Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species
Abstract
:1. Introduction
2. Results
2.1. Chromatographic and Electrophoretic Profiling of T. gracilis Venom
2.2. T. gracilis Venom Proteomic Analysis
2.3. Two Cysteine-Rich Secretory Proteins (CRISPs) of T. gracilis Venom
2.4. Three VEGFs Are Expressed in the T. gracilis Venom Gland
3. Discussion
3.1. T. gracilis Venom Proteome
3.2. Comparison of Venom Composition and Toxicity among Closely Related Species
3.3. Sequence Comparison of T. gracilis Venom CRISPs
3.4. Sequence Comparison of T. gracilis Venom VEGFs
4. Conclusions
5. Materials and Methods
5.1. Chemicals
5.2. Animals and Venom
5.3. Determination of Venom Protein Concentration
5.4. Reverse-Phase High-Performance Liquid Chromatography
5.5. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
5.6. In-Solution Tryptic Digestion and Peptide Identification by Mass Spectrometry
5.7. Molecular Cloning and Sequence Determination of CRISPs and VEGFs
5.8. BLAST Analyses and Sequence Alignments
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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HPLC Fraction /Toxin Family | Protein (Proteoform) Name | Database Accession (NCBI) | Species | Protein Score | Relative Abundance (%) |
---|---|---|---|---|---|
Fraction 6 | |||||
LAAO | L-amino oxidase | gi|538260091 | Ovophis okinavensis | 69.70 | 1.91 |
Fraction 8 | |||||
SVMP III | Metalloprotease PIII [Tgc-PIII] * | gi|335892636 | Trimeresurus gracilis | 34.41 | 0.11 |
Fraction 10 | |||||
VEGF | Tgc-VGFb | OQ614864 | Trimeresurus gracilis | 75.04 | 0.28 |
Fraction 11 | |||||
SVMP I | Metalloprotease PI [Tgc-MP] | gi|335892630 | Trimeresurus gracilis | 50.20 | 0.63 |
Fraction 12 | |||||
SVMP II | Metalloprotease precursor H4, partial | gi|7340946 | Deinagkistrodon acutus | 82.92 | 0.91 |
SVMP I | Metalloprotease PI [Tgc-MP] | gi|335892630 | Trimeresurus gracilis | 71.95 | 0.49 |
LAAO | Chain A Amine oxidase | gi|1186227927 | Bothrops atrox | 132.93 | 0.16 |
Fraction 13 | |||||
DIS | Metalloprotease PIIb [gracilisin] ** | gi|335892632 | Trimeresurus gracilis | 99.53 | 3.13 |
LAAO | Chain A Amine oxidase | gi|1186227927 | Bothrops atrox | 93.75 | 0.65 |
SVMP III | P-III_metalloprotease | gi|547223066 | Ovophis okinavensis | 202.13 | 0.31 |
SVMP III | Metalloproteinase type III 12b | gi|1041577317 | Agkistrodon conanti | 183.99 | 0.22 |
SVMP II | Snake venom metalloprotease precursor | gi|2035122236 | Bothrops jararaca | 127.91 | 0.17 |
SVMP III | Metalloprotease PIII [Tgc-PIII] | gi|335892636 | Trimeresurus gracilis | 147.80 | 0.17 |
SVMP III | Metalloproteinase (type III) 1a | gi|818935191 | Crotalus adamanteus | 130.40 | 0.11 |
Fraction 14 | |||||
CRISP | Serotriflin | gi|1002598708 | Protobothrops mucrosquamatus | 190.38 | 0.46 |
CRISP | Cysteine-rich seceretory protein Og-CRPb, partial [Tgc-CRb] | gi|190195327 | Trimeresurus gracilis | 347.51 | 0.28 |
VEGF | Tgc-VGFb | OQ614864 | Trimeresurus gracilis | 207.39 | 0.21 |
CRISP | CRiSP-Sut-27 | gi|476539526 | Suta fasciata | 52.48 | 0.12 |
Fraction 15 | |||||
VEGF | Tgc-VGFc | OQ614865 | Trimeresurus gracilis | 121 | 1.16 |
VEGF | Tgc-VGFb | OQ614864 | Trimeresurus gracilis | 195.33 | 0.80 |
CRISP | Cysteine-rich seceretory protein Dr-CRPK | gi|190195321 | Daboia russelii | 155.56 | 0.23 |
VEGF | Cadam10_VEGF-1 | gi|1178170176 | Crotalus adamanteus | 85.2 | 0.20 |
