Recombinant Lymnaea stagnalis Lymna-DF-amide 1

What is the chemical structure of Lymna-DF-amide 1?

Lymna-DF-amide 1 belongs to a family of tridecapeptides with the sequence Pro-Tyr-Asp-Arg-Ile-Ser-Asn-Ser-Ala-Phe-Ser-Asp-Phe.NH2. It is characterized by the presence of tyrosine (Tyr) at the second position and asparagine (Asn) at the seventh position of the peptide chain. The peptide is C-terminally amidated, which is essential for its biological activity . This structure is part of the broader lymnaDFamide family that shares the C-terminal Asp-Phe-amide motif with mammalian cholecystokinin (CCK) and gastrin .

How is Lymna-DF-amide 1 detected in neuronal tissues?

Lymna-DF-amide 1 was originally detected using antisera that recognize the biologically active C-termini of cholecystokinin and gastrin . For modern research purposes, multiple complementary techniques are recommended for comprehensive detection:

• Immunohistochemistry: Using antibodies specific to the Asp-Phe-amide terminus, though researchers should be cautious about cross-reactivity with other neuropeptides .

• MALDI-MS (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry): This technique has dramatically decreased the sample amount required for peptide analysis and allows precise molecular identification at varying organizational levels, from whole central nervous system (CNS) to single neurons .

• MALDI-MSI (MALDI-Mass Spectrometry Imaging): Enables visualization of neuropeptide distribution across tissues with high spatial resolution .

When using immunological methods, validation through peptide sequencing or mass spectrometry is strongly recommended to confirm specificity, as antibodies raised against human proteins may show non-specific binding in mollusk tissues .

Where is Lymna-DF-amide 1 localized in Lymnaea stagnalis?

Lymna-DF-amide 1 is primarily expressed in the central nervous system of L. stagnalis. Specifically, it can be found in:

• Multiple ganglia of the CNS, with varying expression patterns

• Specific neuron clusters involved in feeding behavior regulation

• Select neurons within the buccal ganglia

MALDI-MSI analysis has allowed detailed mapping of various neuropeptides, including members of the lymnaDFamide family, across the CNS of L. stagnalis, revealing distinct localization patterns that correspond to functional neural circuits . The expression pattern suggests potential roles in feeding behavior modulation, similar to the functions of cholecystokinin in vertebrates .

What is the evolutionary significance of Lymna-DF-amide 1?

Lymna-DF-amide 1 belongs to a proposed Asp-Phe-amide superfamily of neuropeptides, which includes mammalian cholecystokinin and gastrin . This evolutionary relationship suggests that:

• The Asp-Phe-amide motif represents an ancient signaling mechanism conserved across diverse animal phyla

• The widespread CCK/gastrin immunoreactivity observed in invertebrates may be due to cross-reactivity with members of this superfamily rather than direct homologs

• Despite structural similarities, functional divergence has occurred, as evidenced by lymnaDFamide-1's lack of effect on trout gallbladder, which responds to both CCK and gastrin

This evolutionary conservation makes Lymna-DF-amide 1 valuable for comparative endocrinology studies examining the evolution of neuropeptide signaling systems across vertebrates and invertebrates.

What are the optimal expression systems for producing recombinant Lymna-DF-amide 1?

The choice of expression system depends on research requirements regarding yield, post-translational modifications, and functional considerations:

For basic structural studies and binding assays, E. coli or yeast expression systems provide the best balance of yield and time efficiency . For functional studies requiring proper folding and post-translational modifications, insect or mammalian cell expression is recommended despite lower yields .

How can researchers verify the functionality of recombinant Lymna-DF-amide 1?

Functional verification of recombinant Lymna-DF-amide 1 requires a multi-faceted approach:

• Mass spectrometric characterization: Confirmation of molecular weight and sequence integrity through techniques like MALDI-TOF MS or ESI-MS.

• Structural verification: Circular dichroism (CD) spectroscopy to assess secondary structure elements.

