Recombinant Fasciola hepatica Fatty acid-binding protein type 2

What is Fasciola hepatica Fatty Acid-Binding Protein Type 2 and how does it differ from other FABP types?

Fasciola hepatica Fatty Acid-Binding Protein Type 2 (FABP Type II) is one of several FABP isoforms found in F. hepatica, a parasitic trematode responsible for fascioliasis in livestock and humans. FABP Type II differs from other types (I and III) in its molecular weight, isoelectric point, and amino acid sequence.

According to proteomic analyses, FABP Type II has a molecular mass of approximately 15 kDa with an isoelectric point (pI) around 5.21, while FABP Type I (also referred to as Fh15) has a mass of about 9-10 kDa with a pI of 6.26-6.33, and FABP Type III has a mass of approximately 14.5 kDa with a more basic pI of 7.82 . These differences reflect distinct structural and potentially functional characteristics of the FABP types in F. hepatica.

What is the biological role of FABP Type II in Fasciola hepatica?

FABP Type II, like other FABPs in F. hepatica, plays a crucial role in the parasite's lipid metabolism. Since parasitic trematodes like F. hepatica are unable to synthesize lipids de novo, particularly long-chain fatty acids and cholesterol, they rely on carriers like FABPs to uptake these essential molecules directly from the host .

The biological functions of FABP Type II include:

• Transport of host-derived fatty acids to specific destinations within the parasite

• Potential roles in the parasite's developmental processes

• Possible involvement in modulating host immune responses during infection

These proteins are localized in the tegument and parenchymal cells of F. hepatica, strategically positioned to interact with the host environment .

How is FABP Type II expression regulated during different life stages of Fasciola hepatica?

FABP Type II expression varies significantly across the life cycle of F. hepatica. Proteomic analyses using 2D SDS-PAGE have revealed a dramatic reduction of FABP isoforms, including Type II, in newly excysted juveniles (NEJs) compared to adult worms .

This differential expression suggests that FABP Type II may have stage-specific functions, with greater importance in adult worms that reside in the bile ducts and rely heavily on host-derived lipids. The temporal regulation of FABP expression likely reflects the changing metabolic and immunomodulatory requirements as the parasite develops and migrates through different host tissues .

What are the most effective methods for expressing and purifying recombinant F. hepatica FABP Type II?

The most effective methods for expressing and purifying recombinant F. hepatica FABP Type II involve bacterial expression systems, particularly E. coli. The methodological approach typically includes:

• Gene cloning: The FABP Type II gene sequence (approximately 15 kDa) is amplified from F. hepatica cDNA using PCR with specific primers and cloned into an appropriate expression vector .

• Expression optimization: Transformation into an E. coli expression strain (such as BL21(DE3)) followed by induction with IPTG under optimized conditions (temperature, duration, and concentration).

• Purification strategies:

  • Initial purification using affinity chromatography (His-tag or GST-tag depending on the construct)

  • Further purification by ion-exchange chromatography (given the slightly acidic nature of FABP Type II, pI ≈ 5.21)

  • Final polishing step using size-exclusion chromatography

  • Optional endotoxin removal for immunological studies

• Quality control: Assessment of purity by SDS-PAGE, Western blotting, and mass spectrometry to confirm the molecular weight of 15,327 Da reported in proteomic studies .

This approach has been demonstrated to yield functionally active recombinant FABP proteins, as evidenced by studies with the related Fh15 (Type I) protein .

How can researchers verify the structural integrity and functional activity of recombinant FABP Type II?

Verification of structural integrity and functional activity of recombinant FABP Type II should include multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure characteristics

  • Thermal stability analysis to determine melting temperature

  • Limited proteolysis to confirm proper folding

  • Native PAGE to assess oligomeric state

• Functional activity verification:

  • Lipid binding assays using fluorescent fatty acid analogs (e.g., 1-anilinonaphthalene-8-sulfonic acid)

  • Isothermal titration calorimetry to determine binding affinity constants for different fatty acids

  • Competition assays with natural ligands

• Immunological activity testing:

  • Assessment of interaction with toll-like receptors (TLRs) similar to what has been documented for FABP Type I (Fh15)

  • Measurement of cytokine production (IL-1β, TNFα) in response to FABP stimulation in cell culture models

  • Evaluation of TLR signaling pathway modulation

Research has shown that recombinant forms of F. hepatica FABPs can maintain functional activity similar to their native counterparts, as demonstrated with Fh15 (Type I), which retained its immunomodulatory properties after recombinant production .

