Snake venom metalloproteinases are predominantly responsible for the cytotoxic effects of certain African viper venoms
Posted on: 13 January 2026
Preprint posted on 7 December 2024
Article now published in Toxins at https://www.mdpi.com/2072-6651/17/7/328
Understanding the cytotoxic mechanisms driving snakebite envenoming may help uncover new therapeutics and pharmacological agents.
Selected by Daniel Osorno Valencia
Categories: biochemistry, pathology, pharmacology and toxicology
Updated 12 January 2026 with a postLight by Daniel Osorno Valencia
This preprint has now been published in the peer-reviewed journal Toxins. When looking at the specific questions I raised as part of my preLights post, I notice the following:
Q1 postLight: In the final published version, the dose–response curves still exhibit an abrupt transition in cytotoxicity, with cell viability dropping directly from approximately 100% to 0% between two consecutive concentrations. This issue should be carefully considered, as when dealing with venom-derived proteins and cytotoxicity, small differences in concentration can lead to substantial changes in toxic effects. Moreover, under these conditions, it is not possible to derive a reliable IC₅₀ value, since the analysis effectively relies on only two extreme data points (full viability versus complete loss of viability), rather than on a continuous and gradual dose response relationship. This limitation compromises the robustness and interpretability of the reported cytotoxicity parameters.
Q2 postLight: My concern regarding protein identification has been adequately addressed through the citation of a related study in which complementary molecular approaches, including advanced proteomic tools, were used to identify and characterise venom metalloproteinases. In particular, the work by Wilkinson et al. supports the assignment of the protein families discussed in the present article.
Q3 and Q4 postLight: Regarding Q3 and Q4, these concerns remain unresolved. Although the cited proteomic study strengthens the identification of metalloproteinases in the venoms, it does not demonstrate that the cytotoxic fractions analysed in the present work are free from other cytotoxic proteins, such as L-amino acid oxidases or phosphodiesterases, which may co-elute during size-exclusion chromatography. Moreover, the use of EDTA as an inhibitor remains non-specific, as its chelating activity may suppress multiple Zn²⁺-dependent enzymes. Consequently, the observed reduction in cytotoxicity cannot be attributed exclusively to metalloproteinase inhibition.
Why I think this preprint is important:
In this preprint, the researchers demonstrate that Type III SVMP are the proteins in the venom of the snakes Echis romani and Bitis arietans that are responsible for cytotoxicity effects. Several proteins found in snake venom have shown potential as pharmaceutical agents due to their diverse biological activities. Understanding the cytotoxic mechanisms of these metalloproteinases may help uncover their therapeutic potential and support their development as possible pharmacological agents.
Background:
Snakebite envenoming, the disease caused by the toxins in the bite of a venomous snake, has been classified as a neglected tropical disease since 2017 by the World Health Organization [1]. In Africa and the Middle East alone, up to 580,000 envenomings likely occur each year [2]. Echis romani and Bitis arietans are considered the snake species of most medical importance causing the vast majority of accidents. Their bites can result in severe envenoming or death [3], [4].
Snake venom is composed of a variety of protein families (phospholipase A2 (PLA2), snake venom metalloproteinase (SVMP), snake venom serine protease (SVSP), and l-amino acid oxidase (LAAO) and three finger toxins (3FTx) among others) [5]. The composition of both E.romani and B.arietans venom has been described previously, showing that metalloproteinases are the most abundant family of proteins in these venoms. Metalloproteinases can be classified as P-I, P-II or P-III depending on the presence or absence of non-catalytic ancillary domains and have the capability to degrade the extracellular matrix (EMC)[6]. While the biochemical activities of SVMPs have been described, their specific role in cytotoxicity and potential therapeutic applications remain poorly understood.
This preprint aims to investigate the contribution of metalloproteinases to the cytotoxic effects observed in E. romani and B. arietans venoms, and to explore their potential as key effectors in venom-induced pathology and as candidates for pharmaceutical development.
Key findings:
E.romani and B.arietans venom induce cytotoxicity effect in human epidermal keratinocytes
The authors demonstrated in vitro that both the venom of E.romani and B.arietans show an inhibitory or cytotoxic effect in a human epidermal keratinocytes (HaCaT) cell line. This information was obtained by performing a viability test using the MTT assay.
Geographical variants of SVMP from B. arietans present variation in their cytotoxic effects
The authors isolated two PIIa SVMPs from two B. arietans snakes living in different geographical locations (Nigeria and Tanzania). The authors found significantly higher cytotoxicity in the Tanzanian variant, with an IC50 of 2.7 µg/mL compared to 16.2 µg/mL found in the Nigerian variant, highlighting functional differences between the regional toxin variants.
E.romani and B.arietans fractions presented SVMP activity
The authors found that the fractions isolated by SEC chromatography from both venoms of E. romani and B.arietans presented metalloproteinase activity against the specific substrate, confirming the presence of this family of proteins in the fractions that previously showed cytotoxic effects.
