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Article

Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles

by
Aisha Manuwar
1,*,
Benjamin Dreyer
2,
Andreas Böhmert
2,
Anwar Ullah
3,
Zia Mughal
4,
Ahmed Akrem
5,
Syed Abid Ali
6,
Hartmut Schlüter
2 and
Christian Betzel
7,*
1
Department of Chemistry, University of Engineering and Technology, Lahore 54890, Pakistan
2
Institute of Clinical Chemistry and Laboratory Medicine, Mass Spectrometric Proteomics, University Medical Centre Hamburg-Eppendorf (UKE), Martinistraße 52, 20246 Hamburg, Germany
3
Department of Biosciences, COMSATS University Islamabad, Park Road, Chack Shahzad, Islamabad 45550, Pakistan
4
Pet Centre, University of Veterinary and Animal Sciences, Lahore 54890, Pakistan
5
Botany Division, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan 60800, Pakistan
6
Husein Ebrahim Jamal Research Institute of Chemistry, (International Center for Chemical and Biological Sciences), University of Karachi, Karachi 75270, Pakistan
7
Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, Deutsches Elektronen-Synchrotron, Build. 22a, Notkestr. 85, University of Hamburg, 22603 Hamburg, Germany
*
Authors to whom correspondence should be addressed.
Toxins 2020, 12(11), 669; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12110669
Submission received: 13 September 2020 / Revised: 6 October 2020 / Accepted: 20 October 2020 / Published: 22 October 2020
(This article belongs to the Section Animal Venoms)
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Abstract

:
Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.
Keywords:
Naja naja; Naja oxiana; venom proteome; Ras-GTPase; ankyrin repeat; N-terminal acetylation; extracellular vesicles
Key Contribution: The present study describes a comprehensive overview of the venom proteome of Naja naja and Naja oxiana. A few protein fragments were found to be N-terminal acetylated. The identification of Ras-like proteins in the venom of Naja naja indicates the presence of extracellular vesicles in the venom.

1. Introduction

Pakistan has a particular geographical location and hosts an array of habitats such a, mountains, glaciers, coastal areas, swamps, plane areas, fresh water, and sandy areas [1]. The country is located between two zoogeographical regions (Palearctic and Oriental) and hosts a diverse venomous fauna. Nine habitat zones are recognized according to the distribution of snakes in Pakistan [2]. Seventy-two snake species are known to Pakistan, among which 14 marine and 12 terrestrial are venomous [1]. According to ITIS (Integrated Taxonomic Information System) database there are 29 snake species belonging to the genus Naja [3]. Among these two are found in Pakistan, i.e., Naja naja and Naja oxiana [2]. Both of these snakes are non-spitting cobras [4]. These snakes are shy of humans. However, upon assessing threat they lift the anterior part of their body, display a hood, and if provoked, hiss loudly and sway their hood to frighten their adversary. These snakes attack very furiously, chewing the bitten part. They usually feed on rodents, birds, frogs, lizards, and snakes. They are found in rocky, stony foothills, forests and around the villages [2]. N. naja (black cobra) is known to have variable color and pattern. However, in Pakistan juveniles and young adults tend to be grey with hood marks, but the adult specimens are usually uniformly black. In addition, the throat pattern is obscured in adult snakes, due to pigmentation [4,5]. N. naja is distributed in North West Pakistan, south and desert areas, except most of Baluchistan. N. oxiana (brown cobra) occurs sympatrically in the Northern half of Pakistan with N. naja. Adult N. oxiana is normally brown in color [5,6]. These snakes and their geographical distribution are shown in Figure 1.
Only a few reliable data exist reporting the frequency of morbidity because of snakebites in developing countries. However, it is predicted that snakebite is responsible for a substantial amount of morbidity and mortality in remote areas [7]. The hidden toll of suffering continues to affect the families of the deceased, and patients who survived with crippling deformity [8]. World Health Organization (WHO), included snakebite in its list of “Neglected Tropical disease” in 2007 [9]. Recently, WHO also added snakebite envenoming at high preference in the list of Neglected Tropical disease, in 2017, upon request of some member states of United Nations. The supply of antivenom and snakebite management was declared as a global public health emergency. WHO has included snake antivenom immunoglobulins in the “WHO Model List for Essential Medicines” WHO has also encouraged countries to ensure their national antivenom stocks [10] Despite these efforts, snakebite has not gained attention on international public health agendas [11]. The snakes commonly responsible for clinically significant bites in Pakistan are Bungarus caeruleus (common krait), N. naja (cobra), Daboia russelii (Russel’s viper), and Echis carinatus (Saw-scaled viper) [12]. National Institute of Health, Pakistan, produces around 30,000 vials of polyvalent anti-venom per year. However, the amount of this antivenom is not sufficient and can only treat a fraction of snakebite cases in the country (https://www.nih.org.pk/1255-2/) [13]. To meet the requirement of antivenoms, snake antivenom sera are presently also imported from India. However, studies have shown that Indian antivenoms provide partial neutralization, particularly for N. naja venom [14,15,16]. Although, N. naja and N. oxiana are also prevalent in India, but the venom composition is known to vary within the same species, due to change in geographical and ecological factors [17,18,19,20]. A study reported that Pakistani N. naja is more neurotoxic with lower LD50, then that prevalent in India [8]. Gender, diet, and age of the snake is also known to influence the composition of venoms [21,22,23,24].
Depending on the amount of venom injected, paralysis following cobra bites can occur within several hours, with death ensuing if breathing is not assisted [8]. On average cobras can inject 60 mg of venom in a bite [25]. Cobra venom is a postsynaptic neurotoxin and presents a variety of symptoms like pain, edema, necrosis, respiratory paralysis, headache, cardiac arrest, hypotension, and bleeding wounds [26]. The use of anticholinesterase, such as neostigmine, has been suggested to compensate a cobra bite, in addition to the administration of antivenom [25,26].
Recent scientific advances have paved the way to explore venomous snake composition in detail and various strategies have been evolved to better understand venom components, their function and immunological properties [27]. Genomic and transcriptomic studies have proved to be an invaluable tool in the discovery of the snake venom evolution and proteoform [28,29,30,31,32,33,34]. Consequently, investigations are directed towards the discovery of pharmacologically active snake venom compounds [35,36,37,38]. For example, a recent study reported Mambaquaretin-1 (peptide from green mamba venom), as a promising candidate for the treatment of polycystic kidney disease [39]. Another study described Nubein6.8, a peptide from the venom of N. nubiae, as a promising template for the treatment of human melanoma and ovarian cancer [40].
In the present study, we describe an in-depth comparative proteomic study of two Pakistani snake species of the family elapid and genus Naja, i.e., N. oxiana (brown cobra/Caspian cobra/Central Asian cobra) and N. naja (black cobra/Indian cobra/Spectacled cobra). In Pakistani region both species of adult cobras are melatonic and N. oxiana is commonly known as brown, while N. naja is known as black cobra. These snakes were previously known as Naja n. oxiana and Naja n. karachienis respectively, but now they are named according to the ITIS database [41]. Till now only a few studies have been reported about the proteomics of Pakistani N. naja [42,43,44]. The N. naja venom samples in these studies were collected from Southern Punjab and Sindh Province of Pakistan. These research groups performed pre fractionation of the venom sample either by reverse phase chromatography, 1-dimensional gel electrophoresis (1D gel) or 2-dimensional gel electrophoresis (2 D gel) or a combination of these methods. Further mass spectrometric analysis of peptide fragments obtained from in gel trypsin digestion, was carried out by MALDI TOF/TOF, ion trap or ESI MS. Chanda et al. also reported the venom proteomics of N. naja, from Western and Eastern parts of India [45,46]. In their study of the venom sample from East India, they pre fractionated the crude venom by 1D gel prior to LTQ orbitrap analysis. However, the proteomic analysis of the venom sample from Western India was performed by a combination of fractionation methods and LC- MS/MS was done by QTOF mass spectrometer. Analysis of the comparative statement of the research group showed that pre fractionation of the crude venom by gel filtration chromatography followed by gel electrophoresis, worked best in their hands. The same group reported the proteomic study of South Indian N. naja venom, recently [47].
In this work, they separated the crude venom components by 1D gel electrophoresis. The mass spectrometric analysis of the tryptic peptide was performed on QTOF. The results of this study derive a comparison of common and unique toxins in N. naja venom obtained from all the three different Indian regions. Our results revealed remarkable differences in the relative abundance of the venom components, as compared to the previous studies. In addition, our investigations unveiled new venom components, not reported before in these venoms. The variation in the results could be different geographical of the snakes from which we collected the venom samples. Further, our workflow did not involve any pre fractionation of the venom. Pre fractionation by gel electrophoresis or liquid chromatography might lead to the loss of some low abundant venom components. Also, we used a modern version of the orbitrap mass spectrometer in this work which is very sensitive equipment.
To the best of our knowledge, this is the first report on the proteomic study of Naja oxiana venom. The abbreviations used for proteins and peptides are given in Table 1.

