Recombinant Pelophylax ridibundus Secretogranin-2, partial

How does the molecular structure of Pelophylax ridibundus Secretogranin-2 compare to that in other vertebrate species?

While comprehensive structural analyses of P. ridibundus SgII are not fully documented in current literature, comparative analysis with other vertebrates reveals important insights. SgII typically contains:

• A signal peptide directing it to the secretory pathway

• Multiple dibasic cleavage sites that enable processing into bioactive peptides

• Conserved regions that give rise to secretoneurin, a biologically active neuropeptide

• Domains involved in sorting to regulated secretory pathways

In zebrafish, two paralogues (SgIIa and SgIIb) have been identified, with mutation of both genes resulting in severe reproductive deficits . The Pelophylax genus, with its complex evolutionary history and hybridization events, may similarly possess multiple SgII variants with potentially overlapping functions. The "partial" designation in recombinant P. ridibundus SgII likely indicates work with a specific functional domain rather than the complete protein.

Differential expression patterns of secretogranins observed in chromaffin cells suggest functional specialization among these proteins . For example, some VMAT2 (vesicular monoamine transporter 2)-expressing cells show strong expression of SgIII but negative or hardly detectable SgII expression, indicating non-redundant roles . This differentiation may also exist in amphibian tissues, though direct comparative studies are needed.

What methodological approaches are required for initial characterization of recombinant partial Pelophylax ridibundus Secretogranin-2?

Initial characterization of recombinant partial P. ridibundus SgII should follow a systematic multi-technique approach:

• Sequence verification and mapping:

  • Mass spectrometry to confirm molecular weight and sequence integrity

  • Peptide mapping to identify which domain(s) of the full-length protein are represented

  • N-terminal sequencing to verify proper processing of fusion tags

• Structural analysis:

  • Circular dichroism spectroscopy to assess secondary structure elements

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine oligomeric state

  • Thermal stability assessment using differential scanning fluorimetry

• Functional characterization:

  • Immunolocalization studies to verify subcellular targeting properties

  • Co-immunoprecipitation to identify binding partners

  • Hormone release assays, particularly focusing on luteinizing hormone stimulation capacity

• Cross-species comparative analysis:

  • Alignment with known SgII sequences from related species

  • Testing cross-reactivity with antibodies against SgII from other vertebrates

  • Comparative functional assays with SgII from model organisms like Xenopus

The differential expression patterns observed between SgII and SgIII in adrenal chromaffin cells suggest that it would be valuable to examine whether the partial recombinant P. ridibundus SgII shows similar cell type-specific localization patterns in amphibian tissues.

What expression systems yield the highest functional activity for recombinant Pelophylax ridibundus Secretogranin-2?

The choice of expression system critically impacts the functional activity of recombinant P. ridibundus SgII. Based on research with related secretory proteins, the following systems offer distinct advantages and limitations:

For the partial P. ridibundus SgII, the optimal system depends on which region is being expressed:

• If the partial protein corresponds to the secretoneurin domain (which is typically not heavily glycosylated), E. coli expression may be sufficient .

• If the protein contains regions requiring specific folding or post-translational modifications for activity, mammalian or insect cell systems are preferable.

When using non-mammalian systems, codon optimization for the expression host should be performed, particularly given the potential for rare codons in amphibian sequences.

What purification challenges are specific to Pelophylax secretogranins and how can they be addressed?

Purification of recombinant P. ridibundus SgII presents several challenges stemming from the protein's biochemical properties and amphibian-specific characteristics:

• Aggregation tendency:

  • Challenge: Secretogranins have regions promoting protein-protein interactions

  • Solution: Include mild detergents (0.01-0.05% Tween-20) in buffers; maintain lower protein concentrations; consider arginine (50-200 mM) as a stabilizing agent

• Proteolytic sensitivity:

  • Challenge: Multiple dibasic sites make SgII susceptible to proteolytic cleavage during purification

  • Solution: Include protease inhibitor cocktails; maintain cooler temperatures (4°C); minimize purification duration

• Variable charge distribution:

  • Challenge: Distinct acidic and basic regions affect ion exchange chromatography performance

  • Solution: Implement gradient elution protocols; test multiple pH conditions (typically pH 6.0-7.5)

• Species-specific post-translational modifications:

  • Challenge: Amphibian-specific modifications may affect protein behavior

  • Solution: Use multiple orthogonal purification techniques (affinity, ion exchange, size exclusion)

A recommended multi-step purification strategy would include:

• Affinity chromatography using an appropriate tag (His, GST, or MBP)

• Ion exchange chromatography optimized for the specific pH range

• Size exclusion chromatography as a final polishing step

• Quality control by SDS-PAGE, Western blotting, and mass spectrometry

Researchers should be particularly attentive to differential expression patterns observed between SgII and other secretogranins , as these may reflect biochemical differences that could affect purification behavior.

