude venoms and their selected components, little is known about antifungal properties of elapid snake venoms. In the present study, the proteome of two venoms of red spitting cobra Naja pallida (NPV) and Mozambique spitting cobra Naja mossambica (NMV) was characterized using LC-MS/MS approach, and the antifungal activity of crude venoms against three Candida species was established. A complex response to venom treatment was revealed. NPV and NMV, when used at relatively high concentrations, decreased cell viability of C. albicans and C. tropicalis, affected cell cycle of C. albicans, inhibited C. tropicalis-based biofilm formation and promoted oxidative stress in C. albicans, C. glabrata and C. tropicalis cells. NPV and NMV also modulated ammonia pulses during colony development and aging in three Candida species. All these observations provide evidence that NPV and NMV may diminish selected pathogenic features of Candida species. However, NPV and NMV also promoted the secretion of extracellular phospholipases that may facilitate Candida pathogenicity and limit their usefulness as anti-candidal agents. In conclusion, antifungal activity of snake venoms should be studied with great caution and a plethora of pathogenic biomarkers should be considered in the future experiments.