Purified zymogens reveal mechanisms of snake venom metalloproteinase auto-activation

Snake venom is a complex cocktail of more than 100 proteins and peptides belonging to various toxin families (Oliveira et al., 2022). Although the number and abundance of isoforms within these families vary extensively across different venomous snakes, the snake venom metalloproteinases (SVMPs) are a pathologically important toxin family present in almost all snake venoms. SVMPs play a significant role in the physiological effects resulting from both local and systemic envenoming, such as haemorrhage, coagulopathy, and tissue necrosis (Bittenbinder et al., 2024). In many medically important viperid snake genera such as Echis (saw-scaled vipers), SVMP toxins are the most abundant toxin constituents, on average accounting for 33% of all toxins, although this can be as high as 72% in Echis ocellatus (Wagstaff et al., 2009; Casewell et al., 2014; Tasoulis and Isbister, 2023; Figure 1a).

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SVMPs are complex, structurally variable, cysteine-rich, Zn²⁺-dependent metalloproteinases (MPs) that can be classified into three sub-classes based on domain architecture: PI (comprising MP domain only), PII (MP and disintegrin domains), and PIII (MP, disintegrin-like, and cysteine-rich domains; Figure 1b); representatives of all three sub-classes are present in different viper venoms (Fox and Serrano, 2008; Casewell et al., 2011).

SVMPs are particularly toxic proteins with a wide range of substrates. They are known to degrade the basement membrane and extracellular matrix proteins, as well as several blood clotting factors, most notably fibrinogen, through their MP domain (Asega et al., 2020; Kini and Koh, 2016). Extracellular matrix breakdown and loss in blood capillary wall integrity results in vascular leakage, resulting in both local and systemic haemorrhage (Gutiérrez et al., 2005) and in the spread of other venom toxins (Gutiérrez et al., 2016). In PII SVMPs, the disintegrin domain contains a charged, integrin-binding motif, most commonly an arginine-glycine-aspartate (RGD) motif, which binds, among others, the fibrinogen receptor integrin α_IIbβ₃ (de O Almeida et al., 2023), causing inhibition of platelet aggregation.

Because viper SVMPs often target components of the coagulation cascade with unique specificity and/or lack of reliance on typically essential co-factors, these enzymes are highly valuable for investigating and monitoring the treatment of pathologies related to haemostasis (e.g. factor deficiencies, bleeding disorders; Moore, 2022). In the clinic, the SVMPs RVV-X and Ecarin are both used as standards for coagulation tests (Slagboom et al., 2017). In addition, SVMP-derived disintegrins have been repurposed for use as, or inspiration for the design of, anti-platelet therapies to treat atherothrombosis in the form of unstable angina and myocardial infarction (Oliveira et al., 2022). Thus, a single snake venom toxin family has yielded several translationally valuable tools for diagnosis and treatment of human disease. However, further exploitation of SVMPs is hampered by the considerable challenge in purifying individual SVMP proteins from venom, and the lack of robust, scalable, recombinant toxin production protocols, representing a bottleneck.

Currently, SVMPs are typically purified from venom by size exclusion chromatography (SEC), followed by ion exchange chromatography of the peaks containing SVMPs. Further separation can be achieved by hydrophobic interaction chromatography or reversed-phase high-performance liquid chromatography (RP-HPLC; Wilkinson et al., 2024; Yee et al., 2016). However, complete separation of endogenous functionally active SVMPs is difficult to achieve, because: (i) many venoms contain multiple related SVMP isoforms that share similar physicochemical properties, (ii) the resulting yields of each particular SVMP isoform can be very low, and (iii) the use of some desirable separation approaches (e.g. RP-HPLC) can result in SVMP denaturation. Importantly, this procedure is impeded by the limited access to sufficient amounts of extracted snake venom.

Recombinant expression of SVMPs could resolve the bottleneck but is severely hampered by their cytotoxicity, domain complexity, and high number of cysteine residues that complicate protein folding – for example, the PIII SVMPs contain between 20 and 40 cysteines forming disulfides (Olaoba et al., 2020). Currently, protocols are available for the production of only a select few specific SVMPs (including albocollagenase, bothropasin, and Ecarin; Pinyachat et al., 2011; Assakura et al., 2003; Jonebring et al., 2012). However, these suffer from limitations such as low yield and inactive proteins. Zymogen production of SVMPs has been described before, but it remains generally elusive how the zymogen is processed into the mature, active SVMP. Current protocols describe incubation of the zymogen for >7 days (Jonebring et al., 2012), incubation with other active proteinase toxins (Shimokawa et al., 1996), or the insertion of a TEV protease cleavage site into the SVMP amino acid sequence (Camacho et al., 2019).

