Recombinant Carcinus maenas Carcinustatin-2

What is the basic structure and composition of Carcinustatin-2?

Carcinustatin-2 is a heptapeptide naturally found in the common shore crab (Carcinus maenas) and Jonah crab (Cancer borealis). The peptide has the sequence EAYAFGL (H-Glu-Ala-Tyr-Ala-Phe-Gly-Leu-NH2), with a molecular formula of C37H52N8O10 and a molecular weight of 768.8 Da . This relatively small peptide contains both hydrophilic and hydrophobic amino acid residues, which likely contribute to its biological function in crustacean physiology.

What is currently known about the physiological role of Carcinustatin-2 in Carcinus maenas?

While comprehensive studies on Carcinustatin-2's specific physiological functions are still emerging, research with the green crab Carcinus maenas indicates it belongs to a family of bioactive peptides with potential regulatory roles in crustacean biology . Other carcinustatins, such as carcinustatin-8 (AGPYAFGL-NH2), have demonstrated inhibitory effects on muscle contractions in crayfish and cockroach hindgut, as well as modulation of the pyloric rhythm in the stomatogastric nervous system . By structural analogy, Carcinustatin-2 may participate in similar neuroregulatory or myomodulatory processes in crustaceans.

How does Carcinustatin-2 differ structurally and functionally from other carcinustatins?

Carcinustatin-2 (EAYAFGL) differs from other identified carcinustatins such as carcinustatin-8 (AGPYAFGL-NH2) in its N-terminal sequence . While carcinustatin-8 begins with Ala-Gly, carcinustatin-2 starts with Glu-Ala, introducing a negatively charged residue at the N-terminus. This structural variation likely influences receptor binding specificity, stability, and biological activity. Functional comparison studies between different carcinustatins would provide valuable insights into structure-activity relationships within this peptide family.

What purification strategies yield the highest purity recombinant Carcinustatin-2?

Purification of recombinant Carcinustatin-2 would typically involve a multi-step approach:

• Initial capture using affinity chromatography (if expressed with a tag)

• Tag removal via specific protease cleavage

• Reverse-phase HPLC with acetonitrile-TFA gradients (similar to those used for natural peptide isolation)

• Final polishing using ion-exchange chromatography

To achieve research-grade purity (>95%), optimization of HPLC conditions is critical . Based on the peptide's properties, a gradient from 18% to 36% acetonitrile-TFA over 40 minutes at a flow rate of 1 ml/min would be a suitable starting point, as similar conditions have been successful for related peptides .

How can researchers verify the structural integrity of recombinant Carcinustatin-2?

Verification of recombinant Carcinustatin-2 should employ multiple complementary techniques:

• Mass spectrometry: MALDI-TOF MS to confirm the expected molecular weight of 768.8 Da

• NMR spectroscopy: For complete structural characterization and comparison with synthetic standards

• Circular dichroism: To assess secondary structural elements

• HPLC analysis: To determine purity using established profiles

For NMR analysis, both 1D (1H, 13C) and 2D (COSY, TOCSY, NOESY) experiments should be conducted to fully characterize the peptide structure and compare with theoretical chemical shifts calculated from the known sequence .

What are the optimal storage and handling conditions for Carcinustatin-2 in laboratory settings?

Based on established protocols for similar peptides, recombinant Carcinustatin-2 should be stored at -20°C for up to 1 year in lyophilized form . After reconstitution, the peptide should be refrigerated and used within a limited time period to maintain activity. For reconstitution, the peptide is typically soluble in water, though specific solubility characteristics may vary depending on preparation methods . Researchers should minimize freeze-thaw cycles and consider aliquoting the reconstituted peptide to preserve integrity for long-term studies.

What bioassays can reliably measure Carcinustatin-2 activity?

