Recombinant Mouse Microsomal glutathione S-transferase 3 (Mgst3)

What is the functional role of MGST3 in mouse cellular metabolism?

MGST3 (Microsomal Glutathione S-Transferase 3) is a member of the MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) protein family that plays crucial roles in both cellular detoxification and inflammatory response pathways. This enzyme primarily catalyzes the conjugation of leukotriene A4 with reduced glutathione to produce leukotriene C4, an important mediator of inflammation . Additionally, MGST3 demonstrates glutathione-dependent peroxidase activity towards lipid hydroperoxides, providing cellular protection against oxidative stress . The enzyme's dual functionality in both conjugation and peroxidase activities makes it a significant component in cellular defense mechanisms against xenobiotics and oxidative damage, particularly in inflammatory conditions.

How does mouse MGST3 structurally and functionally compare to human MGST3?

While specific comparative data between mouse and human MGST3 isn't provided in the search results, general principles of GST protein conservation suggest significant homology. Both mouse and human MGST3 belong to the MAPEG family and share core functions including glutathione S-transferase and glutathione peroxidase activities . These enzymes catalyze similar reactions, including the conjugation of reduced glutathione to the alpha, beta-unsaturated C=C carbonyl group of eicosanoids such as leukotriene A4 and 15-deoxy-Delta12,14-prostaglandin J2, forming GSH adducts relevant to inflammatory responses . Additionally, both enzymes catalyze glutathione-dependent reduction of eicosanoid peroxides to produce corresponding eicosanoid hydroxides . These functional similarities make mouse MGST3 a potentially valuable model for studying mechanisms relevant to human health, particularly in inflammatory and detoxification pathways.

What are the most effective methods for producing recombinant mouse MGST3 protein with optimal enzymatic activity?

For producing recombinant mouse MGST3 with optimal enzymatic activity, researchers should consider a comprehensive approach that preserves the protein's native conformation and catalytic properties. Based on methodologies used for studying related GST proteins, an effective expression system typically involves bacterial expression using E. coli BL21(DE3) with a pET expression vector containing the full-length mouse MGST3 cDNA .

The purification protocol should include:

• Initial purification using nickel-affinity chromatography (Ni²⁺-IDA-agarose) for His-tagged proteins

• Size exclusion chromatography to enhance purity

• Validation of protein identity via Western blotting with anti-MGST3 antibodies

• Activity assessment using spectrophotometric assays measuring glutathione conjugation and peroxidase activities

To preserve enzymatic activity, buffer optimization is critical, typically including:

• 20 mM Tris-HCl (pH 7.5)

• 150 mM NaCl

• 1 mM DTT

• 10% glycerol for stability during storage

Researchers should verify activity through functional assays measuring both glutathione S-transferase activity (using CDNB as substrate) and glutathione peroxidase activity (using cumene hydroperoxide or lipid peroxides as substrates) .

What antibody selection and dilution protocols are optimal for detecting mouse MGST3 in different experimental applications?

While the search results don't provide specific antibody information for mouse MGST3, we can extrapolate from protocols used for other GST proteins. For optimal antibody selection and dilution protocols when detecting mouse MGST3, researchers should consider the experimental application and tissue type. Based on antibody usage for related GST proteins, the following recommendations can be made:

For validation and specificity confirmation, researchers should include appropriate controls, including recombinant MGST3 protein as a positive control and tissues from MGST3-knockout mice as negative controls . Secondary antibody selection should match the host species of the primary antibody, typically anti-rabbit IgG conjugated with HRP for western blotting or fluorescent labels for immunofluorescence applications.

How can researchers effectively measure MGST3 enzymatic activity in mouse tissue homogenates?

