Recombinant Viola biflora Cyclotide vibi-D

What is Viola biflora cyclotide vibi-D and how was it discovered?

Viola biflora cyclotide vibi-D is a head-to-tail cyclic peptide isolated from Viola biflora L. (Arctic Yellow-violet, Two-flower violet), a species native to alpine regions of Europe and northern parts of Asia and America. It belongs to the Möbius subfamily of cyclotides, characterized by a cis-proline in loop 5. Vibi-D was discovered through a combined approach of protein isolation and cDNA library screening of V. biflora, as part of a study that identified eleven cyclotides (vibi A-K) from this plant species .

The discovery process involved:

• Initial profiling of plant extracts using liquid chromatography-mass spectrometry (LC-MS)

• Isolation of cyclotide proteins from aqueous plant extracts

• Sequence determination using tandem mass spectrometry

• Parallel screening of a cDNA library using a degenerate primer targeting a conserved AAFALPA motif in the cyclotide precursor ER signal sequence

How does the structure of vibi-D differ from other cyclotides in the Viola genus?

Vibi-D belongs to the Möbius subfamily of cyclotides, which is distinguished by a characteristic cis-proline in loop 5 that creates a twist in the peptide backbone. This contrasts with bracelet cyclotides that lack this feature. The structural characteristics of vibi-D include:

• A circular peptide backbone combined with a cystine knot, forming the cyclic cystine knot (CCK) motif

• Six conserved cysteine residues forming three disulfide bonds

• Notably, vibi-D contains an arginine (Arg) in loop 2, which is believed to be a major contributing factor to its lower cytotoxicity compared to bracelet cyclotides

• Contains a Tyr residue in place of the conserved Trp that is typically found in Möbius cyclotides

The structural differences between vibi-D and other cyclotides directly influence their biological activities and potential research applications.

What are the biological activities associated with vibi-D compared to other V. biflora cyclotides?

Research has demonstrated that vibi-D exhibits distinctive biological activities compared to other cyclotides from the same plant. In cytotoxicity studies using the fluorometric microculture cytotoxicity assay (FMCA) with human lymphoma cell line U-937 GTB, vibi-D showed markedly lower cytotoxicity compared to bracelet cyclotides from the same plant .

Comparative cytotoxicity data for V. biflora cyclotides:

• Bracelet cyclotides (vibi E, G, and H): IC50 values ranging between 0.96 and 5.0 μM

• Möbius cyclotide (vibi-D): Significantly lower cytotoxicity

This reduced cytotoxicity is attributed primarily to the presence of an Arg residue in loop 2, while the Trp/Tyr substitution is thought to play a minor role in the decreased activity. This structural-activity relationship provides valuable insights for researchers designing cyclotide-based therapeutic agents with optimized cytotoxicity profiles .

What expression systems are most effective for recombinant production of vibi-D?

For recombinant production of cyclotides like vibi-D, several expression systems have been investigated, though the optimal system depends on research objectives. Based on cyclotide research approaches, the following systems are recommended:

• Bacterial expression systems (E. coli): Suitable for initial screening and high-yield production, though proper folding of disulfide bonds may require optimization. Fusion partners such as thioredoxin or SUMO are often necessary to improve solubility and facilitate correct folding.

• Plant-based expression systems: These leverage native cyclotide processing machinery. For vibi-D, expression in Viola species would be ideal as they naturally contain the enzymes necessary for cyclization, such as asparaginyl endopeptidases (AEPs) .

• Cell-free expression systems: Useful for rapid production and screening of cyclotide variants without cellular toxicity concerns.

When designing expression constructs, researchers should incorporate:

• The complete cyclotide precursor sequence including the N-terminal pro-region

• Appropriate processing signals for cyclization

• Consideration of the cleavage sites identified in natural cyclotide processing (typically after an Asn/Asp residue at the C-terminal processing site and N-terminal of a Gly residue)

What are the critical factors in ensuring proper cyclization of recombinant vibi-D?

