Next Article in Journal
Microbial-Derived Tryptophan Catabolites, Kidney Disease and Gut Inflammation
Next Article in Special Issue
“Two Cultures in Favor of a Dying Patient”: Experiences of Health Care Professionals Providing Snakebite Care to Indigenous Peoples in the Brazilian Amazon
Previous Article in Journal
In Silico Conformational Features of Botulinum Toxins A1 and E1 According to Intraluminal Acidification
Previous Article in Special Issue
Neurotoxicity and Other Clinical Manifestations of a Common European Adder (Vipera berus) Bite in Romania
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Toxins
Volume 14
Issue 9
10.3390/toxins14090643
toxins-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparison of Protein Variation in Protobothrops mucrosquamatus Venom between Northern and Southeast Taiwan and Association with Human Envenoming Effects

by
Liao-Chun Chiang
1,
Kun-Yi Chien
2,3,4,
Hung-Yuan Su
5,6,7,
Yen-Chia Chen
8,9,10,
Yan-Chiao Mao
10,11,12,* and
Wen-Guey Wu
1,*
1
College of Life Sciences, National Tsing Hua University, Hsinchu City 300, Taiwan
2
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan County 333, Taiwan
3
Clinical Proteomics Core Laboratory, Chang Gung Memorial Hospital, Taoyuan County 333, Taiwan
4
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan County 333, Taiwan
5
Department of Emergency Medicine, E-Da Hospital, Kaohsiung County 824, Taiwan
6
The School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung County 840, Taiwan
7
Department of Safety, Health and Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung County 811, Taiwan
8
Department of Emergency Medicine, Taipei Veterans General Hospital, Taipei City 112, Taiwan
9
School of Medicine, National Yang Ming Chiao Tung University, Taipei City 112, Taiwan
10
Department of Emergency Medicine, National Defense Medical Center, Taipei City 114, Taiwan
11
Division of Clinical Toxicology, Department of Emergency Medicine, Taichung Veterans General Hospital, Taichung City 407, Taiwan
12
College of Medicine, National Chung Hsing University, Taichung City 402, Taiwan
 
add Show full affiliation list
 
remove Hide full affiliation list
*
Authors to whom correspondence should be addressed.
Toxins 2022, 14(9), 643; https://doi.org/10.3390/toxins14090643
Submission received: 12 July 2022 / Revised: 13 September 2022 / Accepted: 16 September 2022 / Published: 18 September 2022
(This article belongs to the Special Issue Snakebite Clinics and Pathogenesis: From Preclinical to Resource Mapping Studies)
Browse Figures
Versions Notes

Abstract

:
Reports of bite from Protobothrops mucrosquamatus (Pmu) are frequent in Taiwan, and its wide-spread distribution and diverse habitats drove us to investigate its envenoming effects and relevant venom variations. We used reversed-phase high-performance liquid chromatography and mass spectrometry to analyze 163 Pmu venom samples collected from northern and southeastern Taiwan. Twenty-two major protein fractions were separated and analyzed, and their contents were determined semi-quantitatively. The results showed that despite the trivial differences in the protein family, there is an existing variation in acidic phospholipases A2s, serine proteinases, metalloproteinases, C-type lectin-like proteins, and other less abundant components in the Pmu venoms. Moreover, clinical manifestations of 209 Pmu envenomed patients hospitalized in northern or southeastern Taiwan revealed significant differences in local symptoms, such as ecchymosis and blistering. The mechanism of these local effects and possibly relevant venom components were examined. Further analysis showed that certain venom components with inter-population variation might work alone or synergistically with others to aggravate the local effects. Therefore, our findings of the venom variation may help one to improve antivenom production and better understand and manage Pmu bites.
Keywords:
Protobothrops mucrosquamatus; inter-population venom variation; snake venom metalloproteinases; phospholipase A2; venom-induced blistering
Key Contribution: Characterization of the inter-population variations of Pmu venom may help one to better understand the pathophysiological effects of envenomation and improve antivenom production and the management of Pmu bites.

1. Introduction

Protobothrops mucrosquamatus (Pmu) is a pit viper widely distributed in southern and southeastern Asia, including north-eastern India, Myanmar, northern Thailand, Laos, Vietnam, southern China, Taiwan, and Japan (Ryukyu islands) [1]. The World Health Organization (WHO) has categorized Pmu as the most medically important venomous snake in Taiwan and China [2]. In addition, it is one of the most common snakebite incidents that have led to morbidity or even mortality in Taiwan [3]. As for its envenoming effects on humans, Pmu bites result in local tissue swelling; pain; ecchymosis; blistering (bullae formation); wound necrosis; compartment syndrome; and systemic effects, such as thrombocytopenia, coagulopathy, rhabdomyolysis, and renal impairment [4]. However, differences in human envenoming effects between the two regions were not compared in those studies.
The composition of Pmu venom is predominately snake venom metalloproteinases (SVMPs) and phospholipases A2 (PLA2s), followed by snake venom C-type lectin-like proteins (Snaclecs), snake venom serine proteinases (SVSPs), cysteine-rich secretory proteins (CRISPs), and L-amino acid oxidases (LAOs) [5,6]. Since the clinical manifestations of the same species snake bites are closely related to the venom composition, intra-specific variations in the profile and amount of each venom component may have resulted in diversified envenoming effects [7,8,9]. Intra-specific venom variations can be further categorized as intra-population (differences within populations; individual variation) and inter-population (differences between populations) variabilities; such variation is common among pit vipers [10,11].
Recently, two retrospective studies related to clinical manifestations of Pmu bites were conducted by two medical centers in Northern and Southeastern Taiwan, respectively [12,13]. Northern and Southeastern Taiwan are separated by the Central Mountain Range (highest peak, 3952 m), and non-alpine snakes, such as Pmu, inhabiting these two biogeographical regions can be considered as two distinct populations. In previous studies, a Taiwanese pit viper, Trimeresurus stejnegeri stejnegeri, was found to show inter-population venom variations, such as PLA2 variation, in the two regions [14,15]. These findings drove us to further investigate inter-population venom variations in Pmu venom.
However, the venom variation of Pmu remains little understood [14], and its association with the effects of human envenoming have not been studied. Therefore, in this study, we first compared Pmu venom components between northern and southeastern Taiwan. Secondly, we examined the clinical manifestations of Pmu envenoming between these two regions. We believe that this study can further assist venom and antivenom research and improve the management of Pmu bites in Taiwan.

