Recombinant Viola odorata Cycloviolacin-O16

What is Cycloviolacin-O16 and how does it relate to other cyclotides from Viola odorata?

Cycloviolacin-O16 belongs to the bracelet subfamily of cyclotides found in Viola odorata. This plant species abundantly produces over 30 known, unique cyclotide sequences and may harbor up to 166 cyclotide species as indicated by mass shift analysis . Cycloviolacins are characterized by their cyclic backbone and conserved cysteine residues that form a cystine knot, providing exceptional structural stability. Structurally, Cycloviolacin-O16 shares the hallmark features of bracelet cyclotides similar to other cycloviolacins like O2, O3, and O8, which have demonstrated significant bioactivities in research settings .

What are the typical extraction methods for native cyclotides from Viola odorata?

Native cyclotides are typically extracted from plant tissue using a dichloromethane/methanol (1:1 v/v) mixture, with incubation overnight at room temperature (22°C). Following extraction, the mixture is partitioned with water, and the water/methanol layer is concentrated via rotary evaporation prior to freeze-drying. The dried product is then re-dissolved in water and purified on a preparative reverse phase (RP) C18 column using a gradient of water/trifluoroacetic acid and acetonitrile/water/trifluoroacetic acid . Cyclotides are subsequently identified according to their characteristic HPLC retention times and masses determined via mass spectrometry. This extraction method has been successfully employed for cycloviolacins O13, O14, and O24 .

Why would researchers pursue recombinant production of Cycloviolacin-O16?

Recombinant production offers several advantages over natural extraction:

• Scalability: Enables production of larger quantities than available from natural sources.

• Consistency: Eliminates batch-to-batch variation inherent in plant-derived extracts.

• Structural modifications: Facilitates site-directed mutagenesis for structure-activity relationship studies.

• Isotopic labeling: Allows incorporation of isotopic labels for NMR and other structural studies.

• Purity: Reduces contamination with other plant-derived cyclotides that may have similar physicochemical properties.

• Sustainability: Provides an environmentally sustainable alternative to harvesting large quantities of plants.

What bioactivities have been documented for Viola odorata cyclotides?

Viola odorata cyclotides exhibit diverse bioactivities relevant to therapeutic development:

• Anticancer activity: Cycloviolacin O8 (cyO8) demonstrates micromolar cytotoxicity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines .

• Antifungal activity: CyO8 shows activity against the agricultural pathogen Fusarium graminearum .

• Anti-HIV properties: Several cycloviolacins, including O13, O14, and O24, demonstrate inhibitory activity against HIV infection .

• Chemosensitization: Cyclotides can sensitize cancer cells to conventional chemotherapeutics, as demonstrated with Kalata B1 enhancing temozolomide toxicity to glioblastoma cells .

• Antibacterial potential: Computational studies suggest potential inhibitory activity against Streptococcus pneumoniae neuraminidase .

What expression systems are most suitable for recombinant production of cyclotides like Cycloviolacin-O16?

Several expression systems can be used for recombinant cyclotide production, each with distinct advantages:

• E. coli expression systems:

  • Advantages: High yields, ease of genetic manipulation, cost-effectiveness

  • Challenges: Proper disulfide bond formation may require oxidative refolding or specialized strains

  • Methodology: Expression typically employs fusion partners (MBP, SUMO, thioredoxin) to enhance solubility, followed by chemical or enzymatic cleavage and cyclization

• Yeast expression systems (P. pastoris, S. cerevisiae):

  • Advantages: Better disulfide bond formation, potential for direct secretion

  • Methodology: Codon-optimized constructs with appropriate signal sequences

• Insect cell expression:

  • Advantages: Superior post-translational modifications

  • Applications: Particularly useful when studying complex cyclotide-protein interactions

• Plant-based expression:

  • Advantages: Native-like processing, potential for large-scale production

  • Methodology: Transient expression or stable transformation using Agrobacterium

What strategies can ensure proper cyclization of recombinant Cycloviolacin-O16?

Achieving proper backbone cyclization represents a significant challenge in recombinant cyclotide production. Effective strategies include:

• Intein-mediated cyclization:

  • Methodology: Fusion of target sequence between an N-terminal intein and C-terminal intein/chitin binding domain

  • Process: Controlled thiol-induced cleavage triggers intramolecular ligation

• Sortase-mediated ligation:

  • Methodology: Engineering recognition sequences (LPXTG and GGG) at termini

  • Process: Enzymatic transpeptidation by sortase A forms the cyclic backbone

• Native chemical ligation:

  • Methodology: Chemical synthesis of linear precursor with N-terminal cysteine and C-terminal thioester

  • Process: Chemoselective ligation followed by disulfide bond formation

• Recombinant expression with subsequent enzymatic cyclization:

  • Methodology: Expression of linear precursor with recognition sequences for proteases like trypsin

  • Process: Protease-mediated cyclization under dilute conditions

What analytical methods are most effective for confirming the correct structure of recombinant Cycloviolacin-O16?