What analytical methods best verify the structural integrity of purified recombinant Pelophylax ridibundus Secretogranin-2?

Verifying the structural integrity of purified recombinant P. ridibundus SgII requires a multi-layered analytical approach that addresses primary sequence, folding, and functional capacity:

• Primary structure verification:

  • Liquid chromatography-mass spectrometry (LC-MS) to confirm molecular weight

  • Peptide mapping with LC-MS/MS after proteolytic digestion to verify sequence coverage

  • Amino acid analysis to confirm composition

• Secondary and tertiary structure analysis:

  • Circular dichroism (CD) spectroscopy to assess α-helical and β-sheet content

  • Intrinsic fluorescence spectroscopy to evaluate tertiary structure if tryptophan residues are present

  • Limited proteolysis coupled with mass spectrometry to identify accessible regions

• Homogeneity assessment:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

  • Analytical ultracentrifugation to verify monodispersity

  • Dynamic light scattering to detect aggregation

• Functional verification:

  • Binding assays with known interaction partners

  • Hormone release assays in relevant cell types, particularly testing for luteinizing hormone stimulation

  • Comparison with synthetic secretoneurin peptide activity

Given the differential expression patterns observed in secretogranins across adrenal chromaffin cells , it would be valuable to develop assays that specifically interrogate the function of the partial recombinant protein in relation to these expression domains.

What cellular assays are most informative for studying Pelophylax ridibundus Secretogranin-2 function?

Based on current understanding of secretogranin biology, the following cellular assays would provide the most informative insights into P. ridibundus SgII function:

• Secretory granule biogenesis assays:

  • Transfection of fluorescently tagged SgII into neuroendocrine cell lines

  • Quantification of secretory granule formation efficiency

  • Co-localization studies with other granule markers

• Hormone secretion assays:

  • Primary pituitary cell cultures or appropriate cell lines

  • Measurement of luteinizing hormone release in response to recombinant SgII or derived peptides

  • Calcium imaging to detect secretagogue activity

• Vesicular transport assessment:

  • Co-localization studies with VMAT2, which shows differential association with secretogranins

  • Live-cell imaging of vesicle trafficking with fluorescently tagged SgII

  • Evaluating effects on catecholamine uptake and storage

• Reproductive cell function assays:

  • Effects on gamete maturation in vitro

  • Analysis of impact on reproductive hormone receptor expression

  • Sperm motility or egg activation studies

The observation that SgII is differentially expressed in chromaffin cells, with some VMAT2-positive cells showing negative or minimal SgII expression , suggests that cell type-specific assays may be particularly informative. Additionally, the zebrafish study demonstrating severe reproductive deficits in SgII double mutants indicates that reproductive cell assays should be prioritized.

How do functional properties of partial Pelophylax ridibundus Secretogranin-2 compare to the full-length protein?

The functional comparison between partial and full-length P. ridibundus SgII depends critically on which domain(s) are represented in the partial protein. Based on findings from related systems:

Research in zebrafish has demonstrated that injection of synthetic secretoneurin peptide enhanced reproductive outcomes in SgII-deficient fish , suggesting that this domain retains significant biological activity independent of the full protein. Similarly, differential expression patterns of SgII and SgIII in chromaffin cells indicate that specific domains may have cell type-specific functions.

For comprehensive functional characterization, researchers should:

• Precisely map the partial protein relative to the full-length sequence

• Compare activity profiles with synthetic peptides representing known bioactive regions

• Evaluate both cellular localization and physiological effects

What are the main technical challenges in working with recombinant Pelophylax ridibundus Secretogranin-2?