2. Results

The venom proteome of N. naja (NN) and N. oxiana (NO) snakes was investigated by mass spectrometric analysis, using a shotgun proteomic approach. We were able to provide an extensive overview of various protein families present in both venoms, based on data base searches and BLAST analysis of the de novo sequenced tandem mass spectra. A total of 735 peptides from NN and 254 peptides from NO were sequenced (Supplementary Table S1 and S2). Subsequently 365 proteins in NN venom (Table 2) and 140 proteins were identified in NO venom (Table 3). The sequences of the protein fragments are listed in Supplementary Tables S1 and S2. The results obtained allowed us to cluster the venom protein content into 34 protein families for N. naja and in 24 protein families for the N. oxiana venom. Figure 2A illustrates the preparation for MS acquisition and Figure 2B represents the strategy applied for data base searches. In the present work, we performed data base search against Serpents, King cobra utilizing Uniprot data base. The venom of Ophiophagus hannah has been well studied and genomic and proteomic data are available in the database [28,48,49]. A recent study showed similarity between the genome of Indian cobra and King cobra [50]. This group analyzed 139 N. naja venom gland toxin genes to identify orthologs in the King cobra. It was determined that 96 genes matched while 43 did not. It was suggested that, although some genes are likely to be unique to Indian cobra, the majority were not annotated in King cobra genome. The possible reason could be its highly fragment assembly. Based on this similarity, we searched our data against King cobra database also. Further, in the data base complete proteome of only King cobra is available. The details of our search against Serpent database are presented in Supplementary Table S3 and S5 while that against King cobra are compiled in Supplementary Table S4 and S6. The results presented and discussed are a conclusion of both data base searches.
A comparative summary of the protein families of the two venoms is presented in Table 1. Figure 3, presents a comparison of the relative abundance of different venom protein families as pie charts. From the pie charts, it can be observed that there are significant differences in the proteome of two snake venoms. In the venom of N. naja, three-finger toxins (3FTx) are more abundant, while in N. oxiana venom, both 3FTXs and snake venom metalloproteinase (SVMPs) are almost equally abundant. In NO, snake venom serine proteases (SVSPs) and phospholipase A2 (PLA2s) are much more abundant than in NN. There are other subtle variations in the relative abundance of protein families between the two venoms. For example, Cysteine-rich Secretory Protein (CRISP) family is much more abundant in NN as compared to NO. Further, NN venom contains 11 protein families, which could not be found in NO venom, listed in Table 1 and highlighted in red color. Whereas NO venom contains serpins, which are absent in NN venom. Figure 3 shows that NN venom is much more versatile and contains a number of different proteins (Table 1). Data base searches revealed that our data provide a deeper insight of the NN and NO venom proteomes. There are several protein families, which have not been reported earlier in NN venom, including western and eastern Indian N. naja. In Table 1, the protein families discovered and reported for the first time in terms of our investigations are shown with check mark (✓). Interestingly previous studies reported PLA2 as the second most abundant protein family found in N. naja venom, and that SVMP was present in relatively low abundance [8,42,43,44,45,46]. In contrast, our data showed SVMP as the second most abundant protein family in N. naja. The venom proteome of N. oxiana displays that, both 3FTXs and SVMP are equally abundant like that of king cobra (Ophiophagus hannah) [51], as illustrated in Figure 3A.
In the present work, three types of posttranslational modification were also observed, i.e., N-terminus pyro-glutamate, methionine oxidation and N-terminal acetylation (N-ace). Pyro-glutamate posttranslational modifications of the venom proteins has been described before and are known to confer stability to the proteins and peptides [52,53,54,55]. However, modification of methionine residues and pyro-glutamate cannot be excluded during sample preparation. Therefore, keeping in view this possibility we have not discussed the observed methionine and pyro-glutamate modifications. The current study is the first description of N-terminal acetylation of venom proteins. In N. naja venom we were able to identify three peptide fragments (Muscarinic toxin-like protein 3, Acid phospholipase A2 and weak neurotoxin 7) containing N-ace modification. Whereas in N. oxiana one peptide (Muscarinic toxin-like protein 3) was identified with N-ace. These sequences have been highlighted with green colour in Supplementary Tables S1 and S2.
In the present work, we have identified a number of proteins like cobra serum albumin, leucine repeat, zinc finger containing protein, venom lectin protein, Ras-like protein. The presence of Ras-like protein demonstrates the presence of extracellular vesicles in the venom of Naja naja. The comparison of our proteomic data with that of N. naja snake both from western and eastern India, reveals that such proteins were not identified in Indian N. naja, Further in Pakistani Naja naja snake we not could identify cholinesterase, butyrylcholinesterase, hyalurinidase and snaclec proteins which were previously reported in Indian N. naja venom [45,46].
Below we briefly describe and discuss the different venom protein families identified.

3. Discussion

3.1. Major Protein Components (Relative Abundance >2%)

3.1.1. Three-Finger Toxins

The detailed proteomic investigations of the, NN and NO snake venoms identified two main types of three-finger toxins, i.e., neurotoxins and cytotoxins. The venom of NN consists of an overall higher abundance and a greater diversity of 3FTXs, as compared to NO (Table 1 and Table 2, Figure 3). Neurotoxins are predominant in both venoms, as compared to cytotoxins, Figure 4. Among the neurotoxins, long, muscarinic, weak, 3FTxs precursor and aminergic toxin families are present in both venoms. In case of NO, a rather low amount of long neurotoxin is present, represented by one neurotoxin, cobratoxin. Whereas, long neurotoxins constitute a major proportion of neurotoxins found in the NN venom. Figure 4 indicates that in NO venom, muscarinic toxins are present in relatively higher amounts as compared to NN venom. It is interesting to note that NN venom contains an aminergic neurotoxin with homology to Dendroaspis angusticeps venom toxin AdTx1. This toxin is known to impair G-protein-coupled receptors [56,57].
Previous studies have shown that 3FTXs make up approximately 56–84% of venom proteome in various species of Naja [58]. However, our results of Pakistani Naja venom samples show a much lower percentage of 3FTXS as compared to other investigations, which is 21% in NN and 16% in NO of the total venom proteins. In contrast to Pakistani N. naja venom proteome, the eastern Indian N. naja venom comprises of 61% 3FTXs and western Indian N. naja contains 68% 3FTXs. Interestingly eastern Indian N. naja lacks short neurotoxins, which are present in both western Indian and Pakistani N. naja snake venom [45,46].
Investigating 3FTXs are not only of interest to characterize their toxicity, but also of great significance for structural studies, as well as for biotechnological, biomedical and evolutionary studies [59,60,61,62,63]. Already, 3FTXs have proven to be an efficient tool to analyze various receptor types, and to study diseases like Parkinson’s disease, myasthenia gravis and cancer [64,65,66,67,68,69,70,71]. The aminergic toxins from mamba venom served as good candidates for protein resurrection methodology [72].

3.1.2. Phospholipase A2

Both Naja snake venom contain PLA2. The percentage abundance of PLA2 enzymes (12.6%) is higher in NO as compared to NN. PLA2s make up 6% of the venom of NN (Table 1). A recent study reported the comparative enzymatic activity of PLA2s in ten different Naja species, with highest activity in N. siamensis and lowest in N. nivea [73]. The venom proteome study of Indian N. naja venom carried out by A. K. Mukherjee research group reported that Indian N. naja contains 20–27% PLA2s [45,46]. This is much higher than the amount of PLA2s present in Pakistani N. naja. A proteomic study of N. kaouthia venom reported PLA2s as one of the most abundant venom proteins [74]. While another study on the venom proteome of N. annulifera did not detect PLA2s [75]. In the venom of N. philippinensis PLA2s made up 22.88% of venom proteome [76]. Another study showed distinct distribution of PLA2s in Afro-Asian cobra venom. Asian spitting cobras showed highest PLA2 activity. Asian non-spitting and African spitting cobras showed moderate activity and low activity was shown by African non-spiting cobras [77].
Table 3 shows that both venom comprise of acidic and basic PLA2s. However, acidic PLA2s are more abundant in the two venoms. Two fragments of phospholipases from NO bear homology to neutral PLA2s paradoxin-like beta chain from Oxyuranus microlepidotus. This protein was found to be one of the most potent presynaptic neurotoxins [78]. Eleven peptide fragments bearing homology to acidic phospholipase in the venom of Naja sputatrix were identified (Table 3). In the Naja naja peptide fragments having homology to acidic PLA2s from the venom of other Naja species were determined (Table 2). Six peptide fragments showed homology to acidic PLA2 from the venom of Pseudonaja textilis. A previous study reported this molecule to have moderate enzymatic activity and procoagulant property and was found to be non-lethal [79]. In the NN venom two peptide fragments matching Basic phospholipase A2 from Bungarus candidus venom and one matching with basic PLA2 with sea krait was identified. While in NO venom only one peptide fragment having homology to a basic PLA2 from Bungarus candidus was found. The activity and specificity of basic phospholipases from Agkistrodon h. blomhiffii and Pakistani N. naja was studied on intact human erythrocytes. Although belonging to two different snake families, similar response was reported for these molecules, from both venoms. Basic PLA2 induced the hydrolysis of membrane phospholipids and total cell hemolysis [80]. Despite the fact that acidic PLA2s are found abundantly in the snake venom, their role is poorly understood [81]. In spite of having high catalytic activity as compared to basic PLA2s, they do not induce toxicity [82]. Studies have suggested acidic PLA2s to participate in prey digestion [83]. Other studies have suggested that acidic PLA2s work synergistically with other venom toxins, as PLA2s, metalloprotease and cytotoxins [84,85,86].
PLA2 is ubiquitously found in nature [87,88]. In mammals, they are known to play important and vital role in many life processes [89,90,91]. On the other hand, snake venom PLA2s are toxic and interfere with a number of physiological processes, upon envenomation [87]. Phospholipase A2 are also known to be responsible for the hepatic injury, inflammation and anticoagulation in a victim [26].

3.1.3. Snake Venom Metalloproteinase

The present study shows that N. naja and N. oxiana snake venom contain significant amounts of metalloproteinases, which are the second most abundant protein family. Proteomics study of other Naja species shows the presence of SVMPs in varying amounts ranging from as low as 0.9% to 16% [74,92,93,94,95,96,97,98,99,100]. Previous proteomic studies reported a lower abundance of SVMP in Pakistani N. naja venom [42,43,44]. Three SVMPs bearing relatively higher homology with snake venom metalloproteinase from N. atra were determined in each of the two venoms. Twenty fragments of SVMPs were detected in N. naja venom, which are homologous to K-like SVMPs from N. atra. 13 Peptide fragments were found to match with SVMPs from N. kaouthia (Table 2). The data shows that in case of N. oxiana venom higher number of peptide fragments match with SVMPs from N. atra venom (Table 3). The eastern Indian N. naja contains only 6% SVMPs in contrast to Pakistani N. naja, which contains 10% of SVMPS. It is interesting to note that western Indian N. naja contains 16% SVMPs as determined by A. K. Mukherjee and his research group [45,46].
SVMPs are found in all advanced snakes and make up the major component of the venom of Crotalid and Viperid snakes [101,102,103,104]. SVMPs are structurally versatile and act on different hemostatic targets of prey upon envenomation [105]. These toxins provoke many systemic changes, such as hemorrhage, acute renal failure, coagulopathy, and/or platelet aggregation inhibition [106]. The SVMPs identified in terms of our investigations, in both of the venoms, belong to subfamily P-III. The P-III SVMPs possess gelatinolytic and hemorrhagic activities [105]. A previous study reported the hemorrhagic response of Pakistani N. naja venom in chicken egg [107]. The determination of a higher amount of SVMPs in both NO and NN venom indicates that there is potential for hemorrhage as a response of NO and NN snakebite envenomation.

3.1.4. L-Amino Acid Oxidase

Snake venom L-Amino acid oxidase (LAAOs) belong to the Flavin monoamine oxidase family and are dependent on FAD group for their activity. These proteins are present in both venoms studied and constitute approximately 4–5% of the venom proteome (Table 1). Peptide fragments bearing sequence similarity to LAAO from different snake venoms were detected and summarized (Table 2 and Table 3). In contrast to our results, studies of western Indian N. naja venom report only 0.31% LAAO. However, Indian N. naja venom contains 3% LAAO, which is similar to that of Pakistani N. naja [45,46]. In terms of our investigations we identified that LAAO from both, NN and NO venom, have homology with LAAO from N. atra venom. LAAO is known to be prevalent in many snake venoms [108] but its role in snake venom envenomation pathology is not clear. A recent study reported that LAAO might contribute to severe tissue disruption. This study suggested that LAAO might elicit its toxicity by catalyzing the intracellular substrates [108]. Different biological activities of the isolated LAAO have been reported like, edema, cytotoxic, antibacterial, antiparasitic, and/or platelet aggregation effects [109,110]. Also some investigations reported antitumor effects of LAAO [111]. LAAO is a glycoprotein and glycosylation is also considered to play a significant role in the toxicity of LAAO, and cause cell death by interacting with the cell surface [112,113].

