Recombinant Gyna lurida Hypertrehalosaemic factor

What is Gyna lurida Hypertrehalosaemic factor and what is its primary physiological function?

Gyna lurida Hypertrehalosaemic factor (Gylur-HTH) is a neuropeptide belonging to the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family. Its primary physiological function is to mobilize trehalose from the fat body into the hemolymph, resulting in elevated hemolymph trehalose levels . This factor plays a central role in insect energy homeostasis, particularly during periods of high energy demand such as flight, starvation, or stress responses. Similar to other cockroach HTHs, Gylur-HTH is synthesized and stored in the corpora cardiaca and released in response to specific physiological triggers .

How does Gylur-HTH compare structurally to other cockroach hypertrehalosaemic factors?

Gylur-HTH, like other cockroach hypertrehalosaemic factors, is likely a decapeptide with characteristic structural features including a pyroglutamate-blocked N-terminus, an amidated C-terminus, aromatic residues at positions 4 and 8, and often a glycine residue at position 9 . Recent mass spectrometry analyses of cockroach HTHs have revealed that many blaberid cockroach HTHs (the family to which G. lurida belongs) share structural similarity with Bladi-HTH and often contain hydroxyproline modifications . The specific primary structure of Gylur-HTH would need to be confirmed using high-resolution mass spectrometry coupled with liquid chromatography to identify any unique modifications or amino acid substitutions.

What methodologies are recommended for recombinant expression of Gylur-HTH?

For recombinant expression of Gylur-HTH, researchers should consider the following methodological approach:

• cDNA cloning: Employ RT-PCR with degenerate primers designed from conserved motifs of AKH/HTH peptides, followed by 5'-RACE and 3'-RACE to obtain full-length cDNA .

• Expression system selection: Bacterial (E. coli), yeast (P. pastoris), or insect cell (Sf9) expression systems may be employed, with insect cells often preferred for proper post-translational modifications.

• Vector construction: Design expression vectors with appropriate tags (His-tag, GST) for purification, and include cleavage sites for tag removal after purification.

• Purification strategy: Implement a multi-step purification process including affinity chromatography, size-exclusion chromatography, and reversed-phase HPLC.

• Verification: Confirm peptide identity and modifications through mass spectrometry and N-terminal sequencing to ensure proper structure including pyroglutamate formation and C-terminal amidation .

How should researchers design bioassays to evaluate the biological activity of recombinant Gylur-HTH?

To evaluate the biological activity of recombinant Gylur-HTH, researchers should implement a comprehensive bioassay design:

• Hemolymph trehalose measurement: Inject recombinant Gylur-HTH into adult cockroaches and collect hemolymph samples at defined time intervals (30, 60, 90 minutes). Treat hemolymph with porcine kidney trehalase to convert trehalose to glucose, then measure glucose using a commercial glucose assay kit .

• Dose-response relationship: Test multiple doses of recombinant Gylur-HTH (0.1-10 pmol) to establish EC50 values and compare with native peptide potency.

• Cross-species activity: Assess activity not only in G. lurida but also in other cockroach species like Periplaneta americana to determine phylogenetic conservation of receptor binding and activation properties .

• Controls: Include appropriate controls: (1) negative control (saline injection), (2) positive control (established HTH like Periplaneta americana HTH), and (3) denatured Gylur-HTH to confirm structure-dependent activity.

• Statistical analysis: Apply appropriate statistical methods (ANOVA with Tukey's HSD post-hoc test) to analyze differences between treatment groups .

What are the recommended approaches for studying Gylur-HTH receptor signaling pathways?

For investigating Gylur-HTH receptor signaling pathways, researchers should consider these methodological approaches:

• Receptor identification and cloning: Clone the Gylur-HTH receptor using degenerate primers based on conserved G protein-coupled receptor (GPCR) motifs from other insect AKH receptors, followed by RACE techniques to obtain full-length receptor cDNA .

• Receptor expression analysis: Determine tissue distribution and developmental expression patterns using semi-quantitative RT-PCR or quantitative RT-PCR, with special attention to fat body expression where highest receptor levels are typically found .

• Functional characterization:

  • Heterologous expression in cell lines (HEK293, CHO-K1)

  • Calcium mobilization assays using calcium-sensitive fluorescent dyes

  • cAMP production measurement using ELISA or reporter assays

  • Receptor binding studies with radiolabeled or fluorescently labeled Gylur-HTH

• RNA interference (RNAi): Design double-stranded RNA targeting the Gylur-HTH receptor (dsHTHR) to knock down receptor expression (1.5 μg dsRNA injection recommended) and confirm knockdown efficiency by RT-PCR .