Researchers working with recombinant P. ridibundus SgII face several technical challenges that must be addressed through careful experimental design:

• Species-specific sequence variation:

  • Challenge: Limited reference genome data for Pelophylax species

  • Solution: Perform transcriptome sequencing to accurately identify SgII variants; consider the complex hybridogenetic nature of Pelophylax frogs

• Protein stability issues:

  • Challenge: Tendency toward aggregation common in secretory proteins

  • Solution: Optimize buffer conditions (pH 6.0-7.0, 150-300 mM NaCl, 5-10% glycerol); incorporate stabilizing agents; avoid freeze-thaw cycles

• Post-translational processing:

  • Challenge: Amphibian-specific processing may differ from mammalian systems

  • Solution: Analyze natural processing products in P. ridibundus tissues; compare with other amphibian species

• Functional assay limitations:

  • Challenge: Lack of validated amphibian-specific assay systems

  • Solution: Develop heterologous assays using appropriate cell types; validate with positive controls from related species

• Antibody cross-reactivity:

  • Challenge: Limited availability of Pelophylax-specific antibodies

  • Solution: Generate custom antibodies against recombinant protein; validate across tissues with appropriate controls

The differential expression patterns of secretogranins observed in adrenal chromaffin cells highlight the importance of developing cell type-specific assays. Additionally, researchers should account for potential redundancy between multiple SgII genes, as observed in zebrafish where the most severe reproductive phenotypes occurred only when both genes were mutated .

How can researchers address contradictory findings when comparing Secretogranin-2 function across species?

Resolving contradictory findings regarding SgII function across species requires systematic approaches that account for evolutionary, methodological, and biological variables:

• Evolutionary context integration:

  • Map phylogenetic relationships between study species

  • Account for gene duplication events (as seen with SgIIa and SgIIb in zebrafish)

  • Consider hybrid genomes in Pelophylax species which may affect gene expression and function

• Standardized methodological framework:

  • Develop consistent experimental protocols across species

  • Use identical recombinant protein production methods when possible

  • Apply uniform functional readouts and quantification approaches

• Tissue and cell-specific analysis:

  • Compare equivalent tissues and cell types

  • Account for differential expression patterns as seen in chromaffin cells

  • Consider developmental and seasonal variations

• Multi-omics data integration:

  • Combine transcriptomic, proteomic, and functional data

  • Identify species-specific regulatory networks

  • Map post-translational processing differences

• Receptor and signaling pathway comparison:

  • Characterize species differences in receptors for SgII-derived peptides

  • Examine conservation of downstream signaling mechanisms

  • Evaluate cofactor requirements across species

The observation that SgII and SgIII have distinct expression patterns in adrenal chromaffin cells highlights the importance of considering protein function in the context of specific cellular environments. Similarly, the complex breeding systems observed in Pelophylax water frogs suggest that reproductive functions of SgII may have species-specific adaptations related to these unique reproductive strategies.

What experimental design considerations are essential when studying the effects of Secretogranin-2 on reproductive function?

Based on current knowledge of SgII's role in reproduction, particularly from zebrafish studies , the following experimental design considerations are essential:

• Comprehensive phenotyping approach:

  • Behavioral assessments (particularly male courtship behaviors)

  • Physiological measurements (hormone levels, particularly luteinizing hormone)

  • Reproductive outcomes (spawning success, fertility rates, embryo survival)

  • Molecular readouts (gene expression changes in reproductive tissues)

• Temporal considerations:

  • Account for seasonal breeding cycles in amphibians

  • Evaluate effects at multiple timepoints in the reproductive cycle

  • Consider both acute and chronic SgII administration

• Dose-response relationships:

  • Test multiple concentrations of recombinant protein or derived peptides

  • Include appropriate vehicle controls

  • Consider route of administration (injection, immersion for aquatic species)

• Sex-specific analyses:

  • Evaluate males and females separately

  • Consider sexually dimorphic responses to SgII

  • Examine effects on both gamete quality and quantity

• Genetic background considerations:

  • Account for the complex hybridogenetic nature of Pelophylax species

  • Consider potential variation in L-E breeding systems

  • Control for ploidy differences that may exist in hybrid populations

• Rescue experiment design:

  • Based on zebrafish studies, include secretoneurin administration

  • Test both preventive and reversal paradigms

  • Include appropriate timing controls

The zebrafish study showing that SgII mutation reduced spawning success from 62% to 6% in double mutants provides a valuable benchmark for expected effect sizes. Additionally, the observation that secretoneurin injection enhanced reproductive outcomes in mutant fish suggests a direct approach for rescue experiments.

How can CRISPR-Cas9 genome editing be optimized for studying Secretogranin-2 function in Pelophylax species?

