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Human Mast Cell Tryptase Is a Potential Treatment for Snakebite Envenoming Across Multiple Snake Species

Anderson, Elizabeth, Stavenhagen, Kathrin, Kolarich, Daniel, Sommerhoff, Christian P., Maurer, Marcus, Metz, Martin·2018·Frontiers in Immunology
DOI 10.3389/fimmu.2018.01532PMC PMC6047305
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Figure 1 Whole venom from six different snakes induces degranulation of human mast cells. LAD2 MCs (A) or primary human skin mast cell (hsMCs) isolated from three healthy donors (B) were stimulated with venom from the six listed snake species. (B) Venom doses were as follows: common lancehead, saw-scaled viper, southern copperhead, red spitting cobra, Russell’s viper, and western diamondback rattlesnake. Negative controls (vehicle) were PBS only. Positive controls (not shown) were Ca ionomycin (1 µM) and caused an average of 55% degranulation in LAD2 (A), and 62, 36, and 8% degranulation in hsMCs from each donor (B). Data are pooled from four independent experiments and measurements were performed in triplicate. Student’s t-test compares venom to vehicle stimulation. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 1 Whole venom from six different snakes induces degranulation of human mast cells. LAD2 MCs (A) or primary human skin mast cell (hsMCs) isolated from three healthy donors (B) were stimulated with venom from the six listed snake species. (B) Venom doses were as follows: common lancehead, saw-scaled viper, southern copperhead, red spitting cobra, Russell’s viper, and western diamondback rattlesnake. Negative controls (vehicle) were PBS only. Positive controls (not shown) were Ca ionomycin (1 µM) and caused an average of 55% degranulation in LAD2 (A), and 62, 36, and 8% degranulation in hsMCs from each donor (B). Data are pooled from four independent experiments and measurements were performed in triplicate. Student’s t-test compares venom to vehicle stimulation. *p < 0.05; **p < 0.01; ***p < 0.001.CC BY · PubMed Central
Figure 2 Lysate from purified human skin mast cells (hsMCs) protects zebrafish from toxicity of cobra venom. Zebrafish embryos aged 48 h post fertilization suspended in 100 µl of fish water received 625 ng red spitting cobra whole venom pre-treated with lysate of purified hsMCs (MC lysate) or PBS (vehicle). Lysate doses were 12-, 6-, or 3- thousand cells per fish. Survival was monitored at regular intervals for 6 h, with complete cessation of heartbeat as the predefined endpoint. Vehicle, lysate alone, or enzyme alone had no effect (data not shown). Data are pooled from three independent experiments, n = 12 fish per group. Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle and to each other. **p < 0.01; ***p < 0.001.
Figure 2 Lysate from purified human skin mast cells (hsMCs) protects zebrafish from toxicity of cobra venom. Zebrafish embryos aged 48 h post fertilization suspended in 100 µl of fish water received 625 ng red spitting cobra whole venom pre-treated with lysate of purified hsMCs (MC lysate) or PBS (vehicle). Lysate doses were 12-, 6-, or 3- thousand cells per fish. Survival was monitored at regular intervals for 6 h, with complete cessation of heartbeat as the predefined endpoint. Vehicle, lysate alone, or enzyme alone had no effect (data not shown). Data are pooled from three independent experiments, n = 12 fish per group. Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle and to each other. **p < 0.01; ***p < 0.001.CC BY · PubMed Central
Figure 3 Purified human tryptase, but not chymase or carboxypeptidase A (CPA), detoxifies snake venoms. Venoms were incubated with 10 µg/ml human tryptase, chymase, or CPA. Treated and untreated venom were then administered to 48 h post fertilization zebrafish in 100 µl of water. Empty circles represent zebrafish receiving venom alone, and solid shapes represent fish receiving enzyme-treated venom. Vehicle or enzyme alone had no effect (data not shown). Data are pooled from three (tryptase) or two (chymase and CPA) independent experiments for n = 15 or 10 fish per group. Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle. **p < 0.01; ***p < 0.001.
