Recombinant Litoria citropa Caerulein-4.1/4.1Y4

What is Caerulein-4.1 and how does it differ from other caerulein peptides in Litoria citropa?

Caerulein-4.1 is one of sixteen caerulein-type peptides isolated from the skin secretions of the Australian Blue Mountains tree frog (Litoria citropa). It has the specific amino acid sequence [pEQDY(SO₃)TGSHMDF-NH₂], distinguishing it from the original caerulein (now renamed caerulein 1.1) which has the sequence [pEQDY(SO₃)TGWMDF-NH₂] . The primary structural difference in caerulein-4.1 compared to caerulein 1.1 is the substitution of "GSH" for "GW" in the middle of the sequence, representing a significant modification that likely affects its biological activity and binding properties . Additionally, caerulein-4.1 belongs to the fourth group of caerulein peptides identified in this species, with each group showing distinctive sequence variations while maintaining the characteristic caerulein backbone structure.

What is the significance of the Y4 designation in Caerulein-4.1Y4?

The Y4 designation in Caerulein-4.1Y4 indicates a specific structural variant of Caerulein-4.1 where phenylalanine (F) replaces methionine (M) at a key position in the peptide sequence. Based on the pattern observed in Litoria citropa skin peptides, all the caerulein peptides are accompanied by associated peptides where phenylalanine replaces methionine . This substitution is significant as it can alter the peptide's physiochemical properties, receptor binding affinity, and biological activity. The replacement of methionine with phenylalanine creates a more hydrophobic and oxidation-resistant variant that may exhibit different pharmacological properties compared to the methionine-containing version, including potentially different potency at cholecystokinin receptors.

How are caerulein peptides naturally produced in Litoria citropa?

Caerulein peptides are naturally produced in specialized skin glands of Litoria citropa as part of their host-defense system. The peptides are synthesized and stored in granular glands (also known as poison glands) located in the dermal layer of the frog skin . When the frog is stressed or threatened, these glands secrete the peptides onto the skin surface as a protective mechanism. The production appears to be regulated by complex neuroendocrine pathways, and some frog species exhibit seasonal variation in their skin peptide profiles . For example, in related species like Litoria splendida and Litoria rothii, the composition of skin peptides changes between summer and winter, with different forms of caerulein being expressed depending on the season . This suggests that environmental factors may influence the expression of these peptides, although specific seasonal variations in Litoria citropa have not been documented in the provided sources.

What expression systems are most effective for recombinant production of Caerulein-4.1/4.1Y4?

Escherichia coli BL21(DE3) represents an effective expression system for recombinant caerulein peptide production due to its well-characterized genetics, rapid growth kinetics, and high protein expression capacity . For optimal production of Caerulein-4.1/4.1Y4, a fed-batch cultivation with tightly controlled process parameters is recommended. The approach should include:

• Expression vector selection: Vectors containing strong inducible promoters (T7 or tac) with appropriate signal sequences for periplasmic targeting

• Culture conditions: Fed-batch cultivation at controlled temperature (typically 30-37°C), pH (6.8-7.2), and dissolved oxygen levels

• Induction strategy: IPTG induction at optimal cell density with careful timing to maximize yield and minimize cell stress

What analytical methods are most appropriate for characterizing recombinant Caerulein-4.1/4.1Y4?

The characterization of recombinant Caerulein-4.1/4.1Y4 requires a multi-faceted analytical approach to confirm identity, purity, and structural integrity. Based on methods used for native caerulein peptides, the following analytical techniques are recommended:

• Mass Spectrometry Analysis:

  • Negative ion electrospray mass spectrometry (ES-MS) for molecular weight determination via [M-H]⁻ ions

  • Positive ion ES-MS analysis of [MH⁺-SO₃]⁺ ions for sequence confirmation through B and Y+2 cleavage ions

• Chromatographic Methods:

  • Reversed-phase HPLC for purity assessment and quantification

  • Ion-exchange chromatography to separate sulfated from desulfated forms

• Structural Verification:

  • Amino acid composition analysis

  • Circular dichroism (CD) spectroscopy for secondary structure assessment

  • 2D NMR spectroscopy for detailed structural confirmation, particularly important for studying hydrophobic and hydrophilic interactions that influence receptor binding

A combination of these methods provides comprehensive characterization of recombinant Caerulein-4.1/4.1Y4, ensuring proper identification of both the sulfated peptide and any desulfated analogues that may form during production or purification.

