Recombinant Bothrops atrox Zinc metalloproteinase atroxlysin-1

What is the basic structure and classification of atroxlysin-1?

Atroxlysin-1 (also known as atroxlysin-I) is a 23kDa P-I metalloproteinase isolated from the venom of Peruvian Bothrops atrox (common name: Jergón or Barba amarilla). It contains 204 amino acid residues and belongs to the class of snake venom metalloproteinases (SVMPs) . Unlike other structurally homologous P-I metalloproteinases, atroxlysin-1 demonstrates significant hemorrhagic activity, making it particularly interesting for toxinological research . The recombinant form available for research has a theoretical molecular weight of 30.4kDa due to the addition of His and Myc tags .

How does the proteolytic activity of atroxlysin-1 compare with other snake venom metalloproteinases?

Atroxlysin-1's proteolytic activity presents several distinctive features compared to other SVMPs:

• Substrate specificity: Atroxlysin-1 demonstrates specific cleavage patterns, targeting the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B-chain .

• Cofactor dependence: Its proteolytic activity toward dimethylcasein is enhanced by Ca²⁺ but inhibited by EDTA, dithiothreitol, excessive Zn²⁺, and alpha2-macroglobulin .

• Extracellular matrix degradation profile: Unlike some other SVMPs, atroxlysin-1 cleaves plasma fibronectin, collagens I and IV, and the triple-helical fragment CB3 of collagen IV, but notably does not degrade laminin-111 .

• Hemorrhagic potential: What distinguishes atroxlysin-1 from other P-I SVMPs is its pronounced hemorrhagic activity, which is directly related to its ability to disrupt basement membrane components and endothelial cell interactions .

What experimental approaches should be considered when studying the hemorrhagic mechanism of recombinant atroxlysin-1?

When investigating the hemorrhagic mechanism of recombinant atroxlysin-1, researchers should implement a multi-faceted experimental approach:

• In vitro proteolytic assays:

  • Test the enzyme's activity against key extracellular matrix proteins including fibronectin, different collagen types, and integrin subunits

  • Determine dose-dependence and time-course of hydrolysis using SDS-PAGE and densitometric analysis

  • Compare activity of the recombinant form against the native toxin to ensure functional equivalence

• Cell-based models:

  • Evaluate effects on endothelial cell monolayer integrity using transwell assays

  • Assess changes in cytoskeletal organization and cellular adhesion

  • Measure integrin expression and activation status after atroxlysin-1 exposure

• In vivo hemorrhagic models:

  • Implement intradermal injection protocols to quantify hemorrhagic lesion diameter

  • Consider microvascular permeability assays using labeled dextran or albumin

  • Utilize intravital microscopy to directly observe microvascular damage in real-time

• Inhibition studies:

  • Test specific inhibitors (EDTA, alpha2-macroglobulin) to confirm metalloproteinase-dependent effects

  • Pre-incubate with anti-atroxlysin-1 antibodies to assess neutralization potential

What are the optimal expression and purification conditions for recombinant atroxlysin-1?

Based on available data and standard practices for recombinant snake venom metalloproteinases, the following expression and purification protocol is recommended:

• Expression system:

  • E. coli is the established host for recombinant atroxlysin-1 expression

  • Consider using BL21(DE3) strain for high-level protein expression

  • Optimize expression using an IPTG-inducible system with varying concentrations (0.1-1.0 mM) and induction temperatures (16-37°C)

• Vector design:

  • Include the expression region encoding amino acids 1-202 of the mature protein

  • Incorporate N-terminal 10xHis-tag and C-terminal Myc-tag for purification and detection

  • Consider codon optimization for E. coli expression

• Purification strategy:

  • Implement metal affinity chromatography using Ni-NTA resin as the primary purification step

  • Follow with size-exclusion chromatography to achieve >85% purity

  • Include reducing agents during purification to maintain proper folding but avoid excessive DTT which can inhibit activity

• Protein refolding:

  • If expressed in inclusion bodies, use controlled refolding with decreasing urea gradients

  • Incorporate Zn²⁺ ions during refolding to ensure proper metalloproteinase activity

  • Monitor proper folding by enzymatic activity assays using synthetic substrates

• Quality control:

  • Confirm purity by SDS-PAGE (target >85%)

  • Verify molecular weight (~30.4 kDa for the tagged recombinant form)

  • Validate activity through proteolytic assays against standard substrates

How are epitopes identified in atroxlysin-1 and what is their significance for neutralizing antibody development?