2. Results and Discussion

2.1. Venom Proteome of Individual Pmu

The individual variation in venom components of 163 adult Pmu venoms is shown in Figure 1. Twenty-two protein peaks (labeled Fr 1–22) were identified for most individual Pmu venoms in the reverse phase high-performance liquid chromatography (RP-HPLC) profiles (Figure 1A–C). Shotgun proteomics with liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis showed 31 venom components, categorized into nine major protein families: SVMP, PLA2, Snaclec, SVSP, CRISP, 5’-nucleotidase (5’-NT), phospholipase B (PLB), glutaminyl-peptide cyclotransferase (QPCT), and LAO. The major venom toxins in each fraction are listed in Table 1. Additionally, detailed proteomics data, including peptide sequences in MS, were provided in the supplementary Table S1. The average amount of the nine protein families of Pmu venoms is shown in Table 2. We found that Pmu venom contained abundant SVMPs (45.1%) and PLA2s (25.0%), followed by Snaclecs (12.8%) and SVSPs (8.23%); however, it contained low contents of 5’-NT (3.85%), LAO (2.37%), CRISP (1.42%), PLB (0.77%), and QPCT (0.46%). These results support the finding of Aird, S. D et al. on Ryukyu Pmu venom proteomics, genomcis, and transcriptomes [6,16] and agree with Villalta, M. et al.’s study using pooled Pmu venom from Taiwan [5].

2.2. Comparison of Inter-Population Variations in Pmu Venom

The inter-population variation in venom protein families of 163 adult Pmu venoms (119 patients in northern and 44 in southeastern Taiwan) was compared in Table 2 and Figure S1. No significant difference was found for dominant protein families, such as SVMP (45.95% vs. 42.76%), PLA2s (25.56% vs. 23.48%), Snaclec (13.15% vs. 11.86%), SVSP (8.26% vs. 8.15%), and other minor protein families. Even though the difference in the average abundance of venom protein families between the two regions was trivial compared to other pit viper species (Table 2), the details of different protein fractions are worth investigating in order to further understand how they may be related to specific clinical manifestations.
The details of different protein fractions in the venom of individual Pmu between populations was compared. The results showed that protein fractions varied between Pmu populations from these two regions (Table 3).
We identified PLA2 or its homolog in five major fractions (1, 2, 4, 5, and 6) eluted from HPLC (Table 1). In contrast to the basic PLA2 homolog (Fr 1, 2, and 5), which did not show variation, acidic PLA2s (Fr 4 and 6) presented a significant variation (Figure 2A, Table 3). The major acidic E6-PLA2 (named Pmu-PLA-I, with anti-platelet activity) [17] was significantly higher in the venom of the northern Pmu population (Fr 6, Figure 2A). Furthermore, another minor acidic R6-PLA2 (TmPL-III) was only present in low amounts in some Pmu venom from both regions (Fr 4, Figure 2A). Notably, the proportion of Pmu venoms containing TmPL-III in the southeastern population was significantly higher than those in the northern region (47.7% vs. 19.3%, p < 0.001) (Table 3). However, in a study by Tsai et al., TmPL-III was present in the northern population but absent in the southwestern population in about 20 Pmu venom samples [18,19]. Therefore, we suggest that the presence of TmPL-III in Pmu venom varies regionally, with more frequent occurrence in the southeastern Pmu population. Although there had been claims about the presence of R49-PLA2, which was found in Chinese Pmu, in Taiwanese Pmu from other research [20], we did not find R49-PLA2 in our 163 venom samples.
SVMPs derived from the PII-protease domains were the most abundant protein family in all Pmu venom samples (Table 2). Proteins of this family were identified from the fraction of 15, 16, 18, 19, 21, 22, and a part of 20 (Table 1). PI-SVMP (Fr 11) and PIII-SVMP (Fr 22) of the Pmu samples also showed inter-population variability. Most Pmu venoms from the two regions contain PIII-SVMP (Fr 22) (northern area: 93.3%, southeastern area: 100%, Table 3), but the relative abundance of PIII-SVMP was significantly higher in the southeastern area (Figure 2C). Furthermore, a higher proportion of Pmu contains PI-SVMP (Fr 11) in the northern region (87.4% vs. 72.7%, p = 0.025, Table 3). Additionally, the analytic result of PI-SVMP (Fr 11) is found to be similar to trimerelysin-2 (trimerelysin-2 like) (accession No. P20165) isolated from the venom of Protobothrops flavoviridis, which may have no or low hemorrhagic activity [21].
As for SVSP variants, mucrofibrase-3 (Fr 9) was consistently present in venoms from both Pmu populations (Table 3), and its relative abundance was similar across the populations (Figure 2B). On the other hand, inter-population variability was found in other SVSPs (Fr 7, 8, and 10). For example, a plasminogen activator similar to TSV-PA (accession No. Q91516, of T. s. stejnegeri venom) was identified in Fr 7. Its relative abundance (Figure 2A) and proportion of presence (82.4% vs. 65.9%, p = 0.025, Table 3) was significantly higher in the northern population. As for Snaclecs, trimecetin identified from Fr 15 and 17 was more prevalent, with higher relative abundance in the northern population (Table 3, Figure 2C). Both LAO enzymes (accession No. T2HRS5) (Fr 13 and 20) and 5’-NT (Fr 14) were more frequent and abundant in the venom of the southeastern population (Table 3, Figure 2B).
In short, we disclosed that the major variations were acidic PLA2 variants (i.e., E6- and R6-PLA2), Snaclec, and metalloproteases of PII-class by analyzing many individual Pmu venoms (Table 3, Figure 2). To produce effective antivenom, guidelines recommended that an adequate number of individual snakes from various regions covering the entire geographic distribution of specific venomous snake species should be collected together [2]. Our findings may help evaluate the pooled snake venoms based on characters of inter-population variation and potentially assist in improving antivenom titer against snake venom from these regions.

2.3. Regional Variation in the Clinical Manifestations of Pmu Envenoming

A total of 209 Pmu envenoming cases, in 149 and 60 patients from northern and southeastern Taiwan, respectively, were compared to investigate the regional variation in clinical manifestations (Table 4). A higher proportion of the Pmu envenoming cases in northern Taiwan developed ecchymosis (75.2% vs. 21.7%, p < 0.001) and blistering (17.5% vs. 5%, p = 0.019). No significant difference was found for local complications, such as tissue necrosis (11.4% vs. 5%, p = 0.154) and compartment syndrome (6% vs. 8.3%, p = 0.549), or in the systemic complications. There were slightly higher proportions of cellulitis (25.5% vs. 13.3%, p = 0.055) and renal impairment (12.3% vs. 5.4%, p = 0.088) in patients from the northern region than those from southeastern area.
Currently, there are only a few studies that examined the difference in snake envenoming effects regarding the geographic locations available, with most of them focusing on the comparison of the severity score and antivenom dosage following bites between regions [7,8,9]. Our study is the first to investigate the clinical manifestation differences of single species snakebite in northern and southeastern Taiwan. The variation in clinical effects is likely to be affected by the variation in venom components, as suggested in two case studies of Crotalus pyrrhus bites in the western and eastern regions of Arizona state in America, respectively [9]. Although specific antivenom dosages used for envenomed patients in the northern and southeastern Taiwan differed (with the mean of 4.4 vials for northern region and 6.4 vials for southeastern area), a solid conclusion for differences in antivenom usage could not be simply drawn based on this data. Thus, further antivenomic evaluation of the neutralization effect on major toxic venom components will be essential for the determination of antivenom administration in managing Pmu bites.