3.1.5. Cobra Venom Factor

Cobra venom factor (CVF) belong to the venom complement C3 homologue family. CVF constitutes approximately 9% of the total proteins identified in both venoms. The identified CVF peptides bear homology mainly to the CVFs from N. kaouthia. Fragments matching to CVF alpha chain and gamma chain were also analyzed. Peptide fragments showing sequence similarity to CVF proteins from other Elapidae and Colubridae have also been identified (Table 2 and Table 3). Proteomic study of Indian N. naja venom showed that it contains only 0.03–1.7% CVF [45,46], which is significantly less compared to our results obtained for the Pakistani N. naja. A venom proteome study of Naja philippinensis showed that it contains less than 4% [76]. The venom of N. ashei contains only 0.12% CVF [99]. Cobra venom factor is a complement activating protein and is functionally and structurally similar to complement component C3b. It is a glycoprotein and herein glycosylation contributes in the immunogenicity of CVF [114,115]. In vivo studies have shown that CVF causes an acute inflammatory injury in the lungs [116]. CVF serves as a gold standard molecule for the evaluation of drugs for trials, to control diseases involving the complement system [117]. A recent study reported CVF as a promising candidate for the treatment of IRI-induced hepatic injury [118]. Our data reveals that CVF is one of the abundant proteins in the venom of Pakistani Naja naja and Naja oxiana (Figure 3). Therefore, these venom can be a good source of isolating CVF for use in biomedical research.

3.1.6. Cysteine-Rich Secretory Protein

Cysteine-rich secretory proteins (CRISPs) have been identified in many animal venoms. These proteins have two domains, a pathogenesis related domain at the N-terminal region and a cysteine rich domain at the C-terminus. Based on sequence homology the CRISP family is classified into four classes, and snake venom CRISPs belong to the group III [119]. CRISPs were found in much higher abundance in N. naja (7%) as compared to N. oxiana (2.8%) and peptide fragments showing similarities to CRISPs from different snake venoms were found in both venoms. However, highest similarity was found with the cysteine-rich venom protein natrin-1(NA-CRVP1) from N. atra. Investigations indicated that NA-CRVP1 could act as inflammatory modulator that could perturb the wound-healing process of a bitten victim by regulating the expression of adhesion molecules in endothelial cells. This study also showed that natrin contains a zinc-binding domain at the N-terminus and elicits its proinflammatory activity in a zinc and heparan-sulfate dependent manner [119]. Natrin has also been reported as a potassium channel blocker and in this context can weakly block muscle contraction [120,121,122,123]. In our study six peptide fragments matching CRISP from N. haje. A study reported this CRISP was found to be non-toxic when administered to crickets [124]. The venom proteome of N. haje contain 10% CRISP [92]. Different species of Naja contain varying amounts of CRISP, from as low as 0.2% to 10% of the total venom proteome. The Indian N. naja contains 1.14–3% CRISPs [45,46].

3.1.7. Snake Venom Serine Proteinase

Snake Venom Serine Proteinase (SVSPs) belong to the peptidase S1 family. N. oxiana venom proteome shows relatively higher abundance of serine proteinases (4%) as compared to N. naja venom, which contains only 2% (Table 1; Figure 3). Both of the venoms contain peptide fragments, which bear homology to tissue-type plasminogen activators from Ophiophagus hannah and the thrombin-like enzyme TLP from Indian N. naja. In addition to this, peptide fragments having sequence similarity to SVSP have also been identified (Table 2 and Table 3). SVSPs have been identified in only few Naja species venom. In western Indian N. naja the SVSPs contributed only 0.69% to venom proteome [46]. N. philippinensis venom proteome consists of 0.35% SVSPs [76]. Previous studies showed that SVSPs are absent in Eastern Indian N.naja venom, while a small percentage (0.03%) was reported for the western Indian snake [45,46].
SVSPs have high specificity towards their substrates. Based on their biological roles, they have also been classified as activators of the fibrinolytic system, procoagulant, anticoagulant and platelet-aggregating enzymes [125]. A few SVSPs, like ancrod and batroxobin have already applications in the treatment of cardiovascular problems, while reptilase serves today as a diagnostic probe for dysfibrinogenemia [126].

3.1.8. Snake Venom Nerve Growth Factor

Snake venom Nerve Growth Factor (NGF) were identified in both venoms but were relatively more abundant in the venom of N. oxiana (4%) as compared to N. naja (2%), Table 3. In both venoms, peptides sharing homology with Ovophis okinavensis, N. sputatrix, and Bitis gabonica NGF were identified (Table 1 and Table 2). In N. naja seven peptides bearing homology with Pseudechis australis were also identified. Further, additional peptide fragments of NGF were also identified in terms of our investigations (Table 2 and Table 3). A previous proteomic study also showed Pakistani N. naja venom to contain 2% NGF [42]. N. naja snake venom from east India contained 3.1% and 1% in western India sample. In the same study N. kaouthia from eastern India was shown to contain 1% NGF [45,46]. N. philippinensis contain only 0.06% NGF [76]. Proteomic analysis of other Naja species venom have also shown them to contain NGF but their relative abundance was not calculated [93,100]. Moroccan cobra venom contains 5% NGF of total venom proteome [92].
Till now not much is known about the contribution and function of NGFs in envenomation. Various bioassays have shown that NGFs have neurotropic activity. Snake venom NGFs have been suggested as a pharmacological tool to study the structure function relationship of human trkA receptor [127]. Studies show that NGFs assert venom toxicity indirectly, either by acting as a carrier of other neurotoxins, which do not have specific recognition sites, like phospholipase or by inducing plasma extravasation at the site of snakebite. NGF is known to coexist with CVF in cobra snake venom, and might be responsible for enhancing the toxic mechanism of CVF in an indirect manner [128]. In 1986, two scientists were awarded a Nobel Prize for their pioneering work, which allowed to explain cell growth regulation. And in context of this investigations Cohen serendipitously discovered NGF from snake venom of Agkistrodon piscivorus [129].

3.1.9. Snake Venom Phosphodiesterase

A lower abundance of snake venom phosphodieterases (PDEs) was determined in both venoms, although relatively higher in N. oxiana, i.e., 3.1%. N. naja venom contains only 1.1% of PDEs. Peptides fragments matching with PDEs from the venom of N. atra, Ovophis okinavensis, and Borikenophis portoricensis were identified in both venom. A recent study determined PDE activity in the venom of ten different species of Naja. All the species showed PDE activity with minor variation [73]. The Indian N. naja venom was reported to contain less than 1 % PDEs, which is similar to Pakistani N. naja.
PDEs are ubiquitously present in snake venom but their activity is higher in Viperidae venom as compared to Elapidae family [130]. In recent years, there has been considerable interest in snake venom PDEs due to their potential applications as pharmacological tool and drug lead. The endonuclease activity of PDEs rendered their use in sequencing of polynucleotides and oligonucleotides [130]. Phosphoribosylation, a protein modification, can also be processed by PDEs [131]. Recent innovative approaches, have utilized snake venom PDEs to digest genomic DNA into single nucleosides to study modifications of DNA [132,133,134].

3.2. Minor Protein Components (Relative Abundance ≤2%)

A large number of low abundant proteins were found in both venoms, particularly in N. naja (Table 2 and Table 3). Ras-like proteins, identified in the venom of N. naja were of particular interest, as they indicate the presence of extra0cellular vesicles in the venom. Snake venom extracellular vesicles (SVEVs) have previously been isolated from the venom of Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis, and Crotalus cerberus oreganus. The size distribution of SVEVs was found to be between approximately 50–500 nm. Proteomic investigations revealed that SVEVs could be assigned to eight different protein classes, such as SVMP, SVSP, and disintegrins [135].
Exosome-like vesicles have also been reported in the venom of Gloydius blomhoffii blomhoffii [136]. In this context extracellular vesicles (EV) are known to carry a diverse cargo of molecules as proteins, DNA, RNA, and/or lipids [137]. Further, EVs are involved in cell-to-cell communication, immune response and apoptotic rescue [138,139] and participate in the maintenance of normal as well as pathophysiological conditions, like cancer [140,141,142]. The proteomic study of extracellular vesicles released from cancer cells have shown the presence of Ras proteins functioning as biomarkers for extracellular vesicles [137,143,144,145]. Studies have shown that Ras proteins are involved in the regulation and assembly of extracellular vesicles cargo [145,146,147,148]. Therefore, the identification of Ras-like proteins indicates the presence of extracellular vesicles in the venom of N. naja. However direct experimental work needs to be done to confirm the presence of such vesicles in the venom. SVEVs in the venom may be involved in another mechanism to secrete membrane proteins like aminopeptidase A and G coupled receptors. SVEVs may also offer an additional route for the envenomation process, thereby facilitating toxins to translocate inside the prey cells.
In the present work, a number of proteins have been identified for the first time in the proteome of these venoms, like G-protein coupled receptors, zinc finger proteins, ankyrin repeat, leucine repeat, Ubiquitin carboxyl-terminal hydrolase and a number of other protein. It can be assumed that these proteins have also a function in the venom. Ankyrin repeats and zinc finger proteins were also identified recently, in the venom of King cobra, Naja annulifera and Micrurus pyrrhocryptus [51,100,149]. A rather old publication reported cobra serum albumin in the venom of cobra snakes [150]. Our data also revealed the presence of cobra serum albumin in the venom of N. naja. It is possible that upon envenomation cobra serum albumin is responsible or supporting the transportation of other venom proteins in the prey serum. Previous studies have reported Cobra blood serum albumin as an antitoxic protein, having the potential to sequester endogenous toxins [151,152]. Cobra serum albumin was also reported in the venom proteome of N. sumatrana [93]. Further, we identified glutathione peroxidase in both venoms. A recent proteomic study also reported the presence of glutathione peroxidase in the venom of Micrurus pyrrhocryptus and N. annulifera [100,149]. It can be speculated that this protein might be involved in protecting the venom gland from oxidative damage. Phospholipase A2 inhibitors, bearing similarity to PLA2 inhibitor isolated from the serum of Elaphe quadrivirgata and Naja kaouthia snakes, were identified in the venom of N. naja. This inhibitor was shown to inhibit the enzymatic activity of all till now known PLA2 enzymes [153,154]. Phospholipase B was also identified in both venom. Only 0.1% constituted the venom proteome of NN while that of NO contained 1.6% of the total venom proteome. Studies have shown that PLBs make up approximately 0.34% of venom components, and in Viperidae venom it varies between 0.23% to 2.5% [155]. Insulin and Transferrin proteins were also identified in the venom of N. naja. Transferrin is a metal binding proteins. Transferrin was also reported before in the venom of P. australis, utilizing two dimensional gel electrophoresis [156]. Snake venom VEGF bearing similarities to that isolated also in Bitis arietans venom, identified in N. naja venom as well. Studies have shown that different variants of snake venom VEGF induce angiogenesis and vascular permeability through different mechanisms [157,158]. Snake venom VEGF are potential candidates for therapeutic angiogenesis [159]. A low abundance of Kunitz type serine protease inhibitors (KSPI) was identified in the venom of both snakes. Snake venom KSPI have the potential to selective inhibit distinct serine proteases [35]. Some of the snake venom KSPI have evolved as potassium channel blockers [160]. BF9 a snake venom KSPI, which act as potassium channel blockers and retain the serine protease inhibitory activity. This bifunctional KSPI was reported in the venom of Bungarus fasciatus [161]. Interestingly another type of serine protease inhibitor, i.e., serpin, was identified in the venom of N. oxiana. Proteins belonging to Ohanin/Vespryn family were found in both venoms. They are small proteins with an average mass of approx. 12 kDa, and are neurotoxic in nature [162]. Further, we could identify 5′-nucleotidase in both venoms. This family of protein is found in different snake venoms [163]. These enzymes play a major role in the release of adenosine upon envenomation, which facilitates prey immobilization. In vivo studies have shown that 5-nucleotidases act synergistically with other venom components like phospholipases, disintegrins to exert a pronounced anticoagulant effect [164]. Aminopeptidase was identified in both N. naja and N. oxiana venoms. Aminopeptidase A activity has been found in the venoms of snakes belonging to Elapidae and Viperidae families. Till now not much is known about the contribution of this enzymes within envenomation pathology [165]. Cystatin, having similarity to cystatin from the venom of N. kaouthia, was identified for the first time in the venom of N. naja in the present study. Cystatin is a cysteine protease inhibitor [166]. Natriuretic peptides were identified in both N. naja and N. oxiana venom. These peptides are known to induce hypotension upon envenomation [167,168]. Cathelicidin was identified in the venom of N. naja, and previous studies have shown it to be potent antimicrobial peptide [169].

