PLA2G7 Human, HEK

What is PLA2G7 and what are its primary biological functions?

PLA2G7 (Phospholipase A2 Group VII) is a secreted enzyme that functions as a calcium-independent phospholipase A2 involved in phospholipid catabolism during inflammatory and oxidative stress responses. At the lipid-aqueous interface, it hydrolyzes the ester bond of fatty acyl groups attached at the sn-2 position of phospholipids. It specifically targets phospholipids with short-chain fatty acyl groups at the sn-2 position, though it can also hydrolyze phospholipids with long fatty acyl chains if they carry oxidized functional groups. A critical function of PLA2G7 is hydrolyzing and inactivating platelet-activating factor (PAF), a potent pro-inflammatory signaling lipid that acts through PTAFR on various innate immune cells .

How does PLA2G7 differ from other phospholipase enzymes?

Unlike many secretory phospholipase A2 enzymes that require millimolar levels of Ca²⁺ for catalysis, PLA2G7 is calcium-independent. Traditional secretory PLA2 enzymes are characterized by their high disulfide bond content and low molecular mass (approximately 14 kDa), whereas PLA2G7 is larger (full-length human PLA2G7 spans amino acids 22-441). Additionally, PLA2G7 exhibits specificity for particular substrates, especially those with oxidized functional groups, making it particularly relevant in contexts of oxidative stress and inflammation .

What is the significance of PLA2G7 in human health and disease?

PLA2G7 plays crucial roles in inflammatory processes and has been implicated in various pathological conditions. As it hydrolyzes and inactivates PAF, it serves as a regulator of inflammatory responses. Research indicates its involvement in granuloma formation and NF-κB signaling pathways, which are essential in immune responses, including those against tuberculosis. Genomic variants in PLA2G7 have been associated with disease resistance/resilience, particularly in the context of immunological challenges like tuberculosis . The enzyme's activity in modulating inflammatory processes makes it relevant to cardiovascular disease, atherosclerosis, and other inflammatory conditions.

Why are HEK293 cells preferred for recombinant human PLA2G7 expression?

HEK293 cells are mammalian cells derived from human embryonic kidney tissue, making them ideal for expressing human proteins with proper post-translational modifications. For complex proteins like PLA2G7, these modifications are crucial for maintaining enzymatic activity and structural integrity. HEK293 cells provide a human-derived cellular environment that supports proper folding, glycosylation patterns, and secretion mechanisms needed for functional PLA2G7. This system yields recombinant proteins that more closely resemble naturally occurring human PLA2G7, with high purity (>95%) and low endotoxin levels (<1 EU/μg), making them suitable for a wide range of research applications including SDS-PAGE and HPLC .

What are the key considerations for optimizing PLA2G7 expression in HEK cells?

Optimizing PLA2G7 expression in HEK cells requires attention to several factors:

• Vector selection: Choose expression vectors with strong promoters compatible with HEK cells (e.g., CMV promoter)

• Codon optimization: Adjust codon usage for optimal expression in human cells

• Signal peptide design: Ensure efficient secretion using appropriate signal sequences

• Transfection efficiency: Optimize transfection conditions using agents compatible with HEK cells

• Culture conditions: Maintain cells at optimal density, temperature, and pH

• Harvest timing: Determine the optimal time post-transfection for protein collection

• Purification strategy: Design purification schemes that preserve enzymatic activity

For research applications requiring high purity, implementing affinity tags (such as the hexahistidine tag visible in the recombinant sequence) facilitates efficient purification while maintaining functional integrity .

How does E. coli-expressed PLA2G7 compare with HEK-expressed PLA2G7?

While both expression systems can produce recombinant human PLA2G7, the proteins differ significantly in several aspects:

While E. coli-expressed PLA2G7 can accurately quantitate in standard assays (as demonstrated with the Quantikine ELISA kit), HEK-expressed protein more closely represents the naturally occurring human enzyme and is preferred for studies examining physiological functions and interactions .

