Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2021 Dec 1;16(12):e0260496. doi: 10.1371/journal.pone.0260496

Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom

Siravit Sitprija 1, Lawan Chanhome 2,*, Onrapak Reamtong 3, Tipparat Thiangtrongjit 3, Taksa Vasaruchapong 2, Orawan Khow 4, Jureeporn Noiphrom 4, Panithi Laoungbua 2, Arissara Tubtimyoy 1, Narongsak Chaiyabutr 2,4, Supeecha Kumkate 1,*
Editor: Rafael Ximenes5
PMCID: PMC8635378  PMID: 34851989

Abstract

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper’s venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

Introduction

Envenomation from snakebites affects over 2.7 million people in tropical and subtropical countries each year, leading to more than 130,000 deaths among victims [1]. Severe injuries and complications from bites also can lead to permanent disabilities and long-term health problems in survivors. In addition, the majority of snakebite victims have been reported within the productive age of the workforce [2]. This creates socio-ecomomic loss as seen in developing countries of Asia and Africa [3].

Currently, there are at least six known species of the venomous Ovophis genus (Family Viperidae, Subfamily Crotalinae) existed according to the phylogenetic and morphological analyses. Five closely related species are distributed across several geographical areas of the Asian mainland [4]. Ovophis tonkinensis occurs in northern Vietnam and southern China; O. zayuensis in southern China (Yunnan), northeastern India and Myanmar; O. makazayazaya in southern China (Sichuan, Yunnan), Taiwan and northern Vietnam; O. convictus is restricted to western Malaysia; and O. monticola is found in Nepal, northeastern India [5], southern China, Myanmar, southern Laos, central Vietnam and northern Thailand [4, 6]. The other member of this genus, O. okinavensis, inhabits Ryukyu Island of Japan [7].

In Thailand, the Asian mountain pit viper O. monticola is found in high-altitude mountains, particularly in the northern province of Chiang Mai [6]. It has a stout body with a short snout. Its triangular head is covered by small, smooth scales rather than large shields. These vipers also exhibit sexual dimorphism in body size, with an average male length of 49 cm and female length of 110 cm. These montane, terrestrial, nocturnal vipers generally live under the forest litter and prey on small mammals [8].

The medical significance of pit viper envenomation primarily relates to the hematotoxic activity on human victims. Severe clinical manifestation includes local damage (e.g., painful oedema, tissue necrosis) and systemic injuries, including haemorrhage, coagulopathy and thrombocytopenia, critically resulting in high mortality and morbidity [5, 9].With advanced proteomic technology, the heterogeneity of snake venoms has progressively been elucidated. For the Ovophis spp., venomic profiles of O. convictus from western Malaysia, O. tonkinensis from northern Vietnam and southern China and Japanese hime habu O. okinavensis from Okinawa, Japan were recently reported. The abundance of four major enzymes namely snake venom serine proteinase (SVSP), phospholipases A2 (PLA2), L-amino acid oxidases (LAAO) and snake venom metalloproteases (SVMP) were dominant within all venoms. Among these enzymatic proteins, SVSP was found in the greatest proportion, accounting for 35–53% of all constituents. The second most abundant enzyme was PLA2 ranging from 19–26%. In addition, various non-enzymatic proteins and peptides including cysteine-rich secretory proteins (CRISP), venom nerve growth factor (VNGF), venom endothelial growth factor (VEGF), kunitz peptides (KUN) and C-type lectins/snaclecs (CTL) were recorded, in varying amounts [10]. However, variation in snake venom composition occurs not only among distinct species but also among different population of the same species, due to ecological niches as well as availability of preys [11].

The present study aims to investigate the protein constituents of venom from the Asian mountain pit viper O. monticola found in Thailand. In addition, since there is no homospecific antivenom to Ovophis spp. venoms currently available, the therapeutic regime for bite victims depends largely on two types of antivenom: pit viper monovalent antivenom, raised against white-lipped green pit viper (Trimeresurus albolabris) venom; and polyvalent antivenom, produced against hematotoxic venom of Calloselasma rhodostoma (Malayan pit viper), Daboia siamensis (Russell’s viper) and T. albolabris. Cross reactivity of O. monticola venom to these readily available antivenoms was therefore evaluated. Compositional profiles of immunoreactive versus non-reactive proteins in O. monticola venom were also clarified. Knowledge gained from this study not only extends the Ovophis spp. venomic database, but also can lead to better management and therapeutic approaches for mountain pit viper envenomation.

Materials and methods

Snakes, venom and antivenoms

All O. monticola pit vipers (Fig 1) were captured in the wild and transferred to Snake Farm, Queen Saovabha Memorial Institute (QSMI) before being quarantined. All procedures were performed following the safety protocol for working with venomous snakes (No. SN 001/2016). Routine snake care and the venom collection was conducted according to the specific protocol. All protocols were approved by the Ethic Committee of the Queen Saovabha Memorial Institute Animal Care and Use (No. QSMI-ACUC-02-2018) in accordance with the guideline of the National Research Council of Thailand. Information about individual snakes used in this study is shown in Table 1.

Fig 1. A wild juvenile Asian mountain pit viper (O. monticola) found in Northern Thailand.

Fig 1

Open in a new tab

The venom of O. monticola was extracted and kept in individual 1.5 ml microcentrifuge tubes. After weighing, the fresh (liquid) venom was immediately frozen at -20°C and lyophilized. The lyophilized venom was then pooled and stored at -20°C until use.

Table 1. Biological and geographical data for all snakes used in the study.

Speciesa Voucher no.b Sexc Snout-Vent Length (cm) Total Length (cm) Localityd
Ovophis monticola QSMI 1441 F 32.0 36.5 Omkoi-Chiang Mai
Ovophis monticola QSMI1443 F 32.0 36.5 Doi Pui-Chiang Mai
Ovophis monticola QSMI 1449 M 34.0 40.0 Omkoi-Chiang Mai
Ovophis monticola QSMI 1469 M 31.0 38.0 Omkoi-Chiang Mai
Ovophis monticola QSMI 1559 M 35.5 43.0 Omkoi-Chiang Mai
Open in a new tab

a The identification of Ovophis monticola was made by specialized veterinarians according to the identification key [8]. Key characters are body coloration and pattern: predominantly tan or reddish-grey with irregular short, black-edged crossbars or blotches along the vertebral ridge, including smaller irregular dark blotches on both sides of the body along the edges of the dorsal scales (Fig 1).

b Voucher no. was attached to each preserved snake after it died.

c Sex: F: Female; M: Male.

d District or subdistrict-province in Thailand where snakes were captured.

Monovalent antivenom against the green pit viper T. albolabris venom (batch no. TA00219; expiry date 08/10/2024) and hematotoxic polyvalent antivenoms (against the venom of C. rhodostoma, D. siamensis and T. albolabris) (batch no. HP 00118; expiry date 16/ 01/2023) produced by QSMI available as a freeze-dried F(ab’)2 form, isolated from horse immunoglobulins were used within their shelf-life. Following reconstitution, each milliliter of monovalent antivenom neutralized 0.7 mg of T. albolabris venom; one milliliter of hematotoxic polyvalent antivenom neutralized 0.7 mg of T. albolabris venom, 1.6 mg of C. rhodostoma venom and 0.6 mg of D. siamensis venom [12].

O. monticola venom preparation and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Crude venom of O. monticola was mixed with lysis buffer (containing of 1% Triton X-100 (Merck, Germany), 1% sodium dodecyl sulfate (SDS) (Merck, Germany), and 1% NaCl (Merck, Germany). The venom was estimated for protein concentration by Quick Start™ Bradford Protein Assay (Bio-Rad, USA). A 30 μg sample of O. monticola venom was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, USA) and stained by Coomassie R-250 solution (Bio-Rad, USA) as previously described [13]. The whole lane of venom was excised into 10 pieces and further subjected to in-gel digestion.

Two-dimensional polyacrylamide gel electrophoresis (2DE)

A 100 μg protein was mixed with IPG sample buffer containing 8 M urea, 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 15 mM dithiothreitol (DTT), and 0.5% IPG sample buffer [14]. Afterwards, the protein solution was rehydrated overnight into a non-linear immobilized pH gradient (IPG) strip (pH 3–10; Amersham Bioscience, USA). Isoelectric focusing (pI) was done using an Ettan IPGphorII instrument (Amersham Bioscience, USA) with the following settings: 30 V for 14 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 3500 V for 1 h, and 8000 V for 18 h. The IPG strips were equilibrated with DTT for 15 min and with iodoacetamine for 15 min. After incubation, the strips were placed onto a 12% SDS-PAGE gel. All three 2DE gels were stained with silver stain and the immunoreactive spots in these gels were excised and pooled for mass spectrometric analysis. Other two 2DE gels were used for immunoblotting.

In-gel digestion

A mixture of 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate was used for de-staining the blue color from gel slides [13]. Venom proteins were reduced by 4mM DTT and incubated at 60°C for 15 min. The reduced proteins were further alkylated by 250 mM iodoacetamine (IAA) (Sigma-Aldrich, USA) and incubated at room temperature for 30 min in dark. The gel pieces were dehydrated by removing all solution and adding 100% ACN (Thermo Scientific, USA). For tryptic digestion, trypsin (Sigma-Aldrich, USA, T6567) in 50 mM ammonium bicarbonate (Sigma-Aldrich, USA) was added to rehydrate the gels, which were then incubated overnight at 37 ˚C. Peptide extraction was performed by adding 100% ACN and incubating for 15 min. The resulting solution was transferred into a new microcentrifuge tube and dried using a centrifugal concentrator (TOMY, Japan). The peptide mixtures were stored at -20°C prior to mass spectrometric analysis.

Mass spectrometric analysis

Venom peptides were dissolved in 0.1% formic acid (Sigma-Aldrich, USA) and subjected to an Ultimate® 3000 Nano-LC systems (Thermo Scientific, USA). The peptides were eluted and infused to a microTOF-Q II (Bruker, Germany). The acquisition was operated by HyStar™ version 3.2 (Bruker, Germany), and the resulting data were processed and converted to mascot generics format (.mgf) files using Compass DataAnalysis™ software version 3.4 (Bruker, Germany). A database search was performed using Mascot Daemon software (Matrix Science, USA) against the NCBI snake database with the following parameters: one missed cleavage site, variable modifications of carbamidomethyl (C) and oxidation (M), 0.8 Da for MS peptide tolerance and 0.8 Da for MS/MS tolerance. The significance threshold was set at 95%. Three biological replications were performed for protein identification.