What are the key molecular features that distinguish recombinant FABP Type II from native FABP Type II?

The key molecular features that distinguish recombinant FABP Type II from its native counterpart include:

• Structural modifications:

  • Presence of affinity tags (His-tag, GST-tag) used for purification

  • Potential additional amino acids from cloning sites

  • Possible differences in post-translational modifications

• Folding characteristics:

  • Minor differences in tertiary structure due to expression in a prokaryotic system

  • Potential variation in disulfide bond formation

• Functional considerations:

  • Generally similar lipid-binding properties, but potentially altered binding kinetics

  • Comparable immunomodulatory functions, as demonstrated with recombinant Fh15 (Type I), which suppressed IL-1β and TNFα expression in macrophages similar to the native protein

• Physical properties:

  • Slightly altered molecular weight due to tagging systems

  • Potential modification of isoelectric point

Studies with recombinant Fh15 have shown that the recombinant form maintains key functional properties of the native protein, suggesting that properly produced recombinant FABP Type II would similarly retain its essential characteristics .

How does FABP Type II modulate host immune responses during Fasciola hepatica infection?

FABP Type II, like other F. hepatica FABPs, likely contributes to immune modulation during infection, though its specific mechanisms may differ from the better-studied FABP Type I (Fh15). Based on current research:

• Cytokine modulation: While specific data for FABP Type II is limited, studies with FABP proteins from F. hepatica demonstrate suppression of pro-inflammatory cytokines such as IL-1β and TNFα in mammalian macrophages and modulation of the TLR4 pathway .

• Inflammatory pathway suppression: F. hepatica FABPs have been shown to suppress LPS-induced TLR4 stimulation and inhibit inflammatory signaling cascades. This effect extends to multiple TLRs in response to bacterial extracts, suggesting a broad spectrum of action .

• Liver fibrosis regulation: F. hepatica infection activates TNF-related pathways that upregulate genes responsible for fibrosis, including IL6, SERPINE1, and TNFRSF1A . FABP Type II may participate in these processes, potentially contributing to the hepatic pathology characteristic of fascioliasis.

• Antigen-presenting cell modulation: Chronic F. hepatica infection has been associated with apoptosis of antigen-presenting cells, mediated through STAT3, APP, and DUSP1 genes . FABPs may contribute to this immunosuppressive environment.

The distinct immunomodulatory properties of FABP Type II warrant further investigation, particularly in comparison to the better-characterized FABP Type I.

What are the potential applications of recombinant FABP Type II in vaccine development against fascioliasis?

Recombinant FABP Type II holds significant potential for vaccine development against fascioliasis due to several advantageous characteristics:

• Antigenic properties and recognition:

  • FABP Type II is recognized by infected host immune systems, as evidenced by co-immunoprecipitation studies using sera from infected sheep

  • Its conservation across the parasite's lifecycle makes it a potential target for interrupting multiple stages of infection

• Immunomodulatory capacity:

  • The ability to modulate host immune responses could be leveraged to design vaccines that overcome parasite-induced immunosuppression

  • Understanding FABP Type II's immunomodulatory mechanisms could inform adjuvant selection for optimal vaccine formulation

• Production advantages:

  • Recombinant FABP Type II can be produced in bacterial expression systems with high yield and purity

  • This addresses the limitations of native protein purification, which is "not cost-beneficial and unsuitable for industrial grade scale-up"

• Cross-species potential:

  • Comparative studies between F. hepatica and F. gigantica have identified species-specific proteins that could be used for differential diagnosis or targeted vaccination

  • FABP Type II could be evaluated for cross-protection against both Fasciola species

How does recombinant FABP Type II compare to other F. hepatica proteins (like cathepsins) as potential therapeutic or diagnostic targets?

