Recombinant Sepia officinalis Ovarian jelly-peptide 1

What is Ovarian jelly-peptide 1 and how was it first identified?

Ovarian jelly-peptide 1 (DQVKIVL) is a regulatory peptide first identified in the cuttlefish Sepia officinalis. It was initially characterized by monitoring HPLC purified fractions using a myotropic bioassay. The peptide belongs to a family of water-borne peptides released by full-grown oocytes (FGO) in the genital coelom and in the lumen of the oviduct of female cuttlefish. It was named an "ovarian jelly-peptide" because of its unique property of forming a jelly-like substance when resuspended in water, which may play an important role in the kinetics of peptide diffusion in the external medium during reproduction.

What is the amino acid sequence of Ovarian jelly-peptide 1?

Ovarian jelly-peptide 1 has the primary structure DQVKIVL, a seven-amino-acid peptide. This sequence was determined through a combination of HPLC purification, mass spectrometry, and bioactivity testing. It shares significant sequence homology with other peptides in the OJP family, particularly in the C-terminal region (KIVL), suggesting evolutionary conservation of functional domains within this peptide family.

How does Ovarian jelly-peptide 1 differ from other peptides in the OJP family?

Ovarian jelly-peptide 1 (DQVKIVL) differs from other peptides in the OJP family primarily in its N-terminal region while maintaining similarities in its C-terminal sequence. For comparison, Ovarian jelly-peptide 2 has the sequence DEVKIVL, differing only in the first two amino acids (DE instead of DQ). Ovarian jelly-peptide 3 (DEVKIVLD) is essentially OJP-2 with an additional aspartic acid residue at the C-terminus. Despite these differences, all three peptides share similar localization in the female genital tract and exhibit comparable biological activities, indicating that the conserved KIVL region may be critical for their function.

What is the biological function of Ovarian jelly-peptide 1 in Sepia officinalis?

The primary biological function of Ovarian jelly-peptide 1 appears to be modulation of contractions in the female genital tract and the main nidamental gland of Sepia officinalis. This myotropic activity suggests that OJP-1 plays a role in coordinating the reproductive process, particularly during egg-laying. The peptide's ability to form a jelly in water likely influences the kinetics of its diffusion in the external medium, potentially allowing for sustained release and extended biological activity. This property may be crucial for regulating reproductive behaviors and physiological processes in the reproductive system of the cuttlefish.

Where is Ovarian jelly-peptide 1 expressed in the cuttlefish?

Tissue mapping studies have shown that Ovarian jelly-peptide 1 and related peptides are strictly localized in the female genital tract of Sepia officinalis. Specifically, these peptides are released by full-grown oocytes (FGO) into the genital coelom and the lumen of the oviduct. This restricted expression pattern further supports their specialized role in reproductive processes. The precise cellular source appears to be the oocytes themselves, which secrete these peptides during development, likely as part of the complex signaling network that coordinates reproductive maturation and egg-laying behaviors.

What expression systems are optimal for producing recombinant Ovarian jelly-peptide 1?

Based on established protocols for similar peptides in the OJP family, yeast expression systems appear to be effective for producing recombinant Ovarian jelly-peptide 1. For instance, recombinant Ovarian jelly-peptide 3 has been successfully produced in yeast systems. When designing an expression system for recombinant OJP-1, researchers should consider incorporating appropriate purification tags that can be cleaved without affecting the native peptide sequence. The small size of OJP-1 (7 amino acids) presents unique challenges, as fusion proteins may be necessary to achieve adequate expression levels. Researchers should validate the final product using a combination of mass spectrometry and bioactivity assays to ensure structural and functional equivalence to the native peptide.

How does the gelation property of recombinant Ovarian jelly-peptide 1 compare to native peptide?

The gelation property of recombinant Ovarian jelly-peptide 1 should theoretically match that of the native peptide if the structural integrity is preserved during the recombinant production process. Native OJPs form a jelly when resuspended in water, a property that likely influences their diffusion kinetics in the marine environment. When working with recombinant OJP-1, researchers should verify this gelation capability as a quality control measure. Factors that may influence gelation include peptide concentration, pH, temperature, and the presence of dissolved salts. Quantitative rheological measurements can be used to compare the viscoelastic properties of native and recombinant peptide gels, providing a metric for functional equivalence.

What analytical methods are best for verifying the structural integrity of recombinant Ovarian jelly-peptide 1?

Multiple complementary analytical techniques should be employed to verify the structural integrity of recombinant Ovarian jelly-peptide 1:

• Mass Spectrometry (MS/MS): Provides precise molecular weight and can confirm the amino acid sequence.

