GSTM4 Antibody

What is GSTM4 and what is its biological function?

GSTM4 (Glutathione S-transferase Mu 4) is a member of the GST family of enzymes that catalyze the conjugation of reduced glutathione to a wide range of exogenous and endogenous hydrophobic electrophiles. This enzyme plays critical roles in:

• Detoxification of electrophilic compounds, including carcinogens and products of oxidative stress

• Catalyzing the conjugation of leukotriene A4 with reduced glutathione to form leukotriene C4

• Transferring glutathionyl groups from glutathione to specific lipid mediators such as 13(S),14(S)-epoxy-docosahexaenoic acid to form maresin conjugate in tissue regeneration 1 (MCTR1)

• Limiting apoptosis through interaction with Apoptosis Signal-regulating Kinase 1 (ASK1) and inhibiting signaling via the c-Jun N-terminal Kinase axis

GSTM4 is particularly notable in cancer research as it has been identified as a major GST specifically expressed in Ewing sarcoma, where it is upregulated by the EWS/FLI oncoprotein via a GGAA microsatellite in the promoter region .

How does GSTM4 expression vary across different tissue types?

Based on the research data, GSTM4 expression shows significant tissue specificity and disease correlation:

• In Ewing sarcoma: GSTM4 ranks as one of the major GSTs expressed, specifically upregulated in approximately half of Ewing sarcoma tumors due to the EWS/FLI oncoprotein

• In ovarian cancer (OC): GSTM4 shows decreased expression in OC samples compared to normal ovary samples at both mRNA and protein levels

• In normal tissues: GSTM4 exhibits very low expression in most non-Ewing sarcoma tissues

RNA-seq analysis of Ewing sarcoma cells demonstrated that GSTM4 ranks fourth among 17 members of the GST family, with GSTP1, GSTO1, and MGST3 being the other prevalent GSTs .

What are the key characteristics of commercially available GSTM4 antibodies?

Research-grade GSTM4 antibodies generally feature the following characteristics:

• Host organisms: Primarily raised in rabbits, with some mouse monoclonal options available

• Clonality: Both polyclonal and monoclonal options exist, with polyclonal being more common

• Immunogen regions: Most commonly target regions within AA 1-218 of the human GSTM4 protein

• Applications: Typically validated for Western Blotting (WB), Immunohistochemistry (IHC), and ELISA, with some also validated for Immunoprecipitation (IP) and Immunocytochemistry (ICC)

• Species reactivity: Primarily human-reactive, with some cross-reactivity to mouse and rat GSTM4

• Storage conditions: Generally stored at -20°C for long-term and 4°C for short-term use and frequent handling

What is the role of GSTM4 in Ewing sarcoma pathogenesis?

GSTM4 plays multiple critical roles in Ewing sarcoma biology and treatment resistance:

• Oncogenic transformation: GSTM4 is required for the oncogenic transformation of Ewing sarcoma cells, as demonstrated by knockdown studies showing inhibition of transformation

• Chemoresistance mechanism: High expression mediates resistance to chemotherapeutic drugs, particularly etoposide

• Prognostic marker: Elevated expression in primary tumors correlates with poor patient outcomes

• Apoptosis regulation: GSTM4 limits apoptosis through interaction with ASK1 and inhibition of c-Jun N-terminal Kinase signaling

These findings have led researchers to explore GSTM4 as a novel therapeutic target using both inhibitors of GSTM4 activity (such as NBDHEX) and GSTM4-activated anti-cancer agents (such as JS-K) .

How can researchers address the contradictory findings regarding GSTM4 expression and cancer prognosis?

Research has revealed seemingly contradictory findings regarding GSTM4 expression and prognosis across different cancer types:

To address these contradictions, researchers should:

• Consider cancer stage-specific effects: Evaluate whether GSTM4's impact varies by disease stage

• Examine dual functions: Investigate GSTM4's role in both cancer initiation and progression phases

  • In cancer initiation: Decreased GSTM4 expression reduces detoxification capacity, potentially promoting carcinogenesis

  • In established cancers: GSTM4 may protect cancer cells from therapeutic agents and oxidative stress

• Analyze microenvironment effects: Examine how GSTM4 affects immune cell infiltration, as GSTM2-4 were found to negatively associate with CD8+ T cells in ovarian cancer

• Standardize methodology: Use consistent analytical approaches and larger cohorts to validate findings

How does GSTM4 contribute to drug resistance mechanisms in cancer?

GSTM4 contributes to drug resistance through several mechanisms:

• Direct detoxification: Conjugates glutathione to chemotherapeutic drugs, promoting their elimination

• Anti-apoptotic activity: Interacts with ASK1 to inhibit stress-induced apoptosis pathways

• Synergistic inhibition: Combined inhibition of GSTM4 with chemotherapeutic agents shows synergistic increases in cytotoxicity:

  • NBDHEX (a GST inhibitor) combined with etoposide showed synergistic cytotoxicity in Ewing sarcoma cells

  • This synergism was most significant with low doses of both drugs, suggesting potential for minimizing side effects

Experimental evidence demonstrated that pharmacological inhibition of GSTM4 using NBDHEX significantly limited cellular proliferation and oncogenic transformation of Ewing sarcoma cells, further supporting its role in treatment resistance .

What are the optimal methods for producing and validating GSTM4 antibodies?