Proper cyclization of recombinant vibi-D requires careful consideration of several biochemical factors:

• Presence of cyclization enzymes: Asparaginyl endopeptidases (AEPs) are critical for cyclotide processing. The V. betonicifolia AEP1 (VbAEP1) shows high sequence similarity (>93%) to other verified ligases from Viola species . For recombinant expression, co-expression with appropriate AEPs or in vitro processing with purified AEPs is recommended.

• pH optimization: Verified ligases from Viola yedoensis (VyPAL1 and VyPAL2) have shown high in vitro backbone cyclization efficiency at pH 5-8 . Similar conditions should be applied for vibi-D cyclization.

• Disulfide bond formation: Proper oxidative folding requires protein disulfide isomerases (PDIs). Two PDIs (VbPDI1-2) have been identified in Viola species with high sequence homology (>74%) to PDIs from other cyclotide-producing plants .

• Construct design: The precursor sequence must include the essential elements for recognition by processing enzymes. The conserved Asn/Asp residue at which cyclization occurs (highlighted in red in cyclotide structures) must be preserved .

Verification of successful cyclization typically requires a combination of mass spectrometry techniques to confirm the absence of linear precursors and presence of the cyclic backbone.

What analytical techniques are most effective for confirming the structure of recombinant vibi-D?

Multiple complementary analytical techniques are recommended for comprehensive structural characterization of recombinant vibi-D:

• Liquid Chromatography-Mass Spectrometry (LC-MS): Essential for initial verification of molecular weight and purity. The choice of ion pairing acid in RP-HPLC can significantly affect retention times and selectivity for cyclotides, with different acids (HCO₂H vs. TFA) providing varied separation patterns .

• Tandem Mass Spectrometry (MS/MS): Critical for sequence confirmation and verification of the cyclic backbone. Fragmentation patterns must be carefully analyzed to confirm the head-to-tail cyclization.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Provides detailed information about the three-dimensional structure, including the characteristic cis-proline in loop 5 that defines Möbius cyclotides.

• Circular Dichroism (CD): Useful for comparing the secondary structure elements of recombinant vs. native vibi-D.

• Enzymatic digestion followed by MS analysis: Selective cleavage followed by MS analysis can provide information about disulfide connectivity and confirm the cyclic cystine knot (CCK) motif.

When analyzing MS data for cyclotides, researchers should account for the cyclotide's unique cyclic nature, which affects fragmentation patterns during MS/MS analysis. Comparison with analytical standards of naturally derived vibi-D is recommended for method validation.

How can researchers distinguish between correctly folded and misfolded recombinant vibi-D?

Distinguishing correctly folded vibi-D from misfolded variants requires a multi-faceted analytical approach:

• Retention time comparison: Correctly folded cyclotides show characteristic retention times in RP-HPLC that differ from misfolded variants. The retention behavior varies based on cyclotide subfamily and net charge - bracelet cyclotides with a net charge of +2 (like vibi H) are most affected by changes in ion pairing acids, while Möbius cyclotides with zero net charge (like vibi C) are least affected .

• Disulfide bond analysis: The correct disulfide connectivity (CysI-CysIV, CysII-CysV, and CysIII-CysVI) is essential for proper folding. This can be verified through partial reduction and alkylation followed by MS analysis.

• Biological activity assays: Correctly folded vibi-D will display characteristic biological activities. The cytotoxicity assay using human lymphoma cell line U-937 GTB has been established for vibi-D and can serve as a functional test .

• Thermal and chemical stability tests: Correctly folded cyclotides exhibit exceptional stability to heat and chemical denaturants due to their cyclic cystine knot motif. Stability tests can help distinguish properly folded structures.

• NMR structural analysis: 2D NMR experiments can confirm the presence of the characteristic structural elements of Möbius cyclotides, including the cis-proline in loop 5.

What cell-based assays are most appropriate for evaluating recombinant vibi-D bioactivity?

Based on established research with vibi-D and related cyclotides, the following cell-based assays are recommended:

• Fluorometric microculture cytotoxicity assay (FMCA): This has been successfully used to evaluate vibi-D's cytotoxicity against human lymphoma cell line U-937 GTB and provides quantitative IC50 values .

• Hemolysis assays: Important for evaluating membrane-disrupting properties, which is a common mechanism of action for many cyclotides.