How can researchers optimize the yield and purity of recombinant Caerulein-4.1/4.1Y4?

Optimizing yield and purity of recombinant Caerulein-4.1/4.1Y4 requires strategic approaches to expression, harvest timing, and purification:

Yield Optimization:

• Culture parameter control: Maintain precise control of pH, temperature, and dissolved oxygen

• Harvest timing optimization: Monitor cell lysis rates, which can reach up to 18.2% during peak expression, and harvest before significant decreases in peptide content occur

• Cell morphology monitoring: Track changes in cell length, diameter, and volume as indicators of expression stress and optimal harvest timing

Purification Strategy:

• Initial capture: Ion exchange chromatography to separate sulfated from desulfated forms

• Intermediate purification: Hydrophobic interaction chromatography

• Polishing: Reversed-phase HPLC for final purity

Quality Control Parameters:

Implementing these approaches can minimize common issues such as desulfation, proteolytic degradation, and heterogeneity in the final peptide product, resulting in higher yields of bioactive Caerulein-4.1/4.1Y4.

How does the structure of Caerulein-4.1/4.1Y4 influence its receptor binding properties?

The structure of Caerulein-4.1/4.1Y4 contains critical elements that determine its receptor binding properties, particularly to cholecystokinin receptors:

Key Structural Elements:

• The tyrosine sulfate moiety (Y-SO₃) at position 4 is crucial for high-affinity receptor binding, as demonstrated by the reduced activity of desulfated analogues

• The GSH (glycine-serine-histidine) sequence distinguishing Caerulein-4.1 from other caeruleins likely alters the spatial orientation of the C-terminal region

• In Caerulein-4.1Y4, the substitution of phenylalanine for methionine creates a more hydrophobic interaction surface

Based on studies of related caerulein peptides, both hydrophobic and hydrophilic interactions are critical for binding to CCK receptors, particularly CCK2R . The phenylalanine variant (Y4) likely exhibits altered receptor selectivity compared to the methionine-containing version, potentially showing differential activation of CCK1R versus CCK2R subtypes. The binding affinity may be approximately 50% of the original caerulein, as seen with similar substitutions in related caerulein variants .

Understanding these structure-activity relationships is essential for designing experiments that accurately assess the pharmacological properties of Caerulein-4.1/4.1Y4 and its potential applications in neurobiological or physiological research.

What experimental design would best evaluate the biological activity of recombinant versus native Caerulein-4.1/4.1Y4?

To rigorously compare recombinant versus native Caerulein-4.1/4.1Y4, a multi-tiered experimental design that evaluates structural, biochemical, and functional parameters is recommended:

Tier 1: Structural Comparison

• High-resolution mass spectrometry to verify identical molecular weights and fragmentation patterns

• 2D NMR spectroscopy to confirm identical three-dimensional structures

• Circular dichroism to assess secondary structural elements

Tier 2: Receptor Binding Analysis

• Competitive binding assays using radiolabeled ligands against both CCK1R and CCK2R receptors

• Surface plasmon resonance (SPR) to determine binding kinetics (kon and koff rates)

• Assessment of binding to isolated receptor domains to identify critical interaction sites

Tier 3: Functional Assays

• In vitro smooth muscle contraction assays:

  • Isolated guinea pig gallbladder strips to measure contractile responses

  • Dose-response curves to calculate EC50 values and maximum responses

  • Comparison with caerulein 1.1 and caerulein 1.2 as reference standards

• Ex vivo secretion models:

  • Pancreatic acinar cell preparations to measure amylase release

  • Gastric tissue preparations to assess acid secretion

• In vivo physiological responses:

  • Pancreatitis induction model in mice

  • Measurement of pancreatic enzyme elevations, edema, and inflammatory markers

This experimental approach would provide comprehensive data to determine whether recombinant Caerulein-4.1/4.1Y4 faithfully reproduces the properties of the native peptide across multiple biological systems and activity parameters.