2.4. Possible Venom Components Contributing to Regional Differences in Clinical Manifestations

Since there were significant differences in ecchymosis and blistering with the high similarity shown in venom components, the results are worthy of further discussion. Ecchymosis is a local hemorrhage near the skin’s surface caused by mechanical injury of the capillary basement membrane and is found to be more common in Pmu envenoming of northern victims (Table 4). SVMPs are considered as the main components in viperid venom that contribute to hemorrhage [22]. SVMPs degrade extracellular matrix proteins in the capillary basement membrane, causing endothelial cell apoptosis [23,24,25] and inducing hemorrhage [26]. P-II and PIII-metalloproteases bind more specifically to the capillary basement membrane than PI-SVMPs, causing higher hemorrhagic activity [26,27,28]. Furthermore, the non-catalytic PLA2 (myotoxic and cytotoxic [29,30]) in viperid venom could induce endothelial cell detachment synergistically with PIII-SVMP [31] and possibly enhance myotoxicity caused by non-catalytic SVSP [32]. Based on the biochemical effects of enzymes and our findings of the variation in venom components and clinical manifestations, we suggest that a higher incidence of venom-induced ecchymosis in patients from the northern region is likely to be caused by PI-SVMP (Fr 11) and PII-SVMP (Fr 15, 18, and 19).
Venom-induced blistering, characterized by the separation of the epidermis and dermis [33], is a clinical feature more frequently observed in patients from northern Taiwan (Table 4). SVMPs, particularly PI-SVMPs, induce the proteolytic cleavage of skin basement membrane components at the dermal–epidermal junction [34,35,36,37,38]. Additionally, the proteolytic action of endogenous matrix metalloproteinases in local tissues activated by inflammatory reactions may be involved in blister formation [37,38]. Moreover, the activation of the complement system induced by PI-SVMPs promotes local tissue inflammation [39]. K49-PLA2 induces cell membrane damage through a non-catalytic mechanism and release inflammatory mediators in tissues [30,40,41,42]. Catalytic D49-PLA2s enhance the release of inflammatory mediators synergistically with K49-PLA2 [29,30]. The high abundance of PI-SVMP (trimerelysin-2 like), PII-SVMP (PMMP-3), and acidic E6-PLA2 in the northern Pmu venom may jointly attribute to more severe local symptoms. Therefore, this information could be important for antivenom manufacturers and clinicians in assessing antivenom efficacy and effectiveness in managing Pmu bites. Although the differences of minor components (i.e., Snaclec, CRISP, LAO, PLB, QPCT, and 5’-NT) in venoms were found between regions, their clinical significance in Pmu envenoming remains undetermined [43,44,45,46]. Furthermore, a proper-designed animal model examining the ecchymosis and blistering effect by the subcutaneous injection of venom component at different doses may be helpful in the determination of the causal relationship between the venom component and the associated clinical effect.

2.5. Limitations

Although we found inter-population variations in venom compositions and suggested the associations of venom variations with clinical manifestations, this study has some limitations. First, the body size, age, and sex of individual snakes; the amount of venom injection; and the health status of patients, which are important factors that may have affected the severity and treatment of envenomation, are difficult to determine in such retrospective studies. Second, it is challenging to separate and analyze snake venom components by RP-HPLC with gel electrophoresis because of their similar molecular weights or hydrophobicity. Furthermore, the method used for protein resolution or separation (mainly re-dissolved or reconstituted venom solution) might cause certain non-protein loss and precipitation of high-molecular-weight constituents, making these components undetectable. Third, the bottom-up venomomics applied in this study may provide incomplete compositional information of venoms, especially for low-abundance components. The top-down mass venomics may eliminate the shortcomings of bottom-up workflows [47,48]. Finally, the data of this study were extracted from medical charts that cover a long period. This introduces the possibility of variations in how the information has been introduced into the charts; that is, different physicians may introduce a different degree of detail in the charts. Additionally, different physicians were likely involved in the therapeutic decisions of these patients. Since this is a retrospective study and has its inherent limitations, results should be interpreted cautiously. Nevertheless, our pioneering study on the investigation of the variation in Pmu venoms and clinical envenoming effects in different regions in Taiwan has strong potential in improving therapeutic implications in snakebite management.

3. Conclusions

We found that there is inter-population variability in the presence of Pmu venom protein fractions 4, 7, 8, 10, 11, 13, 14, 17, 18, and 19 (i.e., PLA2 TmPL-III, SVSP, KN-SVSP, SVSP, PI-SVMP, LAO, 5’-NT, Snaclec, PII-SVMP, and PII-SVMP, respectively). A higher proportion in fractions 7, 11, 17, 18, and 19 (i.e., SVSP, PI-SVMP, Snaclec, PII-SVMP, and PII-SVMP, respectively) was noticed in the northern snakes’ population. Additionally, the amount of relevant protein families is higher in fractions 3, 6, 7, 8, 10, 13, 14, 15, 17, 18, 20, and 22 in the northern snakes’ population even though the presence of certain fractions (i.e., 6, 15, and 20) does not differ between regions.
Furthermore, we found a higher risk of developing ecchymosis and blistering in the northern patients’ population, which is suggested to be associated with the higher abundance of PI-SVMP (trimerelysin-2 like), PII-SVMP (PMMP-3), Snaclec (Trimecetin), SVSP (TSV-PA like), and acidic E6-PLA2 in the Pmu venoms obtained from the same areas. Our study provides objective information on Pmu venoms, and these findings can have therapeutic implications for future antivenom manufacturing processes, efficacy evaluation against these major fractions, and snakebite management in Taiwan.

4. Materials and Methods

4.1. Snake Venoms

Venom samples of 163 adult Pmu were obtained from two regions: Taipei Basin and the surrounding mountain areas in northern Taiwan (n = 119) and East Rift Valley in southeastern Taiwan (n = 44) (Figure 1). Each individual Pmu was manually restrained while venom was milked from both half-erected fangs into a 1.5 mL Eppendorf tube. The fresh yellowish crude venom was lyophilized and stored at –20 °C.

4.2. Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)

Individual Pmu crude venom was reconstituted in ultrapure water containing 0.1% trifluoroacetic acid (TFA, Sigma-Aldrich, MO, USA) and 5% acetonitrile (Mallinckrodt Baker, NJ, USA) and centrifuged at 10,000× g for 10 min. The protein concentration of the supernatant was determined using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, IL, USA). Venom proteins were separated by RP-HPLC following the method of Villalta, M. et al. study [5]. Briefly, 100 μg of venom protein in 10 μL ultrapure water containing 0.1% TFA (solution A) was injected into an ultra-pressure liquid chromatography system (LC-20ADXR, Shimadzu, Kyoto, Japan) equipped with a diode-array detector (SPD-M20A, Shimadzu, Kyoto, Japan). Chromatography was performed using a Phenomenex Jupiter C18 (250 × 4.6 mm, 5 µm particle size, 300 Å pore size) column at 1 mL/min with a linear gradient of 5% solution B (0.1% TFA in acetonitrile) for 5 min, 5–25% solution B for 10 min, 25–45% solution B for 60 min, and 45–70% solution B for 10 min. The effluent was monitored at 215 nm, and eluted peaks were manually collected and lyophilized.
No individual venom samples in this study have all the HPLC-resolvable protein fractions. However, we have identified as many fractions as possible. The isolated fractions were collected and analyzed from representative samples, including Pmu001 (collected from East Rift Valley), Pmu002 (collected from Taipei Basin), and Pmu003 (collected from Taipei Basin). Pmu003, containing the most resolvable fractions, was chosen as the primary reference (Figure 1D). Along with unique fractions obtained from two other samples, all resolvable fractions were subjected to subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry (MS/MS) to identify the proteins.