Indirect enzyme-linked immunosorbent assay (ELISA)

Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenom was assessed by indirect enzyme-linked immunosorbent assay (ELISA) modified from Gawtham and colleagues [15]. Each well of a 96-well Maxisorp Nunc immune plate (Thermo Fisher Scientific, Denmark) was coated with 5 ng of O. monticola venom in 0.05 M carbonate/bicarbonate buffer pH 9.6 (50 μl/well) and kept at 4°C overnight. Plates were washed three times with phosphate-buffered saline (PBS) pH 7.2, blocked by adding 200 μL of PBS containing 2% (w/v) bovine serum albumin (BSA) (Capricorn Scientific GmBH, Ebsdorfergrund, Germany) and incubated for 1.5 h at 37°C. The plates were then washed three times with PBS-0.05% Tween (PBST). They were incubated again for 1 h at 37°C with 50 μL of the serial dilution of either monovalent or polyvalent antivenom (1:105−1:5x107 in 0.2% BSA-PBS). After washing the plate three times with PBST, 50 μL of horseradish peroxidase-conjugated goat anti-horse-IgG (Abcam, Cambridge, UK) in PBST (1:1000) was added into each well and further incubated for another hour at 37°C. Plates were then washed three times with PBST. Fifty microliters of substrate solution (SureBlue TMB microwell peroxidase, Seracare Life Sciences, Milford, MA) was subsequently added to each well, and the plate was kept in the dark for 10 min at room temperature for the reaction to occur. The absorbance at 630 nm was read using a microplate reader (TECAN InfinitePro 200, Switzerland).

Immunoblot analysis

The separated polypeptide spots from 2DE gels were transferred to nitrocellulose membrane for 90 min at 18 V on a Trans-blot semi-dry Transfer CellTM (Biorad) in semi-dry transfer buffer (48 mM Tris and 2.93 g glycine) pH 9.2 containing 20% methanol. The membranes were blocked using 5% (w/v) non-fat milk in PBS for 2 h at room temperature. The membranes were rinsed twice with PBS-T buffer pH 7.4 (8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM NaCl, 2.7 mM KCl and 0.5% v/v Tween) for 30 s each. The blotted membranes were incubated with either monovalent or polyvalent antivenom (1:1000 in 0.2% BSA-PBS). After washing the membrane three times with PBS-T, 50 μL of horseradish peroxidase-conjugated goat anti-horse-IgG (Abcam, Cambridge, UK) in PBS-T (1:2000) was added, and the mixture was incubated for 1 h at ambient temperature under constant agitation. Membranes were washed three times with PBS-T buffer and one time with PBS. Immunogen spots were visualized by detection of peroxidase activity using Ultra TMB-Blotting Solution (ThermoFisher Scientific, UK).

Statistical analysis

Quantitative data are presented as mean ± SEM. Statistical significance between groups was analyzed using standard t-tests or two-way ANOVA followed by the Bonferroni test. Significant p-values are indicated within the figure panels. Error bars indicate SEM.

Results

Proteomic analysis of O. monticola venom

Detectable proteins in venom of O. monticola were between 10–95 kDa (Fig 2A). Intense protein bands at 10, 15, 50 and 72 kDa and faint bands at 26, 28, 30, 34 and 95 kDa were recorded. There were 247 proteins found in O. monticola venom (a list of all proteins is shown in S1 Table). A classification of constituent proteins based on their biological properties is presented in Fig 2B. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant groups included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%) C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). Toxin biosynthesis and other proteins comprised 0.4%. The top 15 unique proteins identified in O. monticola venom are shown in Table 2.

Fig 2. Venomics of Asian mountain pit viper O. monticola from Thailand.

Fig 2

Open in a new tab

(A) Coomassie blue-stained 12% SDS-PAGE of O. monticola venom (30 μg) under reducing conditions. (B) Proteome classification of O. monticola venom; percentages indicate relative abundance (% of total venom proteins) of protein family in snake venom.

Table 2. Fifteen most abundant unique proteins identified in Ovophis monticola venom.

No. Accession no. Protein Score MWa (Da) No. of peptide % Sequence coverage pIb emPAIc
1 sp|P0C2D5.2|OXLAPROFL L-amino-acid oxidase 534 3601 2 80 4.44 14.55
2 sp|O93517.1|VM3S4GLOBR Zinc metalloproteinase/disintegrin 1293 11254 5 48.6 4.42 5.18
3 sp|Q9PRP4.1|VSPFLACMR Thrombin-like enzyme LMR-47 481 3168 2 100 4.31 3.82
4 sp|P0C590.1|VSP2GLOUS Thrombin-like enzyme calobin-2 481 2159 2 100 4.65 3.81
5 sp|C0HLA2.1|VSP3LACMR Thrombin-like enzyme LmrSP-3 335 2942 1 50 4.1 2.22
6 sp|P81478.1|PA2A2TRIGA Acidic phospholipase A2 2 621 13784 4 33.6 4.95 1.73
7 sp|C0HLA3.1|VSP4LACMR Snake venom serine protease LmrSP-4 1090 5841 2 62.3 4.28 1.60
8 sp|Q90W54.1|OXLA_GLOBL L-amino-acid oxidase 2334 57056 19 32.7 6.52 1.37
9 BAA01566.1 Phospholipase A2 621 15697 3 40.6 4.99 1.35
10 pdb|1WVR|A Chain A, Triflin 1099 24782 3 13.6 7.03 1.15
11 sp|Q7ZT99.1|CRVPCROAT Cysteine-rich venom protein catrin 1099 26629 3 19.2 8.42 1.04
12 sp|Q7ZTA0.1|CRVPAGKPI Cysteine-rich venom protein piscivorin 1099 26664 3 26.3 7.83 1.04
13 sp|E5L0E5.1|VSPPAAGKPL Venom plasminogen activator 558 28060 5 12.8 5.78 1.02
14 AAM80563.1 Acidic phospholipase A2 372 15403 3 18.1 5.65 0.95
15 sp|P82896.1|PA2A5TRIST Acidic phospholipase A2 5 427 13870 2 32.8 4.72 0.93
Open in a new tab

aMW: Molecular weight (Dalton).

bpI: isoelectric point.

cemPAI: exponentially modified protein abundance index.

The information of identified proteins including NCBI accession number (Accession no.), protein name (Protein), protein score (Score), molecular weight of protein in Dalton unit (Da), Number of identified peptides (No. of peptide), % sequence coverage of the identified peptides (%Sequence coverage), isoelectric point of protein (pI) and exponentially modified protein abundance index for semi-quantification (emPAI) are demonstrated.

Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenoms by indirect ELISA

Since there is no homospecific antivenom to Ovophis spp. venoms currently available, all pit viper envenoming victims are recommended to receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) to alleviate symptoms [16]. Indirect ELISA was used to determine the cross-reactivity of these antivenoms to O. monticola venom. Hematotoxic polyvalent antivenom exhibited a significantly greater level of immunoreactivity than the monovalent antivenom by 30–50% (up to the dilution 1: 1.6x106), P<0.001 (Fig 3). A titer for the monovalent antivenom was over 1:1.28x107 dilution, while that of hematotoxic polyvalent antivenom was 1:5.12x107 (Fig 3).

Fig 3. Immunoreactivity of O. monticola venom to antivenoms.

Fig 3

Open in a new tab

Cross-reactivity of crude O. monticola venom to monovalent antivenom raised against green pit viper venom and polyvalent antivenom against snake hemotoxins. Data represent the mean ± SEM from two independent experiments; * P < 0.05, ** P < 0.01 and *** P < 0.001.

O. monticola venom protein analysis by two-dimensional electrophoresis (2DE)

In order to explore the protein antigens present in O. monticola venom, crude venom was subjected to 2DE gel electrophoresis. There were 89 spots detected, with pI values ranging from 3 to 10 and MW from 10 to 95 kDa. Within the particular MW regions of 10–15, 30–34, 50 and 72–90 lies the greatest abundance of protein spots (Fig 4A). Using MALDI-TOF/TOF-MS/MS, all protein spots in 2DE gels were identified, and are listed in S2 Table. There were 461 different sequences, which correspond to 173 peptide accession identities.

Fig 4. The 2DE separations and immunoblot analysis of O. monticola venom.

Fig 4

Open in a new tab

(A) 2DE gels stained with silver stain; (B) 2D immunoblot of O. monticola proteins probed with monovalent antivenom and (C) polyvalent antivenom. Matched spots selected for subsequent LC-MS/MS analysis are marked and numbered.

Immunoreactive proteins in O. monticola venom by immunoblot analysis

The immunoblot analysis was performed with either monovalent or polyvalent antivenom to characterize specific protein antigens within the O. monticola venom. Twenty-six immunoreactive spots were detected with monovalent antivenom, with pI values ranging from 3 to 6, and MW ranging from 17 to 95 kDa. Most of these spots were observed at MW 50 to 95 kDa and pI between 3 to 5 (Fig 4B). When probed with hematotoxic polyvalent antivenom, 40 immunoreactive spots were recorded with a broader range of pI values from 3 to 8, and MW ranging from 17 to 95 kDa. A high number of the spots were detected within a MW range of 40–55 kDa (Fig 4C). Comparing the immunoreactive spots obtained from polyvalent antivenom with all protein spots visualized by silver staining (Fig 4A), 49 spots (numbered 24–25, 31–32, 38–40, 42–45, 48–51, 54–73 and 76–89) were not immunologically recognized. These non-reactive spots were grouped according to their MW and pI values into three clusters. Cluster 1 appeared in the MW range from 26 to 43 kDa, with high pI values (7–8); cluster 2 included those with MW ranging from 26 to 43 and with low pI values (3–5); and cluster 3 contained those with low MW from 10 to 26 kDa and with low pI values (3–5).

Identification of immunoreactive and non-reactive peptides in O. monticola venom by LC-MS/MS

LC-MS/MS analysis revealed a total of 202 distinct sequences in O. monticola venom identified within 101 protein types that were immunologically reactive with a polyvenom. All immunoreactive proteins recognized by monovalent and polyvalent antivenom are listed in Table 3. However, from all 173 protein types appearing by silver staining, 72 proteins were left unrecognized by both antivenoms, and are shown in Table 4. Interestingly, the majority of these neglected peptides are well-known members of CTLs (e.g., C-type lectin, snaclec and galactose-binding lectin) and different CRISPs including okinavin, catrin and piscivorin.

Table 3. List of identified proteins in Ovophis monticola venom immunologically reactive with monovalent and polyvalent antivenoms.