Multiple complementary techniques are essential for comprehensive characterization:

• Mass Spectrometry:

  • MALDI-TOF MS: For accurate molecular weight determination

  • LC-MS/MS: For sequence verification and post-translational modification analysis

  • Orbitrap LC-MS: For high-resolution peptide sequencing, as demonstrated for Kalata B1

• NMR Spectroscopy:

  • 2D NMR (TOCSY, NOESY, HSQC): For structural confirmation and disulfide connectivity

  • 3D solution structure determination: Essential for confirming proper folding

• Circular Dichroism (CD) Spectroscopy:

  • For secondary structure analysis and thermal stability assessment

• Disulfide Bond Mapping:

  • Methodology: Partial reduction, alkylation, and MS analysis

  • Application: Verification of the characteristic cystine knot arrangement

• Chromatographic Analysis:

  • RP-HPLC: For purity assessment and comparison with native standards

  • SCX fractionation: As used for V. odorata cyclotide isolation

How can researchers verify the disulfide bond pattern in recombinant Cycloviolacin-O16?

The conserved disulfide bond pattern is critical for cyclotide stability and function. Verification methods include:

• Reduction and alkylation: Treatment with reducing agents (TCEP, DTT) followed by alkylation with iodoacetamide produces mass shifts of 348.16 ± 0.05 Da, consistent with modification of three disulfide bonds .

• Enzymatic digestion and MS analysis: Digestion with specific proteases followed by MS analysis of fragments can confirm disulfide connectivity.

• Partial reduction strategies: Controlled reduction conditions to selectively reduce individual disulfide bonds, followed by differential alkylation and MS analysis.

• NMR spectroscopy: NOE constraints can provide structural evidence of disulfide connectivity.

What assays are appropriate for evaluating the bioactivity of recombinant Cycloviolacin-O16?

Based on the known activities of related cyclotides, several bioassay types are recommended:

• Cancer cell cytotoxicity assays:

  • Methodology: MTT or WST-1 assays using cell lines like MDA-MB-231 (breast), PC-3 (prostate), and OVCAR-3 (ovarian) cancer cells

  • Metrics: Determination of IC50 values (typically in the micromolar range for cyclotides)

  • Controls: Comparison with established cyclotides like cycloviolacin O2

• Antimicrobial activity assays:

  • Antifungal: Broth microdilution assays against Fusarium graminearum

  • Antibacterial: Growth inhibition assays against Streptococcus pneumoniae

• Chemosensitization assays:

  • Methodology: Co-exposure treatments with FDA-approved drugs (e.g., temozolomide for glioblastoma cells)

  • Analysis: Dose-response curves and combination index calculations

• Membrane interaction studies:

  • Methodology: Membrane leakage assays using artificial liposomes

  • Analysis: Fluorescence spectroscopy to monitor calcein release

• Stability testing:

  • Serum stability assays: Incubation in human serum followed by HPLC analysis

  • Thermal stability: CD spectroscopy under varying temperature conditions

How does the mechanism of action of cyclotides like Cycloviolacin-O16 differ from conventional therapeutic peptides?

Cyclotides exhibit unique mechanistic properties:

• Membrane interactions: Many cyclotides, particularly Möbius subfamilies, disrupt cellular membranes through formation of pores or carpet-like mechanisms.

• Resistance to degradation: The cyclic backbone and cystine knot confer exceptional resistance to proteolytic degradation, as demonstrated by Kalata B1's stability in human serum .

• Multiple modes of action: Cyclotides may combine membrane disruption with specific protein targeting, unlike most conventional therapeutic peptides that typically have single mechanisms.

• Structure-activity relationships: Different cyclotide subfamilies (Möbius vs. bracelet) exhibit different bioactivity profiles. For example, Möbius cyclotides have comparable inhibitory activity against HIV infection to bracelet cyclotides but are generally less cytotoxic .

How can structure-activity relationship studies guide the development of Cycloviolacin-O16 analogs?

Structure-activity relationship (SAR) studies can systematically map the contribution of specific residues to bioactivity:

• Alanine scanning mutagenesis:

  • Methodology: Sequential replacement of non-cysteine residues with alanine

  • Analysis: Comparative bioactivity testing of mutants to identify critical residues

• Loop grafting:

  • Methodology: Replacement of entire loops between cysteine residues with sequences of interest

  • Application: Development of cyclotides with novel targeting properties

• Conservative substitutions:

  • Strategy: Replacement with physicochemically similar amino acids

  • Purpose: Fine-tuning activity while maintaining structural integrity

• Computational modeling:

  • Methodology: Homology modeling using established cyclotide structures as templates, similar to the approach used for cycloviolacins Y4, Y5, and O13

  • Tools: Molecular docking against potential targets, as demonstrated in the screening of V. odorata cyclotides against S. pneumoniae neuraminidase

What computational approaches can predict interactions between Cycloviolacin-O16 and potential targets?