Recombinant FABP Type II has several distinctive characteristics when compared to other F. hepatica proteins such as cathepsins:

• Immunogenicity and stability:

  • FABPs are generally more stable than proteolytic enzymes like cathepsins

  • Research with FABP Type I (Fh15) showed that its immunomodulatory effect "was not impaired by a thermal denaturing process" , suggesting FABP Type II may also retain activity under various conditions

  • Cathepsin L1 (FhCatL1) has been identified as "an excellent candidate for commercialized diagnostic assays or vaccine products" , indicating potential competition between these protein families

• Functional mechanisms:

  • FABPs primarily function in lipid transport and immunomodulation

  • Cathepsins serve as proteolytic enzymes involved in tissue invasion, nutrient acquisition, and immune evasion

  • This functional distinction suggests they could be complementary targets in combination therapies or diagnostics

• Expression patterns:

  • Cathepsins show dynamic expression throughout the parasite lifecycle, with specific isoforms (L3 and L4) upregulated during host-parasite interactions

  • FABP Type II shows stage-specific expression with reduced presence in newly excysted juveniles compared to adults

  • These differential expression patterns suggest stage-specific targeting opportunities

• Diagnostic applications:

  • Proteomics studies have identified F. hepatica-specific proteins that exist "in all periods in F. hepatica but not in F. gigantica" , which has implications for species-specific diagnostics

  • Both FABPs and cathepsins appear in excretory-secretory products, making them accessible biomarkers

• Vaccine development potential:

  • Studies indicate that "different preparations of FhCatL1 including recombinant protein or mimotope and either used solitary or in combination with other antigens succeeded to confer efficient protective potentials in different animal models"

  • Combining FABP Type II with cathepsins might provide broader protection against multiple parasite stages

The complementary properties of FABPs and cathepsins suggest potential value in multi-antigen approaches for both diagnostic and therapeutic applications.

What proteomic and transcriptomic approaches are most effective for studying FABP Type II expression and function?

The most effective proteomic and transcriptomic approaches for studying FABP Type II include:

• Proteomic methodologies:

  • 2D SDS-PAGE with selective IPG strips: Using pH 4.7 to 5.9 ranges for optimal resolution of FABP Type II (pI ≈ 5.21)

  • LC-MS/MS shotgun proteomics: For identification and quantification of FABP Type II in complex samples

  • SWATH-MS (Sequential Window Acquisition of all Theoretical Mass Spectra): For quantitative proteomics to evaluate protein expression changes during host-parasite interactions

  • Co-immunoprecipitation (Co-IP): To identify interaction partners of FABP Type II, as demonstrated in studies pulling down proteins from infected sheep sera

• Transcriptomic approaches:

  • RNA-seq: For comprehensive transcriptome analysis and identification of differential FABP expression across parasite life stages

  • Quantitative RT-PCR: For targeted validation of FABP Type II expression patterns

  • Single-cell RNA sequencing: To identify cell-specific expression patterns within parasite tissues

• Integrated multi-omics:

  • Combined proteomics-transcriptomics: To correlate mRNA and protein levels, as demonstrated in studies of F. hepatica eggs

  • Time-course analyses: To track FABP expression changes during development and host interaction

• Bioinformatic analysis techniques:

  • Gene Ontology enrichment: To contextualize FABP Type II within functional networks

  • Pathway analysis using IPA (Ingenuity Pathway Analysis): To identify enriched canonical pathways related to FABP function

  • Principal coordinate analysis (PCoA): For comparative genomics between different Fasciola isolates

Studies incorporating these approaches have successfully identified differential expression patterns of FABPs during parasite development and host interaction, providing valuable insights into their biological functions .

How can researchers effectively analyze the structural differences between FABP Type II and other FABP isoforms?