Questions for the authors:
Q1: Have the authors considered including additional concentration points in the dose–response curve to improve the accuracy of the IC50 calculation and obtain a more unequivocal value? In the cytotoxicity assays (Fig. 1A and B, Fig. 9A and B), cell viability stays close to 100% across several concentrations, but then suddenly drops to 0% at a certain point. This lack of intermediate data points results in a steep curve with no gradual slope, which is essential for reliable interpolation of the IC50 [7]. I suggest including additional concentration points within this critical range, as demonstrated in studies like Pllans-II [8], where a more gradual decline in cell viability can be observed.
Q2: Have the authors explored the idea of using complementary molecular tools like mass spectrometry to ensure a clear and unambiguous identification of protein families? The SDS-PAGE profiles of the fractions presented by the authors (Fig 2 and 3 .B) show multiple bands under reducing conditions, which could represent distinct protein families, degradation products, or separate domains of multidomain proteins such as metalloproteinases.
Q3: The authors attribute the cytotoxic effects to metalloproteinases; however, the presence of multiple bands in the fractions suggests that other proteins may also contribute to this activity. Proteins such as LAAOs (50–70 kDa) [11] or phosphodiesterases (<100 kDa) [12] are known to induce cytotoxic effects [13]. Given this, have the authors considered using additional chromatographic methods, such as ion exchange or affinity chromatography [9,10], to complement SEC and improve protein isolation? Achieving higher purity would help confirm whether the observed cytotoxicity is specifically due to metalloproteinases or if other proteins are involved.
Q4: Taking into consideration the presence of phosphodiesterases (PDEs), which could co-elute with metalloproteinases in the same SEC fractions, and the fact that both enzyme types are zinc-dependent, the use of EDTA as an inhibitor may not specifically target only metalloproteinases. Since EDTA chelates metal ions like Zn²⁺, it could also inhibit PDEs activity. How do the authors distinguish whether the observed inhibition is solely due to the suppression of metalloproteinase activity specifically, rather than that of other zinc-dependent enzymes such as phosphodiesterases?
References:
[1] SNAKEBITE ENVENOMING A strategy for prevention and control. 2019. [Online]. Available: http://apps.who.int/bookorders.
[2] J. M. Gutiérrez, J. J. Calvete, A. G. Habib, R. A. Harrison, D. J. Williams, and D. A. Warrell, “Snakebite envenoming,” Nat Rev Dis Primers, vol. 3, no. 1, Sep. 2017, doi: 10.1038/NRDP.2017.63.
[3] “guidelines for snake envenoming WHO africa”.
[4] A. G. Habib, “Venomous snakes and snake envenomation in Nigeria,” in Toxinology: Clinical Toxinology in Asia Pacific and Africa, Springer Netherlands, 2015, pp. 275–298. doi: 10.1007/978-94-007-6386-9_32.
[5] T. Tasoulis and G. K. Isbister, “A current perspective on snake venom composition and constituent protein families,” Jan. 01, 2023, Springer Science and Business Media Deutschland GmbH. doi: 10.1007/s00204-022-03420-0.
[6] S. E. Gasanov, R. K. Dagda, and E. D. Rael, “Snake Venom Cytotoxins, Phospholipase A2s, and Zn2+- dependent Metalloproteinases: Mechanisms of Action and Pharmacological Relevance,” 2014.
[7] J. L. Sebaugh, “Guidelines for accurate EC50/IC50 estimation,” Pharm Stat, vol. 10, no. 2, pp. 128–134, Mar. 2011, doi: 10.1002/pst.426.
[8] A. Montoya-Gómez et al., “Pllans–II Induces Cell Death in Cervical Cancer Squamous Epithelial Cells via Unfolded Protein Accumulation and Endoplasmic Reticulum Stress,” Molecules, vol. 27, no. 19, Oct. 2022, doi: 10.3390/molecules27196491.
[9] A. Chantry, N. A. Gregson, and P. Glynn, “A Novel Metalloproteinase Associated with Brain Myelin Membranes,” Journal of Biological Chemistry, vol. 264, no. 36, pp. 21603–21607, Dec. 1989, doi: 10.1016/s0021-9258(20)88226-x.
[10] C. P. Bernardes et al., “Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom,” Toxicon, vol. 51, no. 4, pp. 574–584, Mar. 2008, doi: 10.1016/j.toxicon.2007.11.017.
[11] J. F. Wei, H. W. Yang, X. L. Wei, L. Y. Qiao, W. Y. Wang, and S. H. He, “Purification, characterization and biological activities of the l-amino acid oxidase from Bungarus fasciatus snake venom,” Toxicon, vol. 54, no. 3, pp. 262–271, Sep. 2009, doi: 10.1016/j.toxicon.2009.04.017.
[12] I. S. de Oliveira et al., “Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom,” Int J Biol Macromol, vol. 178, pp. 180–192, May 2021, doi: 10.1016/j.ijbiomac.2021.02.120.
[13] C. T. Pan et al., “The evolution and structure of snake venom phosphodiesterase (svPDE) highlight its importance in venom actions,” Elife, vol. 12, Apr. 2023, doi: 10.7554/eLife.83966.
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