• Antimicrobial activity assays: Broth microdilution assays against gram-positive and gram-negative bacteria, as cyclotides are believed to be part of plant defense systems.

• Cell membrane permeabilization assays: Using fluorescent dyes to monitor membrane integrity after cyclotide treatment.

• Cell cycle analysis: Flow cytometry-based analysis to determine if vibi-D affects specific phases of the cell cycle.

When conducting these assays, researchers should:

• Include appropriate controls, including other cyclotides with known activities

• Consider the influence of the Arg residue in loop 2 of vibi-D on its bioactivity

• Account for the lower cytotoxicity of vibi-D compared to bracelet cyclotides when designing dose-response experiments

• Validate findings across multiple cell lines to determine specificity

How can structural modifications of vibi-D be designed to enhance specific bioactivities?

Strategic structural modifications of vibi-D can be designed based on structure-activity relationships observed in cyclotide research:

When designing modifications, researchers should consider:

• The conservation of residues critical for cyclization (typically Asn/Asp in loop 6)

• Maintenance of the six conserved cysteine residues that form the characteristic disulfide bonds

• The impact of modifications on processing by cyclization enzymes if using enzymatic cyclization methods

• The variable loop sizes observed in natural cyclotides, which provide insight into tolerable modifications (e.g., vibe 12 contains 7 residues in loop 3, the longest loop 3 among cyclotides identified in V. betonicifolia)

What is the relationship between vibi-D genetic expression and the plant's ecological adaptations?

The relationship between vibi-D expression and ecological adaptation remains an emerging research area, but several key considerations can guide investigations:

• Defensive role in plant biology: Cyclotides are believed to function as part of plant defense systems. The diversity of cyclotides in Viola biflora (11 identified cyclotides, vibi A-K) suggests they may provide protection against different pests or pathogens in the alpine environment where this plant grows .

• Species-specific expression patterns: While some cyclotides are common across multiple Viola species (e.g., varv A, vitri A, cycloviolacin O2, and O9), others like vibi-D appear to be specific to V. biflora. This indicates a "signature set" of cyclotides that may be adapted to species-specific ecological pressures .

• Transcript abundance vs. protein levels: There is an apparent discrepancy between cyclotide detection at the mRNA and protein levels. In V. biflora, only one of the isolated proteins could be identified as a cDNA clone . This suggests complex post-transcriptional regulation that may be responsive to environmental factors.

• Tissue-specific expression: For comprehensive study of vibi-D expression, sampling should include various plant tissues (leaves, stems, seeds, and roots) as was done in the original transcriptome sequencing of Viola betonicifolia .

Future research could utilize quantitative RT-PCR to monitor vibi-D expression under different environmental stressors to better understand its ecological role.

How do the enzymatic processing pathways for vibi-D differ from other cyclotide subfamilies?

The enzymatic processing of cyclotides involves several specialized enzymes and follows specific pathways that may differ between cyclotide subfamilies:

• Asparaginyl endopeptidases (AEPs): These are crucial for cyclotide processing and can have both proteolytic and ligation activities. In Viola betonicifolia, four main AEPs (VbAEP1-4) were identified, including a peptide asparaginyl ligase (PAL) potentially involved in cyclotide backbone cyclization .

• Processing site preferences: For vibi-D and other cyclotides from the Möbius subfamily, the precursors are likely cleaved after an Asn/Asp residue at the C-terminal processing site and N-terminal of a Gly residue at the N-terminal processing site .

• Protein disulfide isomerases (PDIs): Two PDIs (VbPDI1-2) identified in Viola species are likely involved in cyclotide oxidative folding. These show high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs .

• Subfamily-specific processing differences: While the basic processing machinery is similar, subtle differences in enzyme recognition sites between Möbius and bracelet cyclotides may exist. The presence of acyclotides (linear cyclotide-like peptides that lack the conserved Asn/Asp vital for cyclization) in the same plants provides clues about processing requirements .

Key differences in enzymatic processing between subfamilies remain an important area for future research, particularly in understanding how different cyclization enzymes recognize their substrates and how this might be exploited in recombinant expression systems.