Spot no. Protein/peptide accession Description [Organisms] MW (Da) Monovalent Polyvalent
1 XP_029142019.1 Zinc metalloproteinase-disintegrin-like atrolysin-A, partial [Protobothrops mucrosquamatus] 60272 √ √
  JAS04843.1 Metalloproteinase type III 2b [Crotalus horridus] 68297    
  JAS04684.1 Metalloproteinase type III 1b [Crotalus adamanteus] 67284    
  AAA03326.1 Hemorrhagic toxin a (partial)[Crotalus atrox] 46848    
  GBP06242.1 Disintegrin and metalloproteinase domain-containing protein 12 [Eumeta japonica] 199170    
2 XP_029142019.1 Zinc metalloproteinase-disintegrin-like atrolysin-A, partial [Protobothrops mucrosquamatus] 60272 √ √
  JAS04843.1 Metalloproteinase type III 2b [Crotalus horridus] 68297    
  JAS04684.1 Metalloproteinase type III 1b [Crotalus adamanteus] 67284    
3 sp|Q4VM07.1|VM3VB_MACLB Zinc metalloproteinase-disintegrin-like VLAIP-B (Snake venom metalloproteinase) 68798 √ √
  JAS04447.1 Metalloproteinase type III 7 [Agkistrodon piscivorus conanti] 68638    
  sp|P0DM87.1|VM2_TRIST Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase) 54401    
4 JAS04447.1 Metalloproteinase type III 7 [Agkistrodon piscivorus conanti] 68638 √ √
  sp|Q4VM07.1|VM3VB_MACLB Zinc metalloproteinase-disintegrin-like VLAIP-B 68798    
5 sp|Q4VM07.1|VM3VB_MACLB Zinc metalloproteinase-disintegrin-like VLAIP-B (Snake venom metalloproteinase) 68798 √ √
  sp|P0DM87.1|VM2_TRIST Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase) 54401    
  JAS04675.1 Metalloproteinase type III 5 [Crotalus adamanteus] 69463    
  JAS04447.1 Metalloproteinase type III 7 [Agkistrodon piscivorus conanti] 68638    
  XP_023086434.2 disintegrin and metalloproteinase domain-containing protein 20-like [Piliocolobus tephrosceles] 84212    
6 sp|Q4VM07.1|VM3VB_MACLB Zinc metalloproteinase-disintegrin-like VLAIP-B (Snake venom metalloproteinase) 68798 √ √
  JAS04447.1 Metalloproteinase type III 7 [Agkistrodon piscivorus conanti] 68638    
  sp|P0DM87.1|VM2_TRIST Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase) 54401    
7 - Not identified √ √
8 pdb|1REO|A Chain A, Ahplaao 55097 √ √
  AAQ16182.1 L-amino acid oxidase [Trimeresurus stejnegeri] 58607    
9 pdb|1REO|A Chain A, Ahplaao 55097 √ √
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
10 sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341 √ √
11 pdb|1REO|A Chain A, Ahplaao 55097 - √
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
  sp|P0C2D5.2|OXLA_PROFL L-amino-acid oxidase (Okinawa Habu apoxin protein-1) 3601    
  sp|P0C2D6.1|OXLA_PROMU L-amino-acid oxidase 2929    
12 pdb|1REO|A Chain A, Ahplaao 55097 - √
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
  BAP39915.1 L-amino acid oxidase [Protobothrops elegans] 57339    
  sp|P0DI84.1|OXLA_VIPAA L-amino-acid oxidase 54714    
  sp|C0HJE7.2|OXLA_CRODU L-amino acid oxidase bordonein-L 58882    
  sp|Q4F867.2|OXLA_DABSI L-amino-acid oxidase 46343    
  sp|X2JCV5.1|OXLAA_CERCE L-amino acid oxidase 58520    
  sp|A8QL51.1|OXLA_BUNMU L-amino-acid oxidase 58774    
  sp|P0C2D5.2|OXLA_PROFL L-amino-acid oxidase (Okinawa Habu apoxin protein-1) 3601    
  sp|A0A2U8QPE6.1|OXLA_MICMP L-amino acid oxidase 57079    
  XP_026523888.1 titin isoform X41 [Notechis scutatus] 3637718    
13 pdb|1REO|A Chain A, Ahplaao 55097 - √
  JAS04783.1 L-amino acid oxidase 1b [Crotalus horridus] 58587    
  sp|P0DI84.1|OXLA_VIPAA L-amino-acid oxidase 54714    
  BAP39915.1 L-amino acid oxidase [Protobothrops elegans] 57339    
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
  sp|C0HJE7.2|OXLA_CRODU L-amino acid oxidase bordonein-L 58882    
  JAV01888.1 BATXLAAO1 [Bothrops atrox] 56625    
  sp|Q4F867.2|OXLA_DABSI L-amino-acid oxidase 46343    
  sp|P0C2D5.2|OXLA_PROFL L-amino-acid oxidase (Okinawa Habu apoxin protein-1) 3601    
  sp|X2JCV5.1|OXLAA_CERCE L-amino acid oxidase 58520    
  sp|A0A2U8QPE6.1|OXLA_MICMP L-amino acid oxidase 57079    
14 pdb|1REO|A Chain A, Ahplaao 55097 - √
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
  sp|P0DI84.1|OXLA_VIPAA L-amino-acid oxidase 54714    
  sp|C0HJE7.2|OXLA_CRODU L-amino acid oxidase bordonein-L 58882    
  sp|P0C2D5.2|OXLA_PROFL L-amino-acid oxidase (Okinawa Habu apoxin protein-1) 3601    
  JAV01888.1 BATXLAAO1 [Bothrops atrox] 56625    
  sp|A0A2U8QPE6.1|OXLA_MICMP L-amino acid oxidase 57079    
15 pdb|1REO|A Chain A, Ahplaao 55097 - √
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
  sp|A0A2U8QPE6.1|OXLA_MICMP L-amino acid oxidase 57079    
  XP_026523846.1 Titin isoform X1 [Notechis scutatus] 3675875    
16 pdb|1REO|A Chain A, Ahplaao 55097 - √
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
17 pdb|1REO|A Chain A, Ahplaao 55097 √ √
  JAV01888.1 BATXLAAO1 [Bothrops atrox] 56625    
18 pdb|1REO|A Chain A, Ahplaao 55097 √ √
  AAQ16182.1 L-amino acid oxidase [Trimeresurus stejnegeri] 58607    
  sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341    
  sp|P0DI84.1|OXLA_VIPAA L-amino-acid oxidase 54714    
  JAV01888.1 BATXLAAO1 [Bothrops atrox] 56625    
  sp|A0A2U8QPE6.1|OXLA_MICMP L-amino acid oxidase 57079    
  sp|A8QL51.1|OXLA_BUNMU L-amino-acid oxidase 58774    
  sp|P0C2D5.2|OXLA_PROFL L-amino-acid oxidase (Okinawa Habu apoxin protein-1) 3601    
  XP_026523888.1 Titin isoform X41 [Notechis scutatus] 3637718    
19 sp|A0A024BTN9.1|OXLA_BOTSC L-amino acid oxidase Bs29 56341 √ √
20 BAN82126.1 Serine protease, partial [Ovophis okinavensis] 9035 - √
  JAV51428.1 Serine proteinase 12a [Agkistrodon contortrix contortrix] 28885    
  XP_026529526.1 Microtubule-actin cross-linking factor 1 isoform X1 [Notechis scutatus] 838459    
21 sp|P0C578.1|VSP2_OVOOK Thrombin-like enzyme okinaxobin-2 (Fibrinogen-clotting enzyme) 2310 - √
  JAV51428.1 Serine proteinase 12a [Agkistrodon contortrix contortrix] 28885    
  sp|I2C090.1|VCO3_OPHHA Ophiophagus venom factor (Complement C3 homolog) 183812    
  XP_026526061.1 ALK and LTK ligand 1 [Notechis scutatus] 21543    
  sp|P85109.1|VSP1_GLOBR Thrombin-like enzyme kangshuanmei (Fibrinogen-clotting enzyme) 26415    
  JAG68112.1 Dynamin-binding protein [Boiga irregularis] 90258    
22 JAV51428.1 Serine proteinase 12a [Agkistrodon contortrix contortrix] 28885 - √
  BAN82126.1 serine protease, partial [Ovophis okinavensis] 9035    
  sp|E5L0E5.1|VSPPA_AGKPL Venom plasminogen activator 28060    
  sp|Q5W958.1|VSP20_BOTJA Venom serine proteinase-like HS120 27797    
23 BAN82126.1 Serine protease, partial [Ovophis okinavensis] 9035 √ √
  sp|Q9PSN3.1|VSP2_AGKBI Thrombin-like enzyme bilineobin (Fibrinogen-clotting enzyme/Snake venom serine protease) 26461    
  BAN82122.1 Serine protease, partial [Ovophis okinavensis] 8080    
  pdb|2AIP|A Chain A, Protein C activator 25090    
  sp|C0HLA2.1|VSP3_LACMR Thrombin-like enzyme LmrSP-3 2942    
  ADI47563.1 Serine protease, partial [Echis ocellatus] 27233    
  sp|P0C5B4.2|VSPGL_GLOSH Thrombin-like enzyme gloshedobin(Fibrinogen-clotting enzyme/Snake venom serine protease) 28597    
  sp|Q9DF66.1|VSP3_PROJR Snake venom serine protease 3 28007    
  pdb|1OP0|A Chain A, Venom serine proteinase 25318    
26 JAV51428.1 Serine proteinase 12a [Agkistrodon contortrix contortrix] 28885 √ √
27 XP_029142018.1 Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus] 58843 √ √
  TSK34762.1 Disintegrin and metalloproteinase domain-containing protein 12 [Bagarius yarrelli] 146595    
  XP_032089254.1 ras GTPase-activating-like protein IQGAP1 [Thamnophis elegans] 189690    
28 XP_029142018.1 Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus] 58843 √ √
  sp|P0DM87.1|VM2_TRIST Zinc metalloproteinase-disintegrin stejnitin 54401    