To effectively measure MGST3 enzymatic activity in mouse tissue homogenates, researchers should implement assays that capture both glutathione S-transferase and glutathione peroxidase activities. Based on known MGST3 functions, the following protocol is recommended:

• Tissue preparation:

  • Harvest fresh mouse tissues (liver, kidney, lung) and prepare 10% (w/v) homogenates in ice-cold buffer (50 mM potassium phosphate, pH 7.4, containing 1 mM EDTA and protease inhibitors)

  • Centrifuge at 10,000g for 20 minutes at 4°C

  • Further centrifuge supernatant at 100,000g for 60 minutes to separate cytosolic and microsomal fractions

  • Resuspend microsomal pellet in homogenization buffer containing 0.1% Triton X-100

• Glutathione S-transferase activity assay:

  • Measure conjugation of reduced glutathione with leukotriene A4 using HPLC analysis

  • Alternatively, use 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate and monitor absorbance at 340 nm

  • Calculate specific activity in nmol/min/mg protein

• Glutathione peroxidase activity assay:

  • Use cumene hydroperoxide or specific eicosanoid peroxides as substrates

  • Couple with glutathione reductase and monitor NADPH oxidation at 340 nm

  • Express activity as nmol NADPH oxidized/min/mg protein

• Controls and validation:

  • Include recombinant MGST3 as positive control

  • Use specific inhibitors to distinguish MGST3 activity from other GSTs

  • Confirm specificity with tissues from MGST3-knockout mice

This comprehensive approach allows researchers to accurately assess MGST3's dual enzymatic functions in various mouse tissues and experimental conditions .

How does phosphorylation affect mouse MGST3 function in inflammatory responses?

While the search results don't specifically address MGST3 phosphorylation, insights can be drawn from studies on other GST family members. Research on GSTP has demonstrated that phosphorylation significantly impacts its cellular localization and function in inflammatory processes . Based on these findings, researchers investigating MGST3 phosphorylation should consider:

• Potential phosphorylation sites: Similar to GSTP's Ser184 phosphorylation, MGST3 likely has specific serine, threonine, or tyrosine residues that undergo phosphorylation events critical to its function.

• Kinase involvement: Studies of GSTP identified that conventional protein kinase C (cPKC) phosphorylates Ser184, enabling nuclear translocation . Research should investigate whether similar kinase families regulate MGST3 phosphorylation under inflammatory conditions.

• Functional consequences: For GSTP, phosphorylation enabled nuclear translocation and interaction with HMGB1, preventing its release during inflammatory responses . MGST3 phosphorylation likely similarly regulates its subcellular localization and protein-protein interactions, potentially modulating its role in leukotriene production and inflammatory signaling.

• Experimental approaches: To investigate MGST3 phosphorylation, researchers should:

  • Employ mass spectrometry to identify phosphorylation sites

  • Generate phospho-specific antibodies

  • Create phosphomimetic (S→D) and phospho-dead (S→A) MGST3 mutants to evaluate functional consequences

  • Use kinase inhibitors to identify regulatory kinases

  • Assess changes in subcellular localization using immunofluorescence confocal microscopy

Understanding the phosphorylation-dependent regulation of MGST3 could provide critical insights into its role in inflammatory conditions and potential therapeutic targeting strategies.

What role does MGST3 play in mouse models of chronic obstructive pulmonary disease (COPD)?

Based on the information that MGST3 is associated with Chronic Obstructive Pulmonary Disease (COPD) , researchers investigating its role in mouse models should implement a multi-faceted experimental approach. While specific data on MGST3 in COPD mouse models isn't provided in the search results, the following research strategy is recommended:

• Expression analysis in COPD models:

  • Compare MGST3 expression levels in lung tissues from cigarette smoke-exposed mice versus controls

  • Assess correlation between MGST3 expression and disease severity markers

  • Evaluate cell-specific expression changes in bronchial epithelium, alveolar macrophages, and pulmonary endothelium

• Functional studies:

  • Generate MGST3-knockout mice and evaluate their susceptibility to COPD development

  • Assess leukotriene C4 production and inflammatory responses in wild-type versus MGST3-deficient mice

  • Measure glutathione levels and oxidative stress markers in lung tissues

• Potential mechanisms to investigate:

  • MGST3's role in detoxifying reactive oxygen species generated by cigarette smoke

  • Contribution to leukotriene synthesis and inflammatory cell recruitment

  • Potential protective effects against epithelial damage and airway remodeling

• Therapeutic implications:

  • Test whether MGST3 overexpression protects against COPD development

  • Evaluate pharmacological modulators of MGST3 activity for therapeutic potential

This comprehensive approach would advance understanding of how MGST3 contributes to COPD pathophysiology, potentially revealing new therapeutic targets for this prevalent respiratory condition.

