Recombinant Sarcophaga bullata Ovary-derived ACE interactive factor

What is the primary structure and molecular characteristics of Neb-ODAIF?

Neb-ODAIF is an 11-amino acid peptide with the sequence NKLKPSQWISL and a molecular weight of 1312.7 Da. The peptide shows high sequence similarity to the N-terminal region of dipteran yolk polypeptides (YPs) . When subjected to ACE activity, it undergoes sequential cleavage of two C-terminal dipeptides, generating the fragments Neb-ODAIF 1-9 (NKLKPSQWI) and subsequently Neb-ODAIF 1-7 (NKLKPSQ) . This cleavage pattern is consistent with ACE's typical peptidyl-dipeptidase activity, which removes C-terminal dipeptides from substrate peptides .

How was Neb-ODAIF originally isolated and identified from Sarcophaga bullata?

Neb-ODAIF was first isolated from vitellogenic ovaries of the grey fleshfly Neobellieria bullata (Sarcophaga bullata). Researchers identified it while investigating uncharacterized substances in these ovaries that interact with angiotensin converting enzyme either as substrates or inhibitors . The isolation likely involved tissue extraction followed by chromatographic separation and mass spectrometry for sequence determination. The peptide was subsequently characterized through enzymatic assays with both insect and human ACE to confirm its substrate properties and determine kinetic parameters .

What are the kinetic parameters of Neb-ODAIF interaction with human somatic ACE?

The kinetic parameters of Neb-ODAIF and its cleavage products demonstrate significant affinity for human somatic ACE (sACE). The complete peptide (NKLKPSQWISL) has a Km value of 17 μM for human sACE. The first cleavage product, Neb-ODAIF 1-9 (NKLKPSQWI), shows a Km value of 81 μM. Additionally, the second cleavage product, Neb-ODAIF 1-7 (NKLKPSQ), also interacts with sACE with a Km/i of 90 μM . These affinity constants are comparable to those of physiological ACE substrates, suggesting potential biological significance.

What evolutionary insights can be derived from studying Neb-ODAIF and its interaction with ACE across species?

The ability of both insect and human ACE to recognize and process Neb-ODAIF suggests evolutionary conservation of ACE's substrate recognition mechanisms across diverse phyla. ACE is believed to have evolved from an ancestral single-domain enzyme, with mammalian sACE arising through gene duplication . The sequence similarity between Neb-ODAIF and dipteran yolk polypeptides indicates potential evolutionary connections between reproductive physiology and ACE activity in insects .

Comparative studies of Neb-ODAIF processing across species could provide insights into the evolution of peptide-processing enzymes and their substrate specificities. Furthermore, such studies might illuminate how ACE's physiological roles diversified from primarily reproductive functions in invertebrates to include blood pressure regulation in vertebrates, while maintaining certain conserved activities in reproductive tissues across diverse animal groups .

What is the hypothesized physiological role of Neb-ODAIF in Sarcophaga bullata reproduction?

The physiological role of Neb-ODAIF in Sarcophaga bullata reproduction remains under investigation, but several hypotheses can be proposed based on its characteristics. Given its isolation from vitellogenic ovaries and sequence similarity to yolk polypeptides, Neb-ODAIF may be involved in oocyte development, vitellogenesis, or egg maturation processes . The sequential cleavage by ACE could generate bioactive peptides that regulate specific reproductive processes or signaling pathways.

Insect ACE immunoreactivity has been detected in neurosecretory cells in some insects, suggesting potential neuroendocrine functions . The presence of ACE and its substrates in reproductive tissues indicates possible roles in controlling aspects of reproduction through peptide processing. Further research is needed to determine whether Neb-ODAIF functions as a precursor to bioactive peptides, a regulatory factor in oocyte development, or serves other roles in insect reproduction.

Could Neb-ODAIF have implications for understanding human ACE physiology?

Neb-ODAIF's interaction with human ACE with affinity constants comparable to physiological substrates suggests potential implications for understanding human ACE physiology and developing new therapeutic approaches. The peptide and its derivatives could serve as tools for studying ACE domain specificity, substrate recognition, and inhibitor development .

As human ACE plays critical roles in blood pressure regulation, fluid homeostasis, and male fertility, comparative studies with novel substrates like Neb-ODAIF might reveal previously unrecognized aspects of enzyme function or regulation. Additionally, the structural features that make Neb-ODAIF a good ACE substrate could inform the design of new ACE inhibitors or substrates with therapeutic potential for conditions like hypertension, heart failure, and diabetic nephropathy .

What are the optimal conditions for working with recombinant Neb-ODAIF in enzymatic assays?

When working with recombinant Neb-ODAIF in enzymatic assays, researchers should consider the following optimal conditions:

• Storage and Handling: Store the recombinant protein at -20°C or -80°C for extended storage. Avoid repeated freeze-thaw cycles, as this may compromise activity. Working aliquots can be stored at 4°C for up to one week .