  TSK34762.1 Disintegrin and metalloproteinase domain-containing protein 12 [Bagarius yarrelli] 146595    
29 XP_029142018.1 Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus] 58843    
  sp|P0DM87.1|VM2_TRIST Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase) 54401 √ √
  TSK34762.1 Disintegrin and metalloproteinase domain-containing protein 12 [Bagarius yarrelli] 215963    
  ETE65365.1 putative helicase senataxin, partial [Ophiophagus hannah] 146595    
30 XP_029142018.1 Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus] 58843 √ √
  sp|P0DM87.1|VM2_TRIST Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase) 54401    
31 XP_029142018.1 Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus] 58843 - √
34 JAS05371.1 Serine proteinase 9d [Sistrurus miliarius barbouri] 28266 √ √
  sp|P0DMH6.1|VSP_BOTFO Snake venom serine protease 1729    
  sp|Q8AY78.1|VSP5M_TRIST Snake venom serine protease 5 28117    
  sp|Q8AY79.1|VSPS2_TRIST Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease) 28010    
  sp|Q5W958.1|VSP20_BOTJA Venom serine proteinase-like HS120 27797    
  sp|Q71QH7.1|VSPP_TRIST Snake venom serine protease PA 27933    
  XP_026540424.1 Inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinase 1 isoform X1 [Notechis scutatus] 135476    
35 sp|P0DMH6.1|VSP_BOTFO Snake venom serine protease 1729 √ √
  sp|E5L0E5.1|VSPPA_AGKPL Venom plasminogen activator 28060    
  sp|Q8AY78.1|VSP5M_TRIST Snake venom serine protease 5 28117    
  sp|Q5W958.1|VSP20_BOTJA Venom serine proteinase-like HS120 27797    
  sp|Q71QH7.1|VSPP_TRIST Snake venom serine protease PA 27933    
  sp|Q8AY79.1|VSPS2_TRIST Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease) 28010    
36 JAS05372.1 Serine proteinase 9c [Sistrurus miliarius barbouri] 28221 √ √
  JAS05371.1 Serine proteinase 9d [Sistrurus miliarius barbouri] 28266    
  JAV51414.1 Serine proteinase 8 [Agkistrodon contortrix contortrix] 28242    
  sp|P0DMH6.1|VSP_BOTFO Snake venom serine protease 1729    
  sp|P0C5B4.2|VSPGL_GLOSH Thrombin-like enzyme gloshedobin (Fibrinogen-clotting enzyme/Snake venom serine protease) 28597    
  ADI47574.1 Serine protease, partial [Echis coloratus] 28437    
  sp|Q8AY78.1|VSP5M_TRIST Snake venom serine protease 5 28117    
  sp|Q5W958.1|VSP20_BOTJA Venom serine proteinase-like HS120 27797    
  sp|Q8AY79.1|VSPS2_TRIST Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease) 28010    
  sp|Q8UUJ2.2|VSPUI_GLOUS Snake venom serine protease ussurin; 26184    
  sp|Q71QH7.1|VSPP_TRIST Snake venom serine protease PA 27933    
  XP_032092228.1 Vitelline membrane outer layer protein 1 homolog isoform X1 [Thamnophis elegans] 21236    
  JAI10638.1 Vacuolar protein sorting-associated protein 18 homolog [Crotalus adamanteus] 111967    
37 JAS05372.1 Serine proteinase 9c [Sistrurus miliarius barbouri] 28221 - √
  JAS05371.1 Serine proteinase 9d [Sistrurus miliarius barbouri] 28266    
  sp|P0DMH6.1|VSP_BOTFO Snake venom serine protease 1729    
  JAV51414.1 Serine proteinase 8 [Agkistrodon contortrix contortrix] 28242    
  sp|Q9PT41.1|VSPF5_MACLB Factor V activator (Lebetina viper venom FV activator/Snake venom serine protease 28577    
  ADI47574.1 Serine protease, partial [Echis coloratus] 28437    
  sp|Q8AY78.1|VSP5M_TRIST Snake venom serine protease 5; 28117    
  XP_023086434.2 Disintegrin and metalloproteinase domain-containing protein 20-like [Piliocolobus tephrosceles] 84212    
  sp|Q8AY79.1|VSPS2_TRIST Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease) 28010    
41 - Not identified - √
46 pdb|1BQY|A Chain A, Plasminogen Activator 25590 √ √
  JAS04757.1 Serine proteinase 1f [Crotalus horridus] 28133    
  JAS04429.1 Serine proteinase 13e [Agkistrodon piscivorus conanti] 27985    
  JAS04417.1 Serine proteinase 18b [Agkistrodon piscivorus conanti] 27728    
  JAS04415.1 Serine proteinase 19b [Agkistrodon piscivorus conanti] 27782    
  sp|Q072L7.1|VSP_LACST Snake venom serine protease 27796    
  sp|O13069.1|VSP2_BOTJA Thrombin-like enzyme KN-BJ 2 (Kinin-releasing and fibrinogen-clotting serine protease 2) 26399    
  pdb|4E7N|A Chain A, Snake-venom Thrombin-like Enzyme 28333    
  XP_032089049.1 Spectrin alpha chain, non-erythrocytic 1 [Thamnophis elegans] 263010    
  XP_032064352.1 Zinc finger protein 347-like [Thamnophis elegans] 169270    
  sp|Q9PT41.1|VSPF5_MACLB Factor V activator/Lebetina viper venom FV activatorSnake venom serine protease 28577    
  ADI47574.1 Serine protease, partial [Echis coloratus] 28437    
47 JAS04757.1 Serine proteinase 1f [Crotalus horridus] 28133 √ √
  pdb|1BQY|A Chain A, Plasminogen Activator 25590    
  JAS04417.1 Serine proteinase 18b [Agkistrodon piscivorus conanti] 27728    
  JAS04429.1 Serine proteinase 13e [Agkistrodon piscivorus conanti] 27985    
  JAS04415.1 Serine proteinase 19b [Agkistrodon piscivorus conanti] 27782    
  JAV01826.1 BATXSVSP10 [Bothrops atrox] 28606    
  pdb|4E7N|A Chain A, Snake-venom Thrombin-like Enzyme 26370    
  sp|Q6T5L0.2|VSPSH_GLOSH Alpha-fibrinogenase shedaoenase (Snake venom serine protease) 26399    
  sp|O13069.1|VSP2_BOTJA Thrombin-like enzyme KN-BJ 2 (Kinin-releasing and fibrinogen-clotting serine protease 2) 27876    
  sp|Q71QI0.1|VSP07_TRIST Snake venom serine protease homolog KN7 28703    
  XP_015671556.1 Snake venom serine protease [Protobothrops mucrosquamatus] 28023    
  JAS04671.1 Serine proteinase 3b [Crotalus adamanteus] 28890    
  QHR82809.1 Serine protease 2 [Vipera anatolica senliki] 28084    
  sp|A8QL53.1|VSP1_NAJAT Snake venom serine protease NaSP 31117    
  XP_026523831.1 Integrin alpha-4 [Notechis scutatus] 114850    
52 JAS05359.1 Cysteine-rich secretory protein 1c [Sistrurus tergeminus] 26787 - √
53 ETE67131.1 Keratin, type II cytoskeletal 1, partial [Ophiophagus hannah] 240496 - √
74 sp|A8E2V8.1|PA2A_TRIGS Acidic phospholipase A2 Tgc-E6 15678 √ √
  sp|P0DJJ7.1|PA2A_OVOMO Acidic phospholipase A2 Omo-E6 3261    
  JAV51451.1 Phospholipase A2 1a [Agkistrodon contortrix contortrix] 15952    
  sp|Q6EAN6.1|PA2A_SISTE Acidic phospholipase A2 homolog sistruxin APrecursor 15419    
  XP_032088152.1 Group IIE secretory phospholipase A2-like [Thamnophis elegans] 17310    
  sp|Q7ZTA6.1|PA2AB_CROVV Acidic phospholipase A2 Cvv-E6b 15429    
  AAB28455.1 Phospholipase A2 isozyme III, PLA2-III [Trimeresurus gramineus] 13716    
  JAV01879.1 BATXPLA5 [Bothrops atrox] 15504    
  sp|P06860.1|PA2BX_PROFL Basic phospholipase A2 PL-X 13971    
  AAB28454.1 Phospholipase A2 isozyme IV, PLA2-IV [1] [Trimeresurus gramineus] 13783    
  sp|C0HJC1.1|PA2_BOTLA Acidic phospholipase A2 BlatPLA2 13881    
75 sp|A8E2V8.1|PA2A_TRIGS Acidic phospholipase A2 Tgc-E6 15678 √ √
  sp|P0DJJ7.1|PA2A_OVOMO Acidic phospholipase A2 Omo-E6 3261    
  pdb|1C1J|A Chain A, Basic phospholipase A2 13888    
  JAS04499.1 Phospholipase A2 1s [Agkistrodon piscivorus conanti] 15776    
  sp|P82896.1|PA2A5_TRIST Acidic phospholipase A2 5 13870    
  sp|D6MKR0.1|PA2A6_CROHD Acidic phospholipase A2 CH-E6 15498    
  sp|Q7ZTA6.1|PA2AB_CROVV Acidic phospholipase A2 Cvv-E6b 15429    
  JAV51451.1 Phospholipase A2 1a [Agkistrodon contortrix contortrix] 15952    
  sp|P86907.1|PA2A_BOTAM Acidic phospholipase A2 13858    
  sp|C9DPL5.1|PA2A1_BOTPI Acidic phospholipase A2 BpirPLA2-I 13627    
  sp|C0HLF0.1|PA2_POROP Basic phospholipase A2 14042    
  sp|C0HJC1.1|PA2_BOTLA Acidic phospholipase A2 BlatPLA2 13881    
  sp|P86456.1|PA2A4_BOTAL Acidic phospholipase A2 SpII RP4 13733    
  QHR82796.1 Phospholipase A2 3 [Vipera anatolica senliki] 17437    
Open in a new tab