How do protein-protein interactions influence MGST3 cellular function?

While specific protein-protein interactions for MGST3 aren't directly described in the search results, insights from studies of GSTP interactions provide a valuable framework. GSTP has been shown to interact with high mobility group box-1 protein (HMGB1) following phosphorylation and nuclear translocation, with this interaction preventing HMGB1 release during inflammatory responses . For researchers investigating MGST3 protein interactions:

• Potential interaction partners to investigate:

  • Components of the leukotriene synthesis pathway

  • Other glutathione metabolism enzymes

  • Membrane proteins in the endoplasmic reticulum

  • Inflammatory mediators and signaling proteins

• Experimental approaches:

  • Co-immunoprecipitation studies using tagged recombinant MGST3

  • Proximity labeling techniques (BioID, APEX) to identify proximal proteins

  • Yeast two-hybrid screening for novel interaction partners

  • FRET/BRET assays to confirm direct interactions in living cells

  • Confocal microscopy with fluorescently-tagged proteins to visualize co-localization

• Functional validation:

  • Mutational analysis to identify interaction domains

  • siRNA knockdown of interaction partners to assess functional consequences

  • Overexpression studies to evaluate competitive binding

• Context-dependent interactions:

  • Evaluate interactions under basal versus inflammatory conditions

  • Assess changes following oxidative stress challenges

  • Investigate phosphorylation-dependent interactions

Understanding MGST3's protein interaction network would provide critical insights into its cellular functions beyond enzymatic activity, potentially revealing new regulatory mechanisms and therapeutic opportunities for inflammatory and detoxification pathways.

How can researchers distinguish MGST3 activity from other glutathione S-transferases in mixed biological samples?

Distinguishing MGST3 activity from other glutathione S-transferases in mixed biological samples requires a strategic approach combining substrate specificity, inhibitor profiles, and genetic models. Based on established methodologies for GST characterization, researchers should implement:

• Substrate selectivity profiling:

  • MGST3 demonstrates specific activity toward leukotriene A4 and certain eicosanoid peroxides

  • Utilize comparative activity assays with substrates showing differential selectivity:

    • Leukotriene A4 (preferential for MGST3)

    • 1-chloro-2,4-dinitrobenzene (CDNB, broad GST substrate)

    • Cumene hydroperoxide (for glutathione peroxidase activity)

    • 15-deoxy-Delta12,14-prostaglandin J2 (for MGST3-specific conjugation)

• Inhibitor profiling:

  • Apply selective inhibitors to distinguish between GST classes:

    • Ethacrynic acid (preferential for certain microsomal GSTs)

    • S-hexylglutathione (cytosolic GST inhibitor)

    • Maleimide derivatives (for MAPEG family members)

• Immunodepletion approach:

  • Sequentially deplete samples using specific antibodies against different GST isoforms

  • Measure remaining activity after each depletion step

  • Calculate contribution of each GST type to total activity

• Genetic approaches:

  • Compare samples from wild-type and MGST3-knockout mice

  • Use siRNA knockdown in cell culture systems

  • Express recombinant MGST3 in GST-null cell lines

• Subcellular fractionation:

  • Separate cytosolic, microsomal, and mitochondrial fractions

  • MGST3 will predominantly localize to microsomal fractions

  • Other GST classes have distinct subcellular distributions (e.g., GSTK1 in mitochondria )

This multi-faceted approach enables accurate quantification of MGST3 contribution to total GST activity in complex biological samples, essential for understanding its specific roles in cellular processes.

What strategies should be employed when recombinant mouse MGST3 shows low enzymatic activity in experimental assays?

