Recombinant Lonomia obliqua Unknown protein 2

What are the major recombinant proteins derived from Lonomia obliqua and what are their primary functions?

The major characterized recombinant proteins from Lonomia obliqua include:

• rLosac (recombinant Lonomia obliqua Stuart-factor activator): A hemolin-like protein that functions as Factor X activator with demonstrated neuroprotective and antiapoptotic properties .

• rLopap (recombinant Lonomia obliqua prothrombin activator protease): A lipocalin-like protein that acts as a prothrombin activator with cytoprotective effects .

Both proteins have shown remarkable ability to increase myoblast proliferation, modulate prostaglandin E2 (PGE2) release, affect myogenic regulatory factor (MRF) activities, and inhibit the release of inflammatory mediators like IL-6 in response to inflammatory stimuli . These properties make them promising candidates for modulating tissue regeneration processes, particularly in skeletal muscle injury .

Additional proteins identified in Lonomia obliqua include various serine proteases, phospholipase A2, protease inhibitors, and several unknown proteins that have been detected in proteomic analyses but not yet functionally characterized .

What experimental models are commonly used to study Lonomia obliqua recombinant proteins?

Multiple experimental models have been established to investigate the biological activities of Lonomia obliqua recombinant proteins:

Cell Culture Models:

• C2C12 mouse myoblasts: Used extensively to study effects on skeletal muscle regeneration, proliferation, and myogenic regulatory factors .

• Sf-9 insect cells: Employed to evaluate anti-apoptotic activity .

• HEK-293 human cells: Used for assessing cytoprotective effects .

• V-79 mammalian cells: Utilized in cell survival studies .

• Primary rat hippocampal neurons: Applied in neuroprotection studies against oxidative stress .

• Human Umbilical Vein Endothelial Cells (HUVECs): Used for endothelial cell response studies .

Animal Models:

• Rats have been used to study the effects of envenomation and evaluate antilonomic serum efficacy .

Biochemical and Functional Assays:

• Coagulation assays using chromogenic substrates (e.g., S-2222 for Factor Xa-like activity) .

• Cell viability assays, particularly in the presence of oxidative stressors like H₂O₂ .

• PGE₂ quantification and cyclooxygenase expression analysis .

• Expression analysis of myogenic regulatory factors .

These diverse models enable comprehensive investigation of the multifaceted functions of Lonomia obliqua recombinant proteins.

How is protein identification performed for Lonomia obliqua-derived compounds?

Identification of proteins from Lonomia obliqua involves several complementary techniques:

Sequencing Approaches:

• Edman sequencing has been employed to determine amino acid sequences of proteins isolated from Lonomia obliqua bristle extract (LOCBE) .

• cDNA library screening from various tissues (bristles, tegument, hemolymph) has been used to identify protein-encoding genes .

Mass Spectrometry-Based Proteomics:

• Shotgun proteomic analysis using nanoLC/LTQ-Orbitrap systems has been applied to identify proteins in the hemolymph .

• For protein identification, RAW files of MS-2 spectra are processed using Comet search software .

• Protein databases containing Lonomia entries from NCBI are used as reference for identification .

• Pattern Lab for Proteomics software is employed to identify proteins and peptides based on homology with database entries .

Data Processing:

• Search Engine Processor software filters identified spectra using multiple parameters (Xcorr, DeltaCN, DeltaMass, Peaks Matched, Spec Count Score) to generate a Bayesian score .

• Cutoffs are established to accept 1% false-positive identifications based on reversed database searches .

• Post-processing typically sets identifications to less than 10 ppm of mass variation .

A significant challenge noted in the literature is the limited range of data in databases for this species, with most available sequences derived from transcriptomic studies rather than complete proteomic characterization .

What mechanisms underlie the cytoprotective effects of Lonomia obliqua recombinant proteins?

The cytoprotective effects of Lonomia obliqua recombinant proteins involve several complex mechanisms:

Anti-apoptotic Activity:

• L. obliqua hemolymph and its derived proteins demonstrate inhibition of apoptosis across multiple cell types including insect cells (Sf-9), human cells (HEK-293), and mammalian cells (V-79) .

• These proteins appear to interfere with programmed cell death pathways through mechanisms that remain partially characterized .

Oxidative Stress Protection:

• Chromatographic fractions from L. obliqua provide protection against H₂O₂-induced oxidative stress in rat hippocampal neurons .