What are the main challenges in achieving high yields of correctly folded recombinant vibi-D?

Researchers face several challenges when producing recombinant vibi-D:

• Ensuring proper cyclization: The cyclization step requires specific enzymes (AEPs with ligase activity) that recognize the appropriate cleavage/ligation sites. Co-expression with enzymes like VbAEP1, which shows high homology to known cyclizing enzymes, may be necessary .

• Disulfide bond formation: The correct formation of three disulfide bonds in the specific cyclic cystine knot arrangement is challenging. Expression in systems with oxidizing environments or co-expression with PDIs like VbPDI1-2 can facilitate proper folding .

• Preventing proteolytic degradation: Cyclotides are naturally resistant to proteolysis, but their precursors and improperly folded intermediates may be vulnerable during expression.

• Purification challenges: The hydrophobic nature of many cyclotides can lead to aggregation and loss during purification. The choice of ion pairing acid in RP-HPLC significantly affects cyclotide retention and separation efficiency, with different acids providing varied selectivity patterns for different cyclotide subfamilies .

• Verification of correct structure: Confirming the cyclic structure and correct disulfide bond arrangement requires sophisticated analytical techniques. MS/MS analysis must be carefully interpreted, as the fragmentation patterns of cyclic peptides differ from linear peptides.

Strategies to overcome these challenges include:

• Optimization of expression conditions (temperature, induction time, media composition)

• Exploration of different fusion partners to enhance solubility

• Development of specialized purification protocols that account for the unique properties of cyclotides

• Comprehensive analytical characterization to confirm correct structure formation

How can researchers distinguish between naturally occurring and recombinant vibi-D in analytical studies?

Distinguishing between natural and recombinant vibi-D requires careful analytical approaches:

• Isotope labeling: Incorporating stable isotopes (¹⁵N, ¹³C) during recombinant expression creates a mass shift that is easily detectable by MS without altering biological properties.

• Post-translational modifications: Natural cyclotides may contain PTMs not present in recombinant versions. Detailed mass spectrometric analysis can identify these differences.

• Sequence tags or mutations: Introduction of conservative amino acid substitutions or tags that minimally impact structure but allow discrimination by MS.

• Comparative chromatographic profiles: Subtle differences in retention time may exist between natural and recombinant cyclotides due to minor conformational variations. Using both formic acid and TFA as ion pairing acids in parallel RP-HPLC runs can reveal these differences, as they affect cyclotide retention patterns differently .

• NMR fingerprinting: Solution NMR can detect subtle structural differences that may not be apparent in MS analysis.

When conducting comparative studies, researchers should maintain consistent analytical conditions and include appropriate controls of both natural and recombinant cyclotides analyzed in parallel to minimize technical variation.

What emerging technologies could revolutionize recombinant vibi-D research?

Several cutting-edge technologies hold promise for advancing recombinant vibi-D research:

• CRISPR-based cyclotide engineering: Precision editing of cyclotide genes in plant expression systems to create novel variants with enhanced properties.

• Machine learning approaches: AI-driven prediction of cyclotide structure-activity relationships to guide rational design of vibi-D variants with optimized biological activities.

• Microfluidic cyclotide synthesis and screening: High-throughput platforms for rapid production and testing of cyclotide variants.

• Computational molecular dynamics simulations: Advanced modeling of cyclotide-membrane interactions to better understand mechanisms of action and guide optimization.

• Advanced transcriptomics: Building on the success of transcriptome de novo sequencing for cyclotide discovery , integration with proteomics data can address the observed discrepancies between cyclotide detection at mRNA and protein levels .

• In vitro enzymatic cyclization systems: Development of cell-free systems using purified AEPs with ligase activity, based on enzymes like VbAEP1 that show high sequence similarity to verified cyclizing enzymes .

These technologies could help address current limitations in cyclotide research, such as the apparent discrepancy between cyclotide detection at mRNA and protein levels observed in V. biflora, where only one of the isolated proteins could be identified as a cDNA clone .

How might vibi-D and related cyclotides inform the development of novel peptide-based therapeutics?