The production and validation of GSTM4 antibodies typically involves:

Production Process:

• Recombinant protein expression: Express recombinant GSTM4 protein or peptide fragments (commonly AA 1-218) in bacterial systems such as E. coli

• Purification: Using affinity chromatography, often with Ni-NTA resin for His-tagged proteins

• Immunization: Initial injection with complete Freund's adjuvant, followed by boosting with incomplete Freund's adjuvant (typically 100-200 μg of purified protein)

• Titer testing: Testing antibody reactivity against recombinant peptide by Western blotting

• Final bleeding and purification: Selection of antiserum with highest titer, followed by affinity purification

Validation Methods:

• Western blotting: Using known positive controls and testing specificity across multiple cell lines

• Immunohistochemistry: Testing on formalin-fixed, paraffin-embedded tissues

• Cross-reactivity testing: Evaluation across different species and related GST family members

• Immunoprecipitation: Confirming ability to specifically precipitate the target protein

• RNA knockdown controls: Using cells with GSTM4 knockdown to confirm antibody specificity

What are the recommended dilutions and conditions for using GSTM4 antibodies in various applications?

Based on the research literature, the following application-specific recommendations can be made:

Western Blotting:

• Typical dilution range: 1:500-1:2000

• Blocking buffer: 5% non-fat dry milk in TBST

• Predicted band size: 26 kDa

• Secondary antibody: Anti-host IgG conjugated to HRP at 1:25,000 dilution

Immunohistochemistry:

• Fixation: Formalin-fixed, paraffin-embedded tissues

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer

• Blocking: 3-5% BSA for 15-60 minutes at room temperature

• Primary antibody incubation: Typically 1-3 μg/ml in 2.5% BSA

Flow Cytometry:

• Cell preparation: Fix with 70% ethanol (10 minutes), permeabilize with 0.25% Triton X-100 (20 minutes)

• Blocking: 5% BSA for 1 hour at room temperature

• Antibody concentration: 1-3 μg per million cells in 2.5% BSA

• Incubation: 2-3 hours at room temperature

Immunofluorescence:

• Fixation: 4% paraformaldehyde

• Permeabilization: 0.1% Triton X-100 in PBS for 15 minutes at room temperature

• Blocking: 3% BSA for 15 minutes

• Antibody dilution: 1:25 for fluorophore-conjugated antibodies

How can researchers distinguish between GSTM4 and other closely related GST family proteins?

Distinguishing between GST family members presents significant challenges due to sequence and structural similarities. Researchers should:

• Select highly specific antibodies: Choose antibodies that target unique regions of GSTM4 not conserved in other GST family members

  • RNA-seq analysis shows that multiple GST family members can be expressed simultaneously in the same cells (e.g., GSTM4, GSTP1, GSTO1, and MGST3 are all prevalent in Ewing sarcoma)

• Validate specificity: Test antibodies against recombinant proteins of multiple GST family members to confirm lack of cross-reactivity

  • Surface homology analysis, as performed for GST allergens, can help identify regions with low homology for specific antibody targeting

• Use molecular techniques as complementary approaches:

  • RT-qPCR with isoform-specific primers

  • RNA interference targeting specific GSTM family members

  • Mass spectrometry to distinguish between isoforms based on peptide fragments

• Consider functional assays: Evaluate enzyme activity with substrates preferentially metabolized by GSTM4

• Negative controls: Include samples from tissues known to express minimal GSTM4, particularly for polyclonal antibodies that may have higher cross-reactivity risk

What therapeutic strategies target GSTM4 in cancer treatment?

Research has identified several promising therapeutic strategies targeting GSTM4:

How does GSTM4 expression affect immune cell infiltration in the tumor microenvironment?

GSTM4 has been shown to significantly influence immune cell infiltration in the tumor microenvironment, particularly in ovarian cancer:

• T Cell Populations: GSTM4 expression negatively correlates with CD8+ T cell infiltration, potentially contributing to immune evasion

• Macrophage Regulation: GSTM4 is positively correlated with macrophage M2 infiltration, which typically promotes tumor progression

• Immune Checkpoint Expression: High GSTM4 expression is associated with decreased expression of several immune checkpoints:

  • CTLA4, PDCD1LG2, and TIGIT were significantly reduced in high GSTM4 mRNA expressing tumors

CIBERSORT analysis specifically showed that GSTM4 was correlated with:

• Macrophage M2 populations

• CD8+ T cells

• Memory activated CD4 T cells

This indicates that GSTM4 may influence immunotherapy response through its effects on the tumor immune microenvironment, suggesting potential synergies between GSTM4-targeted therapies and immunotherapies.

What are the technical challenges in developing antibodies that specifically recognize GSTM4 over other GSTM family members?

Developing highly specific antibodies against GSTM4 presents several technical challenges:

• High sequence homology: GSTM family members share significant sequence similarity, making it difficult to identify unique epitopes for antibody generation

  • Research has noted difficulty in finding a GSTM4-specific antibody capable of detecting endogenous levels of the protein

• Conformational similarities: Similar three-dimensional structures between GST family members can result in cross-reactive antibodies, even when targeting regions with sequence differences

  • Structural analysis has shown that GST proteins can have low sequence homology but similar surface structures, or vice versa

• Post-translational modifications: Differences in glycosylation or other modifications between recombinant immunogens and native proteins can affect antibody recognition

• Validation challenges: Comprehensive validation requires:

  • Testing against all GSTM family members

  • Confirming specificity in tissues with different GSTM expression profiles

  • Using knockdown/knockout controls for each GSTM isoform

• Epitope selection strategies:

  • Target unique N-terminal or C-terminal regions

  • Focus on surface-exposed loops with lower conservation

  • Consider using synthetic peptides corresponding to unique GSTM4 regions rather than full-length protein immunogens