3.3. Posttranslational Modifications

In terms of our investigations, we were able to identify N-terminal acetylation (N-ace) for the first time in the snake venom. This posttranslational modification is known to carry and support out different functions in the cell. A most analyzed function of N-ace is the regulation of protein half-life, by labelling proteins for polyubiquitation and thus degradation by the proteasome [170,171]. N-ace modification plays a role in protein folding and protein complex formation [172,173]. Furthermore, studies have shown that N-ace modification mediates the interaction of proteins with membrane and subcellular localization [173]. A probable role of this modification in snake venom proteins could be to stabilize them against proteolytic cleavage, and to assist in distinct protein–protein interactions upon envenomation. In both venoms a peptide fragment bearing homology to muscarinic toxin like-protein 3, from the venom of Naja kaouthia was found to be N- terminal acetylated. Whereas in Naja naja two other peptide fragments were identified to be N-terminal acetylated. One bearing homology to phospholipase A2 and other to a weak neurotoxin 7 (Supplementary Table S1 and S2).

4. Conclusions

Using the MS shotgun approach we could provide a holistic view of the venom profile of the two Pakistani cobra snakes N. naja and N. oxiana. Our data shows for the first time the venom proteome of N. oxiana. The comparative evaluation of the venom proteome of the two snakes reveals differences, as well as similarities in their venom composition. Both snake venoms contain different types of three-finger toxins in their venom, although they exit sympatrically. There are a few toxin families, which were only found in the venom of N. naja, like cystatin, VEGF, TGF, BPP, and Cathelicidin. Therefore, we can suggest, that venom samples from both species should be utilized for the production of effective antivenoms. Also, applying state-of-the-art mass spectrometric tools allowed to identify a number of proteins not known before to be in these venoms, like Ras-GTPase, Ankyrin repeats, leucine repeat, G-protein coupled receptor, zinc finger protein, holiday junction protein, and endonuclease. In this context, identification of Ras-like proteins provided a clue about the presence of extracellular vesicles. These vesicles might function as an additional carrier to transport venom components in the prey upon envenomation. Further, our data highlight N-terminal acetylation of venom proteins for the first time and the results delineate the unique complexity of snake venoms, and open routes for further research to understand function of these molecules upon envenomation.

5. Materials and Methods

5.1. Venom Collection

Venom was milked manually from adult snake species of N. naja (black cobra/Indian cobra/Spectacled cobra) and N. oxiana (brown cobra/Caspian cobra/Central Asian cobra). For the proteomic studies of each species the venom was collected from three adult healthy snakes and pooled. The sex of the snakes was not determined. N. naja snakes were captured from the rural surroundings of Mianwali district, while N. oxiana snakes were caught from Lahore, Punjab province, Pakistan. The venom was freeze dried and kept at −20 °C till further use.

5.2. Sample Preparation for LC-MS/MS

For LC-MS/MS analysis the lyophilized crude venom from N. naja (black cobra) and N. oxiana (brown cobra) was dissolved in 10 mM Triethylamonium bicarbonate (TEAB, Thermo Fisher Scientific), 1% v/w Sodium deoxycholate (SDC, Sigma) buffer. Protein concentration was determined using a bicinchoninic acid protein assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific) and 20 µg of venom protein was tryptically digested. In brief, cysteine residues were reduced for 30 min. in the presence of 10 mM dithiothreitol (DTT, Sigma) at 60 °C and alkylated for 30 min. with 20 mM iodoacetamide (IAA, Sigma) at 37 °C in the dark. Thereafter, sequencing grade trypsin (Promega) was added in a protease/protein ratio of 1:100 at 37 °C to hydrolyze venom proteins overnight. Enzymatic activity was quenched by addition of 1% v/v formic acid (FA, Fluka) and the SDC was precipitated by centrifugation at 16000 g for 5 min. The peptide containing supernatant was vacuum dried and reconstituted in 0.1% FA for LC-MS/MS analysis.

5.3. LC-MS/MS Analysis of the Digested Venom

LC-MS/MS analysis of the venom samples was performed using a nano ACQUITY UPLC® System (Waters, Manchester, UK) coupled to a Hybrid-Quadrupole-Orbitrap mass spectrometer (Q Exactive™, Thermo Fisher Scientific). The LC system was equipped with a reversed phase chromatography (RPC) columns [ACQUITY UPLC® Symmetry C 18 (180 µm i.d × 20 cm, 5 µm particle size, 100 Å pore size, Waters, Manchester, UK) as trap column and a RPC separation column (ACQUITY UPLC® Peptide BEH C-18 (75µm i.d × 20 cm, 1.7 µm particle size, 170 Å pore size, Waters, Manchester, UK) as analytical column. RPC was used with a linear 60 min acetonitrile gradient from 2–30% for peptide separation. (Solvent A: 0.1% FA in water; Solvent B: 0.1% FA in acetonitrile; Flow rate of 250 nL/min).
MS/MS data acquisition was performed in data dependent mode for the 15 most abundant precursor ions. Precursor ions with charge stages between 2+ and 5+ were selected for fragmentation if they exceeded an intensity threshold of 100,000. For MS/MS spectra acquisition the set AGC-target was 100,000 with a maximal ion injection time of 50 ms. Precursor ions were fragmented at a normalized collision energy (NCE) of 25 and the fragment ions were measured with a resolution of 17,500 at 200 m/z. To avoid redundant precursor sampling a dynamic exclusion was applied for 20 s.

5.4. Data Analysis

For protein identification, the generated raw data were processed using the Proteome Discoverer™ Software 2.0.0.802. Database search was performed with the SEQUEST algorithm against an Ophiophagus hannah (txid:8665, King cobra) protein database (UniProt), since it represents the closest sequence database to the analyzed samples. Carbamidomethylation of cysteine was used as fixed modification. Furthermore, oxidation of methionine, conversion of glutamine to pyro-glutamic acid at peptide N-termini, loss of N-terminal methionine and the acetylation of protein N-termini were considered as variable modifications. Precursor and fragment ion tolerance were set at 10 ppm and 0.02 Da, respectively. Peptide-spectra matches with a maximum delta Cn of 0.05 were used by Percolator for FDR estimation.
Unidentified spectra were exported to a new mgf file and de novo sequencing was performed with Novor [174] via DenovoGUI 1.16.2 [175]. Modifications and allowed mass tolerances were identical to the database search approach. Hits with a Novor score above 80 were considered for a protein BLAST approach. Protein BLAST for the sequenced peptides was conducted with the NCBI BLAST p algorithm (2.9.0+) with default settings against non-redundant protein sequences (nr) narrowed down to serpents (taxid: 8570). Alignments were chosen according to the max Score, the query coverage and if the homologous proteins were related to venom activity. With this information, a venom specific peptide database was created to support database searching for further analyses. Similarly, the data search was also performed against Serpents protein data base from UniProt.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [176] with the dataset identifier PXD018726 and 10.6019/PXD018726.
Venom components were classified according to protein families and their relative abundances calculated as, reported previously [51]. Briefly, the proteins analyzed were sorted into different groups of protein families. The relative abundance of each family was calculated as percent of total number of venom proteins detected by the mass spectrometer. The mathematical relationship below was used to calculate the relative abundance of each protein group. Pie chart (Figure 3) and Table 1, presents the percentage relative abundance of proteins.
Number   of   proteins   protein   family   Total   venom   proteins   detected   using   LC − MS / MS   ×   100

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/2072-6651/12/11/669/s1 Table S1: De novo peptide sequencing Naja naja venom. Table S2: De novo peptide sequencing Naja oxiana venom. Table S3: Proteomic data of Naja naja venom searched against Serpents Uniprot protein data base. Table S4: Proteomic data of Naja naja venom searched against King cobra Uniprot protein data base. Table S5: Proteomic data of Naja oxiana venom searched against Serpents Uniprot protein data base. Table S6: Proteomic data of Naja oxiana venom searched against Serpents Uniprot protein data base.

Author Contributions

Conceptualization, A.M. and C.B.; methodology, H.S., B.D., and A.B.; Venom milking, Z.M.; software, H.S., A.M., B.D., and A.B.; validation, H.S., A.M., C.B., S.A.A., and B.D.; formal analysis, A.M., S.A.A., A.U., and A.A.; investigation, A.M., S.A.A., B.D., A.B., A.A., and A.U.; resources, C.B. and H.S.; data curation, A.M. and B.D.; writing—original draft preparation, A.M.; writing—review and editing, All co-authors; supervision, C.B.; project administration, A.M. and C.B.; All authors have read and agreed to the published version of the manuscript.

Funding

This work is in part supported by the Cluster of Excellence ‘The Hamburg Centre for Ultrafast Imaging’ of the Deutsche Forschungsgemeinschaft (DFG)-EXC 1074-project ID 194651731. A part of the project was supported by higher education commission (HEC), Pakistan, (NRPU/R&D/HEC-No: 20-3891) and (HEC-No: 7709/Federal/ NRPU/R&D/HEC/ 2017).