• Reconstitution: Briefly centrifuge the vial before opening to bring contents to the bottom. Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% is recommended for long-term storage .

• Assay Conditions: For ACE activity assays, consider using standard buffer systems such as 50 mM HEPES buffer (pH 7.5) containing 300 mM NaCl and 10 μM ZnCl₂, which supports optimal ACE activity. The reaction temperature should typically be maintained at 37°C for mammalian ACE or adjusted appropriately for insect ACE .

• Detection Methods: HPLC analysis or mass spectrometry can be used to monitor the sequential cleavage of Neb-ODAIF. Alternatively, fluorogenic or chromogenic derivatives could be developed for continuous enzyme assays .

What analytical methods are most effective for studying Neb-ODAIF cleavage products?

Several analytical methods are effective for studying Neb-ODAIF cleavage products:

• Liquid Chromatography-Mass Spectrometry (LC-MS): This provides high-resolution separation and precise molecular weight determination of Neb-ODAIF and its cleavage products (Neb-ODAIF 1-9 and Neb-ODAIF 1-7). MS/MS fragmentation patterns can confirm sequence identity .

• Reversed-Phase HPLC: Using C18 columns with appropriate acetonitrile/water/TFA gradients allows effective separation of the parent peptide from its cleavage products.

• Enzyme Kinetics Analysis: Progress curve analysis with varying substrate concentrations can determine kinetic parameters (Km, Vmax) for each cleavage step .

• Immunoassays: Development of specific antibodies against Neb-ODAIF and its fragments could enable immunological detection methods, though this approach would require additional developmental work.

• Fluorescence Resonance Energy Transfer (FRET): Custom FRET-based substrates derived from Neb-ODAIF sequence could enable real-time monitoring of cleavage with high sensitivity.

The choice of method depends on the specific research question, available equipment, and required sensitivity and specificity.

How might Neb-ODAIF be utilized in the development of novel ACE inhibitors?

Neb-ODAIF could serve as a valuable template for developing novel ACE inhibitors through several approaches:

• Structure-Activity Relationship Studies: Systematic modifications of the Neb-ODAIF sequence could identify critical residues for ACE binding and processing. This could involve alanine scanning, non-natural amino acid substitutions, or terminal modifications to generate peptides with enhanced binding but reduced hydrolysis .

• Transition-State Analogues: Knowledge of how Neb-ODAIF binds to ACE could inform the design of transition-state analogue inhibitors that mimic the geometry of the peptide during catalysis but resist cleavage.

• Domain-Selective Inhibitors: If Neb-ODAIF shows differential processing by the N- and C-domains of human sACE, it could provide insights for developing domain-selective inhibitors, which may have fewer side effects than current ACE inhibitors .

• Peptide-Based Drug Design: Modifications of Neb-ODAIF to enhance stability (such as cyclization or incorporation of D-amino acids) could lead to peptide-based drugs with improved pharmacokinetic properties. The natural sequence serves as a starting point for such modifications.

These approaches require detailed structural and functional analyses of Neb-ODAIF-ACE interactions, potentially including crystallographic studies of the complex .

What experimental approaches could elucidate the in vivo functions of Neb-ODAIF in insect reproductive physiology?

Several experimental approaches could help elucidate the in vivo functions of Neb-ODAIF in insect reproductive physiology:

• RNA Interference (RNAi): If Neb-ODAIF is derived from a precursor protein, RNAi targeting the precursor could reveal phenotypic effects on oogenesis, vitellogenesis, or reproductive success.

• CRISPR/Cas9 Gene Editing: Creating knockout or knockin mutations in genes related to Neb-ODAIF production or processing could provide insights into its physiological role.

• Peptide Administration Studies: Microinjection of synthetic Neb-ODAIF or its fragments into different developmental stages could reveal potential physiological effects.

• ACE Inhibition Studies: Using ACE inhibitors to prevent Neb-ODAIF processing in vivo might reveal whether the intact peptide or its cleavage products are the active forms.

• Immunohistochemistry and In Situ Hybridization: These techniques could determine the precise spatial and temporal expression patterns of Neb-ODAIF and related peptides during insect development and reproduction .

• Receptor Identification: Approaches such as photoaffinity labeling or yeast two-hybrid screens could identify potential receptors for Neb-ODAIF or its fragments.

• Metabolomic and Transcriptomic Analyses: These could reveal downstream pathways affected by Neb-ODAIF administration or deficiency.

These complementary approaches would provide a comprehensive understanding of Neb-ODAIF's role in insect reproductive physiology .

What are the technical challenges in producing and purifying recombinant Neb-ODAIF?

Several technical challenges exist in producing and purifying recombinant Neb-ODAIF:

• Expression System Selection: While E. coli is commonly used for recombinant peptide production , the small size of Neb-ODAIF (11 amino acids) presents challenges for direct expression. Fusion protein approaches (such as GST, MBP, or SUMO tags) may be necessary, requiring additional cleavage and purification steps.