4.3. SDS-PAGE and LC-MS/MS Analysis

Before gel electrophoresis, venom samples in 20 μL Laemmli sample buffer containing 25 mM dithiothreitol (Biosynth AG, Switzerland) were boiled for 5 min and alkylated with 135 mM iodoacetamide (Amersham Biosciences, UK). Then, venom proteins were separated through SDS-PAGE, and the gel was stained with Coomassie brilliant blue. Further preparation of MS samples was conducted by cutting each stained band into a few slices, which were destained in 10% methanol (Mallinckrodt Baker, NJ, USA), dehydrated in acetonitrile, and dried in a SpeedVac. Proteins in the gel were digested by sequencing-grade modified porcine trypsin (20 μg/mL; Promega, Madison, WI, USA) overnight at 37 °C. Then, the tryptic peptides were extracted with 200 μL of 0.1% formic acid (Sigma-Aldrich, MO, USA) and 50% acetonitrile solution, with 70% acetonitrile in ultrapure water, dried in SpeedVac, and finally stored at −80 °C for subsequent processing.
Peptide mixtures were reconstituted with 0.1% formic acid and analyzed by LC-MS/MS using an LTQ Orbitrap Velos mass spectrometer coupled with a nano-LC (Thermo Fisher, San Jose, CA, USA). Further, the sample was loaded into a trap column (Zorbax 300SB-C18, 0.3 × 5 mm; Agilent Technologies, Wilmington, DE) at a flow rate of 0.2 μL/min in solution A (0.1% formic acid in ultrapure water) and separated on a resolving 10-cm analytical C18 column (inner diameter, 75 μm) using a 15-μm tip (New Objective, Woburn, MA, USA). The peptides were eluted using a linear gradient of 0–10% solution B (0.1% formic acid in acetonitrile) for 3 min, 10–30% solution B for 35 min, 30–35% solution B for 4 min, 35–50% solution B for 1 min, 50–95% solution B for 1 min, and 95% solution B for 8 min, with a flow rate of 0.25 μL/min. The Orbitrap’s resolution was set at 30,000, and the ion signal of (Si(CH3)2O)6H+ at 445.120025 (m/z) was used as a lock mass for internal calibration.
For MS scan, the m/z value of scan range was 400–2000 Da. For MS/MS scan, more than 1 × 104 ions were accumulated in the ion trap, and the m/z values selected were dynamically excluded for 180 s. Both MS and MS/MS spectra were acquired using one scan with maximum 1000 and 100 ms fill-times, respectively. MS raw data were analyzed using Proteome Discoverer Software (v.1.4.1.14; Thermo Fisher, San Jose, CA, USA) and searched against a non-redundant Swiss-Prot database of Serpents (downloaded in August 2021, 273,008 entries) using MASCOT. Search parameters for tryptic peptides included one missed cleavage, a mass tolerance of 0.5 Da for fragment ions, fixed cysteine modification with carbamidomethylation, variable methionine oxidation, variable N-terminal acetylation, and variable transformation of N-terminal glutamate or glutamine to pyroglutamic acid. Finally, the criteria used for protein identification were high peptide confidence; the lowest peptide length was five amino acids, and the minimal number of peptides per protein was two.

4.4. Relative Abundance of Protein Families

The relative content of each major protein family was estimated as described by Calvete et al. [49]. Then, the relative content of each LC-eluted protein-peak was calculated based on its peak area using LabSolution (v.5.93, Shimadzu, Kyoto, Japan). For venom protein presenting more than one band in SDS-PAGE, the relative abundance of each protein was estimated by densitometry using GelQuantNET (BiochemLabSolutions Corp., USA). Finally, the abundance of each protein family was calculated as the percentage of the whole venom proteome.

4.5. Clinical Manifestations of Pmu-Envenomed Patients in Two Taiwan Regions

This is a secondary analysis of the retrospective cohort studies [12,13]. All patients enrolled were admitted to two medical centers, Taipei Veterans General Hospital (1991–2006) in northern Taiwan and Hualien Tzu Chi Hospital (2008–2013) in southeastern Taiwan (Figure 3). The culprit snake was identified by examining the snake brought-in by the patient or identifying the snake in a standard picture exhibited in the emergency department [12,13]. The case was excluded in the study if the snake could not be identified [13].
The clinical data collected included patients’ clinical presentation, laboratory analyses, clinical outcomes, and complications. The clinical manifestations of Pmu envenomation were classified into three categories: (1) local symptoms, including ecchymosis (Figure 4A,B) and blistering (bulla formation) (Figure 4B,C); (2) local complications, including cellulitis, wound necrosis (Figure 4D), and compartment syndrome; (3) systemic complications, including thrombocytopenia (with platelet count < 150 × 109/L), coagulopathy (with an international normalized ratio >1.25), renal impairment (with serum creatinine level >1.4 mg/dL), and rhabdomyolysis (with blood creatine kinase level >1000 U/L). The study protocols were approved by the Institutional Review Board of Taipei Veterans General Hospital (95-02-25A) and Hualien Tzu Chi Hospital (IRB102-38).

4.6. Statistical Analysis

The normality of distribution of continuous variables was tested by one-sample Kolmogorov–Smirnov test. Continuous variables with normal distribution were compared using independent samples Student’s t-test and presented as mean ± standard deviation (SD); non-normal variables were compared by the Mann–Whitney U test reported as the median interquartile range (IQR). Categorical variables were compared using the Chi-square test or Fisher’s exact test. We analyzed all the data using the Statistical Package for the Social Sciences v.21.0 (released 2012; IBM Corp., Armonk, NY, USA). A two-tailed p-value of <0.05 was considered statistically significant.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/toxins14090643/s1, Figure S1: Relative abundance of the protein families of individual Pmu venom in the two geographic regions.

Author Contributions

Conceptualization, L.-C.C., W.-G.W. and Y.-C.M.; methodology, L.-C.C. and Y.-C.M.; validation, Y.-C.M., K.-Y.C. and L.-C.C.; formal analysis, L.-C.C.; investigation, L.-C.C.; resources, L.-C.C.; data curation, H.-Y.S., Y.-C.C. and L.-C.C.; writing—original draft preparation, L.-C.C.; writing—review and editing, Y.-C.M.; visualization, L.-C.C.; supervision, L.-C.C. and W.-G.W.; project administration, W.-G.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Foundation for Poison Control, Taiwan (grant number NPCC-201901) and Taichung Veterans General Hospital (grant number TCVGH-1117201). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of the Taipei Veterans General Hospital (VGHIRB No.: 95-02-25A, 13 February 2006) and the Buddhist Tzu Chi General Hospital (REC No.: IRB102-38, 20 June 2014).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Taipei Zoo and Hualien County Fire Department Ji-An Branch for administrative support, and Ming-Jen Tsai, Szu-Lung Chen, Chun-Kai Yang, Kuang-Ting Chen, Chen-Chang Yang, Ya-Fen Zheng, Fen-Shiung Wu, Yu-Jhen Liang, Chien-Chen Huang, Yun-Yi Lin, I-shu Chuang, Hsiu-Chou Chung, Ming Hsin Yu, Wei-Min Wu, and Ming-Yi Lin for technical support.