Acknowledgments

The authors acknowledge financial support by the Cluster of Excellence “Advanced Imaging of Matter” of the Deutsche Forschungsgemeinschaft (DFG)-EXC 2056-project ID 390715994 and BMBF via project 05K16GUA. AM would like to thank Patrick Spencer, (Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares, Avenue Lineu Prestes 2242, São Paulo 05508-000, Brazil) for critically reviewing the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. (A) Geographical distribution of the genus Naja snakes in Pakistan. (B) Naja oxiana (Brown cobra) (C) Naja naja (black cobra).
Figure 1. (A) Geographical distribution of the genus Naja snakes in Pakistan. (B) Naja oxiana (Brown cobra) (C) Naja naja (black cobra).
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Figure 2. (A) Milking of N. naja venom and sample preparation for LC-MS/MS analysis (B) Data base search cycle.
Figure 2. (A) Milking of N. naja venom and sample preparation for LC-MS/MS analysis (B) Data base search cycle.
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Figure 3. Pie chart illustrations highlighting the relative abundance of various protein families in the two venoms. (A) N. oxiana (B) N. naja.
Figure 3. Pie chart illustrations highlighting the relative abundance of various protein families in the two venoms. (A) N. oxiana (B) N. naja.
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Figure 4. Pie chart illustrating a comparative profile of the three-finger toxins present in the two snake venoms. (A) N. oxiana (B) N. naja.
Figure 4. Pie chart illustrating a comparative profile of the three-finger toxins present in the two snake venoms. (A) N. oxiana (B) N. naja.
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Table
Table 1. Comparative evaluation of snake venom protein families in the venom of N. naja and N. oxiana.
Table 1. Comparative evaluation of snake venom protein families in the venom of N. naja and N. oxiana.
Protein FamilyFirst Report in Nn VenomAbbreviation UsedNN (No of Peptides)%AgeNO(No. of Peptides)%Age
Three-Finger toxin 3FTX157214116
Snake venom metalloprotease family SVMP72103915
Cobra venom factor CVF629228.7
Cysteine-rich secretory protein CRISP537 72.8
Phospholipase A2 PLA2466 3212.6
Phospholipase B PLB10.141.6
Phospholipase inhibitor✓CNF-I30.4 --
L-amino-acid oxidase LAAO314145.5
Snake Venom Serine proteinase SP152114.3
Ohanin Oh111.520.8
Kunitz type serine protease inhibitor KSPI14241.6
Nerve Growth Factor NGF121.7114.3
5′-nucleotidase 5-Ntd101.410.4
Serum Albumin✓SA101.4-
Glutathione peroxidase✓GP91.231.2
Phosphodiesterase Pde81.183.1
Aminopeptidase -7141.6
TNF receptor family✓TNF20.331.2
Lectin✓-30.410.4
Natriuretic peptide family NP40.5410.4
Cystatin -40.54-
Cathelicidin✓cath10.1-
N-acetylcholinesterase N-Ache10.110.4
Vascular endothelial growth factor VEGF10.1-
Transforming growth factor✓TGF20.3-
Zinc finger protein✓ZFP60.841.6
Insulin✓In20.3-
Transferrin✓TF20.3-
Ankyrin repeat✓AR20.310.4
Leucine repeat✓LR10.110.4
Endonuclease✓-30.4-
SLRP family✓SLRP20.310.4
Ras-like protein✓Ras50.7-
Serpin -- 10.4
Others -158 37
Total 735 254
Bold text in the first column indicates protein families exclusively identified in N. naja venom. Blue coloured text indicates protein family identified only in N. oxiana. Check mark (✓) in the second column, indicates that this work is the first report of the identification of the corresponding protein families in N.naja venom. The dash sign indicate that the protein family was not identified in the venom.
Table
Table 2. Summary of the venom proteome of Naja naja.
Table 2. Summary of the venom proteome of Naja naja.
S. NoProtein FamilyProteinAccession CodeNumber of Matched PeptidesHomology with a Protein from the Venom of Snake Species
13FTXs (Neurotoxin)Long neurotoxinAHZ088249Micropechis ikaheca
2 P013891Naja anchietae
3 P013902Naja nivea
4 Long neurotoxin homologO934225Naja atra
5 Long neurotoxin 1P256684Naja naja
6 Long neurotoxin 1P013801Hydrophis stokesii
7 Long neurotoxin 1P256743Naja haje haje
8 Long neurotoxin 4P256723Naja naja
9 Long neurotoxin 7O422573Naja sputatrix
10 putative long neurotoxinABX581511Austrelaps labialis
11 putative long neurotoxinABX581631Austrelaps labialis
12 Alpha-neurotoxin NTX-3O573261Naja sputatrix
13 Short neurotoxin 3P014201Naja annulifera
14 Short neurotoxin IIIP592751Naja kaouthia
15 Neurotoxin IIP014276Naja oxiana
16 cobrotoxin bCAA738293Naja atra
17 Cobrotoxin-bP809584Naja atra
18 Alpha-cobratoxinP013914Naja kaouthia
19 kappa-cobrotoxinCAA768461Naja atra
20 Weak toxin 2Q8AY502Bungarus candidus
21 Weak neurotoxin 7P291817Naja naja
22 Weak neurotoxin 10Q802B21Naja sputatrix
23 Weak toxin CM-11P014014Naja haje haje
24 Weak toxin S4C11P014005Naja melanoleuca
25 three-finger toxin precursor, partialADN675724Bungarus multicinctus
26 three-finger toxin precursor, partialADN675829Naja atra
27 three-finger toxin precursor, partialADN675831Naja atra
28 three-finger toxin precursorADN675791Naja atra
29 Muscarinic toxin-like protein 3P824643Naja kaouthia
30 Muscarinic toxin-like proteinQ9W7274Bungarus multicinctus
31 Muscarinic toxin-like protein 2P824636Naja kaouthia
32 Muscarinic toxin-like protein 1P824622Naja kaouthia
33 Muscarinic toxin 38Q2VBN01Ophiophagus hannah
34 Alpha-elapitoxin-Nk2aP013914Naja kaouthia
36 three finger toxin VABX828661Walterinnesia aegyptia
37 Three finger toxin W-VC1IC493Walterinnesia aegyptia
38 Chain A, Putative Ancestral Mamba Toxin 15MG9_A1Dendroaspis angusticeps
393FTXs (cytotoxins)cytotoxin 17, partialBAU2467613Naja naja
40 Cytotoxin Vc-5Q9PS346Naja oxiana
41 Cytotoxin 3aP865394Naja naja
42 Cytotoxin SP15cP6030813Naja atra
43 cardiotoxin 7aAAB369292Naja atra
44 cardiotoxin 7aQ911263Naja atra
46 Cytotoxin 8P865402Naja naja
47 Cytotoxin 1P014471Naja naja
48 Cytotoxin IIP014411Naja oxiana
49 Cytotoxin 5P255172Naja mossambica
50 Cardiotoxin-6Q989651Naja atra
51 Cytotoxin 10P865411Naja naja
52 Cytotoxin homolog 3P014731Naja melanoleuca
53 Cardiotoxin-like basic polypeptide ahP0C5472Naja atra
54 cardiotoxin 1eAAA909604Naja atra
55Venom complement C3-likeVenom factorAAX866415Austrelaps superbus
56 Cobra venom factorQ9113231Naja kaouthia
57 Cobra venom factor gamma chainQ911322Naja kaouthia
58 Cobra venom factor alpha chainQ911322Naja kaouthia
59 cobra venom factor precursorAAA689891Naja kaouthia
60 venom factor-like, partialXP_0250258332Python bivittatus
61 cobra venom factor 1, partialAXL9662013Ahaetulla prasina
62 cobra venom factor, partialAXL952791Spilotes sulphureus
63 cobra venom factor, partialAWX676461Boiga irregularis
64 Ophiophagus venom factorI2C0903Ophiophagus hannah
66Venom Kunitz-type familyKunitz-type serine protease inhibitorP198591Naja naja
67 Kunitz-type serine protease inhibitorP202296Naja naja
68 Kunitz-type serine protease inhibitor isoform 7ACY687031Parasuta nigriceps
69 Kunitz inhibitor b, partialAAL300691Bungarus candidus
70 protease inhibitorAFA900801Daboia siamensis
71 Venom basic protease inhibitor 2P009861Naja nivea
72 Kunitz-type protease inhibitor, partialAWX676601Boiga irregularis
73 papilin-like, partial XP_0250323511Python bivittatus
74 Kunitz inhibitor IABX828671Walterinnesia aegyptia
75natriuretic peptide familyNatriuretic peptide Na-NPD9IX972Naja atra
76 natriuretic peptideADK120011Naja atra
77 natriuretic peptideADK120011Naja atra
78cystatinCystatinE3P6P44Naja kaouthia
79NGF-beta familyVenom nerve growth factor 2Q5YF892Naja sputatrix
80 Venom nerve growth factor 3Q3HXY17Pseudechis australis
81 nerve growth factor, partialAAR245301Bitis gabonica
82 nerve growth factorBAN821424Ovophis okinavensis
83 nerve growth factor beta chain precursorA592181Naja kaouthia
84Ohanin/vespryn family.OhaninP832344Ophiophagus hannah
85 ThaicobrinP828852Naja kaouthia
86 Venom PRY-SPRY domain-containing protein, partialAHZ088034Micropechis ikaheca
87 VesprynAEJ320041Crotalus adamanteus
88Insulin familyInsulin-like growth factor-binding protein 3, partialXP_0250322481Python bivittatus
89 Insulin enhancer protein ISL-1, partialETE721051Ophiophagus hannah
90Snake venom VEGF subfamilySnake venom vascular endothelial growth factor toxin barietinC0K3N11Bitis arietans
91CRISPCysteine-rich venom protein 25P848066Naja haje haje
92 cysteine-rich seceretory protein Ts-CRPMACE735742Trimeresurus stejnegeri
93 Cysteine-rich venom protein mossambinP0DL162Naja mossambica
94 Cysteine-rich venom protein natrin-1Q7T1K616Naja atra
95 Cysteine-rich venom protein ophaninQ7ZT983Ophiophagus hannah
96 cysteine-rich venom protein, partialBAP399571Protobothrops flavoviridis
97 Cysteine-rich venom protein natrin-2Q7ZZN83Naja atra
98 Cysteine-rich seceretory protein Ts-CRPMN-ACE735741Trimeresurus stejnegeri
99 Cysteine-rich venom protein 25-AP848071Naja haje haje
100 HelicopsinP0DJG82Helicops angulatus
101 Cysteine-rich venom protein bucarinP819931Bungarus candidus
102 Cysteine-rich venom protein latiseminQ8JI381Laticauda semifasciata
103 Cysteine-rich venom protein ophaninAAO629961Ophiophagus hannah
104 cysteine-rich secretory protein 4, partialAXL965842Borikenophis portoricensis
105 Cysteine-rich venom protein kaouthin-1P848051Naja kaouthia
106 Cysteine-rich venom protein annuliferin-bP0DL151Naja annulifera
107 Cysteine-rich venom proteinAAP206032Naja atra
108 Cysteine-rich secretory proteinAJB845051Philodryas chamissonis
109 Opharin precursorAAP812921Ophiophagus hannah
110 Cysteine rich secretory protein 2, partialAXL966294Ahaetulla prasina