Here, we establish a novel and generic protocol to access functional PI, PII, and PIII SVMPs in the quality and quantity required for characterisations and show that all three classes of SVMPs can auto-activate in the presence of zinc ions, resulting in distinct outcomes.

We used our MultiBac baculovirus/insect cell expression system (Bieniossek et al., 2012) for recombinant SVMP zymogen production. The MultiBac system is well-established for secretion of proteins, including the C-terminal heavy chain domain of the highly toxic protein, clostridial botulinum neurotoxin (Villaflores et al., 2013). We postulate that this system is highly suitable for SVMP production because (i) folding of the multi-domain SVMP zymogen will be supported by eukaryotic, endogenous chaperones present in insect cells and (ii) secreted SVMPs will undergo cellular protein quality control in the endoplasmic reticulum (ER) and Golgi apparatus for correct disulfide bond formation and glycosylation before secretion, ensuring that the toxins produced are correctly folded, soluble and authentically modified. We show that zymogen expression overcomes SVMP cytotoxicity, and we demonstrate successful production of PI and PII SVMPs from E. ocellatus and a PIII SVMP from E. carinatus sochureki (Figure 1b and c). We establish auto-activation of the SVMP zymogens and concomitant cleavage of the prodomain (all classes), C-terminal tags (PI, PII), and the disintegrin domain (PII). The PIII prodomain is completely degraded by the activated SVMP. We further analyse the cytotoxicity and haemotoxicity of the auto-activated SVMPs in vitro using established casein (Macêdo and Fox, 2016), fibrinogen (Macêdo and Fox, 2016), fluorogenic peptide (Alsolaiss et al., 2022), and insulin degradation (Wilkinson et al., 2024) assays. For the PI SVMP, we show that the substrate specificity of recombinant SVMP is identical to that of the corresponding toxin purified from venom, compellingly validating the utility of our strategy.

Our generic SVMP production protocol thus paves the way to systematically characterise SVMPs and unlock biomedical applications, in haematology, thrombosis and inflammation research and as targets for the discovery of novel snakebite therapeutics.

The characterization of SVMP toxins, critical for effective bioprospecting and the discovery of snakebite therapeutics, is severely hampered by difficulty in isolating the native proteins from venom. Here, we established a generic protocol for efficient recombinant production of the three structurally diverse classes of SVMPs as zymogens, which overcomes this obstacle. We demonstrate expression of PI, PII and PIII SVMP zymogens from Echis species in high quality and quantity using the MultiBac baculovirus/insect cell expression system.

In SVMP zymogens, the highly conserved propeptide sequence which blocks the active site of the MP domain, is clamped in place by the N-terminal prodomain. It should be noted that despite expression as a zymogen, residual cytotoxicity was still observed, evident by the decrease in cell viability throughout expression and reduction in the amount of co-expressed YFP fluorescence due to cell lysis (Figure 2). In agreement with this observation, PI and PIII SVMPs were able to partially activate themselves, resulting in the presence of three characteristic bands in Coomassie-stained SDS gels – the zymogen, mature SVMP, and prodomain. We speculate that this auto-cleavage could be due to the SVMP active site accreting divalent metal ions from the media and buffers used. Additionally, it is likely that the change in pH from 6 (expression) to 8 (purification) allows the activation of SVMPs, as is the case when venom is released from the venom gland (pH 5.4) into the victim (pH 7.4) during envenoming (Mackessy and Baxter, 2006). A complete control of self-activation of SVMP zymogens would be highly desirable. However, previous studies reported that (i) SVMP zymogens are processed within secretory cells of the venom gland (Portes-Junior et al., 2014), and (ii) mature SVMPs accumulate in secretory vesicles during venom production (Carneiro et al., 2002). Accordingly, complete prevention of SVMP zymogen auto-processing will be very difficult to achieve because this would require Zn²⁺ ion depletion within the cells which would result in cytotoxicity.