• Edman Degradation: Useful for N-terminal sequencing verification.

• Acid Hydrolysis: Helps determine amino acid composition.

• HPLC Analysis: Confirms purity and can be compared with elution profiles of native peptide.

• Circular Dichroism (CD): Can provide information about secondary structural elements.

These methods have been successfully applied to characterize native OJPs in previous studies and should be equally applicable to recombinant versions. A combination of these techniques provides comprehensive structural verification and increases confidence in the recombinant product's authenticity.

How can recombinant Ovarian jelly-peptide 1 be labeled for tracking in experimental systems?

For tracking recombinant Ovarian jelly-peptide 1 in experimental systems, several labeling strategies can be employed:

• Fluorescent Labeling: Conjugation with fluorophores at the N-terminus, as the C-terminus may be more critical for biological activity based on sequence conservation patterns in the OJP family.

• Isotopic Labeling: Incorporation of stable isotopes (13C, 15N) during recombinant expression for mass spectrometry tracking.

• Biotin Tagging: Addition of biotin for detection with streptavidin-conjugated reporters.

• Radioactive Labeling: Incorporation of radioactive amino acids for sensitive detection.

When designing labeled OJP-1, researchers should validate that the label does not interfere with the peptide's biological activity or gelation properties. Control experiments comparing labeled and unlabeled peptides are essential to ensure that experimental observations reflect genuine biological phenomena rather than artifacts of the labeling process.

What are the challenges in maintaining recombinant Ovarian jelly-peptide 1 stability?

Maintaining stability of recombinant Ovarian jelly-peptide 1 presents several challenges that researchers must address:

• Storage Conditions: Based on protocols for similar peptides, OJP-1 likely requires storage at -20°C or -80°C for extended periods.

• Freeze-Thaw Cycles: Repeated freezing and thawing should be avoided, as this can lead to degradation and loss of activity.

• Glycerol Addition: Adding glycerol (typically 5-50% final concentration) helps maintain stability during storage.

• Aliquoting: Working aliquots should be prepared to minimize freeze-thaw cycles.

• pH Sensitivity: The peptide may have optimal stability at specific pH ranges that should be determined empirically.

For lyophilized peptide, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, followed by appropriate glycerol addition. Shelf life is approximately 6 months for liquid form at -20°C/-80°C and 12 months for lyophilized form.

What protocols should be followed for reconstitution of lyophilized recombinant Ovarian jelly-peptide 1?

Based on established protocols for similar peptides in the OJP family, the following reconstitution protocol is recommended for lyophilized recombinant Ovarian jelly-peptide 1:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom.

• Reconstitute the peptide in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (with 50% being a common standard).

• Prepare multiple small aliquots to avoid repeated freeze-thaw cycles.

• Store reconstituted aliquots at -20°C or -80°C for long-term storage, or at 4°C for up to one week for working solutions.

When reconstituting the peptide, researchers should be aware that the gelation property of OJPs may affect dissolution rates and homogeneity. Gentle agitation rather than vigorous mixing is recommended to preserve peptide integrity while ensuring complete reconstitution.

How can researchers measure the bioactivity of recombinant Ovarian jelly-peptide 1?

The bioactivity of recombinant Ovarian jelly-peptide 1 can be measured using several approaches based on its known physiological functions:

• Myotropic Bioassay: The primary functional assay involves measuring contractions of isolated female genital tract and nidamental gland tissue from Sepia officinalis in response to the peptide. This can be quantified by measuring the frequency, amplitude, and duration of tissue contractions.

• Receptor Binding Assays: If receptor targets have been identified, competitive binding assays can assess the affinity of recombinant OJP-1 compared to the native peptide.

• Cell-Based Assays: Cellular responses in reproductive tissue cultures can be monitored following peptide application, looking at calcium signaling or other second messenger systems.

• Gelation Kinetics: Quantitative assessment of the peptide's ability to form a jelly in aqueous solution can serve as a functional characterization metric.

Results should be compared with those obtained using native peptide or synthetic standards to validate bioequivalence.

What controls should be included in experiments using recombinant Ovarian jelly-peptide 1?