What are the validated methods for quantifying human PLA2G7 in experimental samples?

Several validated analytical methods are available for quantifying human PLA2G7:

• ELISA: Quantikine ELISA kits demonstrate high precision with intra-assay CVs of 2.3-6.8% and inter-assay CVs of 5.2-9.6%. These assays can detect both recombinant and naturally occurring human PLA2G7 .

• Western Blotting: Using validated antibodies that recognize specific epitopes of human PLA2G7, Western blotting provides semi-quantitative measurement and confirmation of molecular weight .

• Flow Cytometry (FACS): For cellular studies investigating PLA2G7 expression patterns .

• Immunohistochemistry (IHC): For tissue localization studies, with validated antibodies available for paraffin-embedded, frozen sections, and cell culture applications .

• HPLC: For purification and quality assessment of recombinant PLA2G7 .

The choice of method depends on the specific research question, sample type, and required sensitivity.

How can I assess the activity and functional integrity of recombinant PLA2G7?

Assessing the functional integrity of recombinant PLA2G7 involves several complementary approaches:

• Enzymatic Activity Assays: Measure the hydrolysis rate of specific substrates (e.g., PAF or short-chain phospholipids) using colorimetric or fluorometric detection of released fatty acids.

• Substrate Specificity Testing: Evaluate the enzyme's ability to hydrolyze different phospholipid substrates with varying fatty acyl chain lengths and oxidation states.

• Recovery Assessment: Quantify the recovery of PLA2G7 activity after spiking known amounts into different matrices. Published data shows average recovery rates of 92-106% across various sample types, including cell culture media (92%), EDTA plasma (101%), heparin plasma (102%), and serum (106%) .

• Linearity Testing: Determine if the activity remains linear across varying concentrations and conditions.

• Inhibitor Sensitivity: Assess response to known PLA2G7 inhibitors as a specificity confirmation.

• Mass Spectrometry: For detailed structural characterization and confirmation of post-translational modifications.

These methods collectively provide a comprehensive assessment of enzyme functionality.

What quality control parameters should be monitored for PLA2G7 produced in HEK systems?

Key quality control parameters for HEK-produced PLA2G7 include:

• Purity: Minimum 95% purity as determined by SDS-PAGE and HPLC analysis .

• Endotoxin Levels: Should be <1 EU/μg to avoid interfering with downstream applications, especially cell-based assays .

• Protein Concentration: Accurate quantification using validated methods like BCA assay or spectrophotometry.

• Molecular Weight Confirmation: Verification of the expected molecular weight (full-length human PLA2G7 spanning amino acids 22-441) by SDS-PAGE or mass spectrometry .

• Sequence Verification: Confirmation of the amino acid sequence, including any tags (e.g., the C-terminal hexahistidine tag noted in the recombinant protein sequence) .

• Enzymatic Activity: Functional assays demonstrating specific activity against known substrates.

• Batch-to-Batch Consistency: Monitoring inter-batch variation to ensure reproducible experimental results.

• Stability: Assessment of activity retention under storage conditions and after freeze-thaw cycles.

Regular monitoring of these parameters ensures research reproducibility and reliability of results.

How can epigenetic regulation of PLA2G7 be studied in inflammatory disease models?

Studying the epigenetic regulation of PLA2G7 in inflammatory disease models requires a multi-omics approach:

• ChIP-seq Analysis: Chromatin immunoprecipitation sequencing can identify histone modifications (such as H3K4me3 and H3K27me3) associated with PLA2G7 expression changes. Recent research shows that immune response genes demonstrate significant correlation between histone modifications and RNA polymerase II occupancy .

• DNA Methylation Profiling: Techniques such as bisulfite sequencing or methylation arrays can identify methylation patterns in the PLA2G7 promoter and enhancer regions that correlate with expression levels.