Epitope identification in atroxlysin-1 employs multiple complementary approaches with significant implications for antivenom development:

• Methodologies for epitope identification:

  • Spot-synthesis technique: This approach identified two linear epitopes recognized by anti-atroxlysin-1 neutralizing rabbit antibodies located at the N-terminus: Y22NGNSDKIRRRIHQM36 and G55VEIWSNKDLINVQ68 .

  • Computational prediction: This method identified the linear epitope V9DLFIVVDHGMFMKY23, which when synthesized and used for immunization, produced antibodies that reduced atroxlysin-1 enzymatic activity by 70-80% .

• Synthetic peptide design strategies:

  • Multiple epitopes can be combined using glycine spacers (e.g., NGNSDKIRRRIH-GG-GVEIWSNKDLINVQ) to create more effective immunogens .

  • Internal cysteine residues can be replaced by serine to prevent disulfide bond formation during synthesis.

  • Addition of tyrosine residues may be considered for peptides lacking aromatic residues to enable quantification by absorbance at 280 nm.

• Delivery systems for immunization:

  • Liposomal encapsulation of synthetic peptides has proven effective for generating neutralizing antibodies against atroxlysin-1 .

  • The antibodies generated against these epitope-based immunogens have demonstrated capacity to neutralize both enzymatic and hemorrhagic activities of atroxlysin-1.

What are the challenges in developing neutralizing antibodies against recombinant atroxlysin-1?

Developing effective neutralizing antibodies against recombinant atroxlysin-1 presents several research challenges:

• Structural authenticity:

  • Ensuring the recombinant form maintains native epitope conformations despite the presence of purification tags

  • The N-terminal 10xHis-tag and C-terminal Myc-tag may potentially alter protein folding or accessibility of key epitopes

• Cross-reactivity considerations:

  • Balancing specificity with cross-neutralization potential against homologous SVMPs from other Bothrops species

  • Identifying conserved epitopes that may provide broader protection against multiple venom components

• Neutralization assessment methodologies:

  • In vitro neutralization may not correlate perfectly with in vivo protection

  • Researchers must evaluate both enzymatic neutralization (e.g., fibrinogenolytic activity) and biological effect neutralization (hemorrhagic activity)

  • Complete neutralization of hemorrhagic activity requires antibodies targeting specific epitopes that may differ from those needed to neutralize enzymatic activity

• Epitope optimization strategies:

  • Fine-tuning epitope sequences to enhance immunogenicity while maintaining neutralization capacity

  • Potential for combining multiple epitopes (Y22NGNSDKIRRRIHQM36, G55VEIWSNKDLINVQ68, and V9DLFIVVDHGMFMKY23) into a single construct for broader neutralization coverage

How does recombinant atroxlysin-1 contribute to inflammatory reactions and what experimental models best capture this activity?

Atroxlysin-1 plays a significant role in the inflammatory reactions observed following Bothrops atrox envenomation through multiple mechanisms:

• Direct inflammatory mechanisms:

  • Atroxlysin-1 (ATXL) directly induces edema formation in mouse models

  • It promotes leukocyte accumulation in a dose-dependent manner, with significant recruitment observed at dosages as low as 2 μg

  • The inflammatory response involves both polymorphonuclear and mononuclear cell recruitment, with distinct kinetic profiles

• Indirect inflammatory pathways:

  • Atroxlysin-1 generates proinflammatory peptides through the hydrolysis of basement membrane components

  • These fragments can amplify the inflammatory response by activating endogenous signaling pathways

  • Matrigel hydrolysis peptides generated by atroxlysin-1 can independently cause edema (increasing paw size by approximately 30%) and promote leukocyte accumulation (4–5 × 10^6 cells) to the peritoneal cavity