Researchers can effectively analyze structural differences between FABP Type II and other FABP isoforms using a comprehensive multi-method approach:

• Computational structural analysis:

  • Homology modeling: Using solved FABP structures as templates to model FABP Type II and other isoforms

  • Molecular dynamics simulations: To analyze dynamic behavior and ligand binding characteristics

  • Binding pocket analysis: To identify differences in the fatty acid binding sites between isoforms

  • Electrostatic surface mapping: To visualize charge distribution differences that may relate to distinct functions

• Experimental structural determination:

  • X-ray crystallography: To determine high-resolution structures of purified recombinant FABPs

  • Nuclear magnetic resonance (NMR) spectroscopy: For solution structure determination and analysis of protein dynamics

  • Cryo-electron microscopy: Potentially useful for larger FABP complexes with binding partners

• Comparative biochemical analysis:

  • Ligand binding assays: Using fluorescent probes to determine binding specificity and affinity for different fatty acids

  • Thermal shift assays: To compare protein stability between FABP isoforms

  • Limited proteolysis: To identify structural differences in protease accessibility

  • Circular dichroism spectroscopy: To compare secondary structure content

• Functional comparative approaches:

  • Site-directed mutagenesis: To investigate the role of specific amino acids in determining isoform-specific functions

  • Domain swapping experiments: Between FABP types to identify regions responsible for specific functions

  • Structural comparison with host FABPs: To understand host-parasite interactions and potential molecular mimicry

• Immunological structure-function studies:

  • Epitope mapping: To identify isoform-specific antigenic determinants

  • Structural basis of TLR interaction: Comparing how different FABP structures interact with host immune receptors

These approaches can reveal the molecular basis for the distinct properties of FABP Type II compared to Types I and III, as suggested by their different molecular weights (15.3 kDa vs. 9-10 kDa for Type I) and isoelectric points (5.21 vs. 6.26-6.33 for Type I and 7.82 for Type III) .

What in vitro and in vivo experimental models are most appropriate for studying the immunomodulatory effects of recombinant FABP Type II?

Several experimental models are appropriate for studying the immunomodulatory effects of recombinant FABP Type II:

• In vitro cellular models:

  • Murine macrophages: Primary cells or cell lines (e.g., RAW264.7) for studying cytokine suppression, as demonstrated with FABP Type I (Fh15)

  • THP1 Blue CD14 cells: Human monocytic cell line for assessing TLR stimulation and inflammatory responses

  • Mouse primary small intestinal epithelial cells (MPSIEC): To model early intestinal interactions as the parasite crosses the gut barrier

  • Dendritic cells and Treg cells: For evaluating antigen presentation and regulatory T cell induction

  • Hepatic stellate cells: To study the role of FABPs in liver fibrosis development

• Ex vivo tissue models:

  • Precision-cut liver slices: To study hepatic responses in a system that maintains tissue architecture

  • Intestinal organoids: For examining parasite-intestinal epithelium interactions

• In vivo animal models:

  • Sheep and cattle models: Natural hosts with different disease presentations (sheep show more severe pathology than cattle)

  • Mouse models: For preliminary immunological studies and mechanistic investigations

  • Water buffalo: Alternative host model for comparative studies with F. gigantica infection

• Experimental readouts:

  • Cytokine profiling: Measurement of IL-1β, TNFα, IL-6, and IL-10 production

  • TLR activation assays: Assessment of TLR4 and other TLR signaling pathways

  • Gene expression analysis: Evaluation of fibrosis-related genes (TNF, IL6, PLAU, SERPINE1)

  • Immune cell phenotyping: Flow cytometric analysis of antigen-presenting cell and T cell populations

  • Histopathological examination: Assessment of tissue changes in response to FABP Type II treatment

• Comparative experimental design:

  • Time-course experiments: To track immunomodulatory effects across acute and chronic phases

  • Dose-response studies: To determine optimal concentrations for immunomodulation

  • Comparison with native protein: To validate recombinant protein activity

Research has shown significant differences in disease presentation between sheep and cattle, with "fewer DEGs at the acute stage of infection" identified in cattle compared to sheep, suggesting host-specific immune responses that should be considered when designing experiments .