Robust experimental design with recombinant Ovarian jelly-peptide 1 should include the following controls:

• Negative Controls:

  • Buffer-only treatments to establish baseline responses

  • Scrambled peptide sequences with the same amino acid composition but different order

  • Heat-denatured OJP-1 to confirm that activity depends on structural integrity

• Positive Controls:

  • Native OJP-1 isolated from Sepia officinalis (if available)

  • Synthetic OJP-1 prepared by solid-phase peptide synthesis

  • Known myotropic agents for reproductive tissue (when assessing contractile responses)

• Specificity Controls:

  • Other OJP family members (OJP-2, OJP-3) to assess family-specific versus peptide-specific effects

  • Dose-response curves to establish concentration-dependent effects

• Validation Controls:

  • Inhibitors of expected signaling pathways

  • Receptor antagonists if receptors have been identified

How should researchers design experiments to study the myotropic effects of Ovarian jelly-peptide 1?

To study the myotropic effects of Ovarian jelly-peptide 1, researchers should consider the following experimental design elements:

• Tissue Preparation:

  • Isolate segments of female genital tract and main nidamental gland from Sepia officinalis.

  • Maintain tissues in appropriate physiological saline that mimics cephalopod hemolymph.

  • Mount tissues in organ baths equipped with force transducers to measure contractile responses.

• Treatment Protocol:

  • Establish stable baseline contractions before peptide application.

  • Apply recombinant OJP-1 in a concentration range (typically 10^-9 to 10^-6 M).

  • Record responses for sufficient duration to capture both immediate and delayed effects.

• Data Collection:

  • Measure contraction frequency, amplitude, and duration.

  • Quantify area under the curve for integrated response assessment.

  • Document latency to response and recovery dynamics.

• Analysis:

  • Compare responses across different reproductive stages if possible.

  • Analyze dose-dependency of responses.

  • Investigate potential interactions with other reproductive peptides.

This approach has been successfully used to characterize the myotropic effects of native OJPs and should be applicable to recombinant versions.

What approaches can be used to study the receptor interactions of Ovarian jelly-peptide 1?

Several approaches can be employed to study receptor interactions of Ovarian jelly-peptide 1:

• Receptor Binding Assays:

  • Prepare membrane fractions from female reproductive tissues.

  • Use radiolabeled or fluorescently labeled OJP-1 to measure specific binding.

  • Perform competition assays with unlabeled peptide to determine binding affinity (Kd values).

• Signal Transduction Analysis:

  • Monitor second messenger systems (Ca²⁺, cAMP, IP3) in response to OJP-1 application.

  • Use specific inhibitors to identify involved signaling pathways.

  • Employ calcium imaging techniques in tissue preparations or cell cultures.

• Molecular Approaches:

  • Identify candidate receptors through bioinformatic analysis of the Sepia officinalis genome/transcriptome.

  • Express candidate receptors in heterologous systems to confirm binding.

  • Use receptor knockdown approaches (if available) to validate physiological relevance.

• Cross-linking Studies:

  • Use photo-activatable cross-linkers attached to OJP-1 to capture receptor interactions.

  • Identify bound proteins through mass spectrometry.

These approaches would provide complementary information about the molecular targets and mechanisms of action of OJP-1.

How does Ovarian jelly-peptide 1 compare to other reproductive peptides in marine invertebrates?

Ovarian jelly-peptide 1 represents a distinct family of reproductive peptides compared to other known reproductive peptides in marine invertebrates. Unlike many water-borne pheromones that primarily function in mate attraction, OJP-1 appears to have direct myotropic effects on the reproductive tract, suggesting a more mechanistic role in the reproductive process itself. The gelation property of OJP-1 is relatively unusual among reproductive peptides and may represent a specialized adaptation for controlled release in the marine environment.

When compared to other cephalopod reproductive peptides such as SepCRPs (Sepia Capsule Releasing Peptides), OJP-1 has a different structure and localization pattern. SepCRPs contain a conserved SLXKD motif and are involved in egg capsule secretion, while OJP-1 has the sequence DQVKIVL and modulates contractions in the female genital tract. This suggests a division of regulatory functions among different peptide families during the complex process of cephalopod reproduction.

What insights can Ovarian jelly-peptide 1 provide about cephalopod reproductive physiology?

Ovarian jelly-peptide 1 provides several important insights into cephalopod reproductive physiology:

• Compartmentalized Regulation: The restricted localization of OJP-1 to the female genital tract suggests highly specialized and compartmentalized regulatory mechanisms in cephalopod reproduction.

• Oocyte-Derived Signaling: As OJP-1 is released by full-grown oocytes, it represents a form of oocyte-to-reproductive tract communication, highlighting the active role of gametes in coordinating their own transport and processing.