• Integration with Transcriptomics: Correlate epigenetic modifications with RNA-seq data to establish causal relationships between epigenetic states and PLA2G7 expression.

• Pathway Analysis: Tools like Gene Ontology enrichment, Ingenuity Pathway Analysis, and differential expression interaction networks can contextualize PLA2G7 regulation within broader inflammatory pathways .

• GWAS Integration: Incorporate genomic variant data to identify potential genetic influences on epigenetic regulation, as demonstrated in tuberculosis resistance/resilience studies that identified SNPs associated with NF-κB signaling and cytokine receptor interactions .

This integrated approach allows researchers to understand the complex regulatory mechanisms controlling PLA2G7 expression during inflammatory responses.

What role does PLA2G7 play in granuloma formation and host-pathogen interactions?

PLA2G7 has emerging significance in granuloma formation and host-pathogen interactions, particularly in the context of tuberculosis:

Recent multi-omics analyses integrating GWAS data have identified PLA2G7 among the genes that are core to granuloma formation, NF-κB signaling pathways, and cytokine receptor interactions . Granulomas represent organized immune cell structures that form in response to persistent pathogens like Mycobacterium tuberculosis.

The enzyme's role in inflammatory processes makes it a key player in the host immune response. As a regulator of PAF, PLA2G7 can modulate the recruitment and activation of immune cells that participate in granuloma formation. Additionally, its ability to hydrolyze oxidized phospholipids may influence the inflammatory microenvironment within granulomas, potentially affecting bacterial containment and persistence.

Cross-species comparative studies between bovine and human tuberculosis responses have revealed commonalities in host defense mechanisms, including genes involved in granuloma formation that contain SNPs associated with disease resistance/resilience . This evolutionary conservation underscores the fundamental importance of these pathways, including those involving PLA2G7, in antimycobacterial immunity.

Understanding PLA2G7's precise role in these processes could inform novel therapeutic approaches targeting granulomatous diseases.

How can functional genomics approaches be used to study PLA2G7 variants in disease susceptibility?

Functional genomics approaches to study PLA2G7 variants in disease susceptibility involve several sophisticated methodologies:

• Variant Identification and Prioritization: Integrate high-density GWAS data to identify intronic and exonic SNPs within the PLA2G7 gene significantly associated with disease phenotypes. Prior research has identified numerous SNPs associated with disease resistance/resilience in genes involved in key immunological pathways .

• Multi-omics Data Integration: Combine differential expression analysis (DEG), differential expression interaction networks (DEN), combined pathway analysis (CPA), and specialized tools like Ingenuity Pathway Analysis (IPA) to contextualize variant effects .

• Chromatin Accessibility Studies: Use techniques like ATAC-seq to determine if variants affect transcription factor binding sites or chromatin structure around the PLA2G7 locus.

• Epigenetic Profiling: Examine how variants influence histone modifications (H3K4me3, H3K27me3) and RNA polymerase II occupancy at the PLA2G7 gene, which correlate with gene expression changes during immune responses .

• CRISPR-based Functional Validation: Introduce specific variants into cellular models using CRISPR-Cas9 genome editing to directly observe their effects on PLA2G7 expression and function.

• Cross-species Comparison: Compare human and relevant animal model data to identify evolutionarily conserved mechanisms, as demonstrated in tuberculosis studies comparing bovine and human responses .

This comprehensive approach allows researchers to move beyond association to functional understanding of how PLA2G7 variants contribute to disease mechanisms.

What are common challenges in maintaining enzymatic activity of PLA2G7 during purification?

Preserving PLA2G7 enzymatic activity during purification presents several challenges that researchers should address:

• Oxidation Sensitivity: PLA2G7's function in oxidative stress responses makes it vulnerable to oxidation during purification. Include reducing agents like DTT or β-mercaptoethanol in buffers at appropriate concentrations.

• Temperature Stability: Maintain samples at 4°C throughout purification, as temperature fluctuations can lead to conformational changes and activity loss.