• Downstream effector analysis: Monitor key signaling molecules including protein kinase A activation, calcium flux, and subsequent metabolic enzyme regulation.

How can researchers investigate the role of Gylur-HTH in oxidative stress protection mechanisms?

To investigate Gylur-HTH's role in oxidative stress protection, implement this experimental approach:

• Oxidative stress induction: Administer paraquat (PQ) to G. lurida as a model oxidative stressor (empirically determine appropriate dose that induces stress without immediate mortality) .

• Co-injection experiments: Design experiments with the following groups:

  • Control (saline)

  • PQ only

  • Gylur-HTH only

  • PQ + Gylur-HTH

  • PQ + Gylur-HTH in receptor knockdown specimens

  • PQ + Gylur-HTH in hormone knockdown specimens

• Survival assessment: Monitor survival rates over time (up to 96 hours) and analyze using Kaplan-Meier survival curves, log-rank tests for between-group differences, and Kruskal-Wallis rank sum tests for post-hoc comparisons .

• Oxidative damage markers: Measure lipid peroxidation in hemolymph using the TBARS assay 4 hours post-treatment, with samples collected from pools of 8 specimens per treatment group .

• Antioxidant system evaluation: Assess glutathione levels, superoxide dismutase, catalase, and glutathione S-transferase activities in response to treatment combinations.

• Molecular response: Examine expression changes in antioxidant genes using qRT-PCR with BgActin as reference gene for normalization using the 2(-Delta Delta C(T)) method .

What techniques are most effective for studying post-translational modifications of Gylur-HTH?

For characterizing post-translational modifications (PTMs) of Gylur-HTH, researchers should employ these sophisticated analytical techniques:

• High-resolution mass spectrometry: Apply LC-MS/MS analysis with collision-induced dissociation (CID) or electron-transfer dissociation (ETD) fragmentation to identify modifications including pyroglutamate formation, C-terminal amidation, and hydroxyproline modifications .

• PTM-specific enrichment strategies:

  • Titanium dioxide enrichment for phosphorylated peptides

  • Lectin affinity chromatography for glycosylated forms

  • Immunoprecipitation with modification-specific antibodies

• Comparison methodology: Compare native Gylur-HTH (extracted from G. lurida corpora cardiaca) with recombinant versions to identify critical modifications that might be absent in recombinant products.

• Site-directed mutagenesis: Generate variants with altered modification sites to assess functional significance of each PTM.

• Structural analysis: Employ circular dichroism spectroscopy and NMR to determine how modifications affect secondary and tertiary structure.

Table 1: Common Post-Translational Modifications in Insect Neuropeptides

How has Gylur-HTH evolved within the context of cockroach phylogeny?

To investigate the evolutionary history of Gylur-HTH within cockroach phylogeny, researchers should:

• Comparative sequence analysis: Align Gylur-HTH with HTH sequences from other cockroach species across different families (Blattidae, Blaberidae, Ectobiidae) to identify conserved and variable regions .

• Molecular phylogenetics: Construct phylogenetic trees using neighbor-joining or maximum likelihood methods based on aligned sequences to elucidate evolutionary relationships .

• Selection pressure analysis: Calculate dN/dS ratios to determine whether the peptide has undergone purifying selection, positive selection, or neutral evolution.

• Synteny analysis: Compare the genomic context of the HTH gene across cockroach species to identify conserved gene arrangements that might suggest functional constraints.

• Receptor co-evolution: Investigate whether Gylur-HTH receptor has evolved in concert with its ligand by comparing receptor sequences across the same set of species.

Research indicates that within Blaberidae (the family containing G. lurida), many species share structurally similar HTH peptides with close relationship to Bladi-HTH, suggesting evolutionary conservation of this signaling system within the family .

How do the structural features of Gylur-HTH affect its cross-reactivity with receptors from other cockroach species?

To investigate structural determinants of Gylur-HTH receptor cross-reactivity:

• Structure-activity relationship studies: Generate synthetic analogs of Gylur-HTH with systematic amino acid substitutions to identify critical residues for receptor binding and activation.