While specific bioassays for Carcinustatin-2 are not detailed in the search results, functional assays based on related carcinustatins suggest several approaches:

• Muscle contraction assays: Measuring inhibitory effects on isolated crayfish or cockroach hindgut contractions

• Electrophysiological recordings: Evaluating effects on the pyloric rhythm generated by stomatogastric ganglion neurons

• Enzyme inhibition assays: Testing potential protease inhibitory activities

• Cell-based assays: Assessing effects on cellular processes in crustacean cell lines

Dose-response curves should be established using synthetic Carcinustatin-2 as a positive control to validate the activity of recombinant preparations .

How should researchers design experiments to compare native and recombinant Carcinustatin-2?

A comprehensive comparative analysis should address:

• Structural equivalence:

  • Identical molecular weight confirmation by mass spectrometry

  • Matching HPLC retention times

  • Comparable NMR chemical shift profiles

• Functional equivalence:

  • Parallel bioassays at multiple concentrations

  • Side-by-side testing in the same experimental systems

  • Standardized activity units for quantitative comparison

• Stability assessment:

  • Thermal stability comparisons

  • Degradation kinetics in relevant biological matrices

  • Resistance to proteolytic enzymes

Statistical analysis should employ paired designs when possible to minimize experimental variability .

How can Carcinustatin-2 be employed in crustacean ecotoxicology research?

Carcinus maenas has been extensively validated as a reliable estuarine/marine model for ecotoxicology research . Incorporating Carcinustatin-2 into this research framework could:

• Serve as a biomarker for environmental stress responses

• Function as an endpoint for assessing pollutant impacts on neuropeptide expression

• Provide insights into ecotoxicological effects on regulatory peptide systems

• Enable development of molecular assays for environmental monitoring

When designing such studies, researchers should control for confounding factors including gender, size, morphotype, nutritional status, and environmental parameters like temperature and salinity, as these can influence peptide expression and responses .

What techniques can researchers use to investigate Carcinustatin-2's receptor interactions and signaling pathways?

Advanced investigation of Carcinustatin-2's molecular interactions would involve:

• Receptor identification:

  • Photo-affinity labeling with modified Carcinustatin-2

  • Pull-down assays using biotinylated peptide

  • Heterologous expression systems for candidate receptors

• Signaling pathway elucidation:

  • Calcium mobilization assays

  • cAMP/cGMP measurement

  • Phosphorylation studies of downstream effectors

  • Electrophysiological recordings in target tissues

• Structure-activity relationship studies:

  • Alanine scanning mutagenesis

  • N- and C-terminal modifications

  • D-amino acid substitutions

These approaches would provide mechanistic insights into how Carcinustatin-2 exerts its biological effects .

What are the potential applications of Carcinustatin-2 in comparative physiology research?

Carcinustatin-2 offers valuable opportunities for comparative physiology research across different taxonomic groups:

• Evolutionary studies:

  • Comparing peptide function across crustacean species

  • Examining functional conservation between crustacean and insect systems

  • Investigating the evolution of peptide-receptor pairs

• Cross-species functional testing:

  • Testing activity in insect preparations, similar to studies with carcinustatin-8

  • Evaluating effects in vertebrate systems

  • Investigating potential conservation of molecular targets

• Comparative genomics:

  • Analyzing genomic organization of carcinustatin precursor genes

  • Characterizing regulatory elements controlling expression

  • Examining evolutionary relationships with other peptide families

This research would contribute to broader understanding of peptide signaling evolution across invertebrate phyla.

What mass spectrometry approaches are best suited for Carcinustatin-2 detection and quantification?

For comprehensive mass spectrometric analysis of Carcinustatin-2, researchers should consider:

• Qualitative analysis:

  • MALDI-TOF MS for molecular weight confirmation

  • ESI-MS/MS for sequence verification

  • Ion-trap MS for structural characterization

• Quantitative analysis:

  • Multiple reaction monitoring (MRM) for sensitive quantification

  • Standard addition methods using synthetic peptide standards

  • Stable isotope dilution assays for absolute quantification

These approaches have been successfully applied to similar peptides, achieving detection limits in the picomolar range. For MALDI-MS analysis, α-cyano-4-hydroxycinnamic acid serves as an appropriate matrix, with sample preparation involving reconstitution in 10% acetonitrile-TFA .