111Cathelicidin familyCathelicidin-related protein precursorACF210001Naja atra
112TGF-beta familyTransforming growth factor beta-3, partialETE717741Ophiophagus hannah
113 Glial cell line-derived neurotrophic factor, partialETE673241Ophiophagus hannah
114Phospholipase A2Acidic phospholipase A2 3P600454Naja sagittifera
115 85 kDa calcium-independent phospholipase A2, partialETE711582Ophiophagus hannah
116 Acidic phospholipase A2 1P005964Naja kaouthia
117 Acidic phospholipase A2 1Q9W7J46Pseudonaja textilis
118 Basic phospholipase A2 T1-2 A chainP844722Bungarus candidus
119 Acidic phospholipase A2 CQ920865Naja sputatrix
120 Acidic phospholipase A2 1P005983Naja naja
121 Acidic phospholipase A2 2P600441Naja sagittifera
122 Acidic phospholipase A2 1P005964Naja kaouthia
123 Phospholipase A2BAA364031Naja kaouthia
124 Acidic phospholipase A2 beta-bungarotoxin A4 chainP179342Bungarus multicinctus
125 Phospholipase A2-IIIABD240381Daboia russelii russelii
126 Basic phospholipase A2 homolog 1P101171Laticauda colubrina
127 Phospholipase A2AAL555551Hydrophis hardwickii
128 Phospholipase A2P15445 (2WQ5)1Naja naja
129 Phospholipase A2 3P217923Micrurus nigrocinctus
130 Phospholipase A2I precursorBAC776551Bungarus flaviceps
131 Phospholipase a2CAA453721Naja naja
132 Phospholipase A2AAA660291Naja naja
133 Phosphatidylcholine 2-acylhydrolase T1-2 AP844722Bungarus candidus
134Phospholipase B-like familyPhospholipase B-like 1, partialETE595781Ophiophagus hannah
135CNF-like-inhibitor familyPhospholipase A2 inhibitor subunit gamma AQ9PWI41Elaphe quadrivirgata
136 Phospholipase A2 inhibitor beta subunit isoform OMI-2BAAF210491Oxyuranus microlepidotus
137 Phospholipase A2 inhibitor 31 kDa subunitQ7LZI11Naja kaouthia
138SVMP (PIII)Acutolysin e precursorAAD278911Deinagkistrodon acutus
139 Snake venom metalloproteinaseD5LMJ312Naja atra
140 Snake venom metalloproteinaseD3TTC120Naja atra
141 Snake venom metalloproteinaseD3TTC28Naja atra
142 Snake venom metalloproteinase-disintegrin-like mocarhaginQ107497Naja mossambica
143 Snake venom metalloproteinaseQ9PVK75Naja kaouthia
144 Snake venom metalloproteinaseA8QL492Bungarus multicinctus
145 Snake venom metalloproteinaseP829428Naja kaouthia
146 Snake venom metalloprotease(ADAM)ACS749861Philodryas olfersii
147 Snake venom metalloproteinase 27, partialAXL965771Borikenophis portoricensis
148 Disintegrin and metalloproteinase domain-containing protein 21, partialETE71596 2Ophiophagus hannah
149 Microlepidotease-1ABQ011371Oxyuranus microlepidotus
150 Metalloproteinase atrase B, partialADD140361Naja atra
151 Metalloproteinase 7, partialAXL966261Ahaetulla prasina
152 Snake venom metalloproteinaseP0DM461Micrurus corallinus
153 K-like metalloprotease precursor, partialACN500051Naja atra
154Snake venom serine proteinase
(peptidase S1 family)		Tissue-type plasminogen activator, partial ETE66683 3Ophiophagus hannah
155 Tissue-type plasminogen activator-like, partialXP_0250331873Python bivittatus
156 Complement factor B precursorAAR216011Naja kaouthia
157 Thrombin-like enzyme TLPP865452Naja naja
158 Serine endopeptidaseAUS825671Crotalus tigris
159 Snake venom serine protease NaSPA8QL531Naja atra
160 Snake venom serine protease catroxase-1Q8QHK31Crotalus atrox
161 Anionic trypsin-1-likeXP_0074349411Python bivittatus
162 Coagulation factor X isoform 1, partialETE734011Ophiophagus hannah
163 Serine endopeptidaseAUS825521Crotalus scutulatus
1645’-nucleotidase family5-nucleotidaseBAP399725Protobothrops flavoviridis
165 Venom 5’-nucleotidaseA0A2I4HXH5 3Naja atra
166 5’-nucleotidase, partialETE672451Ophiophagus hannah
167 Snake venom 5’-nucleotidaseB6EWW81Gloydius brevicaudus
168AminopeptidaseAminopeptidase N, partialETE610211Ophiophagus hannah
169 Aminopeptidase NBAG825996Gloydius brevicaudus
170Type-B carboxylesterase/lipase N-acetylcholinesteraseAAC599051Bungarus fasciatus
171Phosphodiesterase Snake venom PhosphodiesteraseA0A2D0TC043Naja atra
172 PhosphodiesteraseAHJ808851Macrovipera lebetina
173 Phosphodiesterase, partialAXL965992Borikenophis portoricensis
174 PhosphodiesteraseBAN894252Ovophis okinavensis
175Flavin monoamine oxidase familyL-amino acid oxidase, partialAAZ086201Daboia siamensis
176 L-amino acid oxidase, partialAVX276074Naja atra
177 L-amino-acid oxidaseQ4JHE15Pseudechis australis
178 L-amino-acid oxidaseP0C2D51Protobothrops flavoviridis
179 L-amino-acid oxidaseA8QL511Bungarus multicinctus
180 L-amino-acid oxidaseQ4JHE33Oxyuranus scutellatus scutellatus
181 L-amino acid oxidase, partialAVX276074Naja atra
182 L-amino-acid oxidaseA8QL586Naja atra
183 L-amino-acid oxidaseQ4JHE33Oxyuranus scutellatus scutellatus
184 L-amino acid oxidase precursorAAY896822Pseudechis australis
185 L-amino-acid oxidaseCAQ728941Echis ocellatus
186True venom lectin familyC-type lectin galactose-binding isoformD2YVK12Hoplocephalus stephensii
187 BJcuL precursorAAQ929571Bothrops jararacussu
188Ankyrin SOCS box (ASB) familyAnkyrin repeat and SOCS box protein 7, partialETE638951Ophiophagus hannah
189 Ankyrin repeat domain-containing protein 50, partialETE610411Ophiophagus hannah
190TransferrinTransferrinCAK182212Natrix natrix
191Cobra serum albuminCobra serum albuminS595171Naja kaouthia
192 Serum albumin precursorS595173Naja naja
193 Cobra serum albuminCAA553333Naja naja
194Serum albumin/Alpha-fetoprotein/AfaminAlpha-fetoprotein, partial ETE59846 3Ophiophagus hannah
195Leucine repeatLeucine-rich repeat neuronal protein 4XP_0074247901Python bivittatus
196Small leucine-rich proteoglycan (SLRP) familyDecorin, partial ETE60606 1Ophiophagus hannah
197 Leucine-rich repeat and WD repeat-containing protein, partialETE613231Ophiophagus hannah
198XPG/RAD2 endonuclease familyEndonuclease domain-containing 1 protein, partialETE599392Ophiophagus hannah
199 Deoxyribonuclease-2-alpha, partialETE732061Ophiophagus hannah
200NHS FamilyNHS-like protein 1, partialETE712821Ophiophagus hannah
201G-protein coupled receptorG-protein coupled receptor 161XP_0074282151Python bivittatus
202 Putative G-protein coupled receptorETE615912Ophiophagus hannah
203 Melanocyte-stimulating hormone receptor, partialETE691631 Ophiophagus hannah
204 Latrophilin-2, partialETE735691Ophiophagus hannah
205 Cadherin EGF LAG seven-pass G-type receptor 2, partialETE726211Ophiophagus hannah
206 Putative G-protein coupled receptor, partialETE704001Ophiophagus hannah
207Zinc finger proteinThioredoxin domain-containing protein 11, partialETE721181Ophiophagus hannah
208 Zinc finger protein 91-like isoform X2XP_0074433131Python bivittatus
209 Zinc finger protein 687 isoform X1XP_0250271181Python bivittatus
210 Zinc finger FYVE domain-containing protein 16, partialETE661351Ophiophagus hannah
211 Zinc finger and BTB domain-containing protein 14, partialXP_0265553901Pseudonaja textilis
212 Zinc finger protein 609 isoform X1XP_0074268251Python bivittatus
213Ras-like proteinRas GTPase-activating protein 3, partialETE715701Ophiophagus hannah
214 Rac GTPase-activating protein 1, partialETE618611Ophiophagus hannah
215 Ras-related protein Rap-2a, partialETE666021Ophiophagus hannah
216 RalA-binding protein 1, partialETE678181Ophiophagus hannah
217 Guanylate-binding protein 1-likeXP_0074446321Python bivittatus
218Glutathione peroxidase familyGlutathione peroxidase 3, partialETE688109Ophiophagus hannah
219Protein family not assignedOctapeptide-repeat protein T2, partialETE658341Ophiophagus hannah
220 Atrial natriuretic peptide receptor 2, partialETE584631Ophiophagus hannah
221 Octapeptide-repeat protein T2, partialETE614411Ophiophagus hannah
222 GAS2-like protein 2, partialETE677301Ophiophagus hannah
223 Exocyst complex component 3, partialETE601301Ophiophagus hannah
224 Vacuolar protein sorting-associated protein 54ETE706271Ophiophagus hannah
225 Cohesin subunit SA-2, partialETE63002 Ophiophagus hannah
226 Zona pellucida sperm-binding protein 3 receptor, partialETE595121Ophiophagus hannah
227 Ubiquitin carboxyl-terminal hydrolase 32, partialETE632631Ophiophagus hannah
228 Putative E3 ubiquitin-protein ligase UBR7ETE675031Ophiophagus hannah
229 Mdm2-binding protein, partialETE645331Ophiophagus hannah
230 E3 ubiquitin-protein ligase TTC3, partialETE734511Ophiophagus hannah
231 Protocadherin-23XP_0074256731Python bivittatus
232 Nucleolar complex protein 4-like protein, partialETE598861Ophiophagus hannah
233 Low molecular weight phosphotyrosine protein phosphatase, partialETE667081Ophiophagus hannah
234 Major histocompatibility complex class I-related protein, partialETE568161Ophiophagus hannah
235 Beta-2-microglobulin, partialETE584261Ophiophagus hannah
236 GRAM domain-containing protein 1B, partialETE598751Ophiophagus hannah
237 von Willebrand factor A domain-containing protein 3B, partialETE718981Ophiophagus hannah
238 Homeobox protein PKNOX1, partialXP_0074350141Python bivittatus
239 Homeobox protein prophet of Pit-1, partialETE690181Ophiophagus hannah
240 Homeobox protein cut-like 2, partialETE716121
241 Inosine-uridine preferring nucleoside hydrolase, partialETE689361Ophiophagus hannah
242 Signal recognition particle receptor subunit betaETE611811Ophiophagus hannah
243 Sodium channel protein type 1 subunit alphaXP_0250248921Python bivittatus
244 Small serum protein-4BAJ147091Gloydius blomhoffii blomhoffii
245 Clathrin heavy chain 1, partialETE687391Ophiophagus hannah
246 Neutral amino acid transporter A, partialETE718891Ophiophagus hannah
247 BystinETE675121Ophiophagus hannah
248 Peroxisome biogenesis factor 1-like isoform X1XP_0250321821Python bivittatus