How can researchers effectively study the seasonal variations in caerulein peptide expression and their functional significance?

To effectively study seasonal variations in caerulein peptide expression and their functional significance, researchers should implement a comprehensive longitudinal study design:

Field Sampling Protocol:

• Establish quarterly sampling from wild Litoria citropa populations across multiple locations

• Implement non-lethal skin secretion collection using mild electrical stimulation

• Record environmental parameters (temperature, humidity, photoperiod, precipitation)

• Track reproductive status and behavioral patterns

Analytical Framework:

• Quantitative peptide profiling using LC-MS/MS to identify and quantify all caerulein variants

• Seasonal comparison of sulfated vs. desulfated forms

• Monitoring of caerulein 4.1 vs. 4.1Y4 (Met vs. Phe variant) ratios throughout the year

This approach would build upon observations from related species like Litoria splendida and Litoria rothii, which demonstrate significant seasonal peptide profile changes - including shifts from potent caerulein forms in summer to less active desulfated forms in winter . For L. citropa specifically, researchers should investigate whether caerulein 4.1/4.1Y4 shows similar seasonal substitution patterns.

Functional Significance Assessment:

• Receptor activation assays at CCK1R and CCK2R at different seasonal timepoints

• Antimicrobial activity testing against seasonal pathogens

• Smooth muscle activity comparison between summer and winter peptide profiles

Such comprehensive analysis would reveal whether seasonal variation in caerulein peptide expression represents an adaptive response to changing environmental conditions, predation pressures, or pathogen exposure patterns, providing valuable insights into the ecological and evolutionary significance of these peptide modifications.

What are the most common challenges in recombinant Caerulein-4.1/4.1Y4 production and how can they be overcome?

Researchers working with recombinant Caerulein-4.1/4.1Y4 typically encounter several challenges that require specific troubleshooting approaches:

Challenge 1: Tyrosine Sulfation

• Problem: Inadequate post-translational sulfation of the critical tyrosine residue

• Solution: Consider co-expression with tyrosylprotein sulfotransferase (TPST) or implement chemical sulfation post-purification using established sulfation reagents

Challenge 2: Cellular Stress and Lysis

• Problem: High expression levels causing significant cellular stress (18.2% lysis) and morphological changes

• Solution: Optimize induction conditions (lower temperature, reduced inducer concentration), implement improved feeding strategies, and consider harvest before peak lysis occurs

Challenge 3: Methionine Oxidation

• Problem: Oxidation of methionine in Caerulein-4.1 creating heterogeneity

• Solution: Include antioxidants during purification, purge buffers with nitrogen, and consider working primarily with the Caerulein-4.1Y4 variant which contains phenylalanine instead of the oxidation-prone methionine

Challenge 4: Proteolytic Degradation

• Problem: Degradation of peptide by host cell proteases

• Solution: Include protease inhibitors during lysis, use protease-deficient expression strains, and optimize rapid purification workflows

Challenge 5: Low Recombinant Yields

• Problem: Suboptimal expression due to toxicity or degradation

• Solution: Implement fusion protein strategies (e.g., SUMO, thioredoxin, or MBP fusions) with specific protease cleavage sites to enhance stability and expression while enabling recovery of the native sequence

Each of these challenges requires systematic optimization of expression conditions, careful monitoring of cellular responses, and strategic modifications to purification protocols to achieve consistent, high-quality recombinant Caerulein-4.1/4.1Y4 production.

How can researchers design experiments to investigate structure-activity relationships of Caerulein-4.1/4.1Y4 variants?