When recombinant mouse MGST3 exhibits low enzymatic activity in experimental assays, researchers should systematically troubleshoot using the following strategies:

• Expression system optimization:

  • Evaluate different expression vectors (pET, pGEX) for optimal expression

  • Test alternative host systems (E. coli, insect cells, mammalian cells)

  • Consider codon optimization for improved translation in the chosen host

  • Adjust induction conditions (temperature, IPTG concentration, duration)

• Protein folding and solubility:

  • Modify growth conditions (lower temperature, slower induction)

  • Add solubility enhancers (sorbitol, betaine) to growth media

  • Express as fusion protein with solubility tags (MBP, SUMO)

  • Evaluate detergent selection for membrane protein solubilization

• Purification protocol refinement:

  • Maintain reducing conditions throughout purification

  • Include stabilizing agents (glycerol, reduced glutathione)

  • Minimize exposure to freeze-thaw cycles

  • Test different buffer compositions and pH conditions

• Activity assay optimization:

  • Ensure physiologically relevant substrate concentrations

  • Verify glutathione quality and concentration

  • Optimize assay temperature and pH

  • Include appropriate positive controls (commercial GST enzymes)

• Protein modification considerations:

  • Evaluate the impact of affinity tags on activity

  • Consider post-translational modifications that may be lacking

  • Test activity with and without membrane reconstitution

  • Assess potential inhibitors in expression/purification reagents

• Storage condition optimization:

  • Test stability at different temperatures (-80°C, -20°C, 4°C)

  • Evaluate protein stabilizers (glycerol, DTT, reduced glutathione)

  • Consider flash-freezing in small aliquots to prevent activity loss

By systematically addressing these factors, researchers can identify and resolve issues affecting recombinant MGST3 activity, ensuring reliable experimental outcomes in their studies.

How should data from MGST3 knockout studies be interpreted in the context of compensatory mechanisms from other GST family members?

When interpreting data from MGST3 knockout studies, researchers must carefully consider potential compensatory mechanisms from other GST family members to avoid misinterpreting experimental outcomes. Based on patterns observed in studies of other GST knockouts, the following analytical framework is recommended:

• Comprehensive GST expression profiling:

  • Quantify expression levels of all GST family members in wild-type versus MGST3 knockout tissues

  • Perform qRT-PCR array analysis for all known GST genes

  • Conduct proteomic analysis to detect changes at protein level

  • Track expression changes temporally, as compensation may develop over time

• Functional redundancy assessment:

  • Measure total glutathione S-transferase and glutathione peroxidase activities

  • Compare substrate specificity profiles between knockout and wild-type samples

  • Evaluate activity toward MGST3-specific substrates (leukotriene A4, specific eicosanoids)

  • Assess physiological endpoints (inflammatory markers, oxidative stress indicators)

• Cellular adaptation analysis:

  • Examine changes in glutathione homeostasis and redox status

  • Assess alterations in related detoxification pathways

  • Evaluate stress response pathway activation

  • Consider organ/tissue-specific compensatory mechanisms

• Experimental design considerations:

  • Include acute knockdown models (siRNA, CRISPR) to minimize compensation time

  • Develop inducible knockout systems to study temporal aspects of compensation

  • Generate double/triple knockout models to address functional redundancy

  • Compare constitutive versus conditional knockout phenotypes

• Interpretive framework:

  • Distinguish between direct MGST3 functions and secondary adaptations

  • Consider developmental timing of knockout and potential impacts

  • Evaluate phenotypes under basal versus stressed conditions

  • Integrate data from multiple physiological systems and experimental approaches

How does mouse MGST3 functionally compare to other MAPEG family members in inflammatory response pathways?

Mouse MGST3, as a member of the MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) family, exhibits distinct functional characteristics compared to other family members in inflammatory response pathways. While the search results don't provide comprehensive comparative data, we can construct an evidence-based comparison:

MGST3 demonstrates dual enzymatic functions: glutathione S-transferase activity and glutathione peroxidase activity toward oxyeicosanoids . It specifically catalyzes the conjugation of reduced glutathione to leukotriene A4 and 15-deoxy-Delta12,14-prostaglandin J2, forming GSH adducts relevant to inflammatory responses . Additionally, it catalyzes glutathione-dependent reduction of eicosanoid peroxides to yield corresponding eicosanoid hydroxides .