• Receptor binding assays: Using either:

  • Native tissue preparations from L. stagnalis CNS

  • Heterologous expression systems with cloned Lymnaea receptors

• Electrophysiological recordings: Patch-clamp techniques applied to identified neurons in L. stagnalis ganglia to assess the peptide's effects on membrane properties and synaptic transmission .

• Behavioral assays: Testing the recombinant peptide's effects on feeding behavior in L. stagnalis, as neuropeptides in this model organism have been linked to feeding circuit modulation .

Unlike the peptide identified in search result (GLTPNMNSLFF-NH2), which affects vas deferens contractions, Lymna-DF-amide 1 should be evaluated primarily for effects on feeding circuits based on its structural relationship to CCK/gastrin .

What methodological approaches can be used to study Lymna-DF-amide 1 effects on neuronal circuits?

L. stagnalis offers unique advantages for studying neuropeptide actions on neuronal circuits due to its identified neurons and well-characterized networks. The following methodological approaches are recommended:

• Single-cell MS analysis: Individual neurons (60 pL to 2 nL in volume) can be isolated and analyzed for neuropeptide content using MALDI-MS, providing cellular specificity .

• Subcellular MS imaging: Recent advances allow peptide localization analysis at the subcellular level, revealing compartmentalization of neuropeptides within neurons .

• Combined electrophysiology and peptide application: Intracellular recording from identified neurons during peptide application can determine acute effects on neuronal activity.

• Network analysis: Multi-electrode recordings from feeding, heart, or respiratory networks can assess circuit-level effects of Lymna-DF-amide 1 .

• Peptidogenomics approach: Integration of MS-based peptide discovery with genomic and transcriptomic data using techniques like MERFISH (multiplexed error-robust fluorescence in-situ hybridization) can identify cell types expressing receptors for Lymna-DF-amide 1 .

The heartbeat and feeding networks of L. stagnalis are particularly well-characterized and suitable for studying the modulatory effects of neuropeptides like Lymna-DF-amide 1 .

How does Lymna-DF-amide 1 interact with other neuropeptide systems in Lymnaea stagnalis?

Understanding peptide interactions requires investigation of co-localization, co-release, and functional interactions:

• Co-localization studies: MALDI-MSI can identify neurons containing multiple peptides, including Lymna-DF-amide 1 and other neuropeptides like FMRFamide, myomodulins, or SCPs (small cardioactive peptides) .

• Co-expression analysis: Single-cell transcriptomics combined with proteomics can reveal neurons expressing multiple peptide precursors.

• Receptor cross-talk: Biochemical and electrophysiological approaches can assess how Lymna-DF-amide 1 signaling interacts with other peptidergic systems at the receptor or second messenger level.

• Network integration: Computational modeling based on electrophysiological data can predict how multiple peptides, including Lymna-DF-amide 1, collectively modulate circuit output.

The feeding network in L. stagnalis is regulated by multiple neuropeptides, making it an excellent model for studying peptidergic co-modulation .

What CRISPR/Cas9 approaches can be employed to study Lymna-DF-amide 1 function in vivo?

CRISPR/Cas9 gene editing has been successfully applied to L. stagnalis , offering powerful approaches to study Lymna-DF-amide 1 function:

• Knockout of Lymna-DF-amide 1 precursor gene: Complete elimination of the peptide to assess loss-of-function phenotypes in development, feeding behavior, or neuronal activity.

• Targeted mutations in processing sites: Altering convertase recognition sites to disrupt specific peptide processing while preserving others derived from the same precursor.

• Reporter gene knock-in: Inserting fluorescent reporters to visualize peptide expression patterns with cellular resolution.

• Receptor modification: Editing putative Lymna-DF-amide 1 receptors to assess downstream signaling pathways.

As L. stagnalis is hermaphroditic and can both cross- and self-mate, genetic approaches are facilitated . This reproductive flexibility allows efficient propagation of genetic modifications and creation of stable transgenic lines for long-term studies of Lymna-DF-amide 1 function across generations.

How should researchers optimize extraction protocols for native Lymna-DF-amide 1?

Extraction of native Lymna-DF-amide 1 from L. stagnalis tissues requires careful optimization:

• Tissue preparation: Rapid dissection and flash-freezing of CNS tissue in liquid nitrogen to prevent peptide degradation.