Designing robust structure-activity relationship (SAR) studies for Caerulein-4.1/4.1Y4 requires systematic modification of the peptide structure followed by comprehensive functional assessment:

Experimental Approach:

• Strategic Peptide Library Design:

  • Alanine scanning: Substitute each non-conserved residue with alanine

  • Positional scanning: Create systematic substitutions at the GSH motif that distinguishes Caerulein-4.1

  • Sulfation variants: Compare sulfated Y4 with desulfated forms

  • Met/Phe variants: Direct comparison of Caerulein-4.1 (Met) with Caerulein-4.1Y4 (Phe)

• Receptor Binding and Signaling Analysis:

  • Radiolabeled competitive binding assays against CCK1R and CCK2R

  • BRET/FRET assays to measure G-protein activation

  • Calcium mobilization assays in receptor-expressing cells

  • β-arrestin recruitment assays to assess biased signaling

• Structural Studies:

  • NMR analysis of peptide-receptor interactions

  • Molecular dynamics simulations to identify critical binding interactions

  • X-ray crystallography of peptide-receptor complexes (if feasible)

Data Integration Approach:

This comprehensive approach would generate a detailed pharmacophore model identifying which structural elements are critical for receptor binding, selectivity, signaling pathway activation, and biological activity, enabling rational design of optimized Caerulein-4.1/4.1Y4 variants for specific research applications.

What approaches should be used to resolve contradictory data when comparing activity of recombinant versus native Caerulein-4.1/4.1Y4?

When facing contradictory data between recombinant and native Caerulein-4.1/4.1Y4 activities, researchers should implement a systematic troubleshooting and validation approach:

1. Sample Authentication and Verification:

• Re-verify peptide identity using high-resolution mass spectrometry

• Confirm sulfation status using specific antibodies or sulfation-sensitive analytical methods

• Assess purity by orthogonal chromatographic methods (ion exchange, reversed-phase, size exclusion)

• Check for oxidation, deamidation, or other post-isolation modifications

2. Methodological Refinement:

• Standardize assay conditions (buffers, pH, temperature, incubation times)

• Use multiple biological readouts to measure activity (e.g., receptor binding, calcium flux, smooth muscle contraction)

• Implement positive controls (caerulein 1.1) and negative controls (desulfated variants)

• Establish dose-response relationships across wide concentration ranges

3. Cross-Laboratory Validation:

• Exchange samples between laboratories for independent testing

• Standardize protocols with detailed standard operating procedures

• Implement blinded analysis to eliminate investigator bias

4. Analytical Resolution of Differences:

• Investigate potential structural differences using 2D NMR

• Examine conformational variations via circular dichroism under various conditions

• Consider the influence of post-translational modifications beyond sulfation

• Assess the impact of formulation, storage conditions, and freeze-thaw cycles

5. Biological Context Consideration:

• Evaluate receptor expression levels in different test systems

• Examine the influence of membrane composition on receptor function

• Consider the presence of modulatory factors in different biological preparations

By systematically addressing each of these areas, researchers can identify the source of contradictory results and develop standardized approaches that yield consistent and reliable data on the biological activity of recombinant versus native Caerulein-4.1/4.1Y4, ultimately ensuring that research findings are reproducible and physiologically relevant.

What are the most promising applications of recombinant Caerulein-4.1/4.1Y4 in neuroscience and physiological research?

Recombinant Caerulein-4.1/4.1Y4 offers several promising research applications across neuroscience and physiological research domains:

Neuroscience Applications:

• Receptor Specificity Studies: Caerulein-4.1/4.1Y4's unique structure makes it valuable for investigating the differential activation and signaling of CCK receptor subtypes in neural circuits

• Neuroplasticity Research: The peptide could serve as a tool to study CCK-mediated modulation of synaptic plasticity in learning and memory circuits

• Pain Modulation Pathways: Its potential interaction with pain-processing neural circuits warrants investigation, particularly given the role of CCK in nociception

Physiological Research Applications:

• Pancreatitis Models: The peptide's ability to stimulate pancreatic secretion makes it useful for developing refined experimental models of pancreatitis with potentially more selective effects than conventional caerulein