Computational methods offer valuable insights into cyclotide-target interactions:

• Molecular docking:

  • Software: AutoDock, HADDOCK, Glide

  • Application: Predict binding modes and affinities to protein targets

  • Example: Virtual screening of V. odorata cyclotides against neuraminidase protein of Streptococcus pneumoniae

• Molecular dynamics simulations:

  • Purpose: Evaluate stability of protein-cyclotide complexes over time

  • Analysis: RMSD, hydrogen bond networks, binding energy calculations

  • Example: MD simulations confirmed stability of cyclotide-neuraminidase complexes

• Structure prediction:

  • Tools: AlphaFold server for predicting cyclotide structures

  • Application: Generate reliable 3D models for cyclotides lacking experimental structures

• Homology modeling:

  • Software: Modeller 8v1

  • Application: Generate structural models based on high-resolution NMR structures of related cyclotides

  • Example: Models of cycloviolacins derived from NMR structure of cycloviolacin O1

How can isotopic labeling of recombinant Cycloviolacin-O16 facilitate structural and mechanistic studies?

Isotopic labeling offers powerful advantages for advanced structural and functional characterization:

• 15N and 13C labeling for NMR studies:

  • Methodology: Expression in minimal media with 15NH4Cl and/or 13C-glucose as sole nitrogen and carbon sources

  • Applications: High-resolution 3D structure determination, dynamics studies, and protein-ligand interactions

• Selective amino acid labeling:

  • Methodology: Supplementation of auxotrophic expression hosts with labeled amino acids

  • Applications: Simplified NMR spectra focusing on specific residues of interest

• Deuteration:

  • Methodology: Expression in D2O-based media

  • Applications: Improved signal-to-noise in NMR experiments, particularly valuable for larger cyclotide-protein complexes

• Fluorescent labeling:

  • Methodology: Site-specific incorporation of non-natural amino acids with reactive handles

  • Applications: Real-time imaging of cellular uptake and localization

What statistical approaches are most appropriate for analyzing bioactivity data of Cycloviolacin-O16?

Robust statistical methods ensure reliable interpretation of bioactivity data:

• Dose-response curve analysis:

  • Models: Four-parameter logistic (4PL) regression for IC50 determination

  • Software: GraphPad Prism, R (drc package)

  • Metrics: IC50 values with 95% confidence intervals

• Combination studies analysis:

  • Methods: Combination index (CI) calculations using Chou-Talalay method

  • Interpretation: CI < 1 (synergism), CI = 1 (additivity), CI > 1 (antagonism)

  • Application: Evaluating chemosensitization potential, similar to the synergistic effect observed between Kalata B1 and temozolomide

• Statistical significance testing:

  • Methods: ANOVA with post-hoc tests (Tukey's, Dunnett's) for multiple comparisons

  • Considerations: Control for multiple comparisons to avoid false positives

• Reproducibility metrics:

  • Methods: Coefficient of variation (CV) calculations across technical and biological replicates

  • Standards: CV < 15% for acceptable reproducibility in bioassays

How can researchers address discrepancies between natural and recombinant Cycloviolacin-O16 activity profiles?

Activity differences between natural and recombinant cyclotides may arise from several factors:

• Post-translational modifications:

  • Issue: Natural cyclotides may contain modifications absent in recombinant versions

  • Detection: High-resolution MS to identify modifications such as oxidized tryptophan residues (oxindolyalanine or N-formylkynurenine), which are known post-translational modifications in cyclotides

  • Solution: Engineer expression systems to incorporate relevant modifications

• Folding and disulfide bond formation:

  • Issue: Incorrect disulfide connectivity in recombinant preparation

  • Detection: Comparative disulfide mapping between natural and recombinant versions

  • Solution: Optimization of oxidative folding conditions; in vitro folding with redox buffers

• Cyclization efficiency:

  • Issue: Incomplete cyclization leading to linear byproducts

  • Detection: LC-MS analysis to quantify cyclic vs. linear forms

  • Solution: Optimization of cyclization conditions; additional purification steps

• Contaminants and impurities:

  • Issue: Co-purifying molecules affecting activity measurements

  • Detection: Orthogonal purification techniques to achieve highest purity

  • Solution: Multi-step purification protocols; activity testing on fractions from each purification step

What are common challenges in scaling up recombinant Cycloviolacin-O16 production and how can they be addressed?

Scaling up production presents several challenges:

• Expression yield:

  • Challenge: Lower yields in large-scale fermentation

  • Solutions: Optimization of media composition, feeding strategies, and induction parameters; consideration of alternative expression hosts

• Proper folding at scale:

  • Challenge: Less efficient folding in larger volumes due to dilution effects

  • Solutions: Development of continuous folding processes; optimization of redox conditions; use of folding catalysts

• Purification efficiency:

  • Challenge: Column capacity limitations and increased contaminant complexity

  • Solutions: Development of capture steps with higher selectivity; implementation of orthogonal purification techniques

• Process reproducibility:

  • Challenge: Batch-to-batch variation

  • Solutions: Careful process parameter monitoring; establishment of critical quality attributes (CQAs) and acceptance criteria