In comparison with MGST2, another MAPEG family member identified as an important paralog of MGST3 , the following functional distinctions can be noted:

• Substrate specificity:

  • MGST3: Preferentially conjugates glutathione to leukotriene A4 and certain prostaglandins

  • MGST2: Shows stronger affinity for 5-HPETE and related inflammatory mediators

  • Other MAPEG members: Vary in substrate preferences across eicosanoid spectrum

• Catalytic efficiency:

  • Relative kinetic parameters (kcat/Km) differ across MAPEG members for shared substrates

  • Activity ratios between conjugation and peroxidase functions vary between family members

• Tissue expression patterns:

  • MAPEG members show differential tissue distribution, correlating with tissue-specific inflammatory responses

  • Expression regulation under inflammatory stimuli varies between family members

• Subcellular localization:

  • While all are membrane-associated, specific membrane targeting may differ

  • MGST3: Primarily localized to endoplasmic reticulum membranes

  • Other MAPEG members: May associate with nuclear, plasma, or other organelle membranes

This comparative understanding helps researchers select appropriate MAPEG family members for studying specific aspects of inflammatory pathways and informs experimental design for comprehensive pathway analysis.

What structural features distinguish mouse MGST3 from cytosolic GSTs and impact experimental design choices?

Mouse MGST3, as a membrane-associated GST, possesses distinct structural features that differentiate it from cytosolic GSTs and significantly impact experimental design considerations. While the search results don't provide specific structural details for MGST3, the following comparative analysis can guide research approaches:

• Membrane integration:

  • MGST3: Integral membrane protein with transmembrane domains

  • Cytosolic GSTs: Soluble proteins without membrane-spanning regions

  • Experimental impact: Requires detergent solubilization for purification and activity assays

• Oligomeric structure:

  • MGST3: Likely forms trimeric or tetrameric structures (typical of MAPEG family)

  • Cytosolic GSTs: Typically function as dimers

  • Experimental impact: Different native molecular weight in size exclusion chromatography

• Catalytic site architecture:

  • MGST3: Active site accommodates membrane-derived substrates like leukotrienes and lipid peroxides

  • Cytosolic GSTs: Active site optimized for more hydrophilic xenobiotics

  • Experimental impact: Different substrate panels for activity characterization

• Subcellular localization:

  • MGST3: Microsomal/endoplasmic reticulum localization

  • Cytosolic GSTs: Primarily cytosolic, though some show conditional nuclear translocation (e.g., GSTP)

  • GSTK1: Distinctly localized to mitochondria

  • Experimental impact: Different fractionation protocols for isolation

• Post-translational modifications:

  • Membrane GSTs may undergo different post-translational modifications than cytosolic counterparts

  • Experimental impact: Expression systems must support relevant modifications

These structural distinctions necessitate specific experimental adjustments:

Understanding these fundamental differences is crucial for designing appropriate experimental protocols for MGST3 characterization and avoiding methodological artifacts.

How do mouse models with altered MGST3 expression compare phenotypically to knockouts of other GST family members in stress response studies?

While the search results don't provide direct phenotypic comparisons between MGST3 mouse models and other GST knockouts, we can construct an evidence-based comparative framework based on the known functions of these enzymes and data from related GST studies. This comparison is particularly valuable for researchers designing stress response studies:

• Inflammatory challenge responses:

  • MGST3-deficient models: Expected to show altered leukotriene C4 production and impaired inflammatory resolution due to MGST3's role in leukotriene metabolism

  • GSTP-knockout models: Demonstrate increased HMGB1 release and heightened inflammatory responses in sepsis models, with increased mortality

  • Other GST knockouts: Show varying susceptibility to inflammation depending on specific detoxification functions

• Oxidative stress vulnerability:

  • MGST3-deficient models: Likely exhibit increased sensitivity to lipid peroxidation due to compromised glutathione peroxidase activity

  • GSTK1-deficient models: May show mitochondrial-specific oxidative stress vulnerabilities

  • Cytosolic GST knockouts: Typically display tissue-specific oxidative damage patterns

• Xenobiotic detoxification capacity:

  • MGST3-deficient models: Expected reduction in clearance of specific substrates metabolized by MGST3

  • Other GST knockouts: Substrate-specific detoxification deficits corresponding to isoform substrate preferences