• Homogenization and extraction:

  • Use acidified methanol (methanol:water:acetic acid 90:9:1) to efficiently extract peptides while inhibiting endogenous proteases

  • Sonication in ice-cold conditions to maintain peptide integrity

  • Multiple extraction cycles (minimum 3) to maximize yield

• Purification strategy:

  • Initial separation by C18 reverse-phase chromatography

  • Further purification by ion-exchange chromatography

  • Final purification by analytical HPLC

• Verification:

  • MALDI-TOF MS to confirm molecular weight

  • Tandem MS/MS for sequence verification

  • Comparison with synthetic standards

Yields can be improved by pooling tissue from multiple specimens, with typical yields of 20-50 pmol of purified peptide per CNS .

What are the recommended culture conditions for maintaining Lymnaea stagnalis for neuropeptide research?

Optimal culture conditions for L. stagnalis are critical for consistent neuropeptide expression:

These conditions are based on established protocols for maintaining L. stagnalis in laboratory settings for neuroscience research . Consistent husbandry practices are essential for reproducible neuropeptide studies, as environmental factors can influence neuropeptide expression patterns.

How can researchers address the challenge of antibody cross-reactivity when studying Lymna-DF-amide 1?

Antibody cross-reactivity is a significant challenge in mollusk neuropeptide research:

• Validation requirements:

  • Always verify antibody specificity using Western blotting with recombinant peptides

  • Perform peptide-blocking controls with synthetic Lymna-DF-amide 1

  • Include absorption controls with related peptides to assess cross-reactivity

• Cross-reactivity assessment:

  • Gel electrophoretic separation followed by immunostaining of neural tissue proteins

  • Mass spectrometric analysis of immunoreactive bands to confirm peptide identity

  • Comparison of immunostaining patterns with direct MS detection

• Alternative approaches:

  • Development of custom antibodies against unique epitopes of Lymna-DF-amide 1

  • MS-based label-free quantification as an antibody-independent method

  • In situ hybridization to detect precursor mRNA expression

Research has shown that antibodies against human proteins can produce grossly non-specific results in mollusks, with anti-human aromatase antibodies staining multiple unrelated proteins in L. stagnalis neural tissue . This underscores the importance of rigorous validation when using immunological methods for Lymna-DF-amide 1 detection.

What are the considerations for designing bioassays to measure Lymna-DF-amide 1 activity?

Designing effective bioassays requires careful consideration of physiological relevance:

• Ex vivo tissue preparations:

  • Isolated buccal mass contractions can assess effects on feeding muscles

  • CNS preparations with attached buccal mass allow monitoring of both central pattern generator activity and muscle contractions

  • Semi-intact preparations preserving connections between central neurons and peripheral targets

• Electrophysiological readouts:

  • Intracellular recordings from feeding circuit neurons to assess direct effects

  • Extracellular nerve recordings to monitor network-level activity

  • Muscle fiber recordings to assess neuromuscular effects

• Behavioral assays:

  • Feeding behavior metrics (bites per minute, food consumption rate)

  • Movement tracking for locomotor effects

  • Respiratory behavior monitoring for effects on pneumostome movements

• Dose-response considerations:

  • Start with physiologically relevant concentrations (10⁻⁹ to 10⁻⁶ M)

  • Include time-course measurements to detect both acute and sustained effects

  • Compare with effects of related peptides to establish specificity

Unlike the peptide GLTPNMNSLFF-NH2, which affects vas deferens contractility , Lymna-DF-amide 1 bioassays should focus on feeding-related functions based on its structural similarity to CCK/gastrin and its localization in feeding-related neurons .

How can Lymna-DF-amide 1 research contribute to understanding neuropeptide evolution?

Lymna-DF-amide 1 offers unique insights into neuropeptide evolution:

• Comparative structural analysis: Alignment of Lymna-DF-amide 1 with related peptides across phyla can identify conserved motifs and divergent regions, providing insight into structure-function relationships.