• Contractile Regulation: The myotropic effects of OJP-1 on the female genital tract indicate precise control of mechanical aspects of reproduction, potentially coordinating oocyte movement, fertilization, and egg-laying processes.

• Evolutionary Adaptations: The jelly-forming property suggests adaptations for controlled release of regulatory substances in the aquatic environment, which may be crucial for reproductive success in cephalopods.

These insights contribute to our understanding of the sophisticated regulatory networks that coordinate reproduction in these complex invertebrates.

How might recombinant Ovarian jelly-peptide 1 be used in reproductive biology research?

Recombinant Ovarian jelly-peptide 1 offers several valuable applications in reproductive biology research:

• Comparative Reproductive Physiology: As a tool to investigate similarities and differences in reproductive regulatory mechanisms across cephalopod species and other marine invertebrates.

• Receptor Characterization: To identify and characterize receptors in reproductive tissues, potentially revealing new targets for reproductive manipulation or conservation efforts.

• Evolutionary Studies: To examine the conservation and divergence of reproductive peptides across related species, providing insights into evolutionary adaptations in reproductive strategies.

• Biomimetic Applications: The unique gelation properties could inspire development of controlled-release systems for marine applications or biotechnology.

• Aquaculture Research: Potential applications in improving reproductive success in commercially important cephalopod species by manipulating reproductive processes.

These applications highlight the value of recombinant OJP-1 as both a research tool and a model for understanding peptide-mediated reproductive regulation.

What is the evolutionary significance of Ovarian jelly-peptide 1?

The evolutionary significance of Ovarian jelly-peptide 1 can be examined from several perspectives:

• Peptide Conservation: The identification of related peptides within the OJP family (OJP-1, OJP-2, OJP-3) with conserved C-terminal regions suggests evolutionary pressure to maintain specific functional domains while allowing some N-terminal variation.

• Reproductive Adaptation: The specialized gelation property likely represents an adaptation to the marine environment, allowing for controlled diffusion of regulatory peptides in aqueous media.

• Coordination of Reproduction: The evolution of oocyte-derived peptides that regulate reproductive tract function represents a sophisticated control mechanism that may have contributed to the reproductive success of cephalopods.

• Cephalopod-Specific Innovation: As these peptides appear to be specific to cephalopods, they may represent lineage-specific innovations that contributed to the unique reproductive strategies of these advanced mollusks.

Comparative studies with other marine invertebrates would provide further insights into the evolutionary history and significance of these specialized reproductive peptides.

How does the function of Ovarian jelly-peptide 1 relate to SepCRPs in Sepia officinalis?

Ovarian jelly-peptide 1 and SepCRPs (Sepia Capsule Releasing Peptides) appear to be complementary systems that regulate different aspects of reproduction in Sepia officinalis:

• Structural Differences: OJP-1 (DQVKIVL) and SepCRPs (containing the SLXKD motif) have distinct primary structures, suggesting they evolved independently to serve different functions.

• Temporal Regulation: While both peptide families are involved in egg-laying processes, they may act at different temporal stages. SepCRPs are described as being involved in egg capsule secretion, while OJPs modulate contractions of the female genital tract and may be involved in gamete transport or preparation for fertilization.

• Coordinated Action: Together, these peptide families likely form part of a complex regulatory network that coordinates the multiple processes involved in successful reproduction, from gamete transport to egg encapsulation.

• Source Tissues: Both peptide families are expressed in the female reproductive system but may have different cellular origins, with OJPs being released by full-grown oocytes and SepCRPs potentially derived from other reproductive tissues.

This relationship highlights the complexity of peptidergic regulation in cephalopod reproduction, with multiple specialized peptide families controlling distinct but complementary processes.

How can researchers address difficulties in obtaining consistent gelation with recombinant Ovarian jelly-peptide 1?

Inconsistent gelation of recombinant Ovarian jelly-peptide 1 may be addressed through several approaches:

• Purity Assessment: Ensure peptide purity exceeds 85% using SDS-PAGE or HPLC analysis. Contaminants may interfere with proper self-assembly and gelation.

• Concentration Optimization: Systematically test a range of peptide concentrations (e.g., 0.1-5.0 mg/mL) to identify the critical concentration required for consistent gelation.

• Buffer Composition: Examine the effects of different ionic strengths, pH values, and buffer compositions on gelation properties. Marine-mimicking buffers may provide more native-like conditions.

• Temperature Effects: Investigate gelation behavior at different temperatures, as temperature can significantly affect peptide self-assembly kinetics.

• Aging Time: Allow sufficient time for gel formation and maturation, as some peptide gels require extended periods to reach equilibrium structures.

