Recombinant Vipera berus berus Phospholipase A2

What is the primary structure of Vipera berus berus Phospholipase A2?

VBBPLA2 is a single-chain protein consisting of 122 amino acid residues with 14 cysteine residues in positions characteristic of phospholipase A2 subgroup IIA . The enzyme has a molecular mass of approximately 13,824 Da as determined by MALDI-TOF MS analysis and exhibits a basic isoelectric point (pI) of 9.3, distinguishing it from other viper PLA2s like VLPLA2 from Vipera lebetina which has an acidic pI of 4.3 . The relative enzymatic activity of VBBPLA2 is remarkably high at 1900 μmol/min mg, significantly higher than that of VLPLA2 (882 μmol/min mg) and NNOPLA2 (1200 μmol/min mg) .

How does the genomic organization of VBBPLA2 compare to other viper PLA2s?

The genomic DNA sequences encoding PLA2s from Vipera berus berus contain differences clustered primarily in exons 3 and 5, which potentially alter the biological activities of the enzyme . Single nucleotide differences leading to amino acid substitutions were observed both between genes encoding the same PLA2 and between genes encoding different PLA2s . The distribution and characteristics of PLA2 genes differ according to the species or subspecies, with Vipera berus having a distinct genomic organization compared to Vipera aspis and its subspecies .

What expression systems are most suitable for recombinant VBBPLA2 production?

While the search results do not specifically address expression systems for recombinant VBBPLA2, effective recombinant expression would require a system capable of properly forming the 14 disulfide bonds present in the native enzyme. Mammalian or insect cell expression systems would likely be preferred over bacterial systems due to their superior ability to manage complex disulfide bond formation and post-translational modifications. The choice of expression system should consider:

• Proper folding requirements

• Post-translational modification needs

• Yield requirements

• Downstream purification compatibility

What purification strategies are effective for recombinant VBBPLA2?

Native VBBPLA2 has been purified according to methods described by Križaj et al. . Based on the physicochemical properties of VBBPLA2, an effective purification strategy for the recombinant version would likely include:

• Initial capture using ion exchange chromatography (taking advantage of its basic pI of 9.3)

• Hydrophobic interaction chromatography (as used for native VBBPLA2)

• Verification of purity using SDS-PAGE, isoelectric focusing, and MALDI-TOF MS analysis

• Confirmation of identity through peptide mass fingerprinting after tryptic digestion

What are the major biological activities of VBBPLA2 and how can they be measured?

VBBPLA2 exhibits several distinct biological activities that can be measured through specific assays:

These activities should be evaluated when characterizing recombinant VBBPLA2 to confirm functional similarity to the native enzyme.

How does enzymatic activity correlate with the biological effects of VBBPLA2?

The enzymatic activity of VBBPLA2 is directly related to some, but not all, of its biological effects. Chemical modification of histidine in VBBPLA2 using p-bromophenacylbromide (p-BPB) completely abolished both catalytic activity and inhibitory action on collagen-induced platelet aggregation . This indicates that enzymatic activity is essential for the antiplatelet effects.

These findings reveal that VBBPLA2 has multiple mechanisms of action: some dependent on enzymatic activity and others related to structural features of the protein itself.

What structural elements of VBBPLA2 contribute to its factor Xa binding and anticoagulant effects?

VBBPLA2 belongs to "group M" of snake venom PLA2s that interact with human coagulation factor Xa (FXa) . This interaction occurs with moderate affinity (Kd app 400-830 nM) and inhibits prothrombinase activity with an IC50 of 90-130 nM . The anticoagulant effect is mediated through direct binding to FXa via a non-catalytic, phospholipid-independent mechanism.

Molecular electrostatic potentials calculated at the surface of 3D homology models show a correlation with inhibition of prothrombinase activity, suggesting that electrostatic interactions play a key role in VBBPLA2-FXa binding . Molecular docking simulations further elucidate the specific interaction sites between snake venom PLA2s and FXa .

How can recombinant VBBPLA2 be used to study mechanisms of cancer cell apoptosis?

Recombinant VBBPLA2 represents a valuable tool for studying apoptotic mechanisms in cancer cells. VBBPLA2 induces apoptosis in K-562 leukemic cells at concentrations as low as 0.36 μM, as demonstrated by Annexin-V-FITC staining that reveals phosphatidylserine externalization (a hallmark of early apoptosis) .

Time-course experiments show progression from early apoptosis (Annexin-V positive, PI negative) to late apoptosis/necrosis (Annexin-V and PI positive) with increasing concentration or extended incubation . At higher concentrations (7.23 μM), VBBPLA2 causes extensive cell destruction, but characteristic membrane blebbing remains detectable .

Researchers could use recombinant VBBPLA2 to:

• Identify specific cell death pathways activated in different cancer cell lines

• Compare sensitivity among cancer types

• Develop targeted cancer therapeutics based on VBBPLA2's mechanisms

• Study resistance mechanisms in cancer cells

What are the molecular mechanisms behind VBBPLA2's selective antibacterial activity?

VBBPLA2 demonstrates selective antibacterial activity, completely inhibiting the growth of gram-positive Bacillus subtilis at 36.2 μM while showing no inhibitory effect on gram-negative Escherichia coli . This selectivity presents an interesting research area for understanding bacterial membrane interactions.

The mechanism appears to be independent of VBBPLA2's catalytic activity, suggesting structural features of the protein may be responsible for the antibacterial effect . The selective activity against gram-positive but not gram-negative bacteria suggests the effect may relate to differences in cell wall structure and accessibility.