Table 4. List of non-immunoreactive proteins/peptides in Ovophis monticola venom.

  Protein/peptide accession Description [Organisms] MW (Da) Spot no.
1 AAZ75628.1 Kallikrein-Phi4, partial [Philodryas olfersii] 26827 45, 49, 50
2 BAN82001.1 Galactose binding lectin, partial [Protobothrops flavoviridis] 17654 80, 82, 84, 85
3 BAN82034.1 Serine protease, partial [Protobothrops flavoviridis] 22377 55
4 BAN82147.1 Cysteine rich secretory protein [Ovophis okinavensis] 26920 59, 60, 61, 62
5 BAN82148.1 Galactose binding lectin [Ovophis okinavensis] 18480 80, 82
6 BAN82149.1 C-type lectin alpha subunit [Ovophis okinavensis] 17686 87, 88
7 ETE59238.1 Fascin-3, partial [Ophiophagus hannah] 14941 48
8 ETE60526.1 Trichohyalin, partial [Ophiophagus hannah] 80507 51
9 ETE61374.1 Dynein heavy chain 8, axonemal [Ophiophagus hannah] 284552 72
10 ETE64295.1 Glycerol-3-phosphate acyltransferase 4 [Ophiophagus hannah] 50772 86
11 ETE66458.1 Helicase SRCAP, partial [Ophiophagus hannah] 494261 87
12 ETE70787.1 N6-adenosine-methyltransferase 70 kDa subunit, partial [Ophiophagus hannah] 59797 89
13 JAI12774.1 Leucine-rich repeat-containing protein 7-like [Crotalus adamanteus] 163679 45
14 JAS04407.1 Serine proteinase 6 [Agkistrodon piscivorus conanti] 28115 43,44, 48, 76
15 JAS04411.1 Serine proteinase 2 [Agkistrodon piscivorus conanti] 28333 49, 50, 55, 57,
16 JAS04568.1 Phospholipase A2 1b [Boiga irregularis] 16906 67, 72, 73, 78
17 JAS04670.1 Serine proteinase 3c [Crotalus adamanteus] 28849 45, 49, 50, 54, 55, 56, 57, 58
18 JAS04734.1 Cysteine-rich secretory protein [Crotalus adamanteus] 26612 59, 60, 61, 62
19 JAS04742.1 Serine proteinase 9d [Crotalus horridus] 28299 49
20 JAS04748.1 Serine proteinase 6 [Crotalus horridus] 28594 54, 55, 56, 57
21 JAS05249.1 Serine proteinase 2 [Sistrurus tergeminus] 28326 49
22 JAS05472.1 C-type lectin 2 [Sistrurus miliarius barbouri] 18147 87
23 JAS05484.1 Cysteine-rich secretory protein 1b [Sistrurus miliarius barbouri] 26772 61
24 JAV51425.1 Serine proteinase 15a [Agkistrodon contortrix contortrix] 28940 54, 55, 56, 57
25 JAV51455.1 C-type lectin 9a [Agkistrodon contortrix contortrix] 18657 80
26 pdb|1BK9|A Chain A, Phospholipase A2 13964 72
27 pdb|1GMZ|A Chain A, Phospholipase A2 13850 67
28 pdb|1JZN|A Chain A, Galactose-specific lectin 16281 80, 82
29 pdb|1WVR|A Chain A, Triflin 24782 59, 60, 61, 62
30 pdb|3JR8|A Chain A, Phospholipase A2 bothropstoxin-2 13985 67
31 sp|A0A1I9KNP0.1|VSPH1_VIPAA Vaa serine proteinase homolog 1 28909 49
32 sp|A8QL56.1|VSP1_OPHHA Alpha- and beta-fibrinogenase OhS1 28637 45, 49, 50
33 sp|B0VXW0.1|OXLA_SISCA L-amino-acid oxidase 58532 77
34 sp|B0ZT25.1|VSPH_PROJR Snake venom serine protease homolog 28776 58
35 sp|C0HLA1.1|VSP2_LACMR Thrombin-like enzyme LmrSP-2 (Snake venom serine protease) 3271 25, 32
36 sp|E5AJX2.1|VSP_VIPBN Snake venom serine protease nikobin 28197 45, 49
37 sp|J3S832.1|VSPB_CROAD Snake venom serine proteinase 11 28033 58
38 sp|J3S833.1|VSP2_CROAD Snake venom serine proteinase 2 28298 49
39 sp|K4LLQ2.1|VSP_BOTBA Thrombin-like enzyme barnettobin (Snake venom serine protease) 27567 38, 39
40 sp|O13057.1|VSP2_PROFL Snake venom serine protease 2 28623 56
41 sp|O93421.2|VSPPE_GLOHA Snake venom serine protease pallase 26031 49
42 sp|P0DJG8.1|CRVP_HELAG Helicopsin 2618 61
43 sp|P0DL18.1|CRVP_OVOOK Cysteine-rich venom protein okinavin 3496 59, 60, 61
44 sp|P0DM36.1|LECG_AGKPI C-type lectin APL 16195 80, 82, 84, 85
45 sp|P81114.1|SLA4_TRIAB Snaclec alboaggregin-A subunit beta 14357 81, 83, 86
46 sp|P81176.1|VSP1_GLOBL Thrombin-like enzyme halystase (Snake venom serine protease) 26466 49
47 sp|P82981.1|VSP2_AGKCO Thrombin-like enzyme contortrixobin/Fibrinogen-clotting enzyme (Snake venom serine protease) 25396 54, 55, 56, 57, 58
48 sp|Q27J47.1|VSPPA_LACMU Venom plasminogen activator LV-PA 28044 44
49 sp|Q71QJ4.1|VSP04_TRIST Snake venom serine protease homolog KN4 28685 49, 50, 55, 56, 57
50 sp|Q7SZE2.1|VSPD_GLOUS Bradykinin-releasing enzyme KR-E-1 (Snake venom serine protease) 25335 49
51 sp|Q7T229.1|VSPH_BOTJR Snake venom serine protease homolog 28636 54, 55, 56, 57
52 sp|Q7ZT99.1|CRVP_CROAT Cysteine-rich venom protein catrin 26629 59
53 sp|Q7ZTA0.1|CRVP_AGKPI Cysteine-rich venom protein piscivorin 26664 60, 61, 62
54 sp|Q8AY81.1|VSPST_TRIST Thrombin-like enzyme stejnobin (Fibrinogen-clotting enzyme/Snake venom serine protease) 29309 25, 32
55 sp|Q91053.1|VSP1_GLOUS Thrombin-like enzyme calobin-1 (Snake venom serine protease) 28889 49
56 sp|Q9YGJ2.1|VSP1_GLOHA Snake venom serine protease pallabin 28662 49
57 XP_015671564.1 Snake venom serine protease serpentokallikrein-1 [Protobothrops mucrosquamatus] 88822 56
58 XP_023418723.1 Disintegrin and metalloproteinase domain-containing protein 17 [Cavia porcellus] 92703 64
59 XP_024069019.3 Disintegrin and metalloproteinase domain-containing protein 17 [Terrapene carolina triunguis] 99769 64
60 XP_025414344.1 Disintegrin and metalloproteinase domain-containing protein 9 [Sipha flava] 138126 54
61 XP_026527653.1 Laminin subunit alpha-1 [Notechis scutatus] 331363 80
62 XP_026535629.1 Dynein heavy chain 8, axonemal [Notechis scutatus] 511281 66
63 XP_026540213.1 Regulatory solute carrier protein family 1 member 1 [Notechis scutatus] 37705 80
64 XP_026541175.1 N-acetylated-alpha-linked acidic dipeptidase-like protein [Notechis scutatus] 81911 50
65 XP_028906446.1 Disintegrin and metalloproteinase domain-containing protein 17 [Ornithorhynchus anatinus] 94942 63
66 XP_032078796.1 Laminin subunit alpha-1 [Thamnophis elegans] 339446 85
67 XP_032080246.1 Centromere-associated protein E [Thamnophis elegans] 308791 59
68 XP_032085798.1 60S ribosomal protein L6 isoform X1 [Thamnophis elegans] 30312 42, 81
69 XP_032088226.1 Forkhead-associated domain-containing protein 1 [Thamnophis elegans] 137802 81
70 XP_032091805.1 Glial fibrillary acidic protein [Thamnophis elegans] 52228 88
71 XP_039181676.1 Snake venom serine protease-like isoform X1 [Crotalus tigris] 25811 49
72 XP_039181680.1 Snake venom serine proteinase 12-like [Crotalus tigris] 24539 56
Open in a new tab

Discussion

The protein constituents within the venom of Asian mountain pit viper O. monticola from northern Thailand were investigated in the present study. SDS-PAGE revealed a protein band pattern ranging between MW 10–90 kDa with intense bands representing low MW proteins (10–15 kDa), those of 50 and 72 kDa, and faint bands between 30–50 kDa. This corresponded well to the spot pattern obtained from 2DE, where the clouds of protein spots were observed within MW regions of 10–15, 30–34, 50 and 72–90 kDa. The overall MW range of proteins in O. monticola venom was comparable to those from three other Ovophis species, namely O. convictus, O. tonkinensis and O. okinavensis. The pattern of dominant bands was most similar to the venom of O. tonkinensis found in China [10].

The proteomic profile showed that enzymatic components which are SVMP (36.8%), SVSP (31.1%), PLA2 (12.1%) and LAAO (5.7%) mainly make up the venom of Asian mountain pit viper O. monticola. The overall composition of these major enzymes was comparable to those recently reported from venoms of O. convictus, O. tonkinensis and O. okinavensis, only content proportion seemed to be different. Among these three Ovophis spp., the most abundant proteins were of SVSP (35–53%), followed by PLA2 (19–25%) and LAAO (5–17%). SVMP (11–19%) was detected at a lower percentage than in our venom [10]. Such venom variation in venom composition could would not only be attributed by speciation but also other factors including prey diversity reflecting different ecological habitats [11], snake sex, [17] and age [18, 19]. In addition, the variation in quantity of identified toxin types within snake venom might be resulted from the different quantitative approaches and accompanying calculation methods as well as proteomic database availability [20]. The 2DE indicated that O. monticola venom contained more acidic than basic protein spots. This finding was confirmed by our list of all identified peptides obtained from LC-MS/MS showing that the majority possessed pI values lower than 7. Our spot pattern also confirmed previous 2DE analysis of Trimeresurus sumatranus (another Viperid) venom, in which more proteins were identified in the acidic range than in Elapid venom [21]. The overall acidic properties of 4 main protein groups of vipers greatly contribute to the hemorrhagic and coagulopathic effect on victims [22, 23].

Our results revealed SVMPs as representing more than one-third of the entire O. monticola venom. The greatest amount found in O. monticola was sp|O93517.1|VM3S4_GLOBR Zinc metalloproteinase/disintegrin or disintegrin-like salmosin-4, first identified within Korean Agkistrodon halys brevicaudus snake venom [24]. SVMPs potentially inhibit platelet aggregation and integrin-dependent cell adhesion via interrupting glycoprotein IIb-IIIa/fibrinogen interaction and fibrinogenolysis [25, 26]. Additionally, SVMPs interact with the various types of cellular matrix and exerts the most haemorrhagic effect on hosts [27]. SVSPs were found to be second-most abundant in O. monticola venom. The majority are thrombin-like enzymes including sp|Q9PRP4.1|VSPF_LACMR thrombin-like enzyme LMR-47 and sp|Q9PRP4.1|VSPFLACMR thrombin-like enzyme calobin-2. Known as fibrinogen-clotting enzymes, they are common, and found in large amounts in the venoms of the genera Agkistrodon, Bothrops, Lachesis and Trimeresurus [28]. Thrombin-like enzymes demonstrate strong hydrolytic activity, primarily against triad residues of His57, Asp102 and Ser195 of fibrinogen [29]. Resembling thrombin, they act on blood plasma by forming friable and translucent clots which later are degraded due to non-functional cross-linked structures, leading to coagulopathy and hypofibrinogenemia [30, 31]. However, in terms of enzyme proportion, our findings contrast with previous reports on the transcriptomic analysis of O. okinavensis venom glands, which mainly contained SVSP (93.1%) and relatively little SVMP (4.2%) [32]. It remains unclear whether the microenvironment within the venom gland might preferentially activate or interfere with the functioning of newly synthesized enzymes.

Compared with SVMP and SVSP, we detected smaller amounts of PLA2 and LAAO in O. monticola venom. PLA2 hydrolyzes phospholipids at the sn-2 position, generating fatty acids and lysophospholipids [33]. Group II PLA2 is expressed exclusively in the venoms of the Viperidae [34]. We found both acidic and basic PLA2 subtypes in O. monticola venom. It is noteworthy that the acidic PLA2 (sp|P81478.1|PA2A2TRIGA and sp|P82896.1|PA2A5TRIST were present in high quantities partially contributing to more acidic properties of the venom. These were previously found to trigger oedema [35]. PLA2 elicits inflammatory responses through the overproduction of pro-inflammatory cytokines (such as TNFα, IL-1β and IL-6) largely by immunocompetent cells (monocytes, neutrophils and mast cells) [36].

LAAOs can act in concert with PLA2 in local inflammatory reactions. LAAOs from Calloselasma rhodosthoma venom were shown to induce superoxide anion and hydrogen peroxide production by human neutrophils [37]. The most abundant LAAO in O. monticola venom is sp|P0C2D5.2|OXLA_PROFL, also known as Okinawa habu apoxin protein-1. This protein was first characterized from the venom of Protobothrops flavoridis to induce apoptosis in glioma cells [38]. The roles of both PLA2 and LAAOs in snake venoms are multi-faceted. Their catalytic as well as cytotoxic properties have been extensively investigated for pharmaceutical potential against cancers and other diseases [39].

Due to the unavailability of homospecific antivenom to Ovophis spp. venoms, all pit viper envenoming victims usually receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) [16]. The latter gave considerably higher immunoreactive levels (30–50%) to O. monticola venom proteins than the former. Relatively greater levels of reactivity of the polyvalent antivenom was previously reported with the venoms of C. rhodostoma, Hypnale hypnale and Trimeresurus hageni, and even Trimeresurus albolabris when compared with those of monovalent antivenom [40]. With the combination of 2DE immunoblotting and LC-MS/MS analyses, we found that hematotoxic polyvalent antivenom reacted with a wider range of proteins and peptides accounting for 58% of the entire range of proteins and covering all major enzymatic groups. Nonetheless, we were able to observe that an array of LAAOs and SVSPs did not react with a monovalent antivenom specific only to T. albolabris venom. This finding suggests the shared antigenic epitopes particularly from Ovophis, Calloselasma and Trimeresurus venoms used to generate antivenom. In this context, proteomic analysis of Malayan pit viper C. rhodostoma venom revealed a similar SVMP dominance (41.17%), with other major constituents of snaclec (26.3%) and SVSP (14.9%) [41]. A study of phylogenetic relationships based on geographic distribution and mitochondrial and nuclear gene sequences also demonstrated that O. monticola is less distantly separated from C. rhodostoma than from T. albolabris [42]. Thus, the antivenom against immunogenic epitopes from C. rhodostoma venom should be further investigated for the possible adjunctive treatment of O. monticola bite victims.