What are the current technical challenges in producing high-quality recombinant FABP Type II for research applications?

Several technical challenges exist in producing high-quality recombinant FABP Type II for research:

• Expression optimization challenges:

  • Codon optimization: Adaptation of the F. hepatica FABP Type II gene sequence for optimal expression in prokaryotic systems

  • Solubility issues: Preventing formation of inclusion bodies during high-level expression

  • Folding accuracy: Ensuring proper folding in the bacterial cytoplasm to maintain native structure

  • Yield inconsistencies: Batch-to-batch variation in protein yield and quality

• Purification hurdles:

  • Tag interference: Affinity tags may affect protein structure or function

  • Tag removal: Efficient removal of tags without compromising protein integrity

  • Contaminant elimination: Removing host cell proteins with similar physicochemical properties

  • Endotoxin removal: Critical for immunological studies to avoid LPS contamination that could confound results

• Quality control issues:

  • Structural verification: Confirming proper folding equivalent to native protein

  • Functional validation: Developing reliable assays to verify biological activity

  • Stability assessment: Ensuring consistent stability during storage and experimental conditions

• Scalability considerations:

  • Process optimization: Developing methods suitable for larger-scale production while maintaining quality

  • Cost-effectiveness: Making production economically viable for research purposes

• Specific challenges for FABP Type II:

  • Isoform purity: Ensuring no cross-contamination with other FABP types

  • Post-translational modifications: Addressing any differences from native protein

  • Lipid binding: Controlling the lipid content of the recombinant protein

These challenges echo those faced with FABP Type I (Fh15), where researchers noted that "purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up" , necessitating optimization of recombinant production methods.

How can contradictory research findings about FABP Type II be reconciled through improved experimental design?

Contradictory research findings about FABP Type II can be reconciled through several improvements in experimental design:

• Standardization of protein preparation:

  • Consistent production protocols: Using standardized expression and purification methods

  • Quality control metrics: Implementing rigorous criteria for protein purity and activity

  • Endotoxin testing: Mandatory screening to prevent confounding results in immunological studies

  • Batch documentation: Detailed reporting of production parameters in publications

• Comprehensive characterization:

  • Multi-method validation: Using multiple techniques to verify protein structure and function

  • Activity benchmarking: Establishing standard assays to compare activity between laboratories

• Experimental design improvements:

  • Appropriate controls: Including relevant negative and positive controls in all experiments

  • Dose-response studies: Testing multiple concentrations to identify threshold effects

  • Time-course analyses: Examining temporal dynamics of responses

  • Cell-specific effects: Acknowledging that "discrepancies in TNF production between studies could be due to the differences in cell type populations when evaluated in vitro and in vivo"

• Species and model considerations:

  • Host specificity awareness: Recognizing that "differences in parasite susceptibility between hosts" may affect results

  • Model selection rationale: Clearly justifying the choice of experimental model

  • Cross-species validation: Testing findings in multiple relevant host species

• Detailed reporting practices:

  • Methodological transparency: Complete description of experimental conditions

  • Data sharing: Making raw data available to enable reanalysis

  • Negative results publication: Encouraging reporting of non-significant findings

• Specific reconciliation approaches for FABP Type II:

  • Isoform verification: Ensuring studies are actually examining FABP Type II rather than other isoforms

  • Functional comparisons: Directly comparing FABP Type II with Types I and III in the same experimental system

  • Antigen composition awareness: Acknowledging that "antigen composition employed in each case" can affect immunological outcomes

By implementing these improvements, researchers can better reconcile contradictory findings and build a more consistent understanding of FABP Type II biology and function.

What are the most promising future research directions for recombinant FABP Type II in understanding and controlling fascioliasis?