• Receptor studies: Characterization of Lymna-DF-amide 1 receptors and comparison with vertebrate CCK/gastrin receptors can reveal evolutionary conserved signaling pathways.

• Functional conservation assessment: Comparative studies of Lymna-DF-amide 1 and vertebrate CCK/gastrin effects on analogous physiological processes (e.g., feeding regulation) can identify conserved functional roles.

• Genomic analysis: Integration with genomic data to trace the evolutionary history of the Asp-Phe-amide peptide superfamily across invertebrate and vertebrate lineages .

This research supports the hypothesis that the widespread CCK/gastrin immunoreactivity in invertebrates is due to peptides belonging to a broader Asp-Phe-amide superfamily rather than direct CCK/gastrin homologs .

What are the potential applications of Lymna-DF-amide 1 in neurodegenerative disease research?

L. stagnalis has emerged as a model for studying neurodegenerative diseases , and Lymna-DF-amide 1 research may contribute to this field:

• Neuroprotective potential: Investigation of whether Lymna-DF-amide 1 exhibits neuroprotective properties similar to some mammalian neuropeptides.

• Aging studies: Examination of age-related changes in Lymna-DF-amide 1 expression and function, leveraging L. stagnalis as a model for aging research .

• Learning and memory applications: Assessment of Lymna-DF-amide 1's role in learning and memory processes, which are well-studied in L. stagnalis and relevant to neurodegenerative conditions .

• Synaptic plasticity mechanisms: Investigation of Lymna-DF-amide 1's effects on synaptic strength and plasticity in identified neuronal circuits.

The relatively simple nervous system of L. stagnalis, combined with its capacity for learning and memory, makes it valuable for studying neuropeptide involvement in cognitive processes relevant to neurodegenerative disorders .

How can integration of Lymna-DF-amide 1 research with genomic and transcriptomic data advance peptidogenomics?

Peptidogenomics integration represents a frontier in neuropeptide research:

• Genome-guided peptide discovery: Using the emerging genomic data for L. stagnalis to predict neuropeptide precursors and identify novel members of the lymnaDFamide family.

• Spatially resolved transcriptomics: Application of techniques like MERFISH to map cell-specific expression patterns of Lymna-DF-amide 1 precursors and receptors .

• Regulatory network identification: Integration of transcriptomic data to identify transcription factors and regulatory elements controlling Lymna-DF-amide 1 expression.

• Evolutionary comparative genomics: Cross-species comparison of genomic organization of Asp-Phe-amide peptide genes to identify conserved synteny and regulatory elements.

The integration of MS-based peptide discovery with genomic information along the lines of peptidogenomics will enhance our understanding of neuropeptide signaling networks in L. stagnalis .

What are common challenges in recombinant Lymna-DF-amide 1 expression and how can they be addressed?

Researchers may encounter several challenges when expressing recombinant Lymna-DF-amide 1:

For tridecapeptides like Lymna-DF-amide 1, solid-phase peptide synthesis may offer a practical alternative to recombinant expression, especially when C-terminal amidation is critical for function .

How should researchers interpret contradictory results from different analytical methods when studying Lymna-DF-amide 1?

When faced with contradictory results, systematic troubleshooting is essential:

• Antibody vs. MS discrepancies:

  • Prioritize MS data over immunological methods due to higher specificity

  • Validate antibodies against synthetic standards

  • Consider that antibodies may detect related peptides from the same family

• Functional contradictions:

  • Evaluate dose-response relationships across a wide concentration range

  • Consider that effects may be state-dependent or context-specific

  • Test in different preparations (isolated neurons, ganglia, semi-intact preparations)

• Species differences:

  • Note that L. stagnalis peptides may not cross-react with vertebrate systems

  • LymnaDFamide-1 showed no effect on trout gallbladder despite structural similarity to CCK/gastrin

  • Consider evolutionary divergence in receptor systems

• Technical considerations:

  • Assess peptide stability under experimental conditions

  • Verify peptide identity in each experimental preparation

  • Consider potential contamination with other bioactive peptides

Resolution of contradictions often requires multiple complementary approaches and careful consideration of the biological context in which Lymna-DF-amide 1 functions.