Conflicts of Interest

The authors have no competing interest.

References

  1. Vasaruchapong, T.; Laoungbua, P.; Tangrattanapibul, K.; Chanhome, L. Protobothrops mucrosquamatus (Cantor, 1839), a highly venomous species added to the snake fauna of Thailand (Squamata: Viperidae). Trop. Nat. Hist. 2017, 17, 111–115. [Google Scholar]
  2. Cichutek, K.; Epstein, J.; Griffiths, E.; Hindawi, S.; Jivapaisarnpong, T.; Klein, H.; Minor, P.; Moftah, F.; Reddy, V.; Slamet, L. WHO Expert Committee on Biological Standardization Sixty-seventh report. Tech. Rep. Ser.-World Health Organ. 2017, 1004, 1–591. [Google Scholar]
  3. Mao, Y.C.; Hung, D.Z. Epidemiology of Snake Envenomation in Taiwan. In Toxinology: Clinical Toxinology; Gopalakrishnakone, P., Faiz, S.M.A., Gnanathasan, C.A., Habib, A.G., Fernando, R., Yang, C.C., Eds.; Springer: Dordrecht, The Netherlands, 2013; pp. 1–17. [Google Scholar]
  4. Mao, Y.C.; Liu, P.Y.; Chiang, L.C.; Lee, C.H.; Lai, C.S.; Lai, K.L.; Lin, W.L.; Su, H.Y.; Ho, C.H.; Doan, U.V.; et al. Clinical manifestations and treatments of Protobothrops mucrosquamatus bite and associated factors for wound necrosis and subsequent debridement and finger or toe amputation surgery. Clin. Toxicol. 2020, 59, 28–37. [Google Scholar] [CrossRef] [PubMed]
  5. Villalta, M.; Pla, D.; Yang, S.L.; Sanz, L.; Segura, A.; Vargas, M.; Chen, P.Y.; Herrera, M.; Estrada, R.; Cheng, Y.F.; et al. Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: Keys to understand the variable immune response in horses. J. Proteom. 2012, 75, 5628–5645. [Google Scholar] [CrossRef] [PubMed]
  6. Aird, S.D.; Arora, J.; Barua, A.; Qiu, L.; Terada, K.; Mikheyev, A.S. Population Genomic Analysis of a Pitviper Reveals Microevolutionary Forces Underlying Venom Chemistry. Genome Biol. Evol. 2017, 9, 2640–2649. [Google Scholar] [CrossRef]
  7. Casewell, N.R.; Jackson, T.N.W.; Laustsen, A.H.; Sunagar, K. Causes and Consequences of Snake Venom Variation. Trends Pharmacol. Sci. 2020, 41, 570–581. [Google Scholar] [CrossRef] [PubMed]
  8. Massey, D.J.; Calvete, J.J.; Sánchez, E.E.; Sanz, L.; Richards, K.; Curtis, R.; Boesen, K. Venom variability and envenoming severity outcomes of the Crotalus scutulatus scutulatus (Mojave rattlesnake) from Southern Arizona. J. Proteom. 2012, 75, 2576–2587. [Google Scholar] [CrossRef]
  9. Cochran, C.; Hax, S.; Hayes, W.K. Case reports of envenomation and venom composition differences between two Arizona populations of the Southwestern Speckled Rattlesnake, Crotalus pyrrhus (Cope, 1867). Toxicon 2019, 171, 29–34. [Google Scholar] [CrossRef] [PubMed]
  10. Vélez, S.M.; Salazar, M.; Acosta de Patiño, H.; Gómez, L.; Rodriguez, A.; Correa, D.; Saldaña, J.; Navarro, D.; Lomonte, B.; Otero-Patiño, R.; et al. Geographical variability of the venoms of four populations of Bothrops asper from Panama: Toxicological analysis and neutralization by a polyvalent antivenom. Toxicon 2017, 132, 55–61. [Google Scholar] [CrossRef]
  11. Tan, C.H.; Palasuberniam, P.; Tan, K.Y. 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, 514. [Google Scholar] [CrossRef]
  12. Chen, Y.W.; Chen, M.H.; Chen, Y.C.; Hung, D.Z.; Chen, C.K.; Yen, D.H.; Huang, C.I.; Lee, C.H.; Wang, L.M.; Yang, C.C. Differences in clinical profiles of patients with Protobothrops mucrosquamatus and Viridovipera stejnegeri envenoming in Taiwan. Am. J. Trop. Med. Hyg. 2009, 80, 28–32. [Google Scholar] [CrossRef] [PubMed]
  13. Su, H.Y.; Li, Y.H.; Tang, C.N.; Su, C.I.; Tsai, M.J. Can surgery in patient with Protobothrops mucrosquamatus envenomation be predicted in emergency department? Hong Kong J. Emerg. Med. 2016, 23, 210–219. [Google Scholar] [CrossRef]
  14. Tsai, I.H.; Wang, Y.M.; Chen, Y.H.; Tsai, T.S.; Tu, M.C. Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): Molecular characterization, geographic variations and evidence of multiple ancestries. Biochem. J. 2004, 377, 215–223. [Google Scholar] [CrossRef] [PubMed]
  15. Creer, S.; Malhotra, A.; Thorpe, R.S.; Stöcklin, R.S.; Favreau, P.S.; Hao Chou, W.S. Genetic and ecological correlates of intraspecific variation in pitviper venom composition detected using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing. J. Mol. Evol. 2003, 56, 317–329. [Google Scholar] [CrossRef] [PubMed]
  16. Aird, S.D.; Watanabe, Y.; Villar-Briones, A.; Roy, M.C.; Terada, K.; Mikheyev, A.S. Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis). BMC Genom. 2013, 14, 790. [Google Scholar] [CrossRef]
  17. Tsai, I.H.; Chen, Y.H.; Wang, Y.M. Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers. Biochim. Biophys. Acta 2004, 1702, 111–119. [Google Scholar] [CrossRef]
  18. Tsai, I.H.; Chen, Y.H.; Wang, Y.M.; Liau, M.Y.; Lu, P.J. Differential expression and geographic variation of the venom phospholipases A2 of Calloselasma rhodostoma and Trimeresurus mucrosquamatus. Arch. Biochem. Biophys. 2001, 387, 257–264. [Google Scholar] [CrossRef]
  19. Tsai, I.H.; Wang, Y.M.; Chen, Y.H. Variations of phospholipases A2 in the geographic venom samples of pitvipers. J. Toxicol. Toxin Rev. 2003, 22, 651–662. [Google Scholar] [CrossRef]
  20. Wei, J.F.; Wei, X.L.; Mo, Y.Z.; He, S.H. Induction of microvascular leakage and histamine release by promutoxin, an Arg49 phospholipase A2. Toxicon 2010, 55, 888–896. [Google Scholar] [CrossRef]