The most promising future research directions for recombinant FABP Type II include:

• Structural biology and drug development:

  • High-resolution structural determination: Elucidating FABP Type II's three-dimensional structure to facilitate rational drug design

  • Structure-based design of inhibitors: Developing small molecules that could disrupt FABP Type II function

  • Drug delivery applications: Exploring FABP Type II as a carrier for anthelmintic compounds, leveraging its natural lipid transport function

• Immunology and vaccine development:

  • Multi-antigen vaccine formulations: Combining FABP Type II with other antigens like cathepsins for broader protection

  • Adjuvant optimization: Testing FABP Type II with various adjuvants to enhance protective immunity

  • Immunomodulatory mechanisms: Detailed investigation of how FABP Type II interacts with host immune receptors

  • Epitope mapping and engineering: Identifying and enhancing protective epitopes while removing suppressive ones

• Diagnostic applications:

  • Differential diagnostics: Developing assays to distinguish F. hepatica from F. gigantica based on species-specific FABP features

  • Stage-specific detection: Creating diagnostic tests that can identify different stages of infection

  • Point-of-care test development: Incorporating recombinant FABP Type II into rapid field tests

• Fundamental biology:

  • Interactome mapping: Identifying host and parasite proteins that interact with FABP Type II

  • Developmental regulation: Understanding the mechanisms controlling stage-specific expression

  • Functional genomics: Using CRISPR/Cas9 or RNAi to assess FABP Type II function in vivo

  • Comparative analysis: Investigating FABP Type II orthologs across trematode species

• Translation to control strategies:

  • Overcoming drug resistance: Exploring FABP Type II as a target for new anthelmintics given increasing triclabendazole resistance

  • Immunotherapeutic applications: Using recombinant FABP Type II to modulate host immunity in a therapeutic context

  • One Health approaches: Integrating FABP Type II research into broader control strategies addressing the human-animal interface

• Advanced technological applications:

  • CRISPR-based identification: Developing CRISPR-Cas biosensors for parasite detection

  • Machine learning integration: Using AI to predict FABP-host interactions and optimize vaccine design

  • Systems biology approaches: Modeling FABP Type II within host-parasite interaction networks

These directions align with the need for "novel mechanisms triggering apoptosis during F. hepatica infection" and "better understanding of the immune response against F. hepatica" to develop "novel Fasciola vaccines, where overcoming parasite-immunoregulatory strategies will be key to success" .

How do the molecular and functional properties of FABP Type II compare across different Fasciola species?

The molecular and functional properties of FABP Type II show both conservation and variation across Fasciola species:

Table 1: Comparative properties of FABP Type II across Fasciola species

Comparative proteomics has identified proteins that exist "in all periods in F. hepatica but not in F. gigantica" , suggesting species-specific variations in protein expression patterns. These differences can be exploited for differential diagnosis and species-specific targeting strategies.

Molecular characterization studies of F. hepatica across different geographical regions have shown that "the samples do not exhibit any morphometric variation between departments" and measurements of morphological traits "demonstrate that none of the evaluated characteristics overlap with F. gigantica" , supporting distinct species boundaries while suggesting conservation within F. hepatica.

What are the key differences in experimental outcomes when testing recombinant FABP Type II in different host species models?

Experimental outcomes with recombinant FABP Type II vary significantly across host species models:

Table 2: Comparative experimental outcomes across host species models

Research has demonstrated that "the different presentations of infection in these two species [cattle and sheep]" are "consistent with the different presentations of infection in these two species" . These differences are reflected in transcriptomic analyses showing significantly different patterns of gene expression during infection.

These species-specific differences must be considered when designing experiments with recombinant FABP Type II and interpreting their outcomes.

What comparative data exists on the expression levels of different FABP isoforms across F. hepatica life stages?

Comprehensive data on FABP isoform expression across F. hepatica life stages reveals stage-specific patterns:

What are the optimal laboratory protocols for assessing the immunomodulatory effects of recombinant FABP Type II in cell culture models?