CRISP | Cysteine-rich seceretory protein Og-CRPa [Tgc-CRa] | gi|190195325 | Trimeresurus gracilis | 287.81 | 0.18 |
CRISP | Cysteine-rich secretory protein, partial | gi|2205501413 | Malpolon monspessulanus | 109.71 | 0.13 |
Fraction 16 | |||||
CRISP | Cysteine-rich seceretory protein Dr-CRPK | gi|190195321 | Daboia russelii | 176.09 | 0.88 |
CRISP | Cysteine-rich seceretory protein Bs-CRP | gi|190195305 | Bothriechis schlegelii | 364.81 | 0.69 |
CRISP | Cysteine-rich secretory protein, partial | gi|2205501413 | Malpolon monspessulanus | 128.48 | 0.61 |
CRISP | Cysteine-rich secretory protein TRI1 | gi|123898155 | Trimorphodon biscutatus | 53.33 | 0.26 |
Fraction 17 | |||||
SVSP | Serine proteinase 12a | gi|1180525223 | Agkistrodon contortrix contortrix | 118.97 | 1.34 |
SVSP | Thrombin-like enzyme LmrSP-3 | gi|1714612439 | Lachesis muta rhombeata | 59.05 | 0.75 |
SVSP | Ancrod=thrombin-like alpha-fibrinogenase | gi|247212 | Akistrodon rhodostoma | 89.78 | 0.56 |
SVSP | Venom thrombin-like enzyme, partial | gi|118430266 | Deinagkistrodon acutus | 109.05 | 0.34 |
Fraction 18 | |||||
SVSP | Thrombin-like enzyme collinein-4 | gi|1109550140 | Crotalus durissus collilineatus | 97.45 | 0.39 |
SVSP | Serine proteinase 8b | gi|1041578893 | Sistrurus tergeminus | 120.13 | 0.39 |
SVSP | Thrombin-like enzyme bhalternin; Fibrinogen-clotting enzyme | gi|298351882 | Bothrops alternatus | 107.85 | 0.32 |
SVSP | Snake venom serine protease pallase | gi|158514815 | Gloydius halys | 147.7 | 0.22 |
SVSP | Ancrod-like protein | gi|1334616 | Calloselasma rhodostoma | 104.21 | 0.20 |
SVSP | Serine endopeptidase | gi|1333445426 | Crotalus tigris | 157.2 | 0.18 |
SVSP | Serine proteinase 2 | gi|1041577225 | Agkistrodon conanti | 120.95 | 0.18 |
SVSP | Agkihpin | gi|484358552 | Gloydius halys | 125.98 | 0.16 |
SVSP | Plasminogen-activator subtype serine protease (PA1/2) [Tgc-PAH1/2] | gi|2289393718/2289393720 | Trimeresurus gracilis | 304.56 | 0.14 |
SVSP | Snake venom serine protease precursor | gi|2035122138 | Bothrops jararaca | 172.04 | 0.14 |
Fraction 21 | |||||
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 277.88 | 0.74 |
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 156.88 | 0.65 |
SVSP | Serine proteinase 1 | gi|1041577231 | Agkistrodon conanti | 130.59 | 0.17 |
Fraction 22 | |||||
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 219.86 | 0.50 |
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 151.81 | 0.43 |
SVSP | Serine proteinase 1 | gi|1041577231 | Agkistrodon conanti | 104.59 | 0.23 |
SVSP | Snake venom serine protease serpentokallikrein-2 isoform X1 | gi|1002585685 | Protobothrops mucrosquamatus | 147.93 | 0.14 |
Fraction 23 | |||||
Snaclec | C-type lectin LmsL; Galactose-specific lectin; Mutina | gi|34922643 | Lachesis stenophrys | 220.07 | 0.22 |
Snaclec | Galactose binding lectin | gi|538260107 | Ovophis okinavensis | 204.87 | 0.16 |
Snaclec | Galactose binding lectin, partial | gi|538259813 | Protobothrops flavoviridis | 168.47 | 0.16 |
Snaclec | Chain B Galactose-specific lectin | gi|33357350 | Crotalus atrox | 208.84 | 0.14 |
SVSP | Serine proteinase 8c | gi|1041578891 | Sistrurus tergeminus | 103.02 | 0.12 |
Fraction 24 | |||||
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 172.27 | 0.12 |