• Gastrointestinal Motility Studies: Caerulein-4.1/4.1Y4 could serve as a molecular probe for investigating regional differences in GI tract smooth muscle responsiveness

• Gallbladder Function Research: The peptide's effects on biliary secretion provide opportunities for studying cholecystokinin-mediated regulation of bile production and release

Analytical and Methodological Applications:

• Receptor Binding Assays: Development of labeled Caerulein-4.1/4.1Y4 variants could provide selective tools for CCK receptor characterization

• Biased Signaling Investigation: The peptide may exhibit biased signaling properties at CCK receptors, offering insight into pathway-selective receptor activation

Each of these applications leverages the unique structural and functional properties of Caerulein-4.1/4.1Y4, potentially providing more selective tools than traditional caerulein peptides for investigating specific physiological mechanisms and pathways.

How can advanced computational methods enhance our understanding of Caerulein-4.1/4.1Y4 structure-function relationships?

Advanced computational methods offer powerful approaches to elucidate the structure-function relationships of Caerulein-4.1/4.1Y4:

Molecular Dynamics Simulations:

• Simulating peptide conformational dynamics in solution and membrane environments

• Modeling the effects of sulfation on peptide flexibility and solvent interactions

• Comparing conformational ensembles of Met vs. Phe variants (4.1 vs. 4.1Y4)

Receptor-Peptide Docking and Binding Simulations:

• Detailed binding mode analysis with CCK1R and CCK2R homology models

• Free energy calculations to quantify binding affinity differences

• Identification of key residue interactions responsible for receptor subtype selectivity

Quantitative Structure-Activity Relationship (QSAR) Models:

• Developing predictive models correlating structural features with biological activity

• Virtual screening of potential Caerulein-4.1/4.1Y4 analogs prior to synthesis

• Pharmacophore modeling to identify essential chemical features for activity

Machine Learning Applications:

• Training neural networks on structure-activity data to predict novel peptide properties

• Pattern recognition in molecular interaction data across multiple peptide variants

• Identifying non-obvious correlations between structural features and biological activities

The integration of these computational approaches with experimental data would create a comprehensive model of how Caerulein-4.1/4.1Y4's unique structure determines its receptor binding properties, signaling outcomes, and biological activities. This integrated computational-experimental workflow would significantly accelerate the development of optimized peptide variants for specific research applications while minimizing resource-intensive experimental screening.

What interdisciplinary approaches could advance our understanding of the ecological and evolutionary significance of Caerulein-4.1/4.1Y4?

Understanding the ecological and evolutionary significance of Caerulein-4.1/4.1Y4 requires integrative approaches that span multiple disciplines:

Evolutionary Genomics:

• Comparative analysis of caerulein gene sequences across Litoria species to trace evolutionary relationships

• Investigation of gene duplication and diversification events that led to the four caerulein variants

• Molecular clock analyses to estimate when structural divergence of caerulein-4.1 occurred

Ecological Chemistry:

• Field studies examining predator-prey interactions and how caerulein peptides influence predator behavior

• Analysis of antimicrobial properties against microbes present in the frog's natural habitat

• Seasonal monitoring of peptide expression in relation to environmental conditions and pathogen prevalence

Physiological Ecology:

• Investigation of how caerulein variants affect frog physiology under different environmental conditions

• Comparison of peptide profiles between populations across different habitats and elevations

• Experimental studies on how temperature and humidity affect peptide stability and activity

Behavioral Neuroscience:

• Examination of potential effects of caerulein variants on frog behavioral patterns

• Investigation of whether caerulein-4.1/4.1Y4 serves signaling functions between individuals

• Analysis of potential roles in reproductive behavior or territorial marking

Integrative Data Analysis Framework:

This interdisciplinary approach would reveal whether the structural variations in caerulein-4.1/4.1Y4 represent adaptive responses to specific ecological pressures, providing insights into how molecular evolution of skin peptides contributes to amphibian survival strategies in changing environments, particularly relevant given the global amphibian biodiversity crisis.