Figure 3 Purified human tryptase, but not chymase or carboxypeptidase A (CPA), detoxifies snake venoms. Venoms were incubated with 10 µg/ml human tryptase, chymase, or CPA. Treated and untreated venom were then administered to 48 h post fertilization zebrafish in 100 µl of water. Empty circles represent zebrafish receiving venom alone, and solid shapes represent fish receiving enzyme-treated venom. Vehicle or enzyme alone had no effect (data not shown). Data are pooled from three (tryptase) or two (chymase and CPA) independent experiments for n = 15 or 10 fish per group. Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle. **p < 0.01; ***p < 0.001.CC BY · PubMed Central
Figure 4 MALDI-TOF-MS analysis of venoms treated with purified human tryptase shows degradation of venom proteins. Samples were prepared at concentrations of 10 µg/µl venom and a ratio of 1000:1 venom to tryptase by mass (southern copperhead and common lancehead) or 0.1 µg/µl and a ratio of 10:1 venom to tryptase by mass (western diamondback rattlesnake, Russell’s viper, saw-scaled viper, and red spitting cobra). They were then incubated at 37°C for 30 min, and stored at −20°C. Control was prepared with venom and PBS alone. 17 µl of the venom sample (in PBS) was purified by reversed phase chromatography using C18 Zip TipsTM (Millipore). Mass spectra were acquired in positive ion reflectron mode within the m/z ratio range of m/z 3,500 to m/z 18,000. Panel (A) shows a representative MALDI-TOF-MS spectrum of red spitting cobra venom before and after incubation with purified human tryptase. Five signals (dashed arrows) over the acquired mass range were selected and their signal intensities normalized to the intensity of the doubly charged myoglobin signal (m/z 8,477). The relative signal intensity reduction of several peaks indicates protein degradation by tryptase. Spectra for additional venoms are provided in Figure 5. Comparisons of relative signal intensities for all six venoms are summarized in (B). Arrows indicate a reduced (↓) or increased (↑) relative signal intensity difference (Intens. dif.) of prominent peaks, or no difference (no dif.), with lower peaks indicating degradation of the polypeptide of the corresponding m/z ratio.
Figure 4 MALDI-TOF-MS analysis of venoms treated with purified human tryptase shows degradation of venom proteins. Samples were prepared at concentrations of 10 µg/µl venom and a ratio of 1000:1 venom to tryptase by mass (southern copperhead and common lancehead) or 0.1 µg/µl and a ratio of 10:1 venom to tryptase by mass (western diamondback rattlesnake, Russell’s viper, saw-scaled viper, and red spitting cobra). They were then incubated at 37°C for 30 min, and stored at −20°C. Control was prepared with venom and PBS alone. 17 µl of the venom sample (in PBS) was purified by reversed phase chromatography using C18 Zip TipsTM (Millipore). Mass spectra were acquired in positive ion reflectron mode within the m/z ratio range of m/z 3,500 to m/z 18,000. Panel (A) shows a representative MALDI-TOF-MS spectrum of red spitting cobra venom before and after incubation with purified human tryptase. Five signals (dashed arrows) over the acquired mass range were selected and their signal intensities normalized to the intensity of the doubly charged myoglobin signal (m/z 8,477). The relative signal intensity reduction of several peaks indicates protein degradation by tryptase. Spectra for additional venoms are provided in Figure 5. Comparisons of relative signal intensities for all six venoms are summarized in (B). Arrows indicate a reduced (↓) or increased (↑) relative signal intensity difference (Intens. dif.) of prominent peaks, or no difference (no dif.), with lower peaks indicating degradation of the polypeptide of the corresponding m/z ratio.CC BY · PubMed Central
Figure 5 MALDI-TOF-MS analyses of whole venoms treated with purified human tryptase shows degradation of venom proteins. Venoms from five snake species [(A) common lancehead, (B) saw-scaled viper, (C) southern copperhead, (D) russel’s viper, (E), western diamondback rattlesnake] were treated with tryptase or vehicle control. Spectra were acquired within the mass range of m/z 3,500 to m/z 18,000. Samples contain equal amounts (0.5 µg) myoglobin as an internal reference (m/z 16,952 [M + H]+, m/z 8,476 [M + 2H]2+, m/z 5,651 [M + 3H]3+). Venom-derived signals (dashed arrows) distributed over the whole mass range were selected and normalized to the intensity of the doubly charged myoglobin signal (Figure 4B). The reduction in relative signal intensities of several peaks in tryptase-treated versus untreated venom indicates tryptase-mediated protein degradation.