• Proteolytic Degradation: Add protease inhibitors to all buffers to prevent proteolytic cleavage that could compromise enzyme structure.

• Buffer Optimization: PLA2G7 activity is sensitive to buffer composition; optimize pH, salt concentration, and presence of stabilizing agents.

• Adsorptive Losses: Minimize contact with materials that may adsorb the protein; pre-coat surfaces with BSA or use low-binding materials.

• Aggregation: Monitor protein concentration to prevent aggregation that reduces specific activity; consider adding stabilizers like glycerol.

• Metal Ion Interactions: Though calcium-independent, PLA2G7 activity may be affected by other metal ions introduced during purification; use chelators like EDTA when appropriate.

• Storage Conditions: Establish optimal storage conditions (temperature, buffer composition, additives) to maintain activity during and after purification.

Addressing these factors helps maintain the functional integrity of PLA2G7 throughout the purification process.

How should experimental controls be designed for cell-based assays involving PLA2G7?

Robust experimental controls for cell-based PLA2G7 assays should include:

• Expression Controls:

  • Positive control: Cells transfected with a verified PLA2G7 expression vector

  • Negative control: Cells transfected with empty vector

  • Untransfected control: Baseline cellular background

• Activity Controls:

  • Enzymatic inhibition control: Known PLA2G7 inhibitors at verified concentrations

  • Heat-inactivated enzyme control: Denatured PLA2G7 to establish background

  • Substrate specificity control: Alternative substrates to confirm enzyme specificity

• Antibody Validation Controls (for detection assays):

  • Isotype controls: Match the isotype of the primary antibody

  • Secondary antibody-only control: Evaluate non-specific binding

  • Blocking peptide control: Confirm antibody specificity

• Assay Quality Controls:

  • Inter-assay calibrators: Include standards across multiple experiments

  • Recovery controls: Spike-in experiments with known quantities of PLA2G7

  • Dilution linearity: Serial dilutions to confirm concentration-response relationships

• Biological Relevance Controls:

  • Physiologically relevant inducers: Stimuli known to modulate PLA2G7 expression

  • Time-course controls: Establish temporal dynamics of responses

  • Cell-type specificity controls: Compare responses across relevant cell types

These controls ensure the reliable interpretation of experimental results and facilitate troubleshooting if unexpected outcomes occur.

What strategies can improve reproducibility when measuring PLA2G7 in clinical samples?

Ensuring reproducibility when measuring PLA2G7 in clinical samples requires systematic approaches:

• Standardized Sample Collection:

  • Establish consistent collection protocols (time of day, fasting status)

  • Use standardized collection tubes (EDTA, heparin, or serum as appropriate)

  • Process samples within validated time windows

• Validated Analytical Methods:

  • Use methods with documented precision metrics, such as ELISA kits with established intra-assay (CV 2.3-6.8%) and inter-assay (CV 5.2-9.6%) precision

  • Include calibrators and quality controls in each analytical run

  • Participate in external quality assessment programs when available

• Matrix Considerations:

  • Validate recovery across different sample matrices (serum, EDTA plasma, heparin plasma)

  • Establish expected recovery ranges for each matrix type (e.g., 91-115% for serum, 91-112% for EDTA plasma)

  • Address matrix-specific interference through appropriate sample preparation

• Reference Standards:

  • Use well-characterized reference materials

  • Ensure lot-to-lot consistency of standards and calibrators

  • Establish traceability to international standards when possible

• Data Analysis and Reporting:

  • Pre-define acceptance criteria for analytical runs

  • Apply consistent data processing algorithms

  • Report results with appropriate precision and confidence intervals

• Validation Across Populations:

  • Establish reference ranges specific to relevant demographic groups

  • Account for potential confounding variables (age, sex, comorbidities)

  • Document consistency across diverse patient populations

Implementation of these strategies significantly enhances the reliability and reproducibility of PLA2G7 measurements in clinical research settings.