• Peptide Folding and Stability: Though Neb-ODAIF is a relatively short peptide likely without complex tertiary structure, ensuring proper folding and stability during expression and purification can be challenging. The presence of tryptophan (W) in the sequence may make it susceptible to oxidation.

• Purification Strategies: Size-exclusion chromatography may be less effective for such a small peptide. Reversed-phase HPLC or ion-exchange chromatography may be more suitable final purification steps.

• Yield Optimization: Achieving high yields of functional peptide often requires optimization of expression conditions, including temperature, induction parameters, and culture media composition.

• Quality Control: Ensuring batch-to-batch consistency requires rigorous quality control through mass spectrometry, HPLC, and functional assays to verify sequence integrity and biological activity .

• Alternative Production Methods: For such a small peptide, chemical synthesis might be more efficient than recombinant production, particularly for structure-activity relationship studies requiring multiple variants.

How can researchers address contradictory findings in ACE-Neb-ODAIF interaction studies?

When addressing contradictory findings in ACE-Neb-ODAIF interaction studies, researchers should consider:

• Enzyme Source Variation: Different preparations of ACE (recombinant vs. native, tissue-specific isoforms, different species) may exhibit varying activities. Standardization with well-characterized ACE preparations is essential .

• Assay Condition Differences: Variations in pH, temperature, ionic strength, and presence of chloride ions can significantly affect ACE activity. The C-domain of human ACE is particularly sensitive to chloride concentration . Systematic evaluation of these parameters is necessary.

• Substrate Purity and Integrity: Degradation or oxidation of Neb-ODAIF preparations could affect kinetic measurements. Regular quality control of substrate preparations using mass spectrometry is recommended .

• Analytical Method Sensitivity: Different detection methods vary in sensitivity and specificity. Cross-validation using multiple analytical approaches (e.g., HPLC, mass spectrometry, enzyme-coupled assays) can help resolve discrepancies.

• Domain-Specific Activities: Human sACE contains two domains with distinct substrate preferences. Testing domain-specific mutants or selective inhibitors can clarify which domain is primarily responsible for observed activities .

• Data Analysis Approaches: Different mathematical models for enzyme kinetics analysis can lead to varying parameter estimates. Researchers should clearly report their analytical approach and consider multiple models when appropriate.

By systematically addressing these factors, researchers can reconcile contradictory findings and establish a consistent understanding of ACE-Neb-ODAIF interactions .

What emerging technologies could advance our understanding of Neb-ODAIF biology?

Several emerging technologies could significantly advance our understanding of Neb-ODAIF biology:

• Cryo-Electron Microscopy: High-resolution structural analysis of ACE-Neb-ODAIF complexes could provide insights into binding mechanisms and substrate specificity determinants.

• AlphaFold and Other AI-Based Protein Structure Prediction: These tools could help model interactions between Neb-ODAIF and ACE at atomic resolution, especially when combined with experimental validation.

• Single-Cell Transcriptomics: This could reveal cell-specific expression patterns of genes related to Neb-ODAIF production and processing in insect ovaries, providing insights into its physiological context.

• Peptidomics: Advanced mass spectrometry-based peptidomics could identify natural variants and post-translational modifications of Neb-ODAIF in different physiological states.

• CRISPR-Based Gene Editing: Precise genome editing in Sarcophaga bullata could enable in vivo studies of Neb-ODAIF function through generation of knockout or reporter lines.

• Proximity Labeling Proteomics: Techniques like BioID or APEX2 could identify proteins that interact with Neb-ODAIF in cellular contexts, potentially revealing receptors or downstream effectors.

• Microfluidic Organ-on-a-Chip: This technology could enable studies of Neb-ODAIF function in simplified but physiologically relevant models of insect reproductive systems .

What are the potential applications of Neb-ODAIF research in comparative endocrinology and insect pest management?

Neb-ODAIF research has several potential applications in comparative endocrinology and insect pest management:

• Comparative Endocrinology:

  • Provides insights into the evolution of peptide hormone systems across phyla

  • Illuminates functional conservation and divergence of ACE across species

  • Could reveal novel signaling pathways in reproductive physiology applicable to both invertebrates and vertebrates

  • May uncover fundamental principles of peptide processing and function in neuroendocrine systems

• Insect Pest Management:

  • If Neb-ODAIF plays a critical role in insect reproduction, targeting its production or processing could lead to novel pest control strategies

  • Development of species-specific ACE inhibitors based on Neb-ODAIF structure could disrupt reproductive processes in pest species

  • Understanding the role of ACE in insect physiology could identify new targets for biopesticides with reduced environmental impact

  • Knowledge of insect-specific peptide processing could lead to selective control methods that spare beneficial insects

• Integrated Applications:

  • Combination of structural biology, peptidomics, and functional genomics approaches could accelerate development of both basic comparative endocrinology insights and applied pest management strategies

  • Ecological studies of Neb-ODAIF function across related species could reveal evolutionary adaptations in reproductive strategies with potential applications in conservation biology