• Recommended experimental models:

  • In vivo models:

    • Paw edema assay in mice for quantifying inflammatory swelling

    • Peritoneal cavity leukocyte recruitment assay for cellular inflammatory response assessment

    • Air pouch model for localized inflammatory reaction studies

  • In vitro models:

    • Macrophage peritoneal adherent cells (MPACs) stimulation assay for cytokine production

    • Basement membrane component hydrolysis assays using Matrigel

    • Transwell migration assays for neutrophil chemotaxis studies

What inflammatory mediators are associated with atroxlysin-1 activity and how can they be quantified?

Atroxlysin-1 triggers the production and release of several inflammatory mediators that can be quantified using specific methodologies:

• Key inflammatory mediators:

  • Cytokines: TNF-α has been identified in the supernatant of cells stimulated with atroxlysin-1

  • Eicosanoids: While not specifically reported for atroxlysin-1, B. atrox venom components induce PGE2 and LTB4 production

  • Other mediators: IL-1β, IL-6 have been associated with PI-class SVMPs from B. atrox venom

• Quantification methodologies:

  • ELISA: For precise quantification of cytokines (TNF-α, IL-1β, IL-6) in cell culture supernatants or tissue homogenates

  • Multiplex cytokine assays: For simultaneous measurement of multiple inflammatory mediators

  • RT-qPCR: To evaluate changes in gene expression of inflammatory mediators

  • Western blotting: For protein-level detection of specific inflammatory pathway components

  • Flow cytometry: For cellular phenotyping and intracellular cytokine staining

• Time-course considerations:

  • Inflammatory mediator production follows specific kinetics after atroxlysin-1 exposure

  • For TNF-α detection in cell culture supernatants, collection at 2, 4, 6, and 18 hours post-stimulation is recommended

  • Leukocyte recruitment shows distinct temporal patterns, with assessment at 1, 4, 24, and 48 hours providing comprehensive profiling

How does recombinant atroxlysin-1 differ functionally from other SVMP classes from Bothrops atrox?

Atroxlysin-1 (a PI-class SVMP) exhibits several functional differences when compared to other SVMP classes from Bothrops atrox:

• Structural comparison:

  • Atroxlysin-1 (PI-class): Contains only the metalloproteinase domain with 204 residues and molecular weight of 23kDa (native) or 30.4kDa (recombinant with tags)

  • PIII-class SVMPs (e.g., Batroxrhagin/BATXH): Contain additional disintegrin-like and cysteine-rich domains, with molecular weights typically between 50-70kDa

• Enzymatic activity profiles:

  • Both PI- and PIII-class SVMPs display proteolytic activity against extracellular matrix components

  • Atroxlysin-1 specifically cleaves the alpha-chains of fibrin(ogen) and hydrolyzes fibronectin and collagens I and IV

  • PIII-class SVMPs generally exhibit broader substrate specificity due to their additional domains

• Inflammatory response induction:

  • Both atroxlysin-1 (ATXL) and PIII-class BATXH induce inflammatory reactions characterized by edema and leukocyte accumulation

  • Comparative data on leukocyte recruitment shows similar potency between the two SVMP classes, though with potentially different kinetics and cell type preferences

• Hemorrhagic potential:

  • Unusually for a PI-class SVMP, atroxlysin-1 demonstrates significant hemorrhagic activity

  • PIII-class SVMPs typically exhibit stronger hemorrhagic effects due to their additional domains that enhance targeting to the basement membrane

• Inhibition profile:

  • Atroxlysin-1 activity is enhanced by Ca²⁺ but inhibited by EDTA, dithiothreitol, excessive Zn²⁺, and alpha2-macroglobulin

  • Inhibition profiles may differ between SVMP classes due to structural differences

What are the methodological considerations when comparing native vs. recombinant atroxlysin-1?