Researchers could employ recombinant VBBPLA2 to:

• Investigate membrane binding properties

• Identify specific bacterial targets

• Develop novel antimicrobial strategies based on VBBPLA2's mechanism

• Study bacterial resistance mechanisms

How do PLA2 inhibitors like Varespladib interact with VBBPLA2, and what are the experimental considerations?

Varespladib is an effective inhibitor of snake phospholipase A2 enzymes, including those from Vipera berus . It specifically targets PLA2 activity, making it particularly effective against venoms where PLA2 is a major component—such as V. berus venom, which contains approximately 60% PLA2 by dry mass .

In experimental models, Varespladib has shown protective effects against Viperid venoms, including V. berus venom, by suppressing PLA2 activity . This protection includes increased survival rates and delayed symptom onset in envenomated mice .

When designing experiments with Varespladib and VBBPLA2:

• Consider solubility issues—interaction between DMSO and Varespladib can be complex and affect pharmacological dynamics

• Evaluate multiple administration timepoints, as repetitive administration has shown improved protection in some studies

• Monitor for partial inhibition effects that may prolong intoxication rather than eliminate it

• Consider combination treatments for complex venoms with multiple toxic components

What approaches can be used to develop monoclonal antibodies against recombinant VBBPLA2?

While not specifically addressed in the search results for VBBPLA2, search result discusses the use of recombinant antibody fragments against phospholipase A2 from Crotalus durissus terrificus. Similar approaches could be applied to VBBPLA2:

• Antibody library screening: Non-immune human single-chain fragment variable libraries could be screened against recombinant VBBPLA2

• Selection of specific binders: Identify antibody fragments capable of inhibiting phospholipase activity in vitro

• Functional assessment: Test selected antibodies for their ability to neutralize specific biological activities (anticoagulant, cytotoxic, etc.)

• Epitope mapping: Determine the binding sites of effective antibodies on VBBPLA2

Such antibodies could serve as research tools for studying VBBPLA2 function or potentially as therapeutic agents for V. berus envenomation.

How does VBBPLA2 compare structurally and functionally with PLA2s from other Viperidae species?

Comparative analysis of VBBPLA2 with other viper PLA2s reveals important differences in structure and function:

VBBPLA2 has higher enzymatic activity than both VLPLA2 and NNOPLA2, and greater potency in inhibiting collagen-induced platelet aggregation . In terms of FXa binding, VBBPLA2 falls into "group M" with moderate affinity (Kd app 400-830 nM), whereas some other viper PLA2s show significantly higher affinity (groups VS and S) .

Unlike PLA2s from Vipera ammodytes, VBBPLA2 lacks neurotoxic phospholipases A2 known as Atxs . This likely explains the different clinical manifestations of envenomation between these species.

What genetic variations exist in VBBPLA2 across different V. berus subspecies and populations?

Genetic analysis of V. berus reveals variations in PLA2 genes across different subspecies and populations. Genes encoding neurotoxins from V. berus berus of central France have been characterized, including genes encoding ammodytins I1 and I2, which were previously described in Vipera ammodytes ammodytes .

Three different ammodytin I1 gene sequences have been identified:

• One specific to V. berus berus

• One shared among V. aspis aspis, V. aspis zinnikeri, and neurotoxic V. aspis aspis

• One found in neurotoxic V. aspis aspis (identical to the V. ammodytes ammodytes ammodytin I1 gene)

These genetic variations likely contribute to differences in venom composition and toxicity across different geographic populations of V. berus. Studying these variations in recombinant expression systems could help understand the evolutionary and ecological significance of venom diversity.

What techniques can be used to study the interaction between recombinant VBBPLA2 and phospholipid membranes?

Several advanced techniques could be employed to study VBBPLA2-membrane interactions:

• Surface Plasmon Resonance (SPR): Used in the search results to measure protein-protein interactions , SPR could be adapted to study VBBPLA2 binding to immobilized lipid layers

• Fluorescence-based assays: Monitor changes in membrane integrity or fluidity upon VBBPLA2 binding

• Liposome leakage assays: Measure release of encapsulated fluorescent markers from liposomes of varying composition when exposed to VBBPLA2

• Atomic Force Microscopy: Visualize structural changes in supported lipid bilayers upon VBBPLA2 treatment

• Cryo-electron microscopy: Directly observe VBBPLA2-induced changes in membrane structure

These techniques would provide insights into how recombinant VBBPLA2 interacts with different membrane compositions, potentially explaining its selective effects on different cell types.

What advanced mutagenesis approaches can be used to enhance specific activities of recombinant VBBPLA2?

Based on the structural and functional information available, several mutagenesis approaches could be employed:

• Targeted mutations in exons 3 and 5: These regions contain single nucleotide differences that potentially alter biological activities of PLA2

• Structure-guided mutagenesis: Using molecular docking simulations with FXa to identify critical residues for anticoagulant activity and selectively modify them

• Charge-altering mutations: Modifying surface electrostatic potentials that correlate with prothrombinase inhibition

• Domain swapping: Creating chimeric proteins by exchanging domains between VBBPLA2 and other PLA2s with desirable properties

• Disulfide engineering: Modifying the pattern of disulfide bonds to alter structural stability or flexibility

These approaches could be used to create recombinant VBBPLA2 variants with enhanced specific activities (anticoagulant, antibacterial, anticancer) while reducing undesired effects.