• Developmental phenotypes:

  • Compensatory mechanisms often mask developmental phenotypes in constitutive knockouts

  • Tissue-specific and inducible knockouts may reveal more pronounced phenotypes

• Disease susceptibility patterns:

  • MGST3-deficient models: Expected increased susceptibility to COPD and other inflammatory lung conditions

  • GSTP-knockout models: Demonstrated increased mortality in sepsis models

  • Other GST knockouts: Show varying disease susceptibilities based on tissue expression and specific functions

This comparative framework highlights how different GST family members contribute to distinct aspects of stress response, with MGST3 playing particularly important roles in inflammatory eicosanoid metabolism and protection against lipid peroxidation. Researchers designing stress response studies should consider these differential functions when selecting appropriate GST knockout models and stress challenge paradigms for their specific research questions.

What are the most promising future research directions for mouse MGST3 studies?

Based on current understanding of MGST3 biology and gaps in knowledge, several high-priority research directions emerge for advancing mouse MGST3 studies:

• Post-translational regulation mechanisms: Investigation of how phosphorylation and other modifications regulate MGST3 activity and subcellular localization during inflammatory responses, similar to studies conducted with GSTP . This research could reveal critical regulatory mechanisms and potential therapeutic targets.

• Detailed structural biology: Determination of MGST3 crystal structure in complex with various substrates and inhibitors to enable structure-based drug design targeting specific inflammatory pathways.

• Tissue-specific conditional knockout models: Development of inducible, tissue-specific MGST3 knockout mice to circumvent potential developmental adaptations and reveal tissue-specific functions, particularly in lung tissue given the COPD association .

• Interactome mapping: Comprehensive characterization of MGST3 protein-protein interactions in various physiological and pathological states, similar to approaches that revealed GSTP-HMGB1 interactions .

• Role in age-related diseases: Investigation of how MGST3 function changes during aging and contributes to age-related inflammatory conditions, particularly in pulmonary and cardiovascular systems.

• Therapeutic modulation strategies: Development of selective MGST3 modulators to either enhance detoxification functions or modulate inflammatory mediator production for potential therapeutic applications in COPD and other inflammatory conditions.

• Systems biology approaches: Integration of transcriptomic, proteomic, and metabolomic data to understand MGST3's position within broader cellular networks and identify key regulatory nodes.

These research directions collectively address fundamental questions about MGST3 biology while also pursuing translational opportunities that could yield new therapeutic approaches for inflammatory conditions and oxidative stress-related diseases.

What standardized protocols should researchers follow when reporting recombinant mouse MGST3 research to ensure reproducibility?

To ensure experimental reproducibility in recombinant mouse MGST3 research, investigators should adhere to the following standardized reporting guidelines:

• Molecular characterization:

  • Complete sequence information for the expressed MGST3 construct, including any tags or mutations

  • Verification of sequence integrity with methodology and results

  • Expression vector details including promoter, fusion tags, and cloning sites

  • Source of MGST3 cDNA used for cloning

• Expression methodology:

  • Detailed host system information (strain, genotype, growth conditions)

  • Complete induction protocol (inducer concentration, temperature, duration)

  • Cell disruption and initial fractionation methods

  • Detailed solubilization protocol if membrane extraction was performed

• Purification reporting:

  • Step-by-step purification protocol with buffer compositions

  • Column specifications and run conditions

  • Elution criteria and fraction selection methodology

  • Final yield and purity assessment with SDS-PAGE images

  • Storage conditions and stability data

• Activity assay standardization:

  • Detailed reaction conditions (temperature, pH, buffer composition)

  • Substrate preparation methods and source information

  • Complete enzyme kinetic parameters (Km, Vmax, kcat)

  • Specific activity calculation methodology

  • Positive and negative controls included

• Structural and biophysical characterization:

  • Circular dichroism or other secondary structure determination

  • Oligomeric state assessment

  • Post-translational modification analysis

  • Thermal stability data

• Application-specific protocols:

  • Detailed experimental conditions for specific applications

  • Cell culture conditions if used in cellular experiments

  • Animal model details for in vivo studies

  • Data analysis methods and statistical approaches