Our current study revealed that 72 proteins (42% of venom proteins) were left unrecognized by both antivenoms. The majority of immunologically non-reactive proteins have low molecular mass. They include a number of SVSPs, PLA2 and certain SVMP. The poor immunogenicity of these low molecular venomic proteins has been obviously reported, although some possess high toxicity [43, 44]. In addition, an array of non-enzymatic CTLs such as galactose-binding lectins, snaclec, alboaggregin A, and CRISPs such as triflin, okinavin, catrin, and piscivotin were found unrecognizable by antivenom. This reflects the difference in antigenic abundance between O. monticola venom and those venoms employed to generate horse immunoglobulins, as the proportions of CTLs and CRISPs in our O. monticola venom were only 1.6% and 1.2%, respectively. Nonetheless, their biological impacts on host cells and tissues should not be neglected. In the context of CTLs, alboaggregin A was shown to bind strongly with platelet glycoproteins IB and VI, and hence, activated platelet aggregation [45]. In addition, evidence of enhanced platelet activation and thrombotic microangiopathy-like symptoms has been documented with other related snaclecs [46]. In terms of CRISPs, the unique pdb|1WVR|A chain A triflin, as well as sp|P0DL18.1|CRVP_OVOOK okinavin from the related hime habu O. okinavensis were not reactive with the antivenoms. They have been previously described to have a calcium channel-impairing effect, leading to aberrations in muscle contraction [47, 48]. Considering the pathophysiological effects, our findings address the suite of protein targets which could be additional antigens for future antivenomic design. Furthermore, in order to alleviate the symptoms of mountain pit viper envenomation, these would facilitate the development of specific drug schemes allowing patients to recover more quickly. The proteomic profile of O. monticola venom not only provides insight into the venomic phenotypes reflecting the evolutionary path among Viperid snakes, but also accelerates the discovery of novel candidates for medical and pharmaceutical use.

Supporting information

S1 Raw image

(PDF)

Click here for additional data file. (66.3KB, pdf)
S1 Table. List of all proteins found in Ovophis monticola venom.

(XLSX)

Click here for additional data file. (28.1KB, xlsx)
S2 Table. List of proteins in 89 spots in Ovophis monticola venom.

Crude venom was subjected to 2DE gel. Proteins were separated in the first dimension in the pH range 3–10.

(DOCX)

Click here for additional data file. (69.4KB, docx)

Acknowledgments

We thank David Anderson for manuscript proofreading and the Central Equipment Unit, Faculty of Tropical Medicine, Mahidol University, Thailand for proteomics facilities.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This research was funded by the Center of Excellence on Biodiversity, grant number BDC-PG4-161009.

References

  • 1.Organization WH. Fact sheet [Internet]: World Health Organization. 2021. 17 May 2021. [Google Scholar]
  • 2.Gutierrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite envenoming. Nat Rev Dis Primers. 2017;3:17079. doi: 10.1038/nrdp.2017.79 [DOI] [PubMed] [Google Scholar]
  • 3.Habib AG, Brown NI. The snakebite problem and antivenom crisis from a health-economic perspective. Toxicon. 2018;150:115–23. doi: 10.1016/j.toxicon.2018.05.009 [DOI] [PubMed] [Google Scholar]
  • 4.Malhotra A, Dawson K, Guo P, Thorpe RS. Phylogenetic structure and species boundaries in the mountain pitviper Ovophis monticola (Serpentes: Viperidae: Crotalinae) in Asia. Mol Phylogenet Evol. 2011;59(2):444–57. doi: 10.1016/j.ympev.2011.02.010 [DOI] [PubMed] [Google Scholar]
  • 5.Pandey DP, Chaudhary B, Ram Shrestha B. Documentation of a proven Mountain Pitviper (Ovophis monticola) envenomation in Kathmandu, Nepal, with its distribution ranges: implications for prevention and control of pitviper bites in Asia. J Venom Res. 2021;11:1–6. [PMC free article] [PubMed] [Google Scholar]
  • 6.Chanhome L, Cox MJ, Wilde H, Jintakoon P, Chaiyabutr N, Sitprija V. Venomous snakebite in Thailand. I: Medically important snakes. Mil Med. 1998;163(5):310–7. [PubMed] [Google Scholar]
  • 7.Shibata H, Chijiwa T, Hattori S, Terada K, Ohno M, Fukumaki Y. The taxonomic position and the unexpected divergence of the Habu viper, Protobothrops among Japanese subtropical islands. Mol Phylogenet Evol. 2016;101:91–100. doi: 10.1016/j.ympev.2016.04.027 [DOI] [PubMed] [Google Scholar]
  • 8.Cox MJ, Hoover MF, Chanhome L, Thirakhupt K, Pongcharoen C. A Field Guide to the Venomous Snakes of Mainland ASEAN Nations: published and distributed by Merel J. Cox, Mark F. Hoover, Lawan Chanhome, Kumthorn Thirakhupt, and Chattraphas Pongcharoen; 2018. [Google Scholar]
  • 9.Boda F, Banfai K, Garai K, Curticapean A, Berta L, Sipos E, et al. Effect of Vipera ammodytes ammodytes Snake Venom on the Human Cytokine Network. Toxins (Basel). 2018;10(7). doi: 10.3390/toxins10070259 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Tan CH, Palasuberniam P, Tan KY. Snake Venom Proteomics, Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers, Ovophis convictus, Ovophis tonkinensis, and Hime Habu, Ovophis okinavensis. Toxins. 2021;13(8):514. doi: 10.3390/toxins13080514 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Davies EL, Arbuckle K. Coevolution of Snake Venom Toxic Activities and Diet: Evidence that Ecological Generalism Favours Toxicological Diversity. Toxins (Basel). 2019;11(12). doi: 10.3390/toxins11120711 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Chanhome L, Khow O, Omori-Satoh T, Sitprija V. Capacity of Thai green pit viper antivenom to neutralize the venoms of Thai Trimeresurus snakes and comparison of biological activities of these venoms. J Nat Toxins. 2002;11(3):251–9. [PubMed] [Google Scholar]
  • 13.Kumkate S, Chanhome L, Thiangtrongjit T, Noiphrom J, Laoungboa P, Khow O, et al. Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni). Toxins (Basel). 2020;12(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Danpaiboon W, Reamtong O, Sookrung N, Seesuay W, Sakolvaree Y, Thanongsaksrikul J, et al. Ophiophagus hannah Venom: Proteome, Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv. Toxins. 2014;6(5):1526–58. doi: 10.3390/toxins6051526 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Gowtham YJ, Mahadeswaraswamy YH, Girish KS, K K. Cross-reactivity and neutralization of Indian King cobra (Ophiophagus hannah) venom by polyvalent and monovalent antivenoms. Int Immunopharmacol. 2014;21(1):148–55. doi: 10.1016/j.intimp.2014.04.012 [DOI] [PubMed] [Google Scholar]
  • 16.Asia WHORofS-E. Guidelines for the management of snakebites second edition 2016 22 October 2021.
  • 17.Menezes MC, Furtado MF, Travaglia-Cardoso SR, Camargo AC, Serrano SM. Sex-based individual variation of snake venom proteome among eighteen Bothrops jararaca siblings. Toxicon. 2006;47(3):304–12. doi: 10.1016/j.toxicon.2005.11.007 [DOI] [PubMed] [Google Scholar]
  • 18.Mackessy SP, Sixberry NM, Heyborne WH, Fritts T. Venom of the Brown Treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon. 2006;47(5):537–48. doi: 10.1016/j.toxicon.2006.01.007 [DOI] [PubMed] [Google Scholar]
  • 19.Gibbs HL, Sanz L, Chiucchi JE, Farrell TM, Calvete JJ. Proteomic analysis of ontogenetic and diet-related changes in venom composition of juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri). J Proteomics. 2011;74(10):2169–79. doi: 10.1016/j.jprot.2011.06.013 [DOI] [PubMed] [Google Scholar]
  • 20.Damm M, Hempel BF, Süssmuth RD. Old World Vipers-A Review about Snake Venom Proteomics of Viperinae and Their Variations. Toxins (Basel). 2021;13(6). doi: 10.3390/toxins13060427 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Vejayan J, Shin Yee LP, G., Ambu S, Ibrahim I. Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis. The Journal of Venomous Animals and Toxins including Tropical Diseases. 2010;volume 16 (4):623–30. [Google Scholar]
  • 22.Aung HT, Nikai T, Komori Y, Nonogaki T, Niwa M, Takaya Y. Biological and pathological studies of rosmarinic acid as an inhibitor of hemorrhagic Trimeresurus flavoviridis (habu) venom. Toxins (Basel). 2010;2(10):2478–89. doi: 10.3390/toxins2102478 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Tan NH, Fung SY, Tan KY, Yap MK, Gnanathasan CA, Tan CH. Functional venomics of the Sri Lankan Russell’s viper (Daboia russelii) and its toxinological correlations. J Proteomics. 2015;128:403–23. doi: 10.1016/j.jprot.2015.08.017 [DOI] [PubMed] [Google Scholar]
  • 24.Shin J, Hong SY, Chung K, Kang I, Jang Y, Kim DS, et al. Solution structure of a novel disintegrin, salmosin, from Agkistrondon halys venom. Biochemistry. 2003;42(49):14408–15. doi: 10.1021/bi0300276 [DOI] [PubMed] [Google Scholar]
  • 25.Swenson S, Markland FS Jr. Snake venom fibrin(ogen)olytic enzymes. Toxicon. 2005;45(8):1021–39. doi: 10.1016/j.toxicon.2005.02.027 [DOI] [PubMed] [Google Scholar]
  • 26.Andrews RK, Gardiner EE, Shen Y, Berndt MC. Structure-activity relationships of snake toxins targeting platelet receptors, glycoprotein Ib-IX-V and glycoprotein VI. Curr Med Chem Cardiovasc Hematol Agents. 2003;1(2):143–9. doi: 10.2174/1568016033477559 [DOI] [PubMed] [Google Scholar]
  • 27.Markland FS Jr., Swenson S. Snake venom metalloproteinases. Toxicon. 2013;62:3–18. doi: 10.1016/j.toxicon.2012.09.004 [DOI] [PubMed] [Google Scholar]
  • 28.Kini RM, Rao VS, Joseph JS. Procoagulant proteins from snake venoms. Haemostasis. 2001;31(3–6):218–24. doi: 10.1159/000048066 [DOI] [PubMed] [Google Scholar]
  • 29.Castro HC, Zingali RB, Albuquerque MG, Pujol-Luz M, Rodrigues CR. Snake venom thrombin-like enzymes: from reptilase to now. Cell Mol Life Sci. 2004;61(7–8):843–56. doi: 10.1007/s00018-003-3325-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hutton RA, Warrell DA. Action of snake venom components on the haemostatic system. Blood Rev. 1993;7(3):176–89. doi: 10.1016/0268-960x(93)90004-n [DOI] [PubMed] [Google Scholar]
  • 31.Yokoi H, Sakai A, Kodama T, Magome S, Nagayasu T, Tawara M, et al. Severe hypofibrinogenemia in patients bitten by Gloydius tsushimaensis in Tsushima Island, Nagasaki, Japan, and treatment strategy. Toxicon. 2020;188:142–9. doi: 10.1016/j.toxicon.2020.10.027 [DOI] [PubMed] [Google Scholar]
  • 32.Aird SD, Watanabe Y, Villar-Briones A, Roy MC, Terada K, Mikheyev AS. Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis). BMC Genomics. 2013;14:790. doi: 10.1186/1471-2164-14-790 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kang TS, Georgieva D, Genov N, Murakami MT, Sinha M, Kumar RP, et al. Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS J. 2011;278(23):4544–76. doi: 10.1111/j.1742-4658.2011.08115.x [DOI] [PubMed] [Google Scholar]
  • 34.Lomonte B, Angulo Y, Moreno E. Synthetic peptides derived from the C-terminal region of Lys49 phospholipase A2 homologues from viperidae snake venoms: biomimetic activities and potential applications. Curr Pharm Des. 2010;16(28):3224–30. doi: 10.2174/138161210793292456 [DOI] [PubMed] [Google Scholar]
  • 35.Nakashima K, Nobuhisa I, Deshimaru M, Nakai M, Ogawa T, Shimohigashi Y, et al. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc Natl Acad Sci U S A. 1995;92(12):5605–9. doi: 10.1073/pnas.92.12.5605 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Burin SM, Menaldo DL, Sampaio SV, Frantz FG, Castro FA. An overview of the immune modulating effects of enzymatic toxins from snake venoms. Int J Biol Macromol. 2018;109:664–71. doi: 10.1016/j.ijbiomac.2017.12.101 [DOI] [PubMed] [Google Scholar]
  • 37.Pontes AS, da SSS, Xavier CV, Lacouth-Silva F, Kayano AM, Pires WL, et al. Effect of L-amino acid oxidase from Calloselasma rhodosthoma snake venom on human neutrophils. Toxicon. 2014;80:27–37. doi: 10.1016/j.toxicon.2013.12.013 [DOI] [PubMed] [Google Scholar]
  • 38.Abe Y, Shimoyama Y, Munakata H, Ito J, Nagata N, Ohtsuki K. Characterization of an apoptosis-inducing factor in Habu snake venom as a glycyrrhizin (GL)-binding protein potently inhibited by GL in vitro. Biol Pharm Bull. 1998;21(9):924–7. doi: 10.1248/bpb.21.924 [DOI] [PubMed] [Google Scholar]
  • 39.Hiu JJ, Yap MKK. Cytotoxicity of snake venom enzymatic toxins: phospholipase A2 and l-amino acid oxidase. Biochem Soc Trans. 2020;48(2):719–31. doi: 10.1042/BST20200110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Chaisakul J, Khow O, Wiwatwarayos K, Rusmili MRA, Prasert W, Othman I, et al. A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell’s Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury. Toxins. 2021;13(8):521. doi: 10.3390/toxins13080521 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Tang EL, Tan CH, Fung SY, Tan NH. Venomics of Calloselasma rhodostoma, the Malayan pit viper: A complex toxin arsenal unraveled. J Proteomics. 2016;148:44–56. doi: 10.1016/j.jprot.2016.07.006 [DOI] [PubMed] [Google Scholar]
  • 42.Alencar LRV, Quental TB, Grazziotin FG, Alfaro ML, Martins M, Venzon M, et al. Diversification in vipers: Phylogenetic relationships, time of divergence and shifts in speciation rates. Mol Phylogenet Evol. 2016;105:50–62. doi: 10.1016/j.ympev.2016.07.029 [DOI] [PubMed] [Google Scholar]
  • 43.Correa-Netto C, Teixeira-Araujo R, Aguiar AS, Melgarejo AR, De-Simone SG, Soares MR, et al. Immunome and venome of Bothrops jararacussu: a proteomic approach to study the molecular immunology of snake toxins. Toxicon. 2010;55(7):1222–35. doi: 10.1016/j.toxicon.2009.12.018 [DOI] [PubMed] [Google Scholar]
  • 44.Madrigal M, Pla D, Sanz L, Barboza E, Arroyo-Portilla C, Corrêa-Netto C, et al. Cross-reactivity, antivenomics, and neutralization of toxic activities of Lachesis venoms by polyspecific and monospecific antivenoms. PLoS Negl Trop Dis. 2017;11(8):e0005793. doi: 10.1371/journal.pntd.0005793 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Dörmann D, Clemetson JM, Navdaev A, Kehrel BE, Clemetson KJ. Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib. Blood. 2001;97(4):929–36. doi: 10.1182/blood.v97.4.929 [DOI] [PubMed] [Google Scholar]
  • 46.Long C, Liu M, Tian H, Li Y, Wu F, Mwangi J, et al. Potential Role of Platelet-Activating C-Type Lectin-Like Proteins in Viper Envenomation Induced Thrombotic Microangiopathy Symptom. Toxins (Basel). 2020;12(12). doi: 10.3390/toxins12120749 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yamazaki Y, Koike H, Sugiyama Y, Motoyoshi K, Wada T, Hishinuma S, et al. Cloning and characterization of novel snake venom proteins that block smooth muscle contraction. Eur J Biochem. 2002;269(11):2708–15. doi: 10.1046/j.1432-1033.2002.02940.x [DOI] [PubMed] [Google Scholar]
  • 48.Matsunaga Y, Yamazaki Y, Hyodo F, Sugiyama Y, Nozaki M, Morita T. Structural divergence of cysteine-rich secretory proteins in snake venoms. J Biochem. 2009;145(3):365–75. doi: 10.1093/jb/mvn174 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Rafael Ximenes