Of note, despite the self-cleavage of the PI and PIII zymogens, the majority of the mature SVMP protein remained associated with the prodomain. Interestingly, this did not affect the activity of the SVMPs. According to AlphaFold 3 structure predictions, the prodomain is located on the side of the MP domain, which is opposite to the active site. Upon activation, cleavage of the prodomain releases the propeptide from the active site. This renders the Zn²⁺ ion in the active site free to interact with a substrate. In agreement, we observe that the SVMPs are active even if the prodomain, now no longer covalently linked to the SVMP, remains bound to the MP domain (Figure 3c, Figure 3—figure supplement 2).

SVMP zymogens undergo additional post-translational processing: The PI SVMP construct described here was based on transcriptomic data and corresponds to the UniProt sequence ID: Q2UXQ3. During purification, it underwent C-terminal truncation by ~10 kDa, leading to a smaller protein which corresponds closely to previously characterised PI SVMPs (Sanchez et al., 2010; Bello et al., 2006; Bernardes et al., 2008). Moreover, we observed a general trend of loss of C-terminal tags during purification, activation, and subsequent activity assays for all three SVMP classes.

Here, we show that the recombinant PIII SVMP comprises N-linked glycosylation. We would like to point out that when expressed in insect cells, the glycosylation pattern may be different compared to endogenous SVMPs due to differences in the snake and insect glycosylation pathways. The Sf21 and Hi5 cells used in this study typically produce N-glycans that are trimmed to a core ‘paucimannose’ structure (Man3GlcNAc2), often with an alpha1,6-fucosylation (Watanabe et al., 2002). While snakes can produce more complex, sialylated N-glycans, glycomic studies of native venoms (e.g. Bothrops venoms) have demonstrated that high-mannose and paucimannose structures are also prevalent in native SVMPs (Andrade-Silva et al., 2018). Therefore, the recombinant glycoforms likely represent a subset of the native glycan heterogeneity and not the full diversity found in snake venom toxins. Moreover, Sf21 and Hi5 cells recognise the same N-glycosylation sequon (Asn-X-Ser/Thr) as reptilian cells, and therefore the site-occupancy remains consistent with the native protein, preserving the overall topography of the toxin.

To improve SVMP yields and/or activity, we hypothesised that co-expression of SVMPs with human or snake PDI would aid their proper folding, ensuring the multiple disulfide bonds were correctly formed. Based on transcriptomic data from Echis venom gland (Redureau et al., 2025), PDIs are highly overexpressed in the snake venom gland. However, PDI over-expression in insect cells did not improve protein yields nor enzyme activity. We speculate that the endogenous insect cell PDIs are sufficient for SVMP folding in our setup. Alternatively, a combination of different chaperones may be required for further boosting SVMP production.

SVMP zymogens from all three classes could be auto-activated by the addition of Zn²⁺ ions. While some PI SVMP was processed during purification, any remaining PI zymogen could be cleaved into the mature SVMP and prodomain by incubation with Zn²⁺. The PII zymogen could be activated by the same means, although there was no clear separation into prodomain and mature protein. Instead, we observed two bands corresponding to the disintegrin and prodomain after prolonged incubation with Zn²⁺ (Figure 3e). It is unlikely that the observed disappearance of the MP domain is due to misfolding because the PII zymogen is well expressed with high yields (9–10 mg/l culture) in insect cells and eluted in two clean peaks in SEC corresponding to a monomer and dimer, respectively. In the case of misfolding, we would have expected aggregation and/or degradation during purification, and we would not expect to see degradation of the zymogen in SDS-PAGE analysis only after addition of Zn²⁺ which activates the MP (Figure 3e). Notably, the disintegrin domain of this PII has 100% sequence identity to ocellatusin, a short, monomeric disintegrin of E. ocellatus venom (Smith et al., 2002). Previous studies have identified a discrepancy between the amounts of ocellatusin and its PII-derived MP in the venom, whereby ocellatusin represents 3.9%, and the SVMP less than 0.1% of venom proteins (Wagstaff et al., 2009). In summary, the disintegrin is likely liberated from this PII SVMP and is the more pathogenic component of the SVMP, causing inhibition of platelet aggregation (Figure 5a). In contrast, the PII-derived MP domain is unstable and therefore requires high toxin concentrations to elicit proteolytic activity in our activity assays (Figure 4b and e).