The following optimized laboratory protocols provide a comprehensive framework for assessing the immunomodulatory effects of recombinant FABP Type II in cell culture models:

• Cell preparation and culture conditions:

  • Macrophage isolation and culture:

    • Isolate primary murine peritoneal macrophages or human monocyte-derived macrophages

    • Culture in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics

    • Plate at 1 × 10^6 cells/mL for 24 hours before treatment

  • Cell line maintenance:

    • THP1 Blue CD14 cells should be maintained according to manufacturer protocols

    • Differentiate with PMA (50 ng/mL) for 48 hours before experiments

• FABP Type II preparation:

  • Endotoxin removal: Critical step using polymyxin B columns or commercial endotoxin removal kits

  • Protein quantification: BCA assay with BSA standards

  • Quality control: SDS-PAGE and Western blot verification before each experiment

  • Concentration range: Test 0.1-10 μg/mL in dose-response studies

• Experimental design:

  • Controls:

    • Negative control: Cell culture medium only

    • Positive control: LPS (100 ng/mL) or appropriate TLR ligands

    • Specificity control: Heat-denatured FABP Type II

    • Blocking control: Anti-FABP antibodies co-incubation

  • Time points: 3h, 6h, 12h, 24h, 48h

  • Co-stimulation protocol: Pre-incubate cells with FABP Type II for 2 hours before adding TLR stimulants

• Analytical methods:

  • Cytokine measurement:

    • RNA isolation and qRT-PCR for IL-1β, TNFα, IL-6, IL-10 mRNA levels

    • ELISA for secreted cytokines in culture supernatants

    • Multiplex cytokine assays for comprehensive profiling

  • TLR pathway analysis:

    • TLR reporter cell assays (e.g., THP1-Blue cells)

    • Western blotting for phosphorylation of NF-κB, MAPK pathway components

    • Immunofluorescence for NF-κB nuclear translocation

  • Cell viability and function:

    • MTT or MTS assay for cell viability

    • Flow cytometry for surface marker expression (CD80, CD86, MHC II)

    • Phagocytosis assays to assess functional effects

• Data analysis and interpretation:

  • Normalization: All experimental values normalized to appropriate controls

  • Statistical analysis: ANOVA with post-hoc tests for multiple comparisons

  • Dose-response modeling: EC50 determination for functional endpoints

  • Comparative analysis: Direct comparison with FABP Type I (Fh15) effects under identical conditions

This approach is informed by studies showing that FABP Type I (Fh15) "suppresses the expression of IL-1β and TNFα in murine macrophages and THP1 Blue CD14 cells" and can "suppress the LPS-induced TLR4 stimulation" , providing a methodological framework for similar investigations with FABP Type II.

What quality control measures are essential when validating recombinant FABP Type II for research applications?

A comprehensive quality control framework for validating recombinant FABP Type II includes:

• Physicochemical characterization:

  • Identity verification:

    • Mass spectrometry (MS/MS) confirmation of protein sequence

    • Peptide mapping with coverage of >80% of the sequence

    • Western blot with specific antibodies

  • Purity assessment:

    • SDS-PAGE with densitometry analysis (>95% purity)

    • Size-exclusion chromatography

    • Capillary electrophoresis

  • Physical properties:

    • Confirmation of molecular weight (expected ~15.3 kDa)

    • Verification of isoelectric point (expected pI ~5.21)

    • Dynamic light scattering for aggregation analysis

• Structural integrity validation:

  • Secondary structure analysis:

    • Circular dichroism spectroscopy comparison with native or reference protein

    • Fourier-transform infrared spectroscopy

  • Tertiary structure assessment:

    • Intrinsic fluorescence spectroscopy

    • Nuclear magnetic resonance (for detailed structural analysis)

    • Differential scanning calorimetry for thermal stability

• Functional activity testing:

  • Lipid binding capacity:

    • Fluorescent ligand displacement assays

    • Isothermal titration calorimetry for binding constants

    • Native PAGE with lipid probes

  • Immunological function:

    • TLR4 inhibition assay (as demonstrated for FABP Type I)

    • Cytokine modulation in macrophage cultures

    • Comparison with reference standards or native protein

• Contaminant screening:

  • Endotoxin testing:

    • Limulus amebocyte lysate (LAL) assay (<0.1 EU/μg protein)