• Cross-species bioassays: Test Gylur-HTH activity in heterologous systems by:

  • Measuring trehalose mobilization in different cockroach species after Gylur-HTH injection

  • Expressing receptors from various cockroach species in cell culture and assessing activation by Gylur-HTH

• Molecular modeling: Construct 3D models of Gylur-HTH and related HTHs to identify structural features that might explain differential receptor binding properties.

• Binding assays: Perform competitive binding assays using labeled reference HTH peptides and various concentrations of Gylur-HTH to determine relative binding affinities for different species' receptors.

• Receptor chimeras: Create chimeric receptors with domains from different species' HTH receptors to map species-specific interaction sites.

Research shows that novel decapeptides structurally similar to Bladi-HTH can elicit hypertrehalosaemic responses in Periplaneta americana despite species differences, suggesting some degree of receptor cross-reactivity between blaberid and blattid HTHs .

What are the most effective strategies for optimizing recombinant Gylur-HTH yield and purity?

To optimize recombinant Gylur-HTH production:

• Expression system optimization:

  • Compare bacterial (E. coli), yeast (P. pastoris), and insect cell (Sf9) systems for yield and correct modifications

  • Test different promoters (T7, AOX1, polyhedrin) for optimal expression

  • Evaluate codon optimization based on host organism preferences

• Culture condition optimization:

  • Systematically vary temperature (16-30°C), induction time, and inducer concentration

  • Implement fed-batch or high-density cultivation strategies

  • Test different media formulations to maximize yield

• Fusion protein selection:

  • Compare different fusion partners (His-tag, GST, SUMO, MBP) for improved solubility and yield

  • Evaluate various protease cleavage sites for efficient tag removal

• Purification protocol refinement:

  • Develop multi-step purification combining affinity chromatography, ion-exchange, and reversed-phase HPLC

  • Implement on-column refolding strategies if inclusion bodies form

  • Optimize elution conditions to maximize recovery

• Quality control measures:

  • Employ mass spectrometry to verify intact peptide mass and correct modifications

  • Confirm bioactivity through trehalose mobilization assays

  • Assess stability under various storage conditions

What are the challenges in distinguishing biological activities of recombinant versus native Gylur-HTH, and how can they be addressed?

Challenges in comparing recombinant versus native Gylur-HTH include:

• Post-translational modification differences:

  • Solution: Characterize both peptide forms using high-resolution mass spectrometry to identify modification differences

  • Strategy: Modify expression systems or perform in vitro enzymatic modifications to match native pattern

• Protein folding variations:

  • Solution: Analyze secondary structure using circular dichroism spectroscopy

  • Strategy: Optimize refolding conditions or use chaperone co-expression systems

• Bioactivity comparison methodology:

  • Solution: Develop standardized dose-response curves using trehalose mobilization assays

  • Strategy: Calculate EC50 values for both preparations and establish potency ratios

• Receptor binding kinetics:

  • Solution: Perform surface plasmon resonance (SPR) analysis with purified receptor

  • Strategy: Compare kon, koff, and KD values between native and recombinant peptides

• Biological half-life differences:

  • Solution: Conduct time-course studies measuring peptide clearance rates in vivo

  • Strategy: Develop modified delivery systems to compensate for stability differences

What emerging technologies might enhance future research on Gylur-HTH and related peptides?

Emerging technologies with significant potential for advancing Gylur-HTH research include:

• CRISPR/Cas9 genome editing:

  • Application: Generate G. lurida knockout models lacking HTH or its receptor

  • Advantage: Allow precise investigation of physiological roles without RNAi limitations

• Single-cell transcriptomics:

  • Application: Map HTH receptor expression at cellular resolution across tissues

  • Advantage: Identify previously unknown target cells and potential novel functions

• Cryo-electron microscopy:

  • Application: Determine high-resolution structures of HTH-receptor complexes

  • Advantage: Provide structural insights for rational peptide design

• Optogenetics and chemogenetics:

  • Application: Develop systems for temporally controlled HTH release or receptor activation

  • Advantage: Allow precise investigation of timing-dependent effects

• Biosensors and in vivo imaging:

  • Application: Create fluorescent biosensors for real-time monitoring of HTH levels

  • Advantage: Enable dynamic studies of hormone release and clearance

These emerging technologies will help address fundamental questions about the metabolic regulation and stress response functions of Gylur-HTH that remain challenging with current methodologies.