The recombinant PIII zymogen was fully auto-activated in our experiments by the addition of Zn²⁺ ions (Figure 3f). Interestingly, this PIII SVMP was able to degrade its prodomain despite the latter initially remaining bound to the mature protein according to SEC (Figure 3c). This agrees with the finding that prodomains, which are highly conserved in SVMPs, are rarely identified in the venom proteome (Moura-da-Silva et al., 2016), suggesting that all prodomains might eventually be fully degraded by venom proteases including PIII SVMPs.

Activated recombinant SVMPs were active against bovine casein, a general protease substrate (Figure 4a–c). PI and PIII exhibited strong activity, degrading all bands, while PII was significantly less active, requiring much higher enzyme concentrations. This agrees with our previous observation that bands corresponding to the MP-disintegrin protein or the MP domain alone after activation with Zn²⁺ (Figure 3e) are absent in SDS-PAGE, indicating that the MP domain is likely unstable.

Fibrinogen degradation assays are particularly relevant for SVMPs and indicate their potential haemotoxic activity (Figure 4d–f). The 340 kDa fibrinogen glycoprotein is made up of three pairs of polypeptide chains, which lead to three groups of distinct bands when analysed by reducing SDS-PAGE: the Aα (70–75 kDa), Bβ (65 kDa), and γ (50 kDa) chains. In contrast to the endogenous serine protease thrombin, which cleaves fibrinopeptides A and B off the Aα and Bβ chains, causing self-polymerisation into fibrin fibres and formation of blood clots (Weisel and Litvinov, 2017), the PI, PII, and PIII SVMPs studied here caused a fuller degradation of fibrinogen (Figure 4d–f). All SVMPs were active against both the Aα and Bβ chains of fibrinogen; the PI and PIII showed a preference for the Aα chain and the PII preferentially degraded the Bβ chain of fibrinogen. We conclude that the three SVMPs interfere with clot formation and would thus contribute to an anticoagulant outcome upon envenoming. In agreement, the PIII SVMP cannot cause clot formation in the thrombin clot time assay (Figure 5d), suggesting it is not an Ecarin-like prothrombin activator, which is typically responsible for procoagulant activity seen in such assays with Echis venoms (Figure 5c and e).

The recombinant PIII SVMP was highly active against a fluorogenic peptide substrate, ES010, showing high affinity and specificity for cleavage of the Gly-Leu bond within the peptide (Figure 4g). In this context, we would like to point out that enzyme concentrations and kinetics are based on zymogen concentrations, assuming 100% SVMP activation by Zn²⁺ (Figure 4a–g). If zymogen activations were incomplete or non-linear, our k_cat/K_M calculations could either underestimate or overestimate the PIII SVMP’s catalytic efficiency (Figure 4g).

The SVMP PIII was also active against insulin B (Figure 4h), as was SVMP PI (Figure 4i) which produced identical degradation products to native PI, confirming identical cleavage sites and successful recombinant production of toxin with native-like characteristics, validating our approach.

In addition to the proteolytic activity of the MP domain, PII and PIII SVMPs also include a disintegrin or disintegrin-like domain containing a potential integrin binding motif. In platelet aggregation assays, our RGD-containing PII SVMP inhibited 2-MeS-ADP-induced platelet-aggregation with and without the prodomain present (Figure 5a). The RSECD-containing PIII, in contrast, did not inhibit 2-MeS-ADP-induced platelet aggregation, but this does not exclude other disintegrin-like activity of this domain.

Zymogen expression was previously reported for ADAMs (A Disintegrin and Metalloproteinases) and MMPs (matrix MPs), which are structurally and functionally related to SVMPs. ADAMs and MMPs are involved in cell-cell interactions, ectodomain shedding, and degradation of extracellular matrix components, respectively (Takeda, 2016). ADAMs and MMPs have become prominent targets in drug discovery (Overall and López-Otín, 2002; Craig et al., 2015). For MMPs, zymogen expression in mammalian and insect cells has been reported and enzyme activation was achieved via 4-aminophenylmercuric acetate or trypsin to remove the prodomain (Ogata et al., 1995; Vallon et al., 1997). Moreover, ADAMs have been expressed with their prodomain to aid proper folding and latency (Jones et al., 2010; Chavaroche et al., 2014). The ADAMs prodomain has been removed post-translationally, for example by co-expression of furin (Wong et al., 2015). Interestingly, in agreement with our PI and PIII SVMP observations, ADAM33 zymogen was processed and secreted in Drosophila S2 cells, with the prodomain remaining associated with the catalytic domain after purification. The processing was enhanced by addition of cadmium chloride and zinc chloride (Prosise et al., 2004). Auto-activation of zymogens by Zn²⁺ incubation alone, however, has not been reported to the best of our knowledge.