  21. Maruyama, K.; Kawasaki, T.; Sakai, Y.; Taniuchi, Y.; Shimizu, M.; Kawashima, H.; Takenaka, T. Isolation and amino acid sequence of flavostatin, a novel disintegrin from the venom of Trimeresurus flavoviridis. Peptides 1997, 18, 73–78. [Google Scholar] [CrossRef]
  22. Fox, J.W.; Serrano, S.M. Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases. Toxicon 2005, 45, 969–985. [Google Scholar] [CrossRef]
  23. You, W.K.; Seo, H.J.; Chung, K.H.; Kim, D.S. A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis through its protease and disintegrin-like domains. J. Biochem. 2003, 134, 739–749. [Google Scholar] [CrossRef] [PubMed]
  24. Wu, W.B.; Chang, S.C.; Liau, M.Y.; Huang, T.F. Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells. Biochem. J. 2001, 357, 719–728. [Google Scholar] [CrossRef] [PubMed]
  25. Díaz, C.; Valverde, L.; Brenes, O.; Rucavado, A.; Gutiérrez, J.M. Characterization of events associated with apoptosis/anoikis induced by snake venom metalloproteinase BaP1 on human endothelial cells. J. Cell. Biochem. 2005, 94, 520–528. [Google Scholar] [CrossRef] [PubMed]
  26. Herrera, C.; Escalante, T.; Voisin, M.B.; Rucavado, A.; Morazán, D.; Macêdo, J.K.; Calvete, J.J.; Sanz, L.; Nourshargh, S.; Gutiérrez, J.M.; et al. Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: Clues on the mechanisms of venom-induced hemorrhage. PLoS Negl. Trop. Dis. 2015, 9, e0003731. [Google Scholar] [CrossRef]
  27. Olaoba, O.T.; Karina Dos Santos, P.; Selistre-de-Araujo, H.S.; Ferreira de Souza, D.H. Snake Venom Metalloproteinases (SVMPs): A structure-function update. Toxicon X 2020, 7, 100052. [Google Scholar] [CrossRef]
  28. Baldo, C.; Jamora, C.; Yamanouye, N.; Zorn, T.M.; Moura-da-Silva, A.M. Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: Tissue distribution and in situ hydrolysis. PLoS Negl. Trop. Dis. 2010, 4, e727. [Google Scholar] [CrossRef]
  29. Cintra-Francischinelli, M.; Pizzo, P.; Rodrigues-Simioni, L.; Ponce-Soto, L.A.; Rossetto, O.; Lomonte, B.; Gutiérrez, J.M.; Pozzan, T.; Montecucco, C. Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors. Cell. Mol. Life Sci. CMLS 2009, 66, 1718–1728. [Google Scholar] [CrossRef]
  30. Fernandes, C.A.; Borges, R.J.; Lomonte, B.; Fontes, M.R. A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms. Biochim. Biophys. Acta 2014, 1844, 2265–2276. [Google Scholar] [CrossRef]
  31. Bustillo, S.; García-Denegri, M.E.; Gay, C.; Van de Velde, A.C.; Acosta, O.; Angulo, Y.; Lomonte, B.; Gutiérrez, J.M.; Leiva, L. Phospholipase A(2) enhances the endothelial cell detachment effect of a snake venom metalloproteinase in the absence of catalysis. Chem.-Biol. Interact. 2015, 240, 30–36. [Google Scholar] [CrossRef]
  32. Ogawa, T.; Tobishima, Y.; Kamata, S.; Matsuda, Y.; Muramoto, K.; Hidaka, M.; Futai, E.; Kuraishi, T.; Yokota, S.; Ohno, M.; et al. Focused Proteomics Analysis of Habu Snake (Protobothrops flavoviridis) Venom Using Antivenom-Based Affinity Chromatography Reveals Novel Myonecrosis-Enhancing Activity of Thrombin-Like Serine Proteases. Front. Pharmacol. 2021, 12, 766406. [Google Scholar] [CrossRef] [PubMed]
  33. Warrell, D.A. 75—Venomous and Poisonous Animals. In Manson’s Tropical Infectious Diseases (Twenty-Third Edition); Farrar, J., Hotez, P.J., Junghanss, T., Kang, G., Lalloo, D., White, N.J., Eds.; W.B. Saunders: London, UK, 2014; pp. 1096–1127.e1093. [Google Scholar]
  34. Macêdo, J.K.A.; Joseph, J.K.; Menon, J.; Escalante, T.; Rucavado, A.; Gutiérrez, J.M.; Fox, J.W. Proteomic analysis of human blister fluids following envenomation by three snake species in India: Differential markers for venom mechanisms of action. Toxins 2019, 11, 246. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Jiménez, N.; Escalante, T.; Gutiérrez, J.M.; Rucavado, A. Skin pathology induced by snake venom metalloproteinase: Acute damage, revascularization, and re-epithelization in a mouse ear model. J. Investig. Dermatol. 2008, 128, 2421–2428. [Google Scholar] [CrossRef] [PubMed]
  36. Freitas-de-Sousa, L.A.; Colombini, M.; Lopes-Ferreira, M.; Serrano, S.M.T.; Moura-da-Silva, A.M. Insights into the mechanisms involved in strong hemorrhage and dermonecrosis induced by atroxlysin-Ia, a PI-class snake venom metalloproteinase. Toxins 2017, 9, 239. [Google Scholar] [CrossRef]
  37. Escalante, T.; Rucavado, A.; Pinto, A.F.; Terra, R.M.; Gutiérrez, J.M.; Fox, J.W. Wound exudate as a proteomic window to reveal different mechanisms of tissue damage by snake venom toxins. J. Proteome Res. 2009, 8, 5120–5131. [Google Scholar] [CrossRef]
  38. Gutiérrez, J.M.; Rucavado, A.; Chaves, F.; Díaz, C.; Escalante, T. Experimental pathology of local tissue damage induced by Bothrops asper snake venom. Toxicon 2009, 54, 958–975. [Google Scholar] [CrossRef]
  39. Pidde-Queiroz, G.; Magnoli, F.C.; Portaro, F.C.; Serrano, S.M.; Lopes, A.S.; Paes Leme, A.F.; van den Berg, C.W.; Tambourgi, D.V. P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade. PLoS Negl. Trop. Dis. 2013, 7, e2519. [Google Scholar] [CrossRef]
  40. Costa, S.K.P.; Camargo, E.A.; Antunes, E. Inflammatory action of secretory phospholipases A2 from snake venoms. In Toxins and Drug Discovery; Cruz, L.J., Luo, S., Gopalakrishnakone, P., Eds.; Springer: Dordrecht, The Netherlands, 2017; pp. 35–52. [Google Scholar]