249 Dapper-like 1, partialETE597811Ophiophagus hannah
250 Protein patched-like 2, partialETE720351Ophiophagus hannah
251 Keratin, type II cytoskeletal 1, partialETE671311Ophiophagus hannah
252 Keratin, type II cytoskeletal 6A-likeXP_0074413331Python bivittatus
253 Cytosolic carboxypeptidase 2, partialETE727161Ophiophagus hannah
254 NADH dehydrogenase subunit 4YP_0035407951Hypsiglena ochrorhyncha klauberi
255 Olfactory receptor 2D2-likeXP_0074428541Python bivittatus
256 Histone-lysine N-methyltransferase SETD1B, partialETE636061Ophiophagus hannah
257 Helicase SRCAP, partialETE664581Ophiophagus hannah
258 Tyrosine-protein phosphatase non-receptor type 11-likeXP_0157432351Python bivittatus
259 Glycerol-3-phosphate acyltransferase 4ETE642951Ophiophagus hannah
260 NEDD4-binding protein 1, partialETE717891Ophiophagus hannah
261 Nuclear pore complex protein, partialETE727171Ophiophagus hannah
262 G1/S-specific cyclin-E1, partialETE694191Ophiophagus hannah
263 Copine-3ETE622351Ophiophagus hannah
264 Disks large-like 1, partialETE607751Ophiophagus hannah
265 Tumor necrosis factor receptor superfamily member 11BETE674521Ophiophagus hannah
266 Extracellular matrix protein 1, partialETE630093Ophiophagus hannah
267 Protein PRRC2C isoform X7XP_0250259881Python bivittatus
268 Protein dispatched-like 2, partialETE652801Ophiophagus hannah
269 Cytoplasmic FMR1-interacting protein 1ETE700741 Ophiophagus hannah
270 Sushi domain-containing protein 2 isoform X1XP_0074390941Python bivittatus
271 POU domain, class 2, transcription factor 1, partialETE688871Ophiophagus hannah
272 Vomeronasal type-2 receptor 26-likeXP_0157461721Python bivittatus
273 snRNA-activating protein complex subunit 4, partialETE662571Ophiophagus hannah
274 Small subunit processome component 20-like protein, partiaETE626751Ophiophagus hannah
275 Retrotransposon-derived protein PEG10, partialETE604141Ophiophagus hannah
276 Heterogeneous nuclear ribonucleoprotein RETE700951Ophiophagus hannah
277 Sacsin, partialETE730741Ophiophagus hannah
278 Trafficking protein particle complex subunit 3XP_0074391191Python bivittatus
279 Putative protein C4orf34ETE618481Ophiophagus hannah
280 Sulfate transporter, partialETE722501Ophiophagus hannah
281 Solute carrier family 2, facilitated glucose transporter member 11, partialETE659791Ophiophagus hannah
282 Solute carrier family 25 member 47, partialETE647371Ophiophagus hannah
283 Citrate synthase, mitochondrialETE719021Ophiophagus hannah
284 Separin, partialETE717061Ophiophagus hannah
285 5,6-dihydroxyindole-2-carboxylic acid oxidase, partialETE637591Ophiophagus hannah
286 Protocadherin-15, partialETE731221Ophiophagus hannah
287 Tumor necrosis factor receptor superfamily member 11B isoform X2XP_0250192611Python bivittatus
288 Microtubule-actin cross-linking factor 1, isoforms 1/2/3/5, partialETE722671Ophiophagus hannah
289 Ubiquitin carboxyl-terminal hydrolase CYLDXP_0156801471Protobothrops mucrosquamatus
290 Peroxidasin, partialETE578201Ophiophagus hannah
291 Serine palmitoyltransferase small subunit BXP_0250286241Python bivittatus
292 C-terminal-binding protein 1, partialETE643231Ophiophagus hannah
293 StAR-related lipid transfer protein 13ETE699781Ophiophagus hannah
294 Ty3b-i, partialETE590801Ophiophagus hannah
295 E3 ubiquitin-protein ligase RNF19B, partialETE681531Ophiophagus hannah
296 PDZ domain-containing protein 6, partialETE690931Ophiophagus hannah
297 Nebulin, partialETE709062Ophiophagus hannah
298 Myoferlin, partialETE668701Ophiophagus hannah
299 Protein mago nashi-like 2ETE706121Ophiophagus hannah
300 H(+)/Cl(-) exchange transporter 7, partialETE721341Ophiophagus hannah
301 Membrane cofactor protein-likeXP_0250213162Python bivittatus
302 Holliday junction recognition protein isoform X1XP_0250250011Python bivittatus
303 Adenylate cyclase type 2, partialETE627501Ophiophagus hannah
304 Transmembrane protein, partialETE596101Ophiophagus hannah
305 Transmembrane protein, partialETE582441Ophiophagus hannah
306 Type I inositol 3,4-bisphosphate 4-phosphataseXP_0156861591Protobothrops mucrosquamatus
307 Complement decay-accelerating factor transmembrane isoform, partialETE633848Ophiophagus hannah
308 NACHT, LRR and PYD domains-containing protein 6(Belongs to NLRP family)XP_015679160 1Protobothrops mucrosquamatus
309 Ubiquitin carboxyl-terminal hydrolase 24ETE677251Ophiophagus hannah
310 Epiplakin, partialETE582581Ophiophagus hannah
311 5’ nucleotidase, partialAXL952731Spilotes sulphureus
312 GTP-binding protein 2, partialETE704731Ophiophagus hannah
313 Transmembrane protein 41AXP_0074206931Python bivittatus
314 Serine/threonine-protein kinase TAO2, partialETE670771Ophiophagus hannah
315 Serine/threonine-protein kinase WNK1, partialETE616411Ophiophagus hannah
316 cilia- and flagella-associated protein 57-like, partialXP_0074368521Python bivittatus
317 Lymphocyte antigen 6 complex locus protein G6dETE614521Ophiophagus hannah
318 Histamine H3 receptor, partialETE729721Ophiophagus hannah
319 Glycerol-3-phosphate acyltransferase 1, mitochondrial, partialETE597191Ophiophagus hannah
320 Cleft lip and palate transmembrane protein 1-like protein, partialETE615691Ophiophagus hannah
321 Complement factor B precursorAAR216011Naja kaouthia
322 Selenocysteine lyaseXP_0156691941Protobothrops mucrosquamatus
323 Serine/threonine-protein kinase Nek1, partialETE683061Ophiophagus hannah
324 Collagen alpha-1(IV) chain, partialETE608341Ophiophagus hannah
325 DmX-like protein 2, partialETE638881Ophiophagus hannah
326 Aldehyde dehydrogenase family 3 member B1, partialETE727231Ophiophagus hannah
327 Putative ATP-dependent RNA helicase DHX40, partialETE687401Ophiophagus hannah
328 Immunoglobulin Y2 heavy chain, partialAFR337661Python bivittatus
329 Myomesin-1, partialETE653851Ophiophagus hannah
330 Cyclic AMP-dependent transcription factor ATF-1, partialETE651491Ophiophagus hannah
331 Toll-like receptor 4, partialETE724951Ophiophagus hannah
332 Serine palmitoyltransferase small subunit BXP_0250286241Python bivittatus
333 Histone-lysine N-methyltransferase, H3 lysine-79 specific, partialETE655591Ophiophagus hannah
334 Creatine kinase B-type, partialETE692491Ophiophagus hannah
335 Fibroblast growth factor 3, partialETE693781Ophiophagus hannah
336 RB1-inducible coiled-coil protein 1, partialETE670671Ophiophagus hannah
337 Phosphoinositide 3-kinase regulatory subunit 5, partialETE741441Ophiophagus hannah
338 Cadherin EGF LAG seven-pass G-type receptor 2, partialETE726211Ophiophagus hannah
339 Trafficking kinesin-binding protein 1, partialETE682201Ophiophagus hannah
340 YTH domain family protein 2ETE654641Ophiophagus hannah
341 Vigilin, partialETE619461Ophiophagus hannah
342 39S ribosomal protein L44, mitochondrial, partialETE683991Ophiophagus hannah
343 Pseudouridine-5’-monophosphatase, partialETE716971Ophiophagus hannah
344 Kelch-like protein 13, partialETE719471Ophiophagus hannah
345 Maleylacetoacetate isomeraseETE687521Ophiophagus hannah
346 Neurexophilin-2, partialETE717841Ophiophagus hannah
347 Myocyte-specific enhancer factor 2A isoform X1XP_0074251351Python bivittatus
348 Membrane cofactor protein-like isoform X1XP_0157434251Python bivittatus
349 Ninein-like protein, partialETE701661Ophiophagus hannah
350 Keratin, type I cytoskeletal 19, partialETE702171Ophiophagus hannah
351 Intraflagellar transport protein 88-like proteinETE736571Ophiophagus hannah
352 Complement receptor type 2, partialETE633831Ophiophagus hannah
353 Complement decay-accelerating factor, partialETE595111Ophiophagus hannah
354 Keratin, type II cytoskeletal 5-likeXP_0250305481Python bivittatus
355 7-dehydrocholesterol reductase, partialETE677841Ophiophagus hannah
356 La-related protein 4BETE626711Ophiophagus hannah
357 Intelectin-1a, partial ETE578861Ophiophagus hannah
358 Cation-independent mannose-6-phosphate receptorETE643742Ophiophagus hannah
359 Cerebellin-4ETE652771Ophiophagus hannah
360 C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8, partialASU450321Ophiophagus hannah
361 Neuronal PAS domain-containing protein 2, partialETE636681Ophiophagus hannah
362 Interferon-induced transmembrane protein 10, partialETE669041Ophiophagus hannah
363 Myotubularin-related protein 11, partialETE720681Ophiophagus hannah
364 Tyrosyl-DNA phosphodiesterase 2XP_0265257511Notechis scutatus
365 Phosphoinositide 3-kinase regulatory subunit 5, partialETE741441Ophiophagus hannah
The bold text indicates the proteins identified to have N-terminal acetylation.
Table
Table 3. Summary of the venom proteome of N. oxiana.
Table 3. Summary of the venom proteome of N. oxiana.
S. No.Protein FamilyProteinAccession CodeNumber of Matched PeptidesHomology with Protein from the Venom of Snake Species
13FTX (NeurotoxinNeurotoxin homolog NL1Q9DEQ31Naja atra
2 Short neurotoxin SNTX-1A6MFK61Demansia vestigiata
3 Neurotoxin IIP014271Naja oxiana
4 Cobrotoxin-bP809581Naja atra
5 Alpha-cobratoxinP013913Naja kaouthia
6 Weak toxin 2Q8AY502Bungarus candidus
7 Weak neurotoxin 6O422561Naja sputatrix
8 Weak neurotoxin 7P291812Naja naja
9 Weak toxin S4C11P014001Naja melanoleuca
10 Muscarinic toxin-like protein 3P824644Naja kaouthia
11 Muscarinic toxin-like protein 2P824634Naja kaouthia
12 Muscarinic toxin-like proteinQ9W7271Bungarus multicinctus
13 Three-finger toxin precursor, partialADN675821Naja atra
14 Three-finger toxin precursor, partialADN675821Naja atra
153FTXs (cytotoxins)Cytotoxin Vc-5Q9PS342Naja oxiana
16 Cytotoxin homologP145411Naja kaouthia