• Treatment with chromatographic fractions (0.05% and 0.1%) for 24 hours before H₂O₂ exposure (10 μmol/L) increases cell viability compared to cells exposed to H₂O₂ alone .

• Even with higher oxidative stress (H₂O₂ 30 μmol/L), the 0.05% chromatographic fraction maintained higher cellular viability compared to the positive control .

Prostaglandin Signaling:

• rLosac and rLopap modulate prostaglandin E2 (PGE2) release and affect cyclooxygenase (COX-1 and COX-2) expression .

• Increased expression of the EP4 receptor is observed in proliferating C2C12 cells, suggesting its involvement in mediating PGE2 effects .

Anti-inflammatory Effects:

• Both recombinant proteins inhibit the release of IL-6 and PGE2 induced by IL-1β inflammatory stimulus .

• This anti-inflammatory activity may create an environment more conducive to tissue regeneration and repair.

Complete structural characterization of the proteins responsible for these effects and identification of their specific cellular receptors remain active areas of investigation .

How do rLosac and rLopap modulate myogenic regulatory factors (MRFs) in skeletal muscle regeneration?

The modulation of myogenic regulatory factors by rLosac and rLopap represents a significant mechanism for their effects on skeletal muscle regeneration:

Proliferation Enhancement:

• Both recombinant proteins significantly increase the proliferation of C2C12 mouse myoblasts, a critical initial step in the muscle regeneration process .

• This proliferative effect provides a larger pool of myoblasts available for subsequent differentiation and fusion into myotubes.

MRF Activity Modulation:

• rLosac and rLopap affect the activities of myogenic regulatory factors, which are essential transcription factors controlling skeletal muscle development and regeneration .

• These proteins appear to regulate the temporal expression and activity patterns of MRFs, potentially optimizing the regeneration process.

Prostaglandin Pathway Involvement:

• The proteins modulate prostaglandin E2 (PGE2) release, which has been implicated in myoblast proliferation and differentiation .

• An increased expression of the EP4 receptor was observed in the proliferative phase of C2C12 cells, suggesting this receptor mediates some effects of PGE2 in myoblast proliferation .

Anti-inflammatory Contribution:

• By inhibiting the release of IL-6 and PGE2 induced by IL-1β, these proteins may create a more favorable environment for muscle regeneration by reducing excessive inflammation .

• This balanced inflammatory response likely helps maintain the proper temporal progression of regeneration phases.

These findings suggest that rLosac and rLopap create an optimized cellular environment for muscle regeneration through multiple complementary mechanisms affecting both myoblast behavior and the inflammatory microenvironment.

What methodological approaches can address variability and contradictions in Lonomia obliqua protein studies?

Research with Lonomia obliqua proteins faces several methodological challenges that require systematic approaches:

Source Material Standardization:

• L. obliqua can demonstrate significant qualitative and quantitative variations between specimens from different origins, necessitating careful standardization of source material .

• Researchers should establish detailed collection protocols specifying geographic origin, developmental stage, and storage conditions to minimize variability.

Protein Preparation Consistency:

• Implement standardized extraction, fractionation, and purification protocols to ensure comparable protein preparations across studies.

• Document and report protein concentration, purity assessment (e.g., SDS-PAGE, mass spectrometry), and activity validation for each preparation.

Comprehensive Experimental Design:

• Use multiple complementary techniques to validate findings (e.g., combining genomic, transcriptomic, and proteomic approaches).

• Include appropriate positive and negative controls in all experiments.

• Implement dose-response studies to identify optimal concentrations and potential biphasic effects.

• Assess time-course effects to capture temporal dynamics of protein activities.

Consideration of Synergistic Effects:

• The literature notes that "there may be synergistic action between the substances present in the hemolymph, which may contribute to a greater or lesser effect" .

• Compare isolated proteins with whole extracts or defined protein mixtures to identify potential synergistic or antagonistic interactions.

Ion Concentration Monitoring:

• "Concentration of free ions naturally present in the hemolymph can likewise differ between animals and produce shift in the chromatographic profile" .

• Monitor and report ion concentrations in protein preparations, particularly for studies involving ion-dependent activities.

Cell Type Selection:

• Different cell types may respond differently to the same proteins, as observed with neuronal cells compared to other cell types .

• Test multiple relevant cell types to establish cell type-specific versus universal effects.