    • Endotoxin removal verification

  • Host cell protein quantification:

    • ELISA for E. coli proteins

    • Mass spectrometry for trace contaminants

  • DNA contamination:

    • qPCR for residual DNA (<10 ng/mg protein)

• Stability assessment:

  • Accelerated stability studies:

    • Activity retention at elevated temperatures

    • Freeze-thaw cycle stability

    • pH stability profile

  • Long-term storage stability:

    • Real-time and accelerated conditions

    • Functional activity monitoring over time

    • Degradation product analysis

• Batch consistency:

  • Lot-to-lot comparability:

    • Consistent biological activity between batches

    • Reproducible physicochemical properties

    • Manufacturing process validation

What bioinformatic approaches can be used to predict and analyze potential epitopes and binding sites in FABP Type II?

Several sophisticated bioinformatic approaches can be employed to predict and analyze epitopes and binding sites in FABP Type II:

• B-cell epitope prediction:

  • Sequence-based methods:

    • BepiPred, ABCpred, and SVMTriP for linear epitope prediction

    • DiscoTope and EPCES for conformational epitope prediction

    • Physicochemical property analysis (hydrophilicity, flexibility, accessibility)

  • Structure-based approaches:

    • Molecular dynamics simulations to identify surface-exposed regions

    • ElliPro for protein surface analysis and epitope identification

    • PEPOP for discontinuous epitope prediction

• T-cell epitope prediction:

  • MHC-I binding prediction:

    • NetMHC, IEDB Analysis Resource, and SYFPEITHI

    • Proteasomal cleavage site prediction using PAProC or NetChop

    • TAP binding efficiency prediction

  • MHC-II binding prediction:

    • NetMHCII, IEDB MHC-II binding tool

    • Identification of promiscuous epitopes binding multiple HLA alleles

    • CD4+ T cell epitope analysis focusing on Th2 responses

• Fatty acid binding site analysis:

  • Cavity detection algorithms:

    • CASTp, POCASA, and fpocket for binding pocket identification

    • Volume and hydrophobicity analysis of predicted pockets

    • Comparison with known FABP structures

  • Molecular docking simulations:

    • AutoDock, GOLD, or Glide for modeling fatty acid binding

    • Assessment of binding energy and interaction residues

    • Comparison of binding profiles with other FABP isoforms

• Protein-protein interaction prediction:

  • Interface prediction tools:

    • SPPIDER, PredUs, and WHISCY for interaction surface prediction

    • Hot spot residue identification using KFC2 and HotPoint

    • Conservation analysis of predicted interaction sites

  • TLR interaction modeling:

    • Molecular docking of FABP Type II with TLR4 and other TLRs

    • Identification of key residues for immunomodulatory function

• Cross-reactivity and specificity analysis:

  • Sequence and structural alignment:

    • Comparison with host FABPs to predict cross-reactivity

    • Identification of F. hepatica-specific regions absent in F. gigantica

    • Conservation analysis across Fasciola isolates

  • Homology assessment:

    • Phylogenetic analysis of FABP sequences

    • Structural superposition with related proteins

    • Epitope conservation across trematode species

• Integrated analysis pipelines:

  • Immunoinformatics workflows:

    • Integration of B-cell and T-cell epitope predictions

    • Epitope ranking based on multiple parameters

    • Population coverage analysis for vaccine applications

  • Structural vaccinology approaches:

    • Structure-based epitope selection and optimization

    • Epitope grafting and scaffold design

    • Prediction of epitope accessibility in multimeric assemblies

These bioinformatic approaches can "contribute in improvement of vaccine efficacy via wet-lab approach not only the theoretical aspect" and support "conducting certain preliminary experiments based on the interaction of a prepared antigen with immune-effectors cells or molecules" . The analysis results can guide rational design of experiments and help predict "the interaction of a prepared antigen with immune-effectors cells (macrophages, dendritic cells, Treg cells) or molecules (Interleukin β1, IL-10, MHC classes I and II proteins)" .