SVSP | Plasminogen-activator subtype serine protease (PA3) [Tgc-PA3] | gi|2289393722 | Trimeresurus gracilis | 154.61 | 0.11 |
Fraction 25 | |||||
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 130.75 | 0.27 |
PLA2 | Acidic phospholipase A2 [Tgc-E6] | gi|59727071 | Trimeresurus gracilis | 266.65 | 0.23 |
SVSP | Thrombin-like enzyme | gi|38146946 | Gloydius shedaoensis | 108.67 | 0.14 |
SVSP | Thrombin-like enzyme halystase | gi|3122187 | Gloydius blomhoffii | 100.35 | 0.14 |
PLA2 | Phospholipase A2 precursor | gi|743759444 | Protobothrops tokarensis | 66.77 | 0.13 |
PLA2 | Acidic phospholipase A2 | gi|129420 | Gloydius blomhoffii | 117.08 | 0.13 |
PLA2 | Phospholipase A2 type IIE | gi|384110782 | Dispholidus typus | 83.97 | 0.13 |
SVSP | Serine proteinase 19b | gi|1041577233 | Agkistrodon conanti | 112.35 | 0.12 |
PLA2 | Phospholipase A2 | gi|584481356 | Ovophis makazayazaya | 92.74 | 0.11 |
Fraction 26 | |||||
PLA2 | Acidic phospholipase A2 [Tgc-E6] | gi|59727071 | Trimeresurus gracilis | 444.11 | 1.70 |
PLA2 | Phospholipase A2 isozyme CTs-A3, partial | gi|37785867 | Viridovipera stejnegeri | 136.6 | 1.08 |
PLA2 | Phospholipase A2, partial | gi|538259861 | Protobothrops flavoviridis | 147.33 | 0.90 |
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 135.89 | 0.25 |
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 143.16 | 0.19 |
Fraction 27 | |||||
PLA2 | Acidic phospholipase A2 [Tgc-E6] | gi|59727071 | Trimeresurus gracilis | 300.99 | 0.79 |
PLA2 | Phospholipase A2, partial | gi|538259861 | Protobothrops flavoviridis | 121.88 | 0.53 |
PLA2 | Phospholipase A2 isozyme CTs-A3, partial | gi|37785867 | Viridovipera stejnegeri | 117.83 | 0.40 |
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 102.54 | 0.20 |
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 121.04 | 0.17 |
SVSP | Plasminogen-activator subtype serine protease (PA3) [Tgc-PA3] | gi|2289393722 | Trimeresurus gracilis | 144 | 0.15 |
Fraction 28 | |||||
SVSP | Plasminogen-activator subtype serine protease (PA3) [Tgc-PA3] | gi|2289393722 | Trimeresurus gracilis | 137.28 | 0.28 |
SVSP | Serine proteinase 19b | gi|1041577233 | Agkistrodon conanti | 108.99 | 0.27 |
SVSP | Plasminogen-activator subtype serine protease (PA1/2) [Tgc-PAH1/2] | gi|2289393718/2289393720 | Trimeresurus gracilis | 134.51 | 0.19 |
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 102.15 | 0.17 |
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 96.4 | 0.13 |
Fraction 29 | |||||
LAAO | L-amino-acid oxidase | gi|347602330 | Vipera ammodytes ammodytes | 212.56 | 0.66 |
LAAO | Chain A Ahp-laao | gi|48425312 | Gloydius halys | 199.24 | 0.51 |
LAAO | L-amino acid oxidase | gi|538260091 | Ovophis okinavensis | 275.32 | 0.42 |
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 111.55 | 0.16 |
SVSP | Plasminogen-activator subtype serine protease (PA3) [Tgc-PA3] | gi|2289393722 | Trimeresurus gracilis | 138.9 | 0.13 |
LAAO | BATXLAAO1 | gi|1127252627 | Bothrops atrox | 176.6 | 0.12 |
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 119.14 | 0.12 |
Fraction 30 | |||||
SVMP III | Metalloproteinase, partial | gi|297593822 | Echis carinatus sochureki | 50.52 | 0.31 |
SVMP III | Metalloproteinase-disintegrin-like atrolysin-A, partial | gi|1663479917 | Protobothrops mucrosquamatus | 183.98 | 0.25 |
SVMP III | BATXSVMPIII16 | gi|1127252547 | Bothrops atrox | 74.01 | 0.11 |
Fraction 31 | |||||