By implementing these methodological approaches, researchers can more effectively address variability and apparent contradictions in studies of Lonomia obliqua proteins.

How can proteomic analysis be optimized for novel Lonomia obliqua protein identification?

Optimizing proteomic analysis for novel Lonomia obliqua protein identification requires addressing several technical challenges:

Enhanced Database Development:

• Protein identification in L. obliqua samples is significantly hampered by "the limited range of data contained in the database for this species" .

• Develop custom databases integrating transcriptomic data from various L. obliqua tissues to improve protein sequence coverage.

• Implement de novo sequencing approaches to identify proteins without complete reliance on existing databases.

Sample Fractionation Strategies:

• The research shows that "proteins present in fraction used in this study may differ over those described in the literature using the same chromatography methodology" .

• Employ multidimensional fractionation techniques before mass spectrometry to reduce sample complexity.

• Use orthogonal separation methods (e.g., ion exchange followed by reversed-phase chromatography) to maximize proteome coverage.

Advanced Mass Spectrometry Approaches:

• Utilize high-resolution LC-MS/MS systems like nanoLC/LTQ-Orbitrap for improved detection sensitivity .

• Implement data-independent acquisition (DIA) methods to capture more comprehensive peptide fragmentation patterns.

• Apply targeted proteomics for detection of low-abundance proteins of interest.

Optimized Data Analysis Parameters:

• Fine-tune search parameters including:

  • Mass tolerance for precursor ions (e.g., 50 ppm)

  • Fragment ion tolerance (e.g., 1 Da)

  • Fixed modifications such as cysteine carbamidomethylation

  • Peptide length requirements (e.g., minimum 6 residues)

Peptide Competition Consideration:

• Account for the phenomenon where "in the crude sample, more peptides can compete to the selection of fragmentation within the same cycle of acquisition, and thus, peptides signal with less intensity can be suppressed at the expense of others" .

• Implement strategies like gas-phase fractionation or increased MS/MS acquisition rates to improve coverage of low-abundance peptides.

These optimized approaches would enhance the identification of novel proteins from Lonomia obliqua and contribute to a more comprehensive understanding of its proteome.

What functional assays are essential for characterizing novel recombinant Lonomia obliqua proteins?

A comprehensive functional characterization of novel recombinant Lonomia obliqua proteins requires multiple complementary assays:

Cell Proliferation and Viability Assays:

• MTT or similar colorimetric assays to assess effects on cell viability and proliferation in relevant cell types (e.g., C2C12 myoblasts) .

• BrdU incorporation assays to specifically measure DNA synthesis as an indicator of cell proliferation .

• Real-time cell analysis systems for continuous monitoring of cell proliferation dynamics.

Oxidative Stress Protection Assessment:

• H₂O₂ challenge assays in neuronal and other cell types to evaluate cytoprotective effects .

• Measurement of intracellular ROS levels using fluorescent probes (e.g., DCFDA).

• Analysis of antioxidant enzyme activities (SOD, catalase, GPx) after protein treatment.

Inflammatory Response Modulation:

• Quantification of cytokine production (particularly IL-6) after inflammatory challenge (e.g., IL-1β) .

• Analysis of prostaglandin E2 release and cyclooxygenase expression (COX-1 and COX-2) .

• Assessment of NF-κB pathway activation through reporter assays or phosphorylation status.

Myogenic Factor Regulation:

• RT-qPCR and Western blot analysis of myogenic regulatory factors (MRFs) expression .

• Immunofluorescence staining to visualize myotube formation and assess differentiation capacity.

• Luciferase reporter assays for MRF-dependent transcriptional activity.

Coagulation-Related Activities:

• Chromogenic substrate assays (e.g., S-2222 for Factor Xa-like activity) .

• Prothrombin time (PT) and activated partial thromboplastin time (aPTT) measurements.

• Thromboelastography for comprehensive coagulation profile assessment.

Receptor Binding Studies:

• Analysis of EP4 receptor expression and signaling, given its apparent involvement in mediating some effects in C2C12 cells .

• Competitive binding assays to identify potential receptors for novel proteins.

• Signal transduction pathway activation studies using phosphorylation-specific antibodies.

These functional assays provide a framework for comprehensive characterization of novel recombinant proteins from Lonomia obliqua, enabling better understanding of their potential applications in research and therapeutics.

What are the optimal systems for expressing functional recombinant Lonomia obliqua proteins?