17 Cytotoxin homolog 5VQ9W7161Naja atra
18 Cytotoxin SP15cP603081Naja atra
19 Cytotoxin 8P865402Naja naja
20 Cytotoxin 1P014472Naja naja
21 Cardiotoxin 7aQ911266Naja atra
22 Cardiotoxin 1eAAA909602Naja atra
23Venom Complement C3-likeVenom factorAAX866411Austrelaps superbus
24 Cobra venom factorQ9113210Naja kaouthia
25 A.superbus venom factor 1Q0ZZJ61Austrelaps superbus
26 Cobra venom factor alpha chainQ911321Naja kaouthia
27 Cobra venom factor 1, partialAXL966206Ahaetulla prasina
28 Cobra venom factor, partialAWX676462Boiga irregularis
29 Ophiophagus venom factorI2C0901Ophiophagus hannah
30Venom Kunitz-type familyKunitz-type serine protease inhibitorP202292Naja naja
31 BPTI/Kunitz domain-containing protein-likeXP_0265465101Notechis scutatus
32 Kunitz/BPTI-like toxinXP_0265794671Pseudonaja textilis
33natriuretic peptide familyNatriuretic peptide PaNP-c precursor, partialAAZ828221Pseudechis australis
34NGF-beta familyVenom nerve growth factor 2Q5YF895Naja sputatrix
35 Nerve growth factor, partialAAR245301Bitis gabonica
36 Nerve growth factorBAN821424Ovophis okinavensis
37 Venom nerve growth factor 2Q3HXX91Hoplocephalus stephensii
38ohanin/vespryn family.ThaicobrinP828851Naja kaouthia
39 venom PRY-SPRY domain-containing protein, partialAHZ088031Micropechis ikaheca
40CRISPCysteine-rich venom protein natrin-1Q7T1K63Naja atra
41 Cysteine-rich secretory protein 1, partialAXL966071Ahaetulla prasina
42 Cysteine-rich venom protein ophaninQ7ZT981Ophiophagus hannah
43 Cysteine-rich venom protein, partialBAP399571Protobothrops flavoviridis
44 Cysteine-rich venom protein 2Q7ZZN81Naja atra
45Phosoholipase A2Acidic phospholipase A2 3P600451Naja sagittifera
46 Acidic phospholipase A2 2P005971Naja kaouthia
47 Phospholipase a2CAA453723Naja naja
48 Neutral phospholipase A2 paradoxin-like beta chainQ45Z462Oxyuranus microlepidotus
49 Phospholipase A2AHZ088101Micropechis ikaheca
50 Phospholipase A2AAA66029.11Naja naja
51 Acidic phospholipase A2 2P154451Naja naja
52 Acidic phospholipase A2 1P005966Naja kaouthia
53 Acidic phospholipase A2 1Q9W7J41Pseudonaja textilis
54 Basic phospholipase A2 T1-2 A chainP844721Bungarus candidus
55 Acidic phospholipase A2 CQ9208611Naja sputatrix
56 Acidic phospholipase A2 1P005981Naja naja
57 Acidic phospholipase A2 beta-bungarotoxin A4 chainP179341Bungarus multicinctus
58 Phospholipase A2 3P217921Micrurus nigrocinctus
59Phospholipase BPhospholipase B, partialAXL952741Spilotes sulphureus
60 Phospholipase B1, partialAXL966062Ahaetulla prasina
61 Phospholipase B1, membrane-associatedXP_026537461Notechis scutatus
62SVMPSnake venom metalloproteinaseD3TTC24Naja atra
63 Snake venom metalloproteinaseF8RKW11Drysdalia coronoides
64 Snake venom metalloproteinaseQ9PVK71Naja kaouthia
65 Disintegrin and metalloproteinase domain-containing protein 20, partialETE72945 1Ophiophagus hannah
66 Disintegrin and metalloproteinase domain-containing protein 21, partialETE71596 1Ophiophagus hannah
67 disintegrin and metalloproteinase domain-containing protein 10-like, partialXP_026580760 1Pseudonaja textilis
68 P-III snake venom metalloprotease, partialAHZ088191Micropechis ikaheca
69 Zinc metalloproteinase-disintegrin-like kaouthiagin-likeD3TTC17Naja atra
70 Zinc metalloproteinase-disintegrin-like atrase-AD5LMJ314Naja atra
71 Hemorrhagic metalloproteinase-disintegrin-like kaouthiaginP829422Naja kaouthia
72 metalloproteinase 7, partialAXL966261Ahaetulla prasina
73 metalloproteinase, partialAWX675761Boiga irregularis
74 Snake venom metalloproteinase-disintegrin-like mocarhaginQ107493Naja mossambica
75 Snake venom metalloproteinaseQ9W6M51Deinagkistrodon acutus
76Snake venom serine proteinase
(peptidase S1 family) 		Tissue-type plasminogen activator, partial ETE66683 3Ophiophagus hannah
77 tissue-type plasminogen activator, partialXP_0265446712Notechis scutatus
78 Snake venom serine protease 3O130581Protobothrops flavoviridis
79 Serine protease 27, partialETE646531Ophiophagus hannah
80 Thrombin-like enzyme TLPP865451Naja naja
81 Snake venom serine protease 3AAG107901Protobothrops jerdonii
82 Snake venom serine protease Dav-PAQ9I8X11Deinagkistrodon acutus
83 serine protease 53XP_0265769121Pseudonaja textilis
845’-nucleotidase family5’ nucleotidase, partialAXL952731Spilotes sulphureus
85Aminopeptidaseaminopeptidase N isoform X2XP_0265650374Pseudonaja textilis
86type-B carboxylesterase/lipase acetylcholinesterase XP_026549820 1Notechis scutatus
87Phosphodiesterase PhosphodiesteraseBAN894252Ovophis okinavensis
88 Phosphodiesterase partialALA208531Macropisthodon rudis
89 Phosphodiesterase partialAXL965991Borikenophis portoricensis
90 Ectonucleotide pyrophosphatase/phosphodiesterase family member 3 isoform X2XP_026561286 2Pseudonaja textilis
91 Snake venom PhosphodiesteraseA0A2D0TC042Naja atra
92Flavin monoamine oxidase familyL-amino acid oxidase, partialAVX276077Naja atra
93 L-amino-acid oxidaseXP_0265388304Notechis scutatus
94 L-amino-acid oxidaseQ4JHE31Oxyuranus scutellatus scutellatus
95 L-amino-acid oxidaseQ4JHE11Pseudechis australis
96 L-amino-acid oxidaseA8QL581Naja atra
97True venom lectin familyC-type lectin CalP219631Crotalus atrox
98Glutathione peroxidaseGlutathione peroxidase 3, partialETE688101Ophiophagus hannah
99 Glutathione peroxidase 3 isoform X1XP_0265419081Notechis scutatus
100 Glutathione peroxidase 3 isoform X1XP_0265524061Pseudonaja textilis
101Leucine repeatLeucine-rich repeat and death domain-containing protein 1XP_026543987 1Notechis scutatus
102TNF receptor superfamilyTumor necrosis factor receptor superfamily member 11BXP_026545353 1Notechis scutatus
103 Tumor necrosis factor receptor superfamily member 11BXP_026559377 1Pseudonaja textilis
104 Tumor necrosis factor receptor superfamily member 11BETE67452 1Ophiophagus hannah
105Intermediate filament familyKeratin, type II cytoskeletal 1, partiaETE671311Ophiophagus hannah
106 Keratin, type II cytoskeletal 4-likeXP_0265396581Notechis scutatus
107 Keratin, type II cytoskeletal 5, partialETE590391Ophiophagus hannah
108 Keratin, type II cytoskeletal 5, partialETE590382Ophiophagus hannah
109 Keratin, type II cytoskeletal 1-likeXP_0265731931Pseudonaja textilis
110 Keratin, type I cytoskeletal 19, partialETE702172Ophiophagus hannah
111 Keratin, type I cytoskeletal 18-like isoform X1XP_0265213021Notechis scutatus
112Serpin FamilySerpin B5, partialETE650021Ophiophagus hannah
113Ankyrin repeat domainM-phase phosphoprotein 8, partialETE736521Ophiophagus hannah
114Zinc finger containing proteinsZinc finger protein, partialETE623181Ophiophagus hannah
115 Zinc finger protein, partialETE623031Ophiophagus hannah
116 Zinc finger protein 804AXP_0265525051Pseudonaja textilis
117 Zinc finger SWIM domain-containing protein 6XP_0265728631Pseudonaja textilis
118 Zinc finger MYM-type protein 2 isoform X1XP_0265646701Pseudonaja textilis
119 Zinc finger BED domain-containing protein 1XP_0265226631Notechis scutatus
120NHS FamilyNHS-like protein 1 isoform X1XP_0265613481Pseudonaja textilis
121Protein family not assignedHolliday junction recognition proteinXP_0265197641Notechis scutatus
122 N-acetylgalactosaminyltransferase 7 isoform X1XP_0265554741Pseudonaja textilis
123 PHD finger protein 3XP_0265208991Notechis scutatus
124 Sulfhydryl oxidase 1(contains FAD binding domain)ETE70041 1Ophiophagus hannah
125 C-C chemokine receptor type 10, partialETE652161Ophiophagus hannah
126 Cytosolic carboxypeptidase 2XP_0265211451Notechis scutatus
127 SUMO-specific isopeptidase USPL1 isoform X1XP_0265646461Pseudonaja textilis
128 Protein VPRBPETE703811Ophiophagus hannah
129 Cilia- and flagella-associated protein 97XP_0265536671Pseudonaja textilis
130 lpxK, partialETE684461Ophiophagus hannah
131 Zinc phosphodiesterase ELAC protein 2, partialETE707771Ophiophagus hannah
132 NHS-like protein 1 isoform X1XP_0265613481Pseudonaja textilis
133 Pro-cathepsin HXP_0265651441Pseudonaja textilis
134 C4b-binding protein alpha chain-like isoform X1XP_0265713792Pseudonaja textilis
135 Janus kinase and microtubule-interacting protein 3 isoform X1XP_026566312 1Pseudonaja textilis
136 WD and tetratricopeptide repeats protein 1XP_0265583101Pseudonaja textilis
137 Pro-cathepsin HXP_0265651441Pseudonaja textilis
138 C4b-binding protein alpha chain-like isoform X1XP_0265713792Pseudonaja textilis
139 Janus kinase and microtubule-interacting protein 3 isoform X1XP_026566312 1Pseudonaja textilis
140 WD and tetratricopeptide repeats protein 1XP_0265583101Pseudonaja textilis
The bold text indicates the proteins identified to have N-terminal acetylation.
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Manuwar, A.; Dreyer, B.; Böhmert, A.; Ullah, A.; Mughal, Z.; Akrem, A.; Ali, S.A.; Schlüter, H.; Betzel, C. Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles. Toxins 2020, 12, 669. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12110669

AMA Style

Manuwar A, Dreyer B, Böhmert A, Ullah A, Mughal Z, Akrem A, Ali SA, Schlüter H, Betzel C. Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles. Toxins. 2020; 12(11):669. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12110669

Chicago/Turabian Style

Manuwar, Aisha, Benjamin Dreyer, Andreas Böhmert, Anwar Ullah, Zia Mughal, Ahmed Akrem, Syed Abid Ali, Hartmut Schlüter, and Christian Betzel. 2020. "Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles" Toxins 12, no. 11: 669. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12110669

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