Recombinant Pseudoderopeltis cf. bimaculata JT-2004 Sulfakinin-1

What is Pseudoderopeltis cf. bimaculata JT-2004 Sulfakinin-1 and how is it structurally characterized?

Pseudoderopeltis cf. bimaculata JT-2004 Sulfakinin-1 (PseBi-SK-1) is a neuropeptide isolated from the Harlequin cockroach (Pseudoderopeltis cf. bimaculata). The recombinant form has the amino acid sequence EQFDDYGHMRF and is typically expressed in E. coli systems for research purposes . As a member of the sulfakinin family, it shares structural similarities with other insect sulfakinins and serves as an insect analog to mammalian cholecystokinin (CCK).

When comparing PseBi-SK-1 to other insect sulfakinins, significant sequence conservation is evident, particularly in the C-terminal region:

Like other sulfakinins, PseBi-SK-1 contains a tyrosine residue that can potentially be sulfated, a critical feature for biological activity. Studies with Drosophila sulfakinins have shown that sulfated forms can be approximately 3000-fold more potent than non-sulfated counterparts .

What are the optimal handling conditions for Recombinant PseBi-SK-1?

For optimal experimental results, proper storage and reconstitution of Recombinant PseBi-SK-1 is essential:

Storage recommendations:

• Short-term storage: -20°C

• Extended storage: -20°C or -80°C

• Working aliquots: 4°C for up to one week

• Shelf life: Approximately 12 months for lyophilized form and 6 months for liquid form at -20°C/-80°C

Reconstitution protocol:

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

• Reconstitute 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 the default recommendation)

• Aliquot for long-term storage to avoid repeated freeze-thaw cycles

Researchers should note that repeated freezing and thawing should be avoided as this can lead to protein degradation. The addition of glycerol helps prevent freeze-thaw damage and stabilizes protein structure.

What is the evolutionary relationship between insect sulfakinins and mammalian cholecystokinin?

Sulfakinins are considered the insect analogs of mammalian cholecystokinin (CCK) and gastrin peptides, with several lines of evidence supporting their evolutionary relationship:

• Sequence homology: Both peptide families share key structural features, including conserved C-terminal regions and the presence of sulfated tyrosine residues

• Receptor similarity: Sulfakinin receptors (SKRs) show homology to mammalian CCK receptors. Drosophila sulfakinin receptors (DSK-R1 and DSK-R2) were identified through homology to mammalian cholecystokinin receptors CCKR1 and CCKR2

• Functional conservation: Both peptide families regulate feeding behavior and digestive processes across diverse species

• Signaling mechanisms: Both typically signal through G protein-coupled receptors that activate calcium-dependent pathways

Research on starfish has demonstrated that SK/CCK-type signaling has an ancient evolutionary origin predating the divergence of protostomes (including insects) and deuterostomes (including vertebrates), suggesting this signaling system originated at least 550 million years ago . This conservation across diverse phyla indicates the fundamental importance of these peptides in regulating feeding and digestive processes throughout animal evolution.

How do sulfated and non-sulfated forms of sulfakinins differ functionally?

The functional differences between sulfated and non-sulfated forms of sulfakinins are significant and should be considered in experimental design:

• Receptor binding affinity: In Drosophila, the sulfated form of DSK-1 was approximately 3000-fold more potent than its non-sulfated counterpart, suggesting tyrosine sulfation dramatically increases receptor binding affinity

• Calcium signaling: Sulfated forms typically induce stronger calcium responses upon receptor binding, as seen with the DSK-R1 receptor where the interaction with sulfated [Leu7]-DSK-1S led to dose-dependent intracellular calcium increases with EC50 in the low nanomolar range

• Biological potency: In the starfish Asterias rubens, both sulfated and non-sulfated forms of ArSK/CCK1 and ArSK/CCK2 were detected, with sulfation likely enhancing biological activity

This functional disparity highlights the importance of testing both sulfated and non-sulfated forms in experimental settings. The dramatic difference in potency (3000-fold) between sulfated and non-sulfated forms emphasizes the critical role of post-translational modifications in sulfakinin biology .

What methodologies are effective for studying sulfakinin receptor activation?

Calcium imaging has proven particularly effective for studying sulfakinin receptor activation, as these receptors typically couple to Gq/11 proteins leading to intracellular calcium mobilization:

Experimental approach:

• Cell preparation:

  • Express the putative sulfakinin receptor in mammalian cells (e.g., CHO-K1 cells)

  • Load cells with calcium-sensitive fluorescent dyes (e.g., Fluo-4, Fura-2)

• Stimulation protocol:

  • Apply sulfakinins at multiple concentrations to generate dose-response curves

  • Compare responses to sulfated versus non-sulfated forms

  • Test for receptor desensitization with repeated exposures

• Pharmacological validation:

  • Use pertussis toxin (PTX) to confirm coupling to PTX-insensitive pathways (Gq/11)

  • Apply specific PLC inhibitors to confirm canonical signaling pathway

The DSK-R1 receptor activation was characterized using these approaches, revealing PTX-insensitive signaling pathways that point to Gq/11 involvement in coupling to the activated receptor . In mosquito studies, sulfakinin receptor expressed in CHO-K1 cells responded to sulfakinin stimulation with persistent calcium spikes, which were blockable with receptor antagonist .

What approaches can be used to study sulfakinin effects on feeding behavior?

Based on previous sulfakinin research, several methodological approaches can be employed to study effects on feeding behavior:

• Genetic approaches:

  • RNAi knockdown of sulfakinin or sulfakinin receptor genes

  • Studies in mosquitoes demonstrated that knockdown of sulfakinin or sulfakinin receptor gene with interference RNA increases blood meal intake

• Pharmacological interventions:

  • Microinjection of synthetic sulfakinins

  • In mosquitoes, microinjection of sulfakinin peptides in the thorax inhibited dose-dependently blood meal intake

  • Receptor antagonist administration can reverse these effects

• Quantitative measurements:

  • Blood meal intake in hematophagous insects

  • Feeding duration and frequency analysis

  • Food choice/preference tests

• Combined approaches:

  • Correlate molecular mechanisms with behavioral outcomes

  • Compare effects across different feeding paradigms

The mosquito studies provide an excellent methodological framework, showing that both genetic (RNAi) and pharmacological (peptide injection) approaches can produce consistent and complementary results when studying sulfakinin effects on feeding .

How do different sulfakinins exert distinct effects on different behaviors?

Research has revealed that structurally related sulfakinins can have distinct effects on different behavioral processes, suggesting involvement of separate mechanisms:

• Differential effects on odor preference vs. locomotion:

  • In Drosophila, sulfated and non-sulfated DSK II influenced larval odor preference, while sulfated and non-sulfated DSK I did not

  • Conversely, sulfated and non-sulfated DSK I influenced larval locomotion, while DSK II forms did not

• Quantitative behavioral differences:

  • The effects of sDSK II and nsDSK II on odor preference were statistically different compared to saline (p values 0.00005 and 0.02, respectively)

  • In locomotion assays, larvae treated with sDSK I or nsDSK I crossed significantly fewer lines (32±2 and 31±2, respectively) compared to controls (51±4)

This striking dissociation between the effects of DSK I and DSK II on different behaviors suggests that:

• Different receptor subtypes or splice variants may mediate distinct behavioral effects

• Tissue-specific expression patterns may direct peptides to different neural circuits

• Differential coupling to downstream signaling pathways may produce behavior-specific outcomes

These findings highlight the importance of comprehensive behavioral profiling when studying novel sulfakinins like PseBi-SK-1 .

What challenges exist in distinguishing direct versus indirect effects of sulfakinins?

Distinguishing direct from indirect effects of sulfakinins in behavioral assays presents several methodological challenges:

• Pleiotropic effects:

  • Sulfakinins can affect multiple physiological processes simultaneously

  • Effects on feeding might be secondary to changes in locomotion, as suggested by the distinct effects of DSK I and DSK II in Drosophila

• Receptor distribution:

  • Sulfakinin receptors may be expressed in multiple tissues and neural circuits

  • Central versus peripheral effects may be difficult to separate

• Temporal dynamics:

  • Primary receptor activation may trigger cascading effects over different time scales

  • Immediate versus delayed responses may reflect direct versus indirect mechanisms

Strategies to address these challenges include:

• Site-specific administration (e.g., targeted brain injections versus systemic delivery)

• Tissue-specific genetic manipulations of receptor expression

• Parallel physiological measurements during behavioral assays

The finding that DSK I specifically affects locomotion while DSK II specifically affects odor preference underscores the need for comprehensive experimental designs that can distinguish between different mechanisms of action .

How can cross-species conservation of sulfakinin function be effectively studied?

Examining the conservation of sulfakinin function across species requires multiple complementary approaches:

• Comparative sequence analysis:

  • Alignment of sulfakinin sequences across diverse taxa shows conservation of key features like the sulfated tyrosine residue and C-terminal motifs

• Receptor pharmacology:

  • Expression of receptors from multiple species in common cell backgrounds

  • In starfish, ArSK/CCK1 and ArSK/CCK2 caused dose-dependent contraction of various muscle preparations, similar to effects seen in insects

• Functional conservation testing:

  • Similar inhibitory effects on feeding have been observed across diverse species:

    • In mosquitoes, sulfakinins inhibit blood meal intake

    • In silkworms, sulfakinins suppress food intake

    • In starfish, sulfakinins trigger cardiac stomach retraction and inhibit feeding

• Evolutionary analysis:

  • Studies in starfish demonstrate that SK/CCK-type signaling has an ancient evolutionary origin predating the divergence of protostomes and deuterostomes

The conservation of sulfakinin's role as an inhibitory regulator of feeding across species as diverse as insects and echinoderms suggests this is an evolutionarily ancient and fundamental function of this signaling system .