CRISPR-Cas9 genome editing offers powerful approaches to study SgII function in Pelophylax species, though application in amphibians presents unique challenges:

• Target design considerations:

  • Identify conserved functional domains across secretogranin family

  • Design multiple guide RNAs to account for potential paralogs (as seen in zebrafish)

  • Target early exons to ensure complete loss of function

  • Account for the complex genetics of Pelophylax water frogs

• Delivery optimization:

  • Microinjection protocols for fertilized eggs

  • Adjust injection volume for larger amphibian eggs

  • Consider electroporation for tissue-specific editing in adults

  • Time interventions according to seasonal breeding cycles

• Mutation validation strategy:

  • Develop specific PCR genotyping assays

  • Use T7 endonuclease assay for initial screening

  • Confirm mutations by sequencing

  • Validate at protein level with specific antibodies

• Phenotypic characterization framework:

  • Focus on reproductive parameters identified in zebrafish models :

    • Sexual behavior patterns

    • Spawning success rates

    • Fertility metrics

    • Embryo survival

  • Add amphibian-specific measures related to breeding systems

• Rescue experiment design:

  • Test synthetic secretoneurin administration as demonstrated in zebrafish

  • Evaluate timing requirements for effective rescue

  • Document dose-response relationships

The hybridogenetic nature of many Pelophylax species adds complexity to genome editing approaches, requiring careful genetic analysis and potentially ploidy assessment. Additionally, the differential expression patterns of secretogranins observed in adrenal tissues suggest that tissue-specific editing approaches may be particularly valuable for dissecting cell type-specific functions.

What innovative approaches can uncover the molecular mechanisms linking Secretogranin-2 to luteinizing hormone release?

Uncovering the molecular mechanisms connecting SgII to luteinizing hormone release requires innovative approaches that bridge cellular, molecular, and physiological perspectives:

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize secretory granule dynamics

  • Correlative light and electron microscopy to connect function with ultrastructure

  • Calcium imaging to track secretory responses in real-time

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify proximal interactors of SgII

  • Temporal control of labeling to capture dynamic interactions

  • Cell type-specific expression to map tissue-relevant interactomes

• Single-cell multi-omics:

  • Single-cell RNA sequencing of pituitary cells responding to secretoneurin

  • Spatial transcriptomics to map response gradients

  • Integrated analysis with proteomics data

• Optogenetic and chemogenetic tools:

  • Targeted manipulation of SgII-expressing neurons

  • Temporal control of secretoneurin release

  • Monitoring downstream effects on luteinizing hormone secretion

• Receptor identification strategies:

  • Crosslinking mass spectrometry to identify binding partners

  • Affinity purification with biotinylated secretoneurin

  • Functional screening using CRISPR activation/interference libraries

The observation that SgII shows differential expression with VMAT2 in chromaffin cells suggests potential mechanistic links with monoamine signaling that could be further explored. Additionally, the severe reproductive phenotypes observed in zebrafish double mutants provide a valuable model system for validating mechanisms identified through these approaches.

How do environmental factors influence Secretogranin-2 expression and function in amphibian reproductive cycles?

Understanding environmental influences on SgII expression and function in amphibian reproductive cycles requires integrated approaches spanning molecular, physiological, and ecological perspectives:

• Seasonal expression profiling:

  • Longitudinal sampling across annual breeding cycles

  • Quantification of SgII gene expression and protein levels

  • Correlation with environmental parameters (temperature, photoperiod, rainfall)

  • Assessment of secretoneurin production and processing

• Controlled environmental manipulation:

  • Temperature cycling experiments to mimic seasonal changes

  • Photoperiod manipulation studies

  • Water chemistry alterations relevant to amphibian habitats

  • Combined stressor approaches reflecting climate change scenarios

• Field-laboratory integration:

  • Comparison of wild-caught and laboratory-raised specimens

  • Translocation experiments between different habitat types

  • Non-invasive hormone sampling from natural populations

  • Correlation with breeding success metrics

• Molecular response mechanisms:

  • Promoter analysis for environmental response elements

  • Epigenetic profiling across seasonal changes

  • Identification of temperature-sensitive RNA structures

  • Characterization of stress-responsive post-translational modifications

The complex breeding systems observed in Pelophylax water frogs , including hybridogenetic reproduction and modified L-E systems, represent unique adaptations to environmental niches. Understanding how SgII function contributes to these reproductive strategies under varying environmental conditions may provide insights into amphibian adaptation and resilience to environmental change.

The hybridogenetic nature of many Pelophylax species suggests that environmental factors might differently affect SgII expression from maternal versus paternal genomes, particularly in triploid individuals. This adds another layer of complexity to environmental response studies in these amphibians.