  41. Caccin, P.; Pellegatti, P.; Fernandez, J.; Vono, M.; Cintra-Francischinelli, M.; Lomonte, B.; Gutiérrez, J.M.; Di Virgilio, F.; Montecucco, C. Why myotoxin-containing snake venoms possess powerful nucleotidases? Biochem. Biophys. Res. Commun. 2013, 430, 1289–1293. [Google Scholar] [CrossRef]
  42. Rucavado, A.; Nicolau, C.A.; Escalante, T.; Kim, J.; Herrera, C.; Gutiérrez, J.M.; Fox, J.W. Viperid envenomation wound exudate contributes to increased vascular permeability via a DAMPs/TLR-4 mediated pathway. Toxins 2016, 8, 349. [Google Scholar] [CrossRef]
  43. Arafat, A.S.; Arun, A.; Ilamathi, M.; Asha, J.; Sivashankari, P.R.; D’Souza, C.J.; Sivaramakrishnan, V.; Dhananjaya, B.L. Homology modeling, molecular dynamics and atomic level interaction study of snake venom 5’ nucleotidase. J. Mol. Model. 2014, 20, 2156. [Google Scholar] [CrossRef]
  44. Lazarovici, P.; Marcinkiewicz, C.; Lelkes, P.I. From Snake Venom’s Disintegrins and C-Type Lectins to Anti-Platelet Drugs. Toxins 2019, 11, 303. [Google Scholar] [CrossRef] [PubMed]
  45. Suntravat, M.; Sanchez, O.; Reyes, A.; Cirilo, A.; Ocheltree, J.S.; Galan, J.A.; Salazar, E.; Davies, P.; Sanchez, E.E. Evaluation of Signaling Pathways Profiling in Human Dermal Endothelial Cells Treated by Snake Venom Cysteine-Rich Secretory Proteins (svCRiSPs) from North American Snakes Using Reverse Phase Protein Array (RPPA). Toxins 2021, 13, 613. [Google Scholar] [CrossRef]
  46. Tasoulis, T.; Isbister, G.K. A Review and Database of Snake Venom Proteomes. Toxins 2017, 9, 290. [Google Scholar] [CrossRef] [PubMed]
  47. Toby, T.K.; Fornelli, L.; Kelleher, N.L. Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu. Rev. Anal. Chem. 2016, 9, 499–519. [Google Scholar] [CrossRef] [PubMed]
  48. Ryan, C.M.; Souda, P.; Halgand, F.; Wong, D.T.; Loo, J.A.; Faull, K.F.; Whitelegge, J.P. Confident assignment of intact mass tags to human salivary cystatins using top-down Fourier-transform ion cyclotron resonance mass spectrometry. J. Am. Soc. Mass Spectrom. 2010, 21, 908–917. [Google Scholar] [CrossRef] [PubMed]
  49. Calvete, J.J.; Juárez, P.; Sanz, L. Snake venomics. Strategy and applications. J. Mass Spectrom. 2007, 42, 1405–1414. [Google Scholar] [CrossRef]
Toxins 14 00643 g001 550
Figure 1. HPLC and SDS-PAGE analyses of Pmu venom. In total, 100 µg of the Pmu crude venom protein from (A) Pmu001, (B) Pmu002, and (C) Pmu003 sample was separated by RP-HPLC; (D) each fraction separated in Figure 1C was subjected to the SDS-PAGE analysis under reducing conditions. Molecular mass of the markers (in kDa) are indicated at the gel left.
Figure 1. HPLC and SDS-PAGE analyses of Pmu venom. In total, 100 µg of the Pmu crude venom protein from (A) Pmu001, (B) Pmu002, and (C) Pmu003 sample was separated by RP-HPLC; (D) each fraction separated in Figure 1C was subjected to the SDS-PAGE analysis under reducing conditions. Molecular mass of the markers (in kDa) are indicated at the gel left.
Toxins 14 00643 g001
Toxins 14 00643 g002 550
Figure 2. The relative abundances of HPLC-fractions of the 163 Pmu venoms in the two geographic regions are shown in (A) Fr 1 to 8, (B) Fr 9 to 16, and (C) Fr 17 to 22; the northern population is marked in blue and the southeastern in red. Box plots showing median (line), 25th–75th percentile (box), and 5th-95th percentile (whiskers) of the individual abundance of the 22 fractions, respectively. Significance of the Mann–Whitney test are denoted (* = p < 0.05; ** = p < 0.005; *** = p < 0.001).
Figure 2. The relative abundances of HPLC-fractions of the 163 Pmu venoms in the two geographic regions are shown in (A) Fr 1 to 8, (B) Fr 9 to 16, and (C) Fr 17 to 22; the northern population is marked in blue and the southeastern in red. Box plots showing median (line), 25th–75th percentile (box), and 5th-95th percentile (whiskers) of the individual abundance of the 22 fractions, respectively. Significance of the Mann–Whitney test are denoted (* = p < 0.05; ** = p < 0.005; *** = p < 0.001).
Toxins 14 00643 g002
Toxins 14 00643 g003 550
Figure 3. The collection locales of Pmu venom of northern (blue dots) and southeastern (red dots) populations, and the locales of Taipei Veterans General Hospital (blue triangle) and Hualien Tzu Chi Hospital (red triangle).
Figure 3. The collection locales of Pmu venom of northern (blue dots) and southeastern (red dots) populations, and the locales of Taipei Veterans General Hospital (blue triangle) and Hualien Tzu Chi Hospital (red triangle).
Toxins 14 00643 g003
Toxins 14 00643 g004 550
Figure 4. Local effects of Pmu envenoming. (A) Ecchymosis; (B) blistering; (C) blistering on the skin; and (D) local necrosis on an envenomed finger.
Figure 4. Local effects of Pmu envenoming. (A) Ecchymosis; (B) blistering; (C) blistering on the skin; and (D) local necrosis on an envenomed finger.
Toxins 14 00643 g004
Table
Table 1. Assignment of the HPLC-fractions of Pmu venom based on MS/MS study.
Table 1. Assignment of the HPLC-fractions of Pmu venom based on MS/MS study.
FrcationProtein Name aProtein FamilySpeciesAccession No.
1basic PLA2 homolog, TMV-K49PLA2PmuP22640
2basic PLA2 homologPLA2Pmu~P22640
3cysteine-rich venom protein, TM-CRVPCRISPPmuP79845
4acidic R6-PLA2, TmPL-IIIPLA2PmuQ9I968
5basic N6-PLA2, TrimucrotoxinPLA2PmuQ90W39
6acidic E6-PLA2, TmPL-IPLA2PmuQ91506