When comparing native and recombinant forms of atroxlysin-1, researchers should address several methodological considerations:

• Structural authentication:

  • SDS-PAGE analysis: While native atroxlysin-1 has a molecular weight of 23kDa, the recombinant form with N-terminal His-tag and C-terminal Myc-tag has a higher theoretical MW of 30.4kDa

  • Mass spectrometry: For precise molecular weight determination and peptide mapping

  • Circular dichroism: To compare secondary structure elements between native and recombinant forms

• Activity assays for functional equivalence:

  • Proteolytic activity: Compare hydrolysis rates against standard substrates (dimethylcasein, fibrinogen, fibronectin)

  • Hemorrhagic activity: Intradermal injection in mice to compare minimum hemorrhagic dose

  • Inflammatory response: Evaluate edema formation and leukocyte recruitment potency

• Potential limitations of recombinant form:

  • Tag interference: The N-terminal 10xHis-tag and C-terminal Myc-tag may affect protein folding or substrate accessibility

  • Post-translational modifications: E. coli expression systems lack glycosylation capabilities

  • Folding issues: Proper incorporation of Zn²⁺ and formation of disulfide bonds may vary between native and recombinant forms

• Standardization approaches:

  • Activity normalization: Determine specific activity (units of activity per mg protein) for both forms

  • Calibration curves: Establish dose-response relationships for key activities

  • Reference standards: Include consistent positive controls across experiments

How can recombinant atroxlysin-1 be utilized in therapeutic antibody development?

Recombinant atroxlysin-1 offers several strategic advantages for therapeutic antibody development:

• Epitope-based vaccine design:

  • The identified linear epitopes (Y22NGNSDKIRRRIHQM36, G55VEIWSNKDLINVQ68, and V9DLFIVVDHGMFMKY23) serve as excellent starting points for rational vaccine design

  • Synthetic peptides encompassing these epitopes, when properly formulated (e.g., in liposomes), can elicit antibodies that neutralize both enzymatic and hemorrhagic activities

  • Combined epitope constructs using glycine spacers (e.g., NGNSDKIRRRIH-GG-GVEIWSNKDLINVQ) have demonstrated enhanced immunogenic potential

• Cross-neutralization potential:

  • Antibodies raised against atroxlysin-1 epitopes may cross-react with homologous SVMPs from related Bothrops species

  • This approach could lead to broader-spectrum antivenoms with improved efficacy against multiple snake species

• Novel therapeutic antibody formats:

  • Recombinant monoclonal antibodies targeting specific neutralizing epitopes

  • Bispecific antibodies designed to simultaneously neutralize multiple toxin classes

  • Single-domain antibodies (nanobodies) with enhanced tissue penetration for improved venom neutralization

• Neutralization assessment techniques:

  • In vitro assays: Enzymatic activity inhibition (e.g., fibrinogenolytic activity)

  • Ex vivo assays: Inhibition of platelet aggregation

  • In vivo assays: Protection against hemorrhagic activity in mouse models

What techniques can be employed to analyze the interaction between recombinant atroxlysin-1 and its protein substrates?

Several advanced techniques can effectively characterize the interactions between recombinant atroxlysin-1 and its protein substrates:

• Biophysical interaction analysis:

  • Surface Plasmon Resonance (SPR): For real-time binding kinetics measurement

  • Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of binding

  • Microscale Thermophoresis (MST): For affinity measurements in solution

• Structural interaction mapping:

  • X-ray crystallography: To obtain atomic resolution structures of enzyme-substrate complexes

  • Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): For mapping interaction surfaces

  • Crosslinking Mass Spectrometry: To identify proximal residues at binding interfaces

• Enzymatic cleavage site identification:

  • LC-MS/MS analysis: For identification of cleavage fragments, as demonstrated with Matrigel hydrolysis peptides

  • N-terminal sequencing: To determine precise cleavage sites

  • Synthetic peptide libraries: For comprehensive cleavage site specificity profiling

• Computational approaches:

  • Molecular docking: To predict binding modes with various substrates

  • Molecular dynamics simulations: To analyze dynamic interactions over time

  • Quantitative structure-activity relationship (QSAR) analysis: To correlate structural features with substrate specificity