Selecting the appropriate expression system is critical for producing functional recombinant Lonomia obliqua proteins. Based on successful approaches in the literature:

Bacterial Expression Systems:

• E. coli systems have been used for expressing some Lonomia obliqua proteins, though they may lack post-translational modifications present in native proteins .

• For proteins requiring disulfide bonds, specialized E. coli strains (e.g., SHuffle, Origami) that facilitate disulfide formation in the cytoplasm may be preferable.

• Fusion tags (e.g., His-tag, MBP, GST) can improve solubility and facilitate purification.

Eukaryotic Expression Systems:

• Insect cell expression systems (e.g., Sf9, Sf21, High Five) using baculovirus vectors are particularly suitable for Lonomia-derived proteins, as they can provide insect-specific post-translational modifications.

• Yeast systems (P. pastoris, S. cerevisiae) offer a compromise between bacterial and mammalian systems, providing some post-translational modifications with relatively high yields.

• Mammalian cell systems may be necessary for specific proteins requiring complex mammalian-type modifications or when studying interactions with mammalian targets.

Expression Strategies:

• For lipocalin-like proteins such as Lopap, eukaryotic expression systems are generally preferred to maintain proper folding and potential glycosylation .

• For hemolin-like proteins such as Losac, both bacterial and eukaryotic systems have been successfully employed depending on the specific research requirements .

Optimization Approaches:

• Codon optimization for the selected expression host can significantly improve protein yields.

• Inducible promoter systems allow control over expression timing to minimize potential toxicity.

• Testing multiple constructs with different fusion partners or solubility tags can identify optimal expression conditions.

The literature demonstrates that both rLosac and rLopap have been successfully produced as functional recombinant proteins with their biological activities preserved , validating these expression approaches.

What protein purification strategies are most effective for Lonomia obliqua recombinant proteins?

Effective purification of Lonomia obliqua recombinant proteins typically involves multi-step strategies:

Affinity Chromatography:

• Affinity tags (e.g., His-tag, GST, MBP) enable initial capture of recombinant proteins from complex lysates.

• For lipocalin-like proteins such as Lopap, hydrophobic interaction chromatography may be particularly effective due to their lipid-binding properties .

• For hemolin-like proteins such as Losac, immunoaffinity approaches using specific antibodies may provide high selectivity .

Ion Exchange Chromatography:

• Used as a secondary purification step to remove contaminants based on charge differences.

• The literature notes that "Concentration of free ions naturally present in the hemolymph can differ between animals and produce shift in the chromatographic profile" , suggesting that careful attention to buffer composition is essential.

Size Exclusion Chromatography:

• Valuable as a final polishing step to separate monomeric protein from aggregates and to perform buffer exchange.

• Also provides information about the oligomeric state of the purified protein.

Activity-Based Purification:

• For proteins with enzymatic activity (e.g., protease activity of Lopap), substrate-affinity columns can provide high specificity.

• This approach simultaneously selects for properly folded, functional protein.

Purification Validation:

• SDS-PAGE and Western blotting to assess purity and identity.

• Mass spectrometry to confirm protein identity and detect potential modifications.

• Activity assays to verify that the purified protein retains its functional properties.

• Endotoxin testing for proteins intended for cell culture or in vivo applications.

Establishing reliable purification protocols is essential for consistent results in functional studies and potential therapeutic applications of these recombinant proteins.

How can recombinant Lonomia obliqua proteins be applied in regenerative medicine research?

Recombinant Lonomia obliqua proteins show significant potential for regenerative medicine applications:

Skeletal Muscle Regeneration:

• rLosac and rLopap have demonstrated the ability to increase myoblast proliferation and modulate myogenic regulatory factors, making them valuable tools for investigating muscle regeneration mechanisms .

• Their effects on PGE2 signaling and EP4 receptor expression provide potential targets for enhancing muscle repair processes .

• The research explicitly identifies these proteins as "promising proteins to modulate processes involving tissue regeneration as occurs during skeletal muscle injury" .

Neuroprotection:

• L. obliqua proteins have shown protective effects against oxidative stress in neuronal cells, suggesting applications in neurodegenerative disease research .

• The neuroprotective properties of rLosac in particular could be explored for conditions involving neuronal injury or degeneration .

Anti-inflammatory Applications:

• The ability of these proteins to inhibit the release of inflammatory mediators like IL-6 and PGE2 in response to IL-1β suggests potential in treating inflammatory conditions .