7plasminogen activator, TSV-PASVSPT. stejnegeriQ91516
8kallikrein-CohID-4SVSPC. o. helleriT1DMM6
9beta-fibrinogenase mucrofibrase-3SVSPPmuQ91509
9trimecetin-beta subunitSnaclecPmuQ5FZI5
10beta-fibrinogenase mucrofibrase-2SVSPPmuQ91508
11trimerelysin-2PI-SVMPP. flavoviridisP20165
12mucrocetin-alpha subunitSnaclecPmuQ6TPH0
12trimecetin-beta subunitSnaclecPmuQ5FZI5
13L-amino acid oxidaseLAOP. flavoviridisT2HRS5
14ecto-5’-nucleotidase5’-NTP. elegansA0A077L6M5
15Zn-metalloproteinase, PMMP-3PII-SVMPPmuE9NW28
15trimecetin-beta subunitSnaclecPmuQ5FZI5
16Zn-metalloproteinase, PMMP-3PII-SVMPPmuE9NW28
17trimecetin-beta subunitSnaclecPmuQ5FZI5
18Zn-metalloproteinase, PMMP-3PII-SVMPPmuE9NW28
19Zn-metalloproteinase, PMMP-3PII-SVMPPmuE9NW28
20Zn-metalloproteinase, TM-3PII-SVMPPmuO57413
20trimecetin-alpha subunitSnaclecPmuQ5FZI6
20trimecetin-beta subunitSnaclecPmuQ5FZI5
20phospholipase B-likePLBPmuA0A1W7RER1
20glutaminyl cyclotransferasesQPCTPmuM9ND11
20L-amino acid oxidaseLAOP. elegansA0A077L6L4
21Zn-metalloproteinase, PMMP-1PII-SVMPPmuE9NW26
22Zn-metalloprotease P-IIIa (Fragment)PIII-SVMPP. elegansA0A077L7D6
22flavorasePIII-SVMPP. flavoviridisG1UJB2
a abbreviations: PLA2, phospholipases A2; PI-, PII-, PIII-SVMP, PI-, PII-, or PIII-class of venom metalloproteinase; Snaclec, snake venom C-type lectin like proteins; SVSP, snake venom serine protease; LAO, L-amino acid oxidase; CRISP, cysteine-rich secretory protein; 5’-NT, 5’-nucleotidase; PLB, phospholipase B; and QPCT, glutaminyl-peptide cyclotransferase.
Table
Table 2. Relative abundance of the venom proteins of individual Pmu in the two geographic regions presents by mean value.
Table 2. Relative abundance of the venom proteins of individual Pmu in the two geographic regions presents by mean value.
Protein Family aNorthernSoutheasternTotalp-Value
Sample numbern = 119n = 44n = 163
SVMP, total45.9542.7645.10.001
-PI or PII class43.0939.3442.11<0.001
-PIII class2.863.423.010.011
PLA225.5623.4825.00.033
SVSP8.268.158.230.364
Snaclec13.1511.8612.80.01
CRISP1.112.251.42<0.001
LAO2.152.972.370.01
5’-NT2.637.153.85<0.001
PLB0.740.860.770.172
QPCT0.440.520.460.05
a abbreviations are the same as in Table 1.
Table
Table 3. Presence of the HPLC-fraction in Pmu venom obtained from the two geographic regions.
Table 3. Presence of the HPLC-fraction in Pmu venom obtained from the two geographic regions.
FractionNorthern, n (%) aSoutheastern, n (%)Protein Familyp-Value
1119 (100)44(100)PLA21
217 (14.3)2(4.55)PLA20.103
395 (79.8)39(88.6)CRISP0.192
423 (19.3)21(47.7)PLA2 TmPL-III<0.001
5116 (97.5)42(95.5)PLA20.612
6115 (96.6)40(90.9)PLA20.213
798 (82.4)29(65.9)SVSP0.025
827 (22.7)25(56.8)KN-like SVSP<0.001
9119 (100)44(100)SVSP, Snaclec1
1056 (47.1)37(84.1)SVSP<0.001
11104 (87.4)32(72.7)PI-SVMP0.025
12116 (97.5)42(95.5)Snaclec0.612
1394 (79.0)43(97.7)LAO0.003
1474 (62.2)39(88.6)5’-NT0.001
15119 (100)42(95.5)PII-SVMP, Snaclec0.072
1659 (49.6)17(38.6)PII-SVMP0.214
1793 (78.2)18(40.9)Snaclec<0.001
18100 (84.0)21(47.7)PII-SVMP<0.001
1997 (81.5)29(65.9)PII-SVMP0.035
20119 (100)44(100)SVMP, others b1
2178 (65.6)30(68.2)PII-SVMP0.752
22111 (93.3)44(100)PIII-SVMP0.109
a Values in parentheses are % of the 119 (Northern) or 44 (Southeastern) Pmu samples containing the specific fraction. b Snaclec, PLB, LAO, and QPCT.
Table
Table 4. Comparison of the clinical manifestations of Pmu envenomed patients between the two geographic regions.
Table 4. Comparison of the clinical manifestations of Pmu envenomed patients between the two geographic regions.
RegionNorthernSoutheasternp-Value
Patient numbern = 149(%)n = 60(%)
Local symptoms
Ecchymosis112(75.2)13(21.7)<0.001
Blistering26(17.5)3(5)0.019
Local complications
Cellulitis38(25.5)8(13.3)0.055
Tissue necrosis17(11.4)3(5)0.154
Compartment syndrome9(6)5(8.3)0.549
Systemic complications
Thrombocytopenia18(12.1)8 (n = 57)(14)0.705
Coagulopathy9(6)2 (n = 58)(3.5)0.455
Acute renal impairment8(5.4)7 (n = 57)(12.3)0.088
Rhabdomyolysis17(11.4)3 (n = 27)(5.1)0.964
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Chiang, L.-C.; Chien, K.-Y.; Su, H.-Y.; Chen, Y.-C.; Mao, Y.-C.; Wu, W.-G. Comparison of Protein Variation in Protobothrops mucrosquamatus Venom between Northern and Southeast Taiwan and Association with Human Envenoming Effects. Toxins 2022, 14, 643. https://doi.org/10.3390/toxins14090643

AMA Style

Chiang L-C, Chien K-Y, Su H-Y, Chen Y-C, Mao Y-C, Wu W-G. Comparison of Protein Variation in Protobothrops mucrosquamatus Venom between Northern and Southeast Taiwan and Association with Human Envenoming Effects. Toxins. 2022; 14(9):643. https://doi.org/10.3390/toxins14090643

Chicago/Turabian Style

Chiang, Liao-Chun, Kun-Yi Chien, Hung-Yuan Su, Yen-Chia Chen, Yan-Chiao Mao, and Wen-Guey Wu. 2022. "Comparison of Protein Variation in Protobothrops mucrosquamatus Venom between Northern and Southeast Taiwan and Association with Human Envenoming Effects" Toxins 14, no. 9: 643. https://doi.org/10.3390/toxins14090643

APA Style

Chiang, L. -C., Chien, K. -Y., Su, H. -Y., Chen, Y. -C., Mao, Y. -C., & Wu, W. -G. (2022). Comparison of Protein Variation in Protobothrops mucrosquamatus Venom between Northern and Southeast Taiwan and Association with Human Envenoming Effects. Toxins, 14(9), 643. https://doi.org/10.3390/toxins14090643

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop