Recombinant Periplaneta americana Periviscerokinin-2

What is Periplaneta americana Periviscerokinin-2 and what is its structure?

Periplaneta americana Periviscerokinin-2 (PVK-2) is a myotropic neuropeptide isolated from the abdominal perisympathetic organs of the American cockroach. Its amino acid sequence is Gly-Ser-Ser-Ser-Gly-Leu-Ile-Ser-Met-Pro-Arg-Val-NH2, with an amidated C-terminus that is critical for its biological activity . This peptide is the second neuropeptide identified from insect perisympathetic organs. Besides Periviscerokinin-1, Periviscerokinin-2 is the only putative myotropic neurohormone from the abdominal perisympathetic organs that is effective in the nanomolar range . The peptide contains a C-terminal tripeptide sequence (PRVamide) that shares structural similarity with the pyrokinin C-terminal tripeptide (PRLamide), which contributes to its cross-reactivity with pyrokinin receptors .

What are the primary biological functions of Periviscerokinin-2?

Periviscerokinin-2 primarily functions as a myotropic neurohormone, affecting muscle contractions particularly in the hyperneural muscle of insects. Research demonstrates that it is effective in the nanomolar range, indicating high potency in its biological activities . The peptide is part of the neurohormonal system of the ventral nerve cord, which has been shown to be remarkably different from that of the brain .

The periviscerokinin family, including PVK-2, has been associated with various physiological processes across arthropods. In insects like the American cockroach, it demonstrates myotropic activity on several tissues such as the hyperneural muscle . In some studies, PVK-2 has also demonstrated a low level of pupariation acceleration activity in certain insects, likely due to its C-terminal tripeptide sequence (PRVamide) similarity to pyrokinin peptides . This confirms the hypothesis that the neurohormonal system of the ventral nerve cord is significantly different from that of the brain .

How does Periviscerokinin-2 differ from Periviscerokinin-1?

Periviscerokinin-1 (PVK-1) from Periplaneta americana has the sequence GASGLIPVM-RNa, while Periviscerokinin-2 has the sequence GSSSGLISMPRV-NH2. The key structural differences include:

• PVK-2 has a longer amino acid sequence (12 residues compared to 10 in PVK-1)

• PVK-1 ends with RNamide while PVK-2 ends with RVamide

• Their C-terminal tripeptides differ (RNa vs. PRVa), which affects their interaction with receptors

Despite these differences, both function as myotropic neurohormones from the abdominal perisympathetic organs. Interestingly, the slightly derived CAPA-PVK2s of tick species (Rhipicephalus microplus, R. sanguineus, and Ixodes scapularis) with RNamide at the C-terminal end are similar to the first periviscerokinin identified in insects from P. americana (Pea-PVK-1) . This structural relationship demonstrates the evolutionary conservation of these peptides across different arthropod lineages.

What expression systems are optimal for producing recombinant Periviscerokinin-2?

For recombinant Periviscerokinin-2 production, E. coli expression systems are commonly employed, particularly strains like BL21(DE3)pLysS that offer tight expression control and reduced protease activity. Based on established methodologies for recombinant neuropeptides, the optimal strategy typically involves:

• Vector selection: Vectors with strong inducible promoters like the T7 promoter in pET series vectors (such as pET20b(+)) offer good control over expression

• Fusion tags: Expression as a fusion protein with affinity tags (particularly His-tags) at either N- or C-terminus facilitates subsequent purification

• Induction conditions: IPTG added to a final concentration of 1 mM followed by incubation for approximately 4 hours at 37°C with shaking at 250 rpm

• Cell harvesting: Centrifugation at 4,000 × g at 4°C for 20 minutes followed by lysis in an appropriate buffer using sonication

The expression methodology should be carefully optimized for each specific research application, with particular attention to maintaining the C-terminal amidation that is critical for the biological activity of PVK-2.

What purification methods yield the highest purity of recombinant Periviscerokinin-2?

Achieving high purity of recombinant Periviscerokinin-2 typically requires multi-step chromatography approaches. Based on methodologies used for similar neuropeptides, an effective purification strategy includes:

• Initial capture using Immobilized Metal Affinity Chromatography (IMAC) for His-tagged constructs

• Size-exclusion chromatography to separate monomeric peptide from aggregates

• Reverse-phase HPLC as a polishing step

Quality assessment should include:

• SDS-PAGE to verify purity and apparent molecular weight

• Mass spectrometry to confirm identity

• Circular dichroism (CD) spectroscopy to verify structural integrity

• Dynamic light scattering (DLS) to assess aggregation tendency

• Amino acid analysis for quantitative composition verification

For maximum biological activity, it's crucial to verify that the purified recombinant peptide maintains its C-terminal amidation, as this post-translational modification is essential for receptor recognition and activation.

How can the biological activity of recombinant Periviscerokinin-2 be assessed?

Biological activity of recombinant Periviscerokinin-2 can be assessed through multiple complementary approaches:

• Myotropic activity assays: Using isolated hyperneural muscle from cockroaches (particularly P. americana) as a bioassay system to measure muscle contraction in response to the peptide . This approach directly measures the primary biological function of PVK-2.

• Ex vivo tissue assays: Similar to methods used in tick studies, where tissues like pharynx-esophagus are isolated and exposed to varying concentrations of the peptide (typically 0.1, 0.3, 1, 3, and 10 μM) while recording contractile responses . The methodology involves:

  • Preparing the tissue in physiological saline

  • Recording tissue responses to saline (control) and the peptide at different concentrations

  • Rinsing the tissue between treatments

  • Counting contractions for quantitative analysis

• Comparative activity testing: Comparing the activity of recombinant PVK-2 with chemically synthesized PVK-2 and other related peptides at equivalent concentrations to verify biological equivalence .

• Receptor binding assays: Using cells expressing the cognate receptor to measure binding affinity and activation.

For all assays, appropriate controls should be included, with statistical analysis of dose-dependent responses to establish EC50 values.

What are the structure-activity relationships of Periviscerokinin-2?

Structure-activity relationships (SAR) of Periviscerokinin-2 reveal several critical features that determine its biological function:

• C-terminal amidation: Essential for receptor recognition and biological activity; the amidated form is confirmed to be biologically active through chemical synthesis, bioassay, and comparison of retention times between native and synthetic peptides .

• C-terminal tripeptide (PRVamide): Functions as the core active region, with similarity to the pyrokinin C-terminal tripeptide PRLamide . The periviscerokinin C-terminal tripeptide sequence (PRVamide) is quite similar to the pyrokinin C-terminal tripeptide PRLamide and, accordingly, elicits a lower level pupariation acceleration activity .

• Amino acid substitutions: The relationship between structure and activity can be observed by comparing similar peptides across species. For example, the tick periviscerokinin and pyrokinin family shows conservation in the critical C-terminal regions while allowing variation in other positions .

The table below shows the comparison of periviscerokinin and pyrokinin peptides from different tick species, illustrating the conservation of critical structural elements:

This comparative approach to structure-activity reveals the evolutionary conservation of functional elements across species .

How does Periviscerokinin-2 compare across different arthropod species?

Periviscerokinin-2 shows notable conservation and variation across arthropod species, providing insights into evolutionary relationships:

In cockroaches (Periplaneta americana): The classical PVK-2 has the sequence GSSSGLISMPRV-NH2, functioning as a myotropic neuropeptide .

In ticks (Rhipicephalus species and Ixodes scapularis): Tick periviscerokinin sequences show similarity but feature distinctive variations. For example, R. sanguineus has PVK sequences such as pQLVPVIRNa (CAPA-PVK2) that end with RNa like the P. americana PVK-1 (GASGLIPVM-RNa) .

The CAPA gene encodes both periviscerokinin and pyrokinin families in ticks, suggesting evolutionary relationships . All three tick species studied (R. sanguineus, R. microplus, and I. scapularis) have two PVKs and five PKs encoded by a single capa gene . The slightly derived CAPA-PVK2s of three tick species with RNamide at the C-terminal end are similar to the first periviscerokinin identified in insects from P. americana .

This cross-species comparison provides valuable insights into the evolutionary conservation of these neuropeptide families and their potential functional significance across arthropod lineages.

What are the receptor mechanisms for Periviscerokinin-2?

The receptor mechanisms for Periviscerokinin-2 involve interaction with G-protein coupled receptors (GPCRs) that belong to the pyrokinin/CAPA receptor family. Research on related systems in ticks provides insights into these mechanisms:

• Receptor expression patterns: In Rhipicephalus sanguineus, pyrokinin receptor (PKR) transcripts are most abundant in the pharynx-esophagus, chelicerae, and other feeding-related tissues associated with the capitulum (PECO), with expression levels 3.3-fold higher than in the rest of the body . This tissue-specific expression pattern correlates with the functional activity of these peptides in regulating feeding-related tissues.

• Tissue-specific responses: The pharynx-esophagus of tick species responds with increased contractions to the endogenous PK and PK analogs in a dose-dependent manner, validating the functional significance of these receptor-ligand interactions .

• Quantitative receptor analysis: RT-qPCR methodologies can be used to analyze relative receptor abundance across tissues. For example, in R. sanguineus:

  • Highest expression in feeding-related tissues (PECO)

  • Moderate expression in the synganglion (SynG)

  • Lowest expression in reproductive tissues (ReprT)

The relative quantification of pyrokinin receptor transcripts across different tissues provides important insights into the physiological roles of these neuropeptides and potential targets for intervention .

What role does Periviscerokinin-2 play in insect physiology research?

Periviscerokinin-2 plays several important roles in insect physiology research:

• As a model for neuropeptide signaling systems: PVK-2 exemplifies how small peptide neurohormones can exert potent effects on specific tissues, making it valuable for studying peptidergic signaling mechanisms .