13 Jul 2021

PONE-D-21-18686

Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venoms

PLOS ONE

Dear Dr. Kumkate,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Aug 27 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Rafael Ximenes

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional location information, including geographic coordinates for the data set if available.

3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

4. We understand that you extracted venom from Ovophis monticola for this study. In your Methods section, please provide additional details regarding the source of the animal (Asian Mountain pit viper). Please provide the geographic coordinates and as well as any further details about the animal (e.g., sex, description of appearance) to ensure reproducibility of the analyses.

5. Thank you for stating the following in your Competing Interests section:  

[No]. 

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now 

 This information should be included in your cover letter; we will change the online submission form on your behalf.

6. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

7. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

8.  We note that Figure 1 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a) You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. 

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Referee comments:

In this work, the authors demonstrate by venomics approaches, a comprehensive characterization of the venom proteome and immunocharacterization of Asian Mountain pit viper (Ovophis monticola) venoms, a medically important snake from Thailand, found at high altitude areas of several mountain ranges of East, South and Southeast Asia. Moreover the authors analyzed the immunoreactivity of monospecific (monovalent) and polyspecific (polyvalent) antivenoms against green pit viper Trimeresurus albolabris and hemotoxic venoms, respectively. The authors present a study with satisfactory quality in methodology and data generation. However the manuscript has an inventory of toxins and immunoreactive proteins without coherence with the scientific literature and poor consistency with some cited references. Furthermore, in many topics of the manuscript the authors present a lot of information without any reference. For example, the authors don't compare the results regarding 2D immunoblotting with any previously study. I suggest that this manuscript should be reword to improve the quality about the data presentation. I'm sending some notes

- Abstract

Line 33  ..... venoms was investigated by indirect ELISA..... Line 37.....were subjected to 2D immunoblotting

These related methodologies would be more appropriate if they were introduced together.

- Introduction

. Line 46-50 - The geographical distribution data deserve references. The same for the sentences introducing biological data about pit viper from lines 51-58.... length of 110 cm.

Lines 59: Incidence of pit vipers  bites, including those from mountain pit vipers, have been documented in their known ranges???.

The lack of references should be revised in all manuscript...

-Materials and methods

Line 92 : Snake venom,

- Are there more information about the venom's sample used in this study? Number of specimens? Localities? size/age...

Line 121: ...a non-linear immobilized pH gradient (IPG) strip (pH 3-10 Amersham Bioscience, USA)....

- Why the authors choose the non-linear gradient instead of linear gradient, since the propose were excised immunoreactive spots for mass spectrometric analysis? Just for curious

- Lack references in almost all methods. The references for 2D immunoblotting is required

Results

Line 208: Change Ovophis / Ovophis sp or genera Ovophis

- Are there any published information about neutralize activity of monovalent and polyvalent antivenoms against O. monticola venom? This information should be provided or I suggest that an experiment to determine the lethal dose 50% should be done.

Table 2. List of identified proteins ... immunologically reacted ...

- If the MW of the spots content reacted/ non-reacted proteins could be provided, this information can improve the quality table.

Discussion

The Authors should revise the references from the first paragraph.... For example: the statement about hematotoxic potential of SVMP, SVSP and PLA2 is already a scientific consensus. Therefore, there are many sources specially from the seminal studies that corroborate with this question. However, the Authors present references from venom proteomic analysis of other snakes to support this question. On the other hand, the authors present coherency in the literature proposed as reference to address the mechanism of action of P-III metalloproteinase. I suggest that his paragraph must be reworded.

Line 208: The presence of class P-II metalloproteinase, which possesses metalloproteinase and disintegrin-like domians was also observed.????

Line 303: The combination of SVMP and SVSP observed in O. monticola venom is responsible for prey attack, incapacitation, and digestion.???

Reviewer #2: It is an interesting work to be published in PLOS. This article describes describe proteomics and immuno characterization of snake Ovophis monticola venom. I have the following comments that the authors may like to consider:

Line 95

- Provide the total number of individuals, age, sex, locality (adults or young people, males, females)

- It is important to provide some functional analysis

Discussion:

Despite little published literature on Ovophis monticola:

Please, include in the discussion this reference:

Pandey, D. P., Chaudhary, B., & Ram Shrestha, B. (2021). Documentation of a proven Mountain Pitviper (Ovophis monticola) envenomation in Kathmandu, Nepal, with its distribution ranges: implications for prevention and control of pitviper bites in Asia. Journal of venom research, 11, 1–6.

Mainly about clinical characteristic of poisoning by this specie and lack of specific treatment.

Provide conclusion or future perspectives in the last paragraphy about immunocharacterization results/ envenomation treatment.

Line 527

Exclude the number 75.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Dec 1;16(12):e0260496. doi: 10.1371/journal.pone.0260496.r002

Author response to Decision Letter 0

20 Aug 2021

Responses to reviewers

Reviewer #1:

In this work, the authors demonstrate by venomics approaches, a comprehensive characterization of the venom proteome and immunocharacterization of Asian Mountain pit viper (Ovophis monticola) venoms, a medically important snake from Thailand, found at high altitude areas of several mountain ranges of East, South and Southeast Asia. Moreover the authors analyzed the immunoreactivity of monospecific (monovalent) and polyspecific (polyvalent) antivenoms against green pit viper Trimeresurus albolabris and hemotoxic venoms, respectively. The authors present a study with satisfactory quality in methodology and data generation. However the manuscript has an inventory of toxins and immunoreactive proteins without coherence with the scientific literature and poor consistency with some cited references. Furthermore, in many topics of the manuscript the authors present a lot of information without any reference. For example, the authors don't compare the results regarding 2D immunoblotting with any previously study. I suggest that this manuscript should be reword to improve the quality about the data presentation.