How can researchers develop a sensitive ELISA for Carcinustatin-2 detection in biological samples?

Development of a specific ELISA for Carcinustatin-2 would involve:

• Antibody production:

  • Conjugation of synthetic Carcinustatin-2 to a carrier protein (e.g., KLH)

  • Immunization protocol with appropriate adjuvants

  • Screening for high-affinity antibodies with minimal cross-reactivity

• Assay optimization:

  • Determination of optimal coating conditions and blocking agents

  • Establishing standard curves using synthetic Carcinustatin-2

  • Validation across different sample matrices (hemolymph, tissue extracts)

• Performance evaluation:

  • Assessment of sensitivity, specificity, and reproducibility

  • Cross-reactivity testing with related peptides

  • Recovery experiments in complex biological samples

A well-optimized ELISA would facilitate studies of Carcinustatin-2 expression patterns and regulation in different physiological states .

What are the challenges in distinguishing Carcinustatin-2 from other structurally similar peptides in complex samples?

Differentiation of Carcinustatin-2 from similar peptides presents several challenges:

• Analytical challenges:

  • Similar HPLC retention times for related peptides

  • Mass spectral overlap with isomeric or isobaric peptides

  • Limited fragmentation in MS/MS due to small size

• Technical approaches to overcome these challenges:

  • Two-dimensional chromatography combining orthogonal separation modes

  • High-resolution mass spectrometry with accurate mass measurement

  • Custom immunoaffinity enrichment prior to analysis

  • Application of multiple complementary analytical techniques

• Validation strategies:

  • Synthetic peptide standards as positive controls

  • Negative controls from organisms lacking the target peptide

  • Spiking experiments to assess recovery and specificity

Researchers should implement these approaches to ensure reliable identification and quantification of Carcinustatin-2 in complex biological samples .

How does Carcinustatin-2 compare functionally to similar peptides in other crustacean species?

Comparative analysis of Carcinustatin-2 with related peptides across crustacean species reveals important evolutionary and functional relationships:

These comparative data suggest conservation of C-terminal sequences (particularly -FGL) across related peptides, potentially indicating a shared binding motif for receptor interaction. Functional studies indicate inhibitory effects on hindgut contractions and neuromodulatory actions for several family members . This phylogenetic distribution provides valuable context for investigating Carcinustatin-2's physiological role.

What genomic approaches can advance understanding of Carcinustatin-2 expression and regulation?

Modern genomic approaches offer powerful tools for understanding Carcinustatin-2 biology:

• Transcriptomic analysis:

  • RNA-Seq to quantify expression across tissues and developmental stages

  • Single-cell transcriptomics to identify specific cell types expressing the peptide

  • Differential expression analysis under various physiological challenges

• Genomic characterization:

  • Isolation and sequencing of the precursor gene

  • Promoter analysis to identify regulatory elements

  • Comparative genomics across crustacean species

• Functional genomics:

  • CRISPR/Cas9-mediated gene editing in model crustaceans

  • Reporter gene assays to study promoter activity

  • RNAi for loss-of-function studies

These approaches would address the critical need for complete genome sequencing in Carcinus maenas, identified as essential for cutting-edge research in this field .

What are the most promising therapeutic or biotechnological applications for recombinant Carcinustatin-2?

While maintaining focus on basic research applications rather than commercial aspects, several promising research directions for recombinant Carcinustatin-2 emerge:

• Physiological research tools:

  • Selective modulators of crustacean digestive physiology

  • Probes for studying neuromuscular junction function

  • Tools for investigating peptidergic signaling mechanisms

• Potential biotechnological applications:

  • Development of selective bioassays for environmental monitoring

  • Models for peptide-based therapeutic development

  • Templates for designing peptidomimetic compounds

• Comparative endocrinology:

  • Investigating evolutionary conservation of peptide signaling systems

  • Exploring functional convergence across invertebrate phyla

  • Developing new model systems for neuropeptide research

These research applications leverage Carcinustatin-2's unique structural and functional properties while adhering to the academic research focus of this guide.