Vibi-D's unique properties offer valuable insights for therapeutic peptide development:

• Stability template: The exceptional stability of cyclotides due to their cyclic cystine knot motif makes them attractive scaffolds for drug development. Vibi-D's Möbius subfamily characteristics can be particularly valuable for applications requiring specific conformational constraints .

• Tunable cytotoxicity: The observed lower cytotoxicity of vibi-D compared to bracelet cyclotides (attributed primarily to the Arg in loop 2) provides a model for designing cyclotide-based therapeutics with controlled cytotoxicity profiles .

• Grafting applications: The variable loop sizes observed in natural cyclotides (e.g., vibe 12 with 7 residues in loop 3) demonstrate the scaffold's adaptability for grafting bioactive peptide sequences .

• Hybrid design strategies: The existence of hybrid cyclotides that combine elements of Möbius and bracelet subfamilies suggests possibilities for creating chimeric therapeutics with custom properties .

• Processing enzyme engineering: Insights from cyclotide processing enzymes like VbAEP1 can inform the development of improved enzymatic methods for producing cyclic peptide therapeutics .

The high variability of cyclotide sequences in V. betonicifolia showcases the cyclotide structure as an adaptable scaffold and highlights their importance as a combinatorial library with implications for both plant defense and therapeutic applications .

What is the most effective protocol for extracting and purifying native vibi-D for comparative studies?

Based on established cyclotide research methodologies, the following protocol is recommended for native vibi-D extraction and purification:

• Initial extraction:

  • Collect fresh plant material from Viola biflora

  • Extract with MeOH/H₂O mixture

  • Remove chlorophyll and other lipophilic compounds

  • Prepare an aqueous extract for further processing

• Initial profiling:

  • Analyze extract by LC-MS to obtain a fingerprint of protein expression

  • Identify peaks corresponding to potential cyclotides based on characteristic mass ranges (2,500-4,000 Da)

• Chromatographic separation:

  • Use reversed-phase high-performance liquid chromatography (RP-HPLC)

  • Test both formic acid and trifluoroacetic acid as ion pairing agents, as they significantly affect cyclotide retention and selectivity

  • Note that bracelet cyclotides with net charge +2 (like vibi H) are most affected by the choice of ion pairing acid, while Möbius cyclotides with zero net charge (like vibi C) are least affected

• Mass spectrometric verification:

  • Confirm the identity of vibi-D using high-resolution mass spectrometry

  • Verify the cyclic nature through MS/MS sequencing

• Storage considerations:

  • Lyophilize purified vibi-D for long-term storage

  • For solution storage, use acidified aqueous solutions (pH 2-3) to minimize degradation

This protocol enables isolation of native vibi-D for direct comparison with recombinant versions, allowing researchers to verify structural and functional equivalence.

What experimental design best assesses the structure-activity relationship of vibi-D variants?

A comprehensive experimental design for studying structure-activity relationships of vibi-D variants should include:

• Systematic sequence variations:

  • Focus on the Arg in loop 2, which has been identified as a major contributor to vibi-D's lower cytotoxicity

  • Explore the impact of Trp/Tyr substitutions, which appear to play a minor role in activity

  • Generate variants with modifications in each loop while maintaining the core cyclic cystine knot structure

• Structural characterization:

  • Comprehensive analysis using LC-MS, MS/MS, and NMR to confirm correct folding

  • CD spectroscopy to assess secondary structure elements

  • Thermal and chemical stability assessments

• Activity assays:

  • Use the fluorometric microculture cytotoxicity assay (FMCA) with human lymphoma cell line U-937 GTB as a primary screening method, as this has been established for vibi-D

  • Expand testing to multiple cell lines to determine specificity

  • Include membrane interaction studies to understand mechanism of action

• Comparative analysis:

  • Include control bracelet cyclotides (like vibi E, G, and H) with known IC₅₀ values (0.96-5.0 μM) for comparison

  • Establish dose-response curves for all variants

  • Perform statistical analysis to quantify structure-activity relationships

• Advanced biophysical characterization:

  • Surface plasmon resonance to measure binding affinities to potential targets

  • Isothermal titration calorimetry to determine thermodynamic parameters of interactions

  • Molecular dynamics simulations to understand structural dynamics