Response: We thank the reviewer for suggestion. We rewrote introduction and discussion to improve data presentation. Relevant references were cited in every part to enhance literature coherence. Biological and geographical details regarding all snakes were also provided in materials and methods (Table 1; page 5, line 105).

- Abstract

Line 33 ..... venoms was investigated by indirect ELISA..... Line 37.....were subjected to 2D immunoblotting

These related methodologies would be more appropriate if they were introduced together.

Response: We edited an abstract according to reviewer’s suggestion.

- Introduction

. Line 46-50 - The geographical distribution data deserve references. The same for the sentences introducing biological data about pit viper from lines 51-58.... length of 110 cm.

Lines 59: Incidence of pit vipers bites, including those from mountain pit vipers, have been documented in their known ranges???.

The lack of references should be revised in all manuscript...

Response : We rewrote introduction with relevant references cited in a revised version of manuscript; ref no.6 and 8).

-Materials and methods

Line 92 : Snake venom,

- Are there more information about the venom's sample used in this study? Number of specimens? Localities? size/age...

Response: Biological and geographical information regarding all snakes is provided as a table in materials and methods (Table 1; page 5 line 105).

Line 121: ...a non-linear immobilized pH gradient (IPG) strip (pH 3-10 Amersham Bioscience, USA)....

- Why the authors choose the non-linear gradient instead of linear gradient, since the propose were excised immunoreactive spots for mass spectrometric analysis? Just for curious

Response: According to our previous experiments, we found that the non-linear immobilized pH gradient (IPG) could provide the well separation of protein in venoms. Therefore, we chose the NL strip for this research.

- Lack references in almost all methods. The references for 2D immunoblotting is required.

Response: In a revised materials and methods of manuscripts, we provided relevant references for all methods used. For 2DE immunoblotting, a reference is doi: 10.3390/toxins6051526 as reference page 7, line 137 (ref no.14)

Results

Line 208: Change Ovophis / Ovophis sp or genera Ovophis

- Are there any published information about neutralize activity of monovalent and polyvalent antivenoms against O. monticola venom? This information should be provided or I suggest that an experiment to determine the lethal dose 50% should be done.

Table 2. List of identified proteins ... immunologically reacted ...

- If the MW of the spots content reacted/ non-reacted proteins could be provided, this information can improve the quality table.

Response: We added a column of MW of proteins in a revised Table 2. In this particular study, we focused mainly on providing the inventory of all protein constituents of Asian mountain pit viper O. monticola venom. Those with antigenic properties reacted with monovalent and hematotoxic polyvalent antivenom were given. However, we did not perform neutralizing experiments.

Discussion

The Authors should revise the references from the first paragraph.... For example: the statement about hematotoxic potential of SVMP, SVSP and PLA2 is already a scientific consensus. Therefore, there are many sources specially from the seminal studies that corroborate with this question. However, the Authors present references from venom proteomic analysis of other snakes to support this question. On the other hand, the authors present coherency in the literature proposed as reference to address the mechanism of action of P-III metalloproteinase. I suggest that his paragraph must be reworded.

Line 208: The presence of class P-II metalloproteinase, which possesses metalloproteinase and disintegrin-like domians was also observed.????

Line 303: The combination of SVMP and SVSP observed in O. monticola venom is responsible for prey attack, incapacitation, and digestion.???

Response: We thank the reviewer for comments on discussion. We rewrote discussion by updating and improving literature coherence.

Reviewer #2:

It is an interesting work to be published in PLOS. This article describes describe proteomics and immuno characterization of snake Ovophis monticola venom. I have the following comments that the authors may like to consider:

Line 95

- Provide the total number of individuals, age, sex, locality (adults or young people, males, females)

- It is important to provide some functional analysis

Response: Biological and geographical information regarding all snakes is provided as a table in materials and methods (Table 1; page 5 line 105). In this particular study, we focused mainly on providing the inventory of all protein constituents of Asian mountain pit viper O. monticola venom. Those with antigenic properties reacted with monovalent and hematotoxic polyvalent antivenom were given. However, we did not perform the experiments to analyse the functions.

Discussion:

Despite little published literature on Ovophis monticola:

Please, include in the discussion this reference:

Pandey, D. P., Chaudhary, B., & Ram Shrestha, B. (2021). Documentation of a proven Mountain Pitviper (Ovophis monticola) envenomation in Kathmandu, Nepal, with its distribution ranges: implications for prevention and control of pitviper bites in Asia. Journal of venom research, 11, 1–6.

Mainly about clinical characteristic of poisoning by this specie and lack of specific treatment.

Provide conclusion or future perspectives in the last paragraphy about immunocharacterization results/ envenomation treatment.

Line 527

Exclude the number 75.

Response: We cited the above literature as a reference in a revised version of manuscript (ref no.5).

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file. (16.4KB, docx)

Decision Letter 1

Rafael Ximenes

6 Oct 2021

PONE-D-21-18686R1Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venomsPLOS ONE

Dear Dr. Kumkate,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Rafael Ximenes

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Based on the new round of peer-round some minor concerns have arisen. I recommend a minor revision before acceptance of the manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors rewrote the manuscript and provide relevant references and endeavored to comply with all referee requests.I just emphasize that only Ovophis don't belong to a taxonomic system. This task dont were revide for authors. However. The authors present a study with high quality in methodology, data generation and data analysis, producing significant contribution for toxinology field. Thus, this work is suitable for publication on PLOS ONE.

Reviewer #3: The article highlights the spectrometry-based proteomics and the immunoreactivity of the venom of the Ovophis monticola viper. It is a work based on an excellent premise and the methodologies are very well defined. I recommend the work for publication. However, some minor revisions are needed.

1. In the session "Two-dimensional polyacrylamide gel electrophoresis (2DE)" (Material and Methods), the authors state that a 100 µg sample of venom was used. However, since the protein concentrations were measured using the Lowry method, please review whether these 100 µg are venom or protein.

2. In the session “Proteomic analysis of O. monticola venom” (Results), the first paragraph deals with a methodological aspect.

3. The same observation applies to the following topics. Instead of repeating the concepts of the methodology, the objective could be presented if evaluating that result, since this information is not present in the text.

4. In “Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenoms by indirect ELISA”, the information “Since there is no homospecific antivenom to Ovophis venoms currently available, all pit viper envenoming victims usually receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) to alleviate symptoms” is applicable to the discussion, requiring references.

5. Figures and tables should be self-explanatory. In the case of table 2, a legend stating the meaning of MW, PI, emPI must be provided.

6. Discussion: What is the toxinological consequence related to the fact that the venom has more acidic than basic protein spots? How does this interfere with symptoms related to accident? Is this positive or negative regarding the immunoreactivity of the venom, especially with the polyvalent serum? Present references.

7. Why are some references in numerical format (ex (22) (23)) and others are in nominal format (ex Damm et al, 2021)?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Carlos Correa-Netto

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Dec 1;16(12):e0260496. doi: 10.1371/journal.pone.0260496.r004

Author response to Decision Letter 1

9 Nov 2021

Additional Editor Comments (if provided):

Based on the new round of peer-round some minor concerns have arisen. I recommend a minor revision before acceptance of the manuscript.

Reviewers' comments:

Reviewer #1: The authors rewrote the manuscript and provide relevant references and endeavored to comply with all referee requests. I just emphasize that only Ovophis don't belong to a taxonomic system. This task dont were revide for authors. However. The authors present a study with high quality in methodology, data generation and data analysis, producing significant contribution for toxinology field. Thus, this work is suitable for publication on PLOS ONE.

Response: We corrected Ovophis to Ovophis spp. as suggested by the reviewer (e.g. lines 68, 83, 91, 288, 305, 313, 396)

Reviewer #3: The article highlights the spectrometry-based proteomics and the immunoreactivity of the venom of the Ovophis monticola viper. It is a work based on an excellent premise and the methodologies are very well defined. I recommend the work for publication. However, some minor revisions are needed.

1. In the session "Two-dimensional polyacrylamide gel electrophoresis (2DE)" (Material and Methods), the authors state that a 100 µg sample of venom was used. However, since the protein concentrations were measured using the Lowry method, please review whether these 100 µg are venom or protein.

Response: : It was 100 �g of protein. We corrected the sentence in line 135.

2. In the session “Proteomic analysis of O. monticola venom” (Results), the first paragraph deals with a methodological aspect.

Response: We rewrote this paragraph according to the reviewer’s suggestion.

3. The same observation applies to the following topics. Instead of repeating the concepts of the methodology, the objective could be presented if evaluating that result, since this information is not present in the text.

Response: We revised result session and eliminated repetition particularly on methodology.

4. In “Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenoms by indirect ELISA”, the information “Since there is no homospecific antivenom to Ovophis venoms currently available, all pit viper envenoming victims usually receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) to alleviate symptoms” is applicable to the discussion, requiring references.

Response: We clarified this point by providing a reference (line 232, ref no. 16 )

5. Figures and tables should be self-explanatory. In the case of table 2, a legend stating the meaning of MW, PI, emPI must be provided.

Response: We added the explanation in the table 2 legend (lines 238-241, 244-246) and the definition of abbreviation is provided as in a legend as suggested.

6. Discussion: What is the toxinological consequence related to the fact that the venom has more acidic than basic protein spots? How does this interfere with symptoms related to accident? Is this positive or negative regarding the immunoreactivity of the venom, especially with the polyvalent serum? Present references.

Response: In this revised version of manuscript, we discussed the above points raised by the reviewer with relevant references, lines 323-327, 391-395. In addition, to make table 4 more readable, we added a column of MW of non-immunoreactive peptides.

7. Why are some references in numerical format (ex (22) (23)) and others are in nominal format (ex Damm et al, 2021)?

Response: We corrected all references appeared in text in numerical format according to Plos guideline.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file. (15.9KB, docx)

Decision Letter 2

Rafael Ximenes

11 Nov 2021

Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venom

PONE-D-21-18686R2

Dear Dr. Kumkate,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Rafael Ximenes

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Rafael Ximenes

19 Nov 2021

PONE-D-21-18686R2

Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venom

Dear Dr. Kumkate:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Rafael Ximenes

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Raw image

    (PDF)

    Click here for additional data file. (66.3KB, pdf)
    S1 Table. List of all proteins found in Ovophis monticola venom.

    (XLSX)

    Click here for additional data file. (28.1KB, xlsx)
    S2 Table. List of proteins in 89 spots in Ovophis monticola venom.

    Crude venom was subjected to 2DE gel. Proteins were separated in the first dimension in the pH range 3–10.

    (DOCX)

    Click here for additional data file. (69.4KB, docx)
    Attachment

    Submitted filename: Response to reviewers.docx

    Click here for additional data file. (16.4KB, docx)
    Attachment

    Submitted filename: Response to reviewers.docx

    Click here for additional data file. (15.9KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES