Purified zymogens reveal mechanisms of snake venom metalloproteinase auto-activation
- School of Biochemistry, University of Bristol, United Kingdom
- Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, United Kingdom
- School of Cellular and Molecular Medicine, University of Bristol, United Kingdom
- Lancaster University, United Kingdom
- Department of Biotechnology and Bioinformatics, Faculty of Chemistry, Rzeszow University of Technology, Poland
- Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257 CNRS-Aix-Marseille Université, France
- Bristol Medical School, University of Bristol, United Kingdom
- Max Planck Bristol Centre for Minimal Biology, School of Chemistry, University of Bristol, United Kingdom
Figures
Figure 1 with 1 supplement
Snake venom metalloproteinase (SVMP) classification and abundance in Echis venom.
(a) Pie charts displaying Echis ocellatus and Echis carinatus sochureki venom composition, with SVMP coloured in cyan (Oliveira et al., 2022). (b) Simplified schematic of mature PI, PII, and PIII SVMP architecture, showing metalloproteinase domain (MP, cyan), disintegrin domain (Dis, green), and cysteine-rich domain (C-rich, magenta). (c) AlphaFold 3 predicted structural models of PI and PII SVMPs from E. ocellatus, and PIII SVMP from E. carinatus sochureki. Domain names and colour coding as in panel b. Zn2+ ions (orange) in the active site are shown as spheres.
Figure 1—figure supplement 1
Schematic outlining snake venom metalloproteinase (SVMP) production and characterisation strategy.
(a) Design of the expression construct for secretion of SVMP zymogens, comprising the native signal sequence, the endogenous prodomain with propeptide, the SVMP, and a C-terminal Avi- and octa-histidine tags for biotinylation and affinity purification, respectively. (b) Baculovirus insect cell expression timeline (adapted from Figure 3 in Trowitzsch et al., 2010). Plasmids encoding the SVMP zymogen constructs are integrated into EMBacY baculoviral DNA via Tn7 transposition. Positive clones are identified by blue/white screening. EMBacY baculoviral DNA (single white colonies) is isolated and used to transfect Sf21 cells. Media containing initial virus (V0) is removed from the wells and used for infecting insect cell cultures (25 ml volume) in an Erlenmeyer shaker flask. Protein production is followed by the yellow fluorescent protein (YFP) fluorescence signal and by western blot (WB) using anti-His antibodies. Infected Hi5 cell cultures in shaker flasks are split every 24 hr until cell proliferation arrest (PA) occurs. Media containing amplified virus (V1) is removed ∼48 hr after PA, and fresh medium is replenished instead. Cells are harvested when the YFP signal has reached a plateau (typically after 3–4 days). Protein production is analysed by SDS-PAGE. Baculovirus-infected insect cell (BIIC) stocks are prepared for long-term storage of viruses. The whole procedure takes less than 2 weeks. (c) SVMP zymogens are secreted and purified from the medium using three consecutive chromatography steps. Activation is accomplished by incubation with Zn2+, the concentration of Zn2+ required for full activation of the zymogen has to be optimised for each individual SVMP. Activated SVMPs are then used for activity assays, including protein degradation, cleavage of fluorogenic peptide, blood clotting, and platelet aggregation assays (for more details see Materials and methods). The amount of SVMP required for each assay has to be optimised.
Figure 2 with 2 supplements
PI, PII, and PIII snake venom metalloproteinase (SVMP) zymogen expression.
(a) Above: Schematic of PI SVMP zymogen with N-terminal prodomain (Pro, salmon), propeptide (purple). Below: left: two views of AlphaFold 3 prediction of PI zymogen model; right: zoom in on active site Zn2+ ion coordinated by histidines and propeptide cysteine. Expression of SVMP zymogens monitored by (b) cell viability, (c) yellow fluorescent protein (YFP) fluorescence (PIII harvested on day 4) and (d) western blot analysis using HRP-conjugated anti-Penta-His antibody. SNP: supernatant + pellet, SN: supernatant, M: media (10 x concentrated). Cyan: PI SVMP, green: PII SVMP, magenta: PIII SVMP. SVMP zymogen expression was repeated five times.
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Figure 2—source data 1
Original file for western blot analysis displayed in Figure 2d.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file containing original western blot for Figure 2d, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig2-data2-v1.zip
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Figure 2—source data 3
Excel file containing YPF fluorescence reads and cell viability counts for Figure 2b and c.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
Optimisation of snake venom metalloproteinase (SVMP) expression using mature SVMP, broad-spectrum inhibitor, SVMP active site mutants, and propeptide-SVMP fusion protein to overcome SVMP cytotoxicity.
(a) Expression of mature SVMPs monitored by cell viability (left), yellow fluorescent protein (YFP) fluorescence (middle), and western blot analysis of His-tagged protein (right). SNP: supernatant + pellet; SN: supernatant, M: medium. Grey bars: non-toxic protein control expression, cyan: PI SVMP, and magenta: PIII SVMP. (b) Expression of mature PIII SVMP in the presence of 0 μM (magenta), 3 μM (lilac), or 6 μM (purple) Marimastat. Expression is monitored by cell viability (left), YFP fluorescence (middle), and western blot analysis of His-tagged protein (right). (c) Expression of SVMP active site mutants monitored by cell viability (left) and YFP fluorescence (middle). Right: western blot analysis of expressed His-tagged protein following Ni-NTA purification. I: input; FT: flowthrough; E1, E2: elution fractions. (d) SVMP propeptide-fusion expression monitored by cell viability (left), YFP fluorescence (middle), and western blot analysis of His-tagged protein (right). Grey bars: non-toxic protein expression control, cyan: PI SVMP, and magenta: PIII SVMP. HRP-conjugated anti-Penta-His antibody was used for all western blots. Experiments were performed at least in duplicate.
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF file containing original western blots for Figure 2—figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Excel file containing YPF fluorescence reads and cell viability counts for Figure 2—figure supplement 1a–d.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig2-figsupp1-data3-v1.xlsx
Figure 2—figure supplement 2
AlphaFold 3 structural predictions of SVMP zymogens.
Structural predictions of SVMP zymogens of (a–c) PI, (d-f) PII, and (g–i) PIII class, performed by AlphaFold 3 (Abramson et al., 2024) in the presence of Zn2+, along with the corresponding predicted Local Distance Difference Test (pLDDT) (b, e, h) and predicted Aligned Error (pAE) (c, f, i) plots.
Figure 3 with 7 supplements
Size exclusion chromatography (SEC) purification of snake venom metalloproteinase (SVMP) zymogens and auto-activation.
Size exclusion chromatograms and SDS-PAGE of (a) PI zymogen (PIzymΔC lacks C-terminal tags), (b) PII zymogen, and (c) PIII zymogen after IMAC and IEX. PIIIzym partial cleavage separates the prodomain (Pro) and the mature PIII. Elution volumes of molecular weight (MW) calibration markers are indicated in the chromatograms as black arrows. (d) Activation of PI zymogen into metalloproteinase and prodomain. C (control): no 18 hr incubation. (e) Activation of PII zymogen into metalloproteinase-disintegrin and disintegrin domain (prodomain cannot be definitively identified). After 1 week, only 8 kDa and 23 kDa bands remain. (f) Activation of PIII zymogen into prodomain and mature PIII. At higher Zn2+ concentrations, the prodomain is degraded. C (control): no 18 hr incubation. SVMP zymogen purifications and activations were repeated five times.
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Figure 3—source data 1
Original files for original SDS-PAGE gels displayed in Figure 3a–f.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-data1-v1.zip
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Figure 3—source data 2
PDF file containing original SDS-PAGE gels for Figure 3a–f, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-data2-v1.zip
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Figure 3—source data 3
Excel sheet containing the chromotograms for the affinity (IMAC), IEX and SEC purification of PI SVMP zymogen shown in Figure 3a.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-data3-v1.xlsx
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Figure 3—source data 4
Excel sheet containing the chromotograms for the affinity (IMAC), IEX and SEC purification of PIII SVMP zymogen shown in Figure 3c.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-data4-v1.xlsx
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Figure 3—source data 5
Excel sheet containing the chromotograms for the affinity (IMAC), IEX and SEC purification of PIII SVMP zymogen shown in Figure 3c.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-data5-v1.xlsx
Figure 3—figure supplement 1
PI snake venom metalloproteinase (SVMP) C-terminal auto-cleavage before and after dialysis, following IMAC purification.
(a) SDS-PAGE and (b) western blot using HRP-conjugated anti-Penta-His antibody of sample before and after dialysis, following IMAC purification. (c) Top five LC-MS/MS (liquid chromatography-tandem mass spectrometry) hits of before dialysis sample. Proteins were identified using the Sequest search engine against the UniProtKB Trichoplusia ni (Hi5) database containing sequence for the protein of interest (PI SVMP). Keratins and trypsin were excluded as common contaminants. (d) PI SVMP protein sequence coverage by LC-MS/MS. Peptides identified by MS that provide unique sequence information are shown as horizontal bars below the amino acid sequence. Residues detected in the analysis are highlighted in red, covering 56% of the protein sequence.
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Figure 3—figure supplement 1—source data 1
Original files for SDS-PAGE and western blot analysis displayed in Figure 3—figure supplement 1a and b.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file containing original SDS gel and western blot for Figure 3—figure supplement 1a and b, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp1-data2-v1.zip
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Figure 3—figure supplement 1—source data 3
Excel sheet comprising the MS results for all peptides identified in the PI SVMP protein sample for Figure 3—figure supplement 1c.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp1-data3-v1.xlsx
Figure 3—figure supplement 2
IMAC purification of secreted snake venom metalloproteinases (SVMPs) from insect cell media.
Chromatograms and reducing SDS-PAGE of IMAC purification of (a) PIΔC and (b) PIII SVMP.
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Figure 3—figure supplement 2—source data 1
Original files for SDS-PAGE analysis displayed in Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
PDF file containing original SDS gels for Figure 3—figure supplement 2, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp2-data2-v1.zip
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Figure 3—figure supplement 2—source data 3
Excel sheet comprising the data of the chromatograms of the IMAC purification of PIΔC and PIII SVMP shown in Figure 3—figure supplement 2a and b.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp2-data3-v1.xlsx
Figure 3—figure supplement 3
Size exclusion chromatography (SEC) purification and auto-activation of PIΔC snake venom metalloproteinase (SVMP) zymogen, following IMAC and IEX.
Size exclusion chromatograms and SDS-PAGE of (a) metalloproteinase (MP) domain of PIΔC and (b) MP domain and prodomain (Pro) of PIΔC. (c) PIΔC activity against casein with no prior addition of Zn2+ ions (left) and with prior incubation with 450 μM of Zn2+ (right, see also Figure 4a). Titration of PIΔC SVMP (concentration range indicated) into 23 μM casein, followed by incubation at 37 °C for 18 hr. 5 μl reaction run on a reducing SDS-PAGE gel.
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Figure 3—figure supplement 3—source data 1
Original files for SDS-PAGE analysis displayed in Figure 3—figure supplement 3.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp3-data1-v1.zip
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Figure 3—figure supplement 3—source data 2
PDF file containing original SDS gels for Figure 3—figure supplement 3, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp3-data2-v1.zip
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Figure 3—figure supplement 3—source data 3
Excel sheet comprising the data for the size exclusion chromatograms of PIΔC MP alone and of PIΔC MP with prodomain shown in Figure 3—figure supplement 3a and b.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp3-data3-v1.xlsx
Figure 3—figure supplement 4
Size exclusion chromatography (SEC) purification of PII snake venom metalloproteinase (SVMP) zymogen, following IMAC and IEX.
(a) Size exclusion chromatogram and (b) reducing and non-reducing SDS-PAGE of SEC peaks 2 and 3. (c) Western blot analysis using HRP-conjugated anti-Penta-His antibody of sample before and after activation of PII SVMP.
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Figure 3—figure supplement 4—source data 1
Original files for SDS-PAGE analysis and western blot displayed in Figure 3—figure supplement 4b and c.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp4-data1-v1.zip
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Figure 3—figure supplement 4—source data 2
PDF file containing original SDS-PAGE analysis and western blot for Figure 3—figure supplement 4b and c, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp4-data2-v1.zip
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Figure 3—figure supplement 4—source data 3
Excel sheet comprising the data for the SEC of PII SVMP zymogen shown in Figure 3—figure supplement 4a.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp4-data3-v1.xlsx
Figure 3—figure supplement 5
Glycosylation test of recombinant snake venom metalloproteinases (SVMPs).
Purified (a) PI, (b) PII, and (c) PIII SVMP zymogens were treated with PNGase F. A shift to lower molecular weight (MW) is observed in bands corresponding to PIII SVMP zymogen and mature PIII SVMP (MP-Dis-Crich) in panel c.
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Figure 3—figure supplement 5—source data 1
Original files for SDS-PAGE analysis displayed in Figure 3—figure supplement 5.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp5-data1-v1.zip
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Figure 3—figure supplement 5—source data 2
PDF file containing original SDS-PAGE analysis for Figure 3—figure supplement 5, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp5-data2-v1.zip
Figure 3—figure supplement 6
Size exclusion chromatography (SEC) chromatograms of PII and PIII snake venom metalloproteinase (SVMP) zymogens produced in the presence and the absence of human and snake protein disulfide isomerases (PDI).
(a) Top five LC-MS/MS hits of PIII + sPDI IMAC flowthrough. Proteins were identified using the Sequest search engine against the UniProtKB Trichoplusia ni (Hi5) database containing sequences for the proteins of interest (PIII SVMP and snake PDI). Keratins and trypsin have been excluded as common contaminants. (b) PII SVMP zymogen and (c) PIII SVMP zymogen were co-expressed with human (magenta) or snake (purple) PDIs. SEC chromatograms were aligned with the control where no PDI was expressed (cyan). (d) Fibrinogen degradation assays in the presence of Zn2+ and increasing amounts of SVMPs PII, PII + hPDI (human PDI) and PII + s PDI (snake PDI). (e) Fibrinogen degradation assays in the presence of Zn2+ and increasing amounts of SVMPs PIII, PIII + hPDI and PIII + sPDI. Degradation assays were performed four times for the PII SVMP + PDI assays and six times for the PIII SVMP + PDI assays.
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Figure 3—figure supplement 6—source data 1
Original files for SDS-PAGE analysis displayed in Figure 3—figure supplement 6b–e.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp6-data1-v1.zip
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Figure 3—figure supplement 6—source data 2
PDF file containing original SDS-PAGE analysis for Figure 3—figure supplement 6b–e, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp6-data2-v1.zip
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Figure 3—figure supplement 6—source data 3
Excel sheets comprising all peptides identifiied by LC-MS/MS of PIII + sPDI sample (shown in panel a), and data for size exclusion chromatograms shown in Figure 3—figure supplement 6b and c.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp6-data3-v1.xlsx
Figure 3—figure supplement 7
Auto-activated PIzymΔC size exclusion chromatography (SEC).
(a) Size exclusion chromatogram and (b) corresponding Coomassie-stained SDS gel of activated PIzymΔC, confirming metalloproteinase (MP) domain and prodomain (Pro) remain bound together, despite the protein being active.
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Figure 3—figure supplement 7—source data 1
Original file for SDS-PAGE analysis displayed in Figure 3—figure supplement 7b.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp7-data1-v1.zip
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Figure 3—figure supplement 7—source data 2
PDF file containing original SDS-PAGE analysis for Figure 3—figure supplement 7b, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp7-data2-v1.zip
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Figure 3—figure supplement 7—source data 3
Excel table of sixe exclusion chromatogram of auto-activated PIΔC shown in Figure 3—figure supplement 7a.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig3-figsupp7-data3-v1.xlsx
Figure 4 with 1 supplement
In vitro snake venom metalloproteinase (SVMP) activity and substrate specificity.
Casein degradation in the presence of increasing amounts of (a) PI, (b) PII, and (c) PIII SVMPs. Orange arrowheads: domains αS1, αS2, β, and κ. Fibrinogen degradation in the presence of increasing amounts of (d) PI, (e) PII, and (f) PIII. Orange arrowheads: fibrinogen chains Aα, Bβ, and γ. C (control): incubation in the presence of 5 mM EDTA. (g) PIII SVMP (20 nM) activity towards the fluorogenic peptide substrate ES010. (h, i) Degradation of insulin B by (h) PIII and (i) recombinant (blue) and Echis ocellatus PI (red). Full-length (FL) insulin and degradation products (*) are marked above the peaks. All degradation assays were repeated three times. The fluorogenic peptide assay shows the result of three independent repeats.
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Figure 4—source data 1
Original files for original SDS-PAGE gels displayed in Figure 4a–f.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig4-data1-v1.zip
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Figure 4—source data 2
PDF file containing original SDS-PAGE gels for Figure 4a–f, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig4-data2-v1.zip
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Figure 4—source data 3
Excel sheets comprising the data for the fluorogenic peptide substrate ES010 degradataion assay using PIII SVMP (g) and the data for the RP-HPLC runs of insulin B degradation by PIII SVMP (h) and by recombinant and native PI SVMP (i) shown in Figure 4g, h and i.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Snake venom metalloproteinase (SVMP) activity assays and blood coagulation tests.
(a) PI, PII, and PIII SVMP activity (40 nM each) towards the fluorogenic peptide substrate ES010 (100 µM). Data points represent the mean of four individual values recorded over two independent technical replicates, and error bars represent SD. The rate of reaction was determined by calculating the slope of the fluorescence intensity versus time curve.(b) Degradation of insulin B by PII SVMP (above) and insulin B only (negative control, below). Full-length (FL) insulin is marked. See Figure 4h and i PI and PIII SVMP degradation tests, respectively. (c) Citrated human blood was spiked with the recombinant PI SVMP (left) and the recombinant PII SVMP (right). Change in absorbance (cyan) is plotted on the left Y axis, rate of change (first derivative: blue; second derivative: pink) is plotted on the right Y axis. See Figure 5d for PIII SVMP for human blood coagulation test.
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Figure 4—figure supplement 1—source data 1
Excel sheets comprising the data for the fluorogenic peptide substrate ES010 degradataion assays using PI and PII SVMPs (a), the data for the RP-HPLC runs of insulin B degradation by PII SVMP and the insulin B control (b), and the data for the blood clotting experiments with PI and PII SVMPs (c) shown in Figure 4—figure supplement 1a–c.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig4-figsupp1-data1-v1.xlsx
Figure 5
Snake venom metalloproteinase (SVMP) blood coagulation, platelet aggregation, and cytotoxicity.
(a) Inhibition of platelet aggregation by PI, PII (mature and zymogen), and PIII. Control: PRP with buffer. Data are presented as the mean of % aggregation ± SEM. A two-way ANOVA was conducted, followed by simple effects analysis (Dunnett post-test) to compare column means (SVMPs) within each row (concentrations). ****: p≤0.0001, **: p≤0.01. The assay was carried out in experimental duplicate and biological triplicate. (b-d) Thrombin clot time assays using the ACL TOP coagulation analyser. Citrated human blood was spiked with (b) thrombin, (c) a pool of native Echis ocellatus PIII SVMPs, and (d) the recombinant PIII SVMP. Change in absorbance (blue) is plotted on the left Y axis, and rate of change (first derivative: orange; second derivative: red) is plotted on the right Y axis. Assays were repeated three times. (e) Coagulation profiles of the recombinant SVMPs and E. ocellatus venom in bovine plasma over 30 min at an absorbance of 595 nm. Data points represent the mean of three individual values ± SD. The experiment was repeated two times. (f) MTT (3-(4,5-dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide) cytotoxicity assay showing viability compared to the buffer-only control of HaCaT cells after 24 hr incubation with activated PI, PII, and PIII SVMPs. Bars show the results of two independent repeats.
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Figure 5—source data 1
Excel sheets comprising the original data used to generate graphs shown in Figure 5a–f.
- https://cdn.elifesciences.org/articles/109112/elife-109112-fig5-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Echis ocellatus) | Group I snake venom metalloproteinase | UniProt | UniProt ID: Q2UXQ3 | |
| Gene (E. ocellatus) | Venom metalloprotease PII-SVMP EOC00006 | UniProt | UniProt ID: A0A3G1E3U2 | |
| Gene (Echis carinatus sochureki) | E. carinatus sochureki Metalloproteinase | UniProt | UniProt ID: E9JG34 | |
| Gene (Homo sapiens) | Human protein disulfide-isomerase | UniProt | UniProt ID: P07237 | |
| Strain, strain background (Trichoplusia ni) | Hi5 insect cells (BTI-TN-5B1-4) | Thermo Fisher Scientific | Cat number: B85502 | Insect cell lines for recombinant protein production |
| Strain, strain background (Spodoptera frugiperda) | Sf21 insect cells (IPLB-Sf-21-AE) | Expression systems | Cat number: 94–003 F | Insect cell lines for recombinant protein production |
| Genetic reagent (plasmid) | pACEBac1 | Geneva Biotech | Baculovirus transfer plasmid used in the MultiBac system | |
| Genetic reagent (plasmid) | pIDC | Geneva Biotech | Baculovirus transfer plasmid used in the MultiBac system | |
| Cell line (Escherichia coli) | DH10 with EMBacY | Geneva Biotech | Modified baculovirus genome maintained in DH10 strain | |
| Cell line (H. sapiens) | HaCaT | AddexBio | Cat number: T0020001 RRID:CVCL_0038 | Cells derived from a 62-year-old Caucasian male donor’s skin epidermis |
| Biological sample (E. ocellatus) | Echis ocellatus venom | Liverpool School of Tropical Medicine | Venom extracted from specimens held at the herpetarium facility. | |
| Biological sample (Bos taurus) | Citrated bovine plasma | Biowest | Cat number: S0260-500 | |
| Antibody | Rabbit Monoclonal anti-PDI antibody | Cell Signalling Technologies | Cat number: 3501 | diluted 1:1,000 in PBS |
| Antibody | IRDye 680RD Goat Polyclonal Anti-Rabbit IgG | LICORbio | Cat number: 926–68071 | diluted 1:10,000 in PBS |
| Recombinant DNA reagent | PI | Genscript | Synthesised and inserted into the plasmid pACEBac1. Codon optimised for Spodoptera frugiperda. | |
| Recombinant DNA reagent | PII | Genscript | Synthesised and inserted into the plasmid pACEBac1. Codon optimised for Spodoptera frugiperda. | |
| Recombinant DNA reagent | PIII | Genscript | Synthesised and inserted into the plasmid pACEBac1. Codon optimised for Spodoptera frugiperda. | |
| Recombinant DNA reagent | hPDI | Genscript | Synthesised and inserted into the plasmid pIDC. Codon optimised for Spodoptera frugiperda. | |
| Recombinant DNA reagent | sPDI from E. ocellatus | Genscript | Synthesised and inserted into the plasmid pIDC. Codon optimised for Spodoptera frugiperda. | |
| Peptide, recombinant protein | PNGase F | Promega | Cat number:V4831 | |
| Commercial assay or kit | Penta-His HRP Conjugate Kit | QIAGEN | Cat number: 34460 | diluted 1:2000 in PBS |
| Commercial assay or kit | SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Scientific | Cat number: 34580 | |
| Commercial assay or kit | Gel Filtration HMW Calibration Kit | Cytiva | Cat number: 28403841 | |
| Commercial assay or kit | Gel Filtration LMW Calibration Kit | Cytiva | Cat number: 28403842 | |
| Commercial assay or kit | Thrombin Time | HemosIL | Cat number: 0009758515 | |
| Commercial assay or kit | MTT Cell Viability Assay Kit | Biotium | Cat number: 30006 | |
| Chemical compound, drug | Marimastat | APExBIO | Cat number: A4049 | |
| Chemical compound, drug | 2-MeS-ADP | Tocris | Cat number: 1624 | |
| Chemical compound, drug | Mca-KPLGL-Dpa-AR-NH2 Fluorogenic Peptide Substrate | R&D systems Inc | Cat number: ES010 | |
| Software, algorithm | AlphaFold 3 | ColabFold | RRID:SCR_028034 | Protein 3D structure prediction |
| Software, algorithm | UCSF ChimeraX | UCSF Resource for Biocomputing, Visualization, and Informatics | RRID:SCR_015872 | 3D structure visualization and figure generation |
| Software, algorithm | Origin | OriginLab Corporation | RRID:SCR_014212 | Data analysis and graphing |
| Software, algorithm | GraphPad Prism 10 | GraphPad Software, Inc | RRID:SCR_002798 | Data analysis and graphing |
| Other | ESF 921 | Expression Systems | Cat number: 96-001-01 | Insect Cell Culture Media |
| Other | DMEM high glucose with GlutaMAX | Thermo Fisher Scientific | Cat number: 10566016 | Supplemented with 2 mM sodium pyruvate, 100 IU/ml penicillin, 250 µg/ml streptomycin, 10% Fetal Bovine Serum, 1% GlutaMAX |
| Other | Casein from bovine milk | Sigma-Aldrich | Cat number: C7078 | |
| Other | Fibrinogen from human plasma | Sigma-Aldrich | Cat number: F3879 | |
| Other | HiTrap IMAC FF 5 ml | Cytiva | Cat number: 17092104 | Connected to an AKTA Pure system |
| Other | HiTrap Q XL 5 ml | Cytiva | Cat number: 17515901 | Connected to an AKTA Pure system |
| Other | Superdex 200 increase 10/300 GL | Cytiva | Cat number: 28990944 | Connected to an AKTA Pure system |
| Other | HiLoad 16/600 Superdex 75 pg | Cytiva | Cat number: 28989333 | Connected to an AKTA Pure system |
| Other | Amicon Ultra Centrifugal Filter, 30 kDa MWCO | Millipore | Cat number: UFC9030 | |
| Other | Amicon Ultra Centrifugal Filter, 10 kDa MWCO | Millipore | Cat number: UFC9010 |
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Purified zymogens reveal mechanisms of snake venom metalloproteinase auto-activation
eLife 15:RP109112.
https://doi.org/10.7554/eLife.109112.3