While ADAMs and MMPs are well characterised in the literature, comparable insights into SVMPs were largely absent to date, due to lack of means for recombinant SVMP protein production. Our generic protocol for recombinant SVMP zymogen production of all three classes overcomes their high cytotoxicity and allows, for the first time, to study auto-activation revealing important differences between the SVMP classes. Furthermore, our protocol will unlock the production of many additional hitherto inaccessible SVMPs, allowing in-depth characterisation of their post-translational processing, enzymatic activity, substrate specificity, cyto- and haemotoxicity, and enable the evaluation of potential biomedical applications to develop new diagnostics and treatments for blood clotting disorders. Our study paves the way to engineer existing and new SVMPs for improved and novel applications in haematology, thrombosis, and inflammation research and other important areas in which SVMPs can play a crucial role. Importantly, the recombinantly produced, tagged SVMPs can serve as antigens for the future selection and evolution of neutralising antibodies as a basis for next-generation snakebite treatment to combat snakebite envenomation.

All other chemical reagents were purchased from Sigma-Aldrich/ Merck.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Sophie Hall

   School of Biochemistry, University of Bristol, Bristol, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3176-6004

2. Iara Aime Cardoso

   School of Biochemistry, University of Bristol, Bristol, United Kingdom

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0288-4706

3. Mark C Wilkinson

   Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Liverpool, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

4. Maria Molina Carretero

   School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

5. Srikanth Lingappa

   School of Biochemistry, University of Bristol, Bristol, United Kingdom

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0129-5512

6. Bronwyn Rand

   School of Biochemistry, University of Bristol, Bristol, United Kingdom

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0008-0997-6217

7. Dakang Shen

   School of Biochemistry, University of Bristol, Bristol, United Kingdom

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0008-5327-1185

8. Johara Boldrini-França

   School of Biochemistry, University of Bristol, Bristol, United Kingdom

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

9. Richard Stenner

   School of Biochemistry, University of Bristol, Bristol, United Kingdom

   Contribution

   Methodology

   Competing interests

   No competing interests declared

10. Stefanie Kate Menzies

    1. Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
    2. Lancaster University, Lancaster, United Kingdom

    Contribution

    Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9273-9296

11. Georgia Balchin

    School of Biochemistry, University of Bristol, Bristol, United Kingdom

    Contribution

    Investigation

    Competing interests

    No competing interests declared

12. Konrad Kamil Hus

    1. School of Biochemistry, University of Bristol, Bristol, United Kingdom
    2. Department of Biotechnology and Bioinformatics, Faculty of Chemistry, Rzeszow University of Technology, Rzeszow, Poland

    Contribution

    Investigation

    Competing interests

    No competing interests declared

13. Renaud Vincentelli

    Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257 CNRS-Aix-Marseille Université, Marseille, France

    Contribution

    Investigation

    Competing interests

    No competing interests declared

14. Andrew Mumford

    School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom

    Contribution

    Supervision, Writing – original draft

    Competing interests

    No competing interests declared

15. Alastair Poole

    Bristol Medical School, University of Bristol, Bristol, United Kingdom

    Contribution

    Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-0868-297X

16. Nicholas R Casewell

    Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Liverpool, United Kingdom

    Contribution

    Supervision, Writing – original draft

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8035-4719

17. Imre Berger

    1. School of Biochemistry, University of Bristol, Bristol, United Kingdom
    2. Max Planck Bristol Centre for Minimal Biology, School of Chemistry, University of Bristol, Bristol, United Kingdom

    Contribution

    Conceptualization, Supervision, Methodology, Writing – original draft

    For correspondence

    imre.berger@bristol.ac.uk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7518-9045

18. Christiane Schaffitzel

    School of Biochemistry, University of Bristol, Bristol, United Kingdom

    Contribution

    Conceptualization, Supervision, Methodology, Writing – original draft, Writing – review and editing

    For correspondence

    christiane.berger-schaffitzel@bristol.ac.uk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1516-9760

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