What controls are essential for robust sulfakinin research?

For rigorous experiments with sulfakinins like PseBi-SK-1, several control conditions should be implemented:

• Peptide-specific controls:

  • Test both sulfated and non-sulfated forms

  • Include truncated peptide fragments (e.g., MRFNH₂ as used in Drosophila studies)

  • Test structurally related peptides (e.g., human CCK-8 and gastrin-II were found inactive when tested with DSK-R1)

• Dose controls:

  • Establish complete dose-response relationships

  • In receptor activation studies, the EC50 for [Leu7]-DSK-1S was in the low nanomolar range

• Genetic/pharmacological validation:

  • Use receptor antagonists to block peptide effects

  • In mosquito studies, sulfakinin-induced inhibition of blood meal intake was reversible with receptor antagonist

• Multiple behavioral assays:

  • Assess multiple behaviors to determine specificity of effects

  • The Drosophila studies exemplify this approach by testing effects on both odor preference and locomotion

These comprehensive controls help establish the specificity and reliability of observed sulfakinin effects. The striking dissociation between DSK I effects on locomotion and DSK II effects on odor preference in Drosophila highlights the importance of testing multiple behavioral endpoints .

What techniques can resolve contradictory findings in sulfakinin research?

To address contradictory findings in sulfakinin signaling research, several methodological approaches can be employed:

• Standardize experimental conditions:

  • Use consistent peptide preparations (ensuring correct post-translational modifications)

  • The 3000-fold potency difference between sulfated and non-sulfated forms demonstrates the critical importance of sulfation status

• Receptor characterization:

  • Identify all receptor subtypes and splice variants

  • Characterize signaling pathways using multiple approaches

  • Drosophila studies identified two sulfakinin receptors, DSK-R1 and DSK-R2, with potential functional differences

• Multi-level analysis:

  • Combine genetic, pharmacological, and physiological approaches

  • In mosquitoes, both RNAi knockdown and peptide injection produced consistent results regarding feeding inhibition

• Consider context:

  • Examine effects under different physiological states

  • Evaluate developmental stage and sex differences

The finding that different sulfakinins have distinct effects on specific behaviors demonstrates the importance of comprehensive characterization when investigating these neuropeptides .

How might sulfakinin research translate to vector control strategies?

Research on sulfakinins has revealed potential applications for vector control, particularly for blood-feeding insects that transmit disease:

• Blood meal regulation:

  • In mosquitoes, activation of the G protein-coupled sulfakinin receptor inhibits blood meal intake

  • This could potentially be exploited to control both mosquito reproduction and disease transmission

• Potential intervention strategies:

  • Development of stable sulfakinin analogs or receptor-specific agonists

  • Genetic approaches targeting the sulfakinin signaling system

  • Integration with existing vector control methods

• Mechanistic understanding:

  • Sulfakinin receptor expressed in mammalian cells responds to stimulation with persistent calcium spikes

  • This provides a molecular target for potential intervention strategies

As stated in the mosquito research: "These data together suggest that activation of the Gq protein-coupled sulfakinin receptor inhibits blood meal intake in female A. aegypti mosquitoes and could serve as a strategic node for the future control of A. aegypti mosquito reproduction/population and disease transmission" .

What emerging technologies could advance sulfakinin research?

Several cutting-edge technologies and approaches could significantly advance sulfakinin research:

• CRISPR-Cas9 gene editing:

  • Generate receptor knockouts to study loss-of-function phenotypes

  • Create tagged receptor variants for localization studies

  • Introduce specific mutations to study structure-function relationships

• Advanced imaging techniques:

  • Calcium imaging has already proven valuable for studying receptor activation

  • Expanding to in vivo calcium imaging during behavior could link cellular activity to behavioral outcomes

• Optogenetic and chemogenetic approaches:

  • Temporal control of sulfakinin receptor activation

  • Cell-type specific manipulation of sulfakinin signaling

• Computational modeling:

  • Structure-based design of selective receptor agonists/antagonists

  • Prediction of peptide-receptor interactions based on evolutionary conservation

• Single-cell transcriptomics:

  • Detailed mapping of receptor expression patterns

  • Identification of downstream signaling components

These technologies could help resolve current knowledge gaps and provide more detailed insights into the mechanisms through which sulfakinins like PseBi-SK-1 regulate various physiological and behavioral processes.