SVMP III | Metalloprotease PIII [Tgc-PIII] | gi|335892636 | Trimeresurus gracilis | 307.13 | 0.46 |
SVMP III | Metalloproteinase-disintegrin-like atrolysin-A, partial | gi|1663479917 | Protobothrops mucrosquamatus | 236.99 | 0.40 |
Fraction 32 | |||||
SVMP III | Metalloprotease PIII [Tgc-PIII] | gi|335892636 | Trimeresurus gracilis | 202.57 | 0.37 |
SVMP III | Metalloproteinase-disintegrin-like atrolysin-A, partial | gi|1663479917 | Protobothrops mucrosquamatus | 162.6 | 0.12 |
LAAO | L-amino acid oxidase | gi|538260091 | Ovophis okinavensis | 181.72 | 0.11 |
Fraction 34 | |||||
SVMP I | P-II_metalloprotease *** | gi|547223068 | Ovophis okinavensis | 217.81 | 5.79 |
SVMP I | Metalloprotease PI [Tgc-MP] | gi|335892630 | Trimeresurus gracilis | 362.99 | 3.88 |
SVMP I | P-II metalloprotease, partial *** | gi|547223015 | Protobothrops flavoviridis | 189.92 | 0.20 |
SVMP III | MDC-6d | gi|1829138061 | Crotalus atrox | 167.81 | 0.22 |
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 165.77 | 0.11 |
Fraction 35 | |||||
SVMP I | Metalloprotease PI [Tgc-MP] | gi|335892630 | Trimeresurus gracilis | 360.09 | 19.26 |
SVSP | Kallikrein-like serine protease (KN1) [Tgc-KN1] | gi|2289393706 | Trimeresurus gracilis | 119.16 | 0.15 |
SVSP | Kallikrein-like serine protease (KN4) [Tgc-KN4] | gi|2289393712 | Trimeresurus gracilis | 105.69 | 0.11 |
Fraction 36 | |||||
SVMP III | Metalloproteinase, partial | gi|1773624963 | Dispholidus typus | 62.96 | 0.38 |
SVMP II | Tgc-PIIc | OK482650 | Trimeresurus gracilis | 308.73 | 0.10 |
SVMP II | Metalloproteinase type II 6c | gi|1041579264 | Sistrurus miliarius barbouri | 127.08 | 0.10 |
Fraction 37 | |||||
SVMP II | Snake venom metalloprotease precursor | gi|2035122167 | Bothrops jararaca | 106.31 | 0.16 |
SVMP II | Tgc-PIIc | OK482650 | Trimeresurus gracilis | 206.13 | 0.12 |
SVMP III | Metalloproteinase type III 9b | gi|1041579226 | Sistrurus miliarius barbouri | 75.23 | 0.11 |
Fraction 38 | |||||
SVMP II | Tgc-PIIc | OK482650 | Trimeresurus gracilis | 220.3 | 0.11 |
Fraction 39 | |||||
SVMP III | Metalloprotease P-III 3, partial | gi|675402421 | Protobothrops flavoviridis | 137.47 | 0.76 |
SVMP III | Metalloproteinase (type III) 6a | gi|1180525232 | Agkistrodon contortrix contortrix | 222.51 | 0.68 |
SVMP III | Metalloproteinase type III 12b | gi|1041577317 | Agkistrodon conanti | 206.17 | 0.60 |
SVMP III | P-III_metalloprotease | gi|547223066 | Ovophis okinavensis | 318.96 | 0.51 |
SVMP II | Snake venom metalloprotease precursor | gi|2035122236 | Bothrops jararaca | 111.17 | 0.42 |
SVMP III | Metalloproteinase type III 5a | gi|1041579244 | Sistrurus miliarius barbouri | 230.38 | 0.33 |
SVMP III | Snake venom metalloprotease precursor | gi|2035122126 | Bothrops jararaca | 115.29 | 0.19 |
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Tse, T.-C.; Tsai, I.-H.; Chan, Y.-Y.; Tsai, T.-S. Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species. Toxins 2023, 15, 408. https://doi.org/10.3390/toxins15070408
Tse T-C, Tsai I-H, Chan Y-Y, Tsai T-S. Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species. Toxins. 2023; 15(7):408. https://doi.org/10.3390/toxins15070408
Chicago/Turabian StyleTse, Tsz-Chun, Inn-Ho Tsai, Yuen-Ying Chan, and Tein-Shun Tsai. 2023. "Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species" Toxins 15, no. 7: 408. https://doi.org/10.3390/toxins15070408