How can researchers address peptide degradation issues when working with recombinant Carcinustatin-2?

Peptide degradation presents significant challenges in Carcinustatin-2 research. Implement these strategies to minimize degradation:

• Storage optimization:

  • Maintain lyophilized peptide at -20°C

  • Add protease inhibitor cocktails to reconstituted solutions

  • Store in small aliquots to minimize freeze-thaw cycles

• Handling procedures:

  • Use low-binding tubes and pipette tips

  • Prepare working solutions immediately before use

  • Keep samples on ice during processing

• Formulation considerations:

  • Test stabilizing excipients (trehalose, albumin, glycerol)

  • Optimize buffer composition and pH

  • Consider modified derivatives with improved stability

Monitoring sample integrity via analytical HPLC or MS at different timepoints can help establish optimal handling protocols for specific experimental conditions.

What strategies can overcome challenges in achieving sufficient yield of bioactive recombinant Carcinustatin-2?

Optimizing production of bioactive recombinant Carcinustatin-2 requires addressing several technical challenges:

• Expression optimization:

  • Test multiple fusion partners (SUMO, thioredoxin, ubiquitin)

  • Evaluate different promoter systems and expression conditions

  • Consider codon optimization for the host organism

• Folding and processing:

  • Optimize cleavage conditions for fusion tag removal

  • Implement refolding protocols if necessary

  • Validate proper disulfide bond formation if modified versions are produced

• Activity preservation:

  • Minimize exposure to extreme pH and temperatures

  • Evaluate different buffer systems for maximal stability

  • Implement quality control testing at each production step

Systematic optimization of these parameters can significantly improve both yield and biological activity of the recombinant peptide.

How should researchers interpret contradictory results between different functional assays of Carcinustatin-2?

When faced with contradictory results across different functional assays, researchers should:

• Systematic analysis of discrepancies:

  • Evaluate assay sensitivity and specificity limitations

  • Consider differences in experimental conditions (temperature, pH, ionic strength)

  • Assess potential interfering factors in different assay systems

  • Examine peptide stability under specific assay conditions

• Methodological refinement:

  • Implement multiple complementary assays

  • Standardize positive and negative controls across experiments

  • Develop concentration-response relationships in each assay

  • Establish time-course studies to capture kinetic differences

• Biological interpretation:

  • Consider context-dependent effects based on target tissue or cell type

  • Evaluate potential for multiple receptor interactions with different affinities

  • Assess species-specific differences if cross-species assays are employed

This systematic approach will help reconcile apparent contradictions and develop a more complete understanding of Carcinustatin-2's biological activities.

What online databases and resources are most valuable for Carcinustatin-2 research?

Researchers investigating Carcinustatin-2 should utilize these specialized resources:

• Peptide databases:

  • PepBank: Repository of peptide sequences and bioactivity data

  • Antimicrobial Peptide Database: For potential antimicrobial properties

  • EROP-Moscow: Database of regulatory oligopeptides

• Structural resources:

  • PDB (Protein Data Bank): For structural models if available

  • PEP-FOLD: Web server for peptide 3D structure prediction

  • Peptide property calculators for physicochemical parameters

• Genomic and transcriptomic resources:

  • Marine invertebrate genomic databases

  • Transcriptome data from Carcinus maenas

  • Comparative crustacean genomic resources

These databases provide valuable context for Carcinustatin-2 research within the broader framework of crustacean neuropeptides and bioactive peptides.

What collaborative research networks exist for advancing Carcinustatin-2 research?

Several research networks and collaborative frameworks support work on crustacean peptides like Carcinustatin-2:

• Academic consortia:

  • Marine invertebrate neurobiology networks

  • Crustacean endocrinology research groups

  • Comparative peptidome analysis initiatives

• Resource sharing platforms:

  • Repositories for plasmids and expression vectors

  • Reagent sharing networks for antibodies and standards

  • Bioinformatic tool development collaborations

• Scientific meetings and workshops:

  • Crustacean Society conferences

  • Invertebrate neuropeptide workshops

  • Marine model organism symposia