These findings establish accessory-domain-mediated substrate recognition as a key determinant of SVMP function, informing rational antivenom design and support a model in which PIII SVMP accessory domains enhance substrate positioning and catalytic efficiency while selectively restricting access to non-cognate substrates.
Abstract
Snake venom metalloproteinases (SVMPs) are major drivers of pathology following viper envenomation and represent important targets for the development of next-generation recombinant antivenoms. PIII SVMPs are among the most potent haemorrhagic toxins and contain disintegrin-like (Dis) and cysteine-rich (C-rich) accessory domains. Despite their biomedical importance, the mechanistic roles of these accessory domains in substrate recognition and catalysis remain poorly understood. We produced recombinant full-length and domain-deletion variants of two functionally distinct PIII SVMPs: the broadly proteolytic, cytotoxic cPIII and the highly specific prothrombin activator Ecarin. Proteins were expressed as latent zymogens in insect cells, auto-activated by Zn²⁺, and analysed using enzymatic, blood clotting, and cell-based assays. Progressive removal of the C-rich and Dis domains reduced zymogen auto-activation and markedly diminished catalytic activity in both toxins. In cPIII, domain deletion caused a stepwise loss of proteolytic and cytotoxic activity without altering substrate preference. In Ecarin, removal of the accessory domains strongly impaired prothrombin activation, and thus plasma clotting, demonstrating a critical role in substrate recognition. Conversely, deletion of the C-rich domain increased fibrinogenolytic activity, revealing a substrate-dependent gatekeeping function. Deglycosylation showed that N-linked glycans modulate SVMP activity in a construct-dependent manner. Recombinant Ecarin closely recapitulated the biochemical properties of the native venom-derived toxin. Our data support a model in which PIII SVMP accessory domains enhance substrate positioning and catalytic efficiency while selectively restricting access to non-cognate substrates. These findings establish accessory-domain-mediated substrate recognition as a key determinant of SVMP function, informing rational antivenom design.
Snake venom metalloproteinases are major determinants of viperid venom pathology, but the specific contribution of their metalloprotease domain to toxin function remains insufficiently defined. Here, we produced and characterized the metalloprotease domain of Pictolysin-III, a P-III metalloproteinase from Bothrops pictus, using two heterologous expression systems: Pichia pastoris KM71H and Escherichia coli BL21 Star™ (DE3). Codon-optimized constructs containing the prodomain, an N-terminal His-tag, and a TEV cleavage site were generated, sequence-verified, expressed, purified by Ni-NTA chromatography, and activated by TEV processing. Both recombinant proteins were immunoreactive and catalytically active but showed host-dependent biochemical and biological profiles. The P. pastoris-derived protein, rMD-Pp, displayed a higher apparent molecular mass and greater final recovery than the E. coli-derived protein, rMD-Ec. Relative to native Pictolysin-III, rMD-Pp retained 59.44 ± 0.56% azocaseinolytic activity, whereas rMD-Ec retained 23.49 ± 0.79%. rMD-Pp also showed functional preservation under acidic conditions, fibrino(geno)lytic activity, and hemorrhagic activity. In contrast, rMD-Ec showed reduced proteolytic performance and no visible hemorrhagic lesion at the tested doses up to 4 µg. Exploratory molecular dynamics simulations of theoretical glycosylated and non-glycosylated models suggested comparable global compactness, with higher calculated solvent exposure and localized flexibility, particularly around the omega-loop region. Real-time cell analysis showed distinct cell-line-dependent impedance profiles in RMF-621, MCF-7, and MDA-MB-231 cells. Overall, these findings demonstrate that the metalloprotease domain constitutes an active functional unit of Pictolysin-III. However, the two recombinant preparations differed in their biochemical and biological profiles, with rMD-Pp more closely reproducing the functional properties of the native toxin than rMD-Ec.
A. Roque, D. Torrejón, Alex Proleón et al.· Biomedicine & pharmacotherap...· 0 citations
Structural analysis reveals that ASPRV1-14 possesses distinctly hydrophobic S2/S2' pockets, dictating a strict requirement for hydrophobic residues at the P2/P2' positions of substrates and explaining its resistance to most HIV-1 PR inhibitors, except indinavir.
Xueqian Feng, Zi-Lian Chen, Chao Lan et al.· Acta Biochimica et Biophysic...· 0 citations
Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.
Janish Kumar, Jyotisha, Rahila Qureshi et al.· International Journal of Bio...· 0 citations
Abstract Granzymes are a family of trypsin-like serine proteinases (TLSP) produced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. They play critical roles in immune defense by inducing diverse forms of programmed cell death (e.g., apoptosis, pyroptosis, and necroptosis) in infected or abnormal cells. Here, we review current information on the structural mechanisms underlying the activation, substrate specificity, and inhibition of the five human granzymes (GzmA, GzmB, GzmH, GzmK, and GzmM). We highlight how specific variations of the canonical trypsin fold allow these five proteinases to display entirely distinct substrate specificities. We emphasize the role of allosteric mechanisms, such as exosites and dimerization, in expanding their substrate recognition profiles far beyond simple P1 cleavage site requirements. We discuss the rapidly growing evidence on the contribution of dysregulated granzyme activity to various inflammatory and autoimmune diseases. Finally, we explore emerging therapeutic strategies targeting granzymes, aiming to either enhance their beneficial cytotoxic effects (e.g., in cancer immunotherapy and infection) or inhibit their pathological roles in chronic inflammation and autoimmunity. Despite significant progress, open questions remain regarding the full spectrum of physiological granzyme substrates, their precise synergistic actions in vivo, and the translational challenges of applying in vitro findings to complex clinical contexts.
José Luis Gardeazábal-Torbado, E. Estébanez-Perpiñá, Pablo Fuentes-Prior· Biological chemistry· 0 citations
The Hom family and canilysin are defined as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.
A. Rodríguez-Banqueri, T. Goulas, Marina Girbal-González et al.· Journal of Molecular Biology· 0 citations
L-amino acid oxidase (LAAO) is a major component of snake venoms and contributes to several pathological effects associated with envenomation. However, the identification of immunodominant B-cell epitopes within this toxin remains poorly explored. In this study, native LAAO from Bothrops atrox venom was purified and used to generate polyclonal antibodies in rabbits. Anti-LAAO IgG antibodies were purified and employed in SPOT synthesis-based epitope mapping using a peptide array comprising 122 overlapping pentadecapeptides covering the complete amino acid sequence of B. atrox LAAO (GenBank: ALL27300.1). Five antigenic regions distributed throughout the N-terminal, central, and C-terminal portions of the toxin were identified. Subsequent bioinformatic analyses, including epitope prediction and structural evaluation, identified PEP1 (VGEVNKDPGVLEYPVKPSEVGKS) as the most promising candidate. This peptide was synthesized and evaluated as an immunogen in mice. Immunization with PEP1 elicited antibodies capable of recognizing both the synthetic peptide and native LAAO, as well as cross-reacting with crude B. atrox venom. Sequence alignment demonstrated that this epitope is highly conserved among LAAOs from different snake species. These findings identify a conserved and immunoreactive linear B-cell epitope of LAAO, providing a basis for future studies exploring its use in immunogen design and antivenom development.
Thamyres C Silva de Assis, Tamara G Fernandes Costa, L. Lopes-de-Souza et al.· Toxicon· 0 citations