• Mechanical Influences: Minimize mechanical disturbances during gelation, as shear forces can disrupt nascent gel networks.

Systematic documentation of conditions that produce consistent results will help establish reliable protocols for future experiments.

What are the known limitations of working with recombinant Ovarian jelly-peptide 1?

Researchers working with recombinant Ovarian jelly-peptide 1 should be aware of several limitations:

• Structural Authenticity: Ensuring that recombinant versions maintain the same folding and self-assembly properties as native peptides can be challenging, particularly regarding the gelation property.

• Post-translational Modifications: Native OJPs may have undiscovered post-translational modifications that are absent in recombinant versions, potentially affecting function.

• Storage Stability: Small peptides are often susceptible to degradation, requiring careful storage conditions and potentially limiting shelf life.

• Batch Variability: Production inconsistencies between batches may affect experimental reproducibility, necessitating rigorous quality control.

• Physiological Relevance: In vitro behavior may not fully recapitulate in vivo functionality, particularly regarding concentration-dependent effects and complex tissue interactions.

• Species Specificity: Results obtained with Sepia officinalis OJP-1 may not be generalizable to other cephalopod species without validation.

Understanding these limitations is crucial for designing robust experiments and interpreting results accurately.

How can researchers validate that recombinant Ovarian jelly-peptide 1 has native-like properties?

To validate that recombinant Ovarian jelly-peptide 1 possesses native-like properties, researchers should employ a multi-faceted approach:

• Structural Comparison:

  • Compare mass spectrometry profiles between native and recombinant peptides

  • Analyze circular dichroism spectra to assess secondary structure similarities

  • Compare HPLC elution profiles

• Functional Bioassays:

  • Conduct parallel myotropic assays with native and recombinant peptides at multiple concentrations

  • Compare dose-response curves for statistical equivalence

  • Measure EC50 values for both peptide forms

• Physical Properties:

  • Compare gelation kinetics and rheological properties of the resulting gels

  • Assess temperature and pH dependencies of gelation

  • Examine gel microstructure using microscopy techniques

• Biological Distribution:

  • Use labeled peptides to compare tissue distribution patterns

  • Assess receptor binding profiles

Statistical comparison of these parameters between native and recombinant peptides provides quantitative validation of biological equivalence.

What are the key considerations when designing peptide analogs of Ovarian jelly-peptide 1?

When designing peptide analogs of Ovarian jelly-peptide 1 for structure-function studies, researchers should consider:

• Sequence Conservation: The C-terminal region (KIVL) is conserved across the OJP family, suggesting functional importance. Modifications in this region should be strategic and conservative.

• N-terminal Variability: The N-terminus shows natural variation within the OJP family (DQ vs. DE), suggesting this region may tolerate modifications while maintaining some functionality.

• Gelation Properties: Changes that might affect the peptide's ability to form a jelly in water should be carefully considered, as this property may be essential for proper function.

• Charge Distribution: The peptide contains charged residues (D, K) that likely contribute to both folding and receptor interactions. Charge-preserving substitutions may be less disruptive.

• Secondary Structure Propensities: Modifications that dramatically alter predicted secondary structure should be avoided unless specifically testing structural hypotheses.

• Bioactivity Prediction: In silico modeling based on known structure-activity relationships of related peptides can guide rational design.

A systematic alanine scan (replacing each residue with alanine) provides a foundation for understanding the contribution of individual amino acids to function.

How can researchers investigate potential synergistic effects between Ovarian jelly-peptide 1 and other OJPs?

To investigate potential synergistic effects between Ovarian jelly-peptide 1 and other members of the OJP family, researchers could employ the following approaches:

• Combination Studies:

  • Test OJP-1 alone, other OJPs alone, and defined combinations at various ratios

  • Construct isobolograms to quantitatively assess synergy, additivity, or antagonism

  • Compare effects of sequential versus simultaneous application

• Physiological Measurements:

  • Monitor multiple parameters (contraction frequency, amplitude, duration) to detect synergistic effects on different aspects of tissue response

  • Measure concentration-response relationships for combinations versus individual peptides

• Biochemical Approaches:

  • Investigate receptor occupancy with competitive binding assays using combinations of peptides

  • Examine downstream signaling pathway activation with peptide combinations

  • Assess calcium mobilization patterns in response to peptide combinations

• Statistical Analysis:

  • Apply formal mathematical models of drug synergy to peptide combination data

  • Use three-dimensional response surface methodology to comprehensively map interaction effects