Figure 5 MALDI-TOF-MS analyses of whole venoms treated with purified human tryptase shows degradation of venom proteins. Venoms from five snake species [(A) common lancehead, (B) saw-scaled viper, (C) southern copperhead, (D) russel’s viper, (E), western diamondback rattlesnake] were treated with tryptase or vehicle control. Spectra were acquired within the mass range of m/z 3,500 to m/z 18,000. Samples contain equal amounts (0.5 µg) myoglobin as an internal reference (m/z 16,952 [M + H]+, m/z 8,476 [M + 2H]2+, m/z 5,651 [M + 3H]3+). Venom-derived signals (dashed arrows) distributed over the whole mass range were selected and normalized to the intensity of the doubly charged myoglobin signal (Figure 4B). The reduction in relative signal intensities of several peaks in tryptase-treated versus untreated venom indicates tryptase-mediated protein degradation.CC BY · PubMed Central
Figure 6 Skin mast cell lysate and purified human tryptase protects zebrafish from cobra venom when administered as a therapy. (A) 48 h post fertilization (hpf) zebrafish received 625 ng red spitting cobra whole venom in 100 µl each. 1, 5, or 10 min after addition of venom, zebrafish were treated with lysate from 12 thousand purified human skin mast cells per fish. Data are pooled from three independent experiments, with n = 4 zebrafish per group, each. (B) 48 hpf zebrafish received 625 ng red spitting cobra whole venom in 100 µl each. Between 0 and 40 min after addition of venom, zebrafish were treated with 10 µg/ml purified human tryptase. Data are pooled from six independent experiments with n = 5 embryos per group, each. Lysate, tryptase, or vehicle alone had no effect (data not shown). Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle. ***p < 0.001.
Figure 6 Skin mast cell lysate and purified human tryptase protects zebrafish from cobra venom when administered as a therapy. (A) 48 h post fertilization (hpf) zebrafish received 625 ng red spitting cobra whole venom in 100 µl each. 1, 5, or 10 min after addition of venom, zebrafish were treated with lysate from 12 thousand purified human skin mast cells per fish. Data are pooled from three independent experiments, with n = 4 zebrafish per group, each. (B) 48 hpf zebrafish received 625 ng red spitting cobra whole venom in 100 µl each. Between 0 and 40 min after addition of venom, zebrafish were treated with 10 µg/ml purified human tryptase. Data are pooled from six independent experiments with n = 5 embryos per group, each. Lysate, tryptase, or vehicle alone had no effect (data not shown). Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle. ***p < 0.001.CC BY · PubMed Central
Figure 7 Active recombinant human tryptase β detoxifies snake venoms. Venoms were incubated with 10 µg/ml active human recombinant tryptase β and then administered to 48 h post fertilization zebrafish in 100 µl of water. Controls were inactive recombinant tryptase β and PBS (vehicle). Vehicle or enzymes alone had no effect (data not shown). Data are pooled from three to four independent experiments for n = 15–20 fish per group. Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle. ***p < 0.001.
Figure 7 Active recombinant human tryptase β detoxifies snake venoms. Venoms were incubated with 10 µg/ml active human recombinant tryptase β and then administered to 48 h post fertilization zebrafish in 100 µl of water. Controls were inactive recombinant tryptase β and PBS (vehicle). Vehicle or enzymes alone had no effect (data not shown). Data are pooled from three to four independent experiments for n = 15–20 fish per group. Kaplan–Meyer survival analysis, Log-rank (Mantel-Cox) test compares treatments to vehicle. ***p < 0.001.CC BY · PubMed Central

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Metadata

Authors
Anderson, E., Stavenhagen, K., Kolarich, D., Sommerhoff, C. P., Maurer, M., & Metz, M.
Venue
Frontiers in Immunology
Publisher
Frontiers Media SA
Published
2018-07-09
Volume
9
ISSN
1664-3224
DOI
10.3389/fimmu.2018.01532
PMC
PMC6047305
Cited by
30
References
0
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)
Funders
  • Deutsche Forschungsgemeinschaft (ME 2668/3-2, ME 2668/2-1, SO 249/1-1)
  • Else Kröner-Fresenius-Stiftung
  • Max-Planck-Gesellschaft

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Anderson, E., Stavenhagen, K., Kolarich, D., Sommerhoff, C. P., Maurer, M., & Metz, M. (2018). Human Mast Cell Tryptase Is a Potential Treatment for Snakebite Envenoming Across Multiple Snake Species. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2018.01532