• For understanding neurohormonal regulation: The finding that PVK-2 is one of only two putative myotropic neurohormones from the abdominal perisympathetic organs that is effective in the nanomolar range confirms the hypothesis that the neurohormonal system of the ventral nerve cord is remarkably different from that of the brain .

• In comparative endocrinology: Studying homologous peptides across species (like the comparison between cockroach PVKs and tick PVKs) provides insights into the evolution of neuropeptide systems .

• As a target for pest management research: Understanding the specific roles of neuropeptides like PVK-2 in regulating critical physiological processes could potentially lead to the development of highly selective pest control strategies that target specific neuropeptide receptors .

The study of PVK-2 also contributes to our broader understanding of the neurobiology of insects and other arthropods, particularly the complex interplay between different neuropeptide families and their diverse physiological roles.

How is Periviscerokinin-2 related to allergenic proteins in cockroach allergy research?

While Periviscerokinin-2 itself is not identified as a major cockroach allergen, research on Periplaneta americana allergens provides context for understanding how different cockroach proteins contribute to allergic responses:

• Allergen classification: Unlike PVK-2, major cockroach allergens include proteins like tropomyosin (Per a 7) and other proteins designated as Per a 1 through Per a 10 (excluding Per a 6) . These allergens have been systematically characterized for their immunogenic properties.

• Component-resolved diagnosis (CRD): Studies using recombinant allergens have established the frequencies of sensitization to different Periplaneta allergens in allergic populations. For example, in one study the sensitization frequencies were: Per a 1: 16%, Per a 3: 41%, Per a 4: 18%, Per a 7: 28%, Per a 8: 24%, and Per a 9: 50% .

• Recombinant protein production methodology: The expression and purification methods used for recombinant cockroach allergens provide valuable technical insights that can be applied to the production of other P. americana proteins, including neuropeptides like PVK-2. These methods typically include:

  • Expression in E. coli as non-fusion or tagged proteins

  • Multistep chromatography purification

  • Characterization by SDS-PAGE, circular dichroism spectroscopy, and mass spectrometry

Understanding the broader proteome of P. americana, including both allergenic proteins and neuropeptides like PVK-2, contributes to a more comprehensive picture of cockroach biology and potential applications in both allergy research and basic neurobiology.

What are potential applications of recombinant Periviscerokinin-2 in tick control research?

Recombinant Periviscerokinin-2 and related peptides show promising applications in tick control research based on several lines of evidence:

• Target tissue identification: Research has demonstrated that pyrokinin/periviscerokinin peptides affect the pharynx-esophagus, lateral dilator pharynx muscles, and likely the cheliceral muscles in ticks . These tissues are directly involved in the feeding process, making them attractive targets for feeding disruption strategies.

• Receptor expression patterns: The pyrokinin receptor (PKR) is most abundant in feeding-related tissues associated with the capitulum in ticks like R. sanguineus . This specific localization suggests that targeting this receptor system could potentially disrupt feeding behavior.

• Functional evidence: Dose-dependent myotropic activity of pyrokinin peptides and analogs on the pharynx-esophagus of tick species provides direct evidence of their potential to interfere with feeding mechanisms .

• Receptor silencing effects: Research on the related periviscerokinin receptor in Rhipicephalus microplus has shown that silencing this receptor in females reduces survival, weight, and reproductive output . This demonstrates the critical role of this signaling system in tick physiology.

The tick mouthparts represent an attractive target for interfering with tick feeding and therefore blocking pathogen transmission. Understanding the role of neuropeptides like PVK-2 in regulating these structures could lead to novel approaches for tick control and prevention of tick-borne diseases.

What methodological challenges exist in studying Periviscerokinin-2 receptor systems?

Studying Periviscerokinin-2 receptor systems presents several methodological challenges that researchers must address:

• Receptor specificity challenges: The structural similarity between periviscerokinin and pyrokinin peptides can lead to cross-reactivity at their respective receptors. Researchers must carefully design experiments to distinguish between receptor subtypes and their specificities .

• Tissue preparation complexity: For functional assays, the preparation of intact tissues like the pharynx-esophagus requires careful microdissection to maintain tissue viability. The methodology involves:

  • Dissecting under physiological saline

  • Maintaining appropriate temperature

  • Standardizing measurement techniques for contractile responses

• Quantitative expression analysis: When studying receptor expression patterns using RT-qPCR, researchers must carefully select appropriate reference genes for normalization. Studies have used genes like elongation factor 1-alpha and ribosomal protein S4 for this purpose .

• Statistical analysis considerations: For comparing receptor expression across tissues, appropriate statistical methods must be applied. One approach is to use one-way ANOVA followed by Tukey's multiple comparisons test to determine significant differences in relative receptor abundance .

• Receptor localization challenges: Future research should address the precise localization of receptors using immunolocalization techniques to definitively identify the target sites of these peptides in feeding-related tissues .

Overcoming these methodological challenges is essential for advancing our understanding of how periviscerokinin peptides interact with their receptors and regulate important physiological processes in insects and ticks.