• Balancing inflammation is critical in many regenerative processes, making these proteins valuable research tools for optimizing the inflammatory phase of tissue repair.

Cell Survival Enhancement:

• The cytoprotective and anti-apoptotic properties documented across multiple cell types indicate broad potential for enhancing cell survival in various regenerative medicine applications .

• These properties could be particularly valuable in cell transplantation therapies where initial cell survival is often limited.

Tissue Engineering:

• The effects on cell proliferation and survival suggest applications in improving cell expansion for tissue engineering applications.

• Their ability to modulate the cellular microenvironment could be exploited to enhance scaffold-based tissue engineering approaches.

Future research should focus on elucidating the precise molecular mechanisms of these effects and developing delivery systems for targeted application of these proteins in specific regenerative medicine contexts.

What are the most promising directions for identifying novel functions of uncharacterized Lonomia obliqua proteins?

Several strategic approaches can accelerate the discovery of novel functions for uncharacterized Lonomia obliqua proteins:

Integrative Omics Approaches:

• Combine proteomic data with transcriptomic and genomic information to identify uncharacterized proteins of interest .

• Correlate protein expression patterns with specific tissues (bristles, hemolymph, tegument) or developmental stages to prioritize candidates for functional studies.

• Apply computational prediction tools to identify potential functional domains and homology to known proteins.

High-Throughput Functional Screening:

• Develop recombinant expression libraries of uncharacterized proteins for systematic functional screening.

• Implement cell-based assays to detect effects on key processes like cell proliferation, apoptosis resistance, or inflammatory response.

• Screen for interactions with known cellular receptors or signaling pathways.

Targeted Investigation of "Unknown Proteins":

• The research mentions "Unknown protein 3" in the proteomic analysis , suggesting the existence of multiple uncharacterized proteins.

• Focus on proteins uniquely present in chromatographic fractions with demonstrated biological activities.

• Prioritize proteins that demonstrate structural similarities to known bioactive proteins like Lopap and Losac.

Comparative Analysis with Other Venomous Species:

• Examine functional similarities with proteins from related lepidopterans or other venomous arthropods.

• Apply phylogenetic approaches to identify evolutionarily conserved functional domains.

Structure-Function Studies:

• Determine three-dimensional structures of uncharacterized proteins through X-ray crystallography or cryo-EM.

• Use structural information to guide targeted mutagenesis for function identification.

• Apply molecular docking approaches to predict potential binding partners or substrates.

Translational Research Focus:

• Investigate effects on tissue regeneration processes beyond skeletal muscle, such as wound healing, cardiac repair, or nerve regeneration.

• Explore potential immunomodulatory properties relevant to autoimmune or inflammatory conditions.

These approaches would significantly advance our understanding of Lonomia obliqua's proteome and potentially lead to the discovery of novel bioactive proteins with therapeutic applications.

What are the major knowledge gaps in Lonomia obliqua recombinant protein research?

Despite significant advances, several critical knowledge gaps remain in our understanding of Lonomia obliqua recombinant proteins:

Structural Characterization:

• Complete three-dimensional structures of key proteins like rLosac and rLopap have not been fully elucidated, limiting structure-based functional analysis and optimization .

• The search results note that "the structure of the anti-apoptotic effects responsible protein remains without being elucidated" .

Receptor Identification:

• Specific cellular receptors mediating the effects of Lonomia obliqua proteins remain largely unidentified, though the EP4 receptor has been implicated in some effects .

• The literature acknowledges that "it is still unknown antiapoptotic protein interaction with specific cellular receptors" .

Signaling Pathway Elucidation:

• Detailed molecular mechanisms and signaling pathways underlying the observed effects on cell proliferation, anti-apoptosis, and anti-inflammatory actions require further characterization.

• Understanding these pathways is essential for optimizing potential therapeutic applications.

Uncharacterized Proteins:

• Proteomic analyses have identified numerous proteins that remain uncharacterized, including several "Unknown proteins" mentioned in the search results .

• These represent a substantial untapped resource for potential novel bioactive compounds.

In Vivo Efficacy:

Post-Translational Modifications:

• The impact of post-translational modifications on recombinant protein function compared to native proteins requires systematic investigation.

• These modifications may significantly affect activity, stability, and receptor interactions.

Addressing these knowledge gaps would significantly advance the field and potentially lead to novel therapeutic applications for these fascinating bioactive proteins.