ENO1 Mouse

What is ENO1 and what is its primary function in mice?

ENO1 (alpha-enolase) is a glycolytic enzyme that catalyzes the conversion of 2-phospho-pyruvate to phosphoenolpyruvate in the glycolytic pathway. In mice, beyond its metabolic function, ENO1 has been identified as an autoantigen in various autoimmune conditions and demonstrates immunomodulatory properties when administered exogenously . Mouse ENO1 (GI: 158853992) shares significant homology with human ENO1, making mouse models valuable for studying ENO1-related human pathologies .

Methodological approach: Researchers studying ENO1 function should consider both its enzymatic role in glycolysis and its non-canonical functions in immune regulation, particularly in autoimmune disease models.

How is recombinant mouse ENO1 produced for research purposes?

Recombinant mouse ENO1 for research purposes is typically produced through the following process:

• RNA extraction from mouse liver, followed by RT-PCR to obtain ENO1 cDNA

• Cloning into an expression vector (e.g., pET15b) containing a histidine tag

• DNA sequence verification

• Transfer into Escherichia coli for protein production

• Protein purification using affinity chromatography

• Solubilization in appropriate buffer (2M urea, 50 mM Phosphate, 5 mM HCl-Tris)

Critical quality control steps include:

• Verification of non-carbamylation (using methods like OxiSelect Protein Carbamylation ELISA Kit)

• Removal of lipopolysaccharides (LPS) by affinity chromatography (e.g., Endotrap)

• LPS titration to ensure levels below detection limits (<1 EU/mL)

Note: Recombinant ENO1 produced through this method is typically not enzymatically active as it cannot catalyze the transformation of 2-phospho-pyruvate into phosphoenolpyruvate .

What mouse models are commonly used to study ENO1 function?

Several mouse models are employed to study ENO1 function in different disease contexts:

Methodological approach: Selection of the appropriate mouse model should be guided by the specific aspect of ENO1 function being investigated. For autoimmune studies, the CIA model in DBA/1 mice has strong experimental validation .

What are the mechanisms by which prophylactic ENO1 injection reduces arthritis severity in CIA mouse models?

The mechanisms by which prophylactic ENO1 injection reduces arthritis severity in CIA mouse models include:

• Reduction in anti-collagen II (anti-CII) antibodies: ENO1-treated mice show significantly reduced production of anti-CII IgG antibodies, which are known to be pathogenic in the CIA model .

• Histological improvements: ENO1 treatment (100 μg) significantly reduces synovitis, cartilage resorption, and bone erosion scores in mouse joints .

• No shift toward Th2 response: ENO1 does not induce a shift toward a Th2 response, as evidenced by:

  • Unchanged IgG1/IgG2a ratio of anti-CII antibodies

  • Similar IL-4 serum levels compared to control groups

• Dose-dependency: Effects are dose-dependent, with 100 μg ENO1 showing significant effects while 10 μg does not provide protection against arthritis .

• Route of administration significance: Intraperitoneal injection is effective, while intravenous or subcutaneous routes show no effect .

Methodological approach: Researchers investigating ENO1's protective mechanisms should examine multiple parameters, including antibody production, cytokine profiles, and histological outcomes, using appropriate controls for each.

How does ENO1 influence immune cell populations in mouse models of autoimmune disease?

While the complete picture of how ENO1 influences immune cell populations remains to be fully elucidated, several observations provide insights:

• Cytokine production: In the CIA model, ENO1 treatment does not significantly alter serum levels of IL-4 and IL-6, the only detectable cytokines among 8 tested (IFNα, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, and TNFα) .

• Antibody production: ENO1 treatment reduces anti-CII antibody levels without changing the IgG1/IgG2a ratio, suggesting effects on B cell responses without skewing T helper cell phenotypes .

• Joint inflammation: Histological analyses show reduced inflammatory infiltrates in ENO1-treated mice, indicating potential effects on immune cell recruitment or activation at inflammation sites .

Methodological approach: Researchers should employ comprehensive immune phenotyping including flow cytometry of various lymphoid tissues, functional assays of isolated immune cells, and cytokine profiling to fully characterize ENO1's immunomodulatory effects.

What are the methodological considerations for studying ENO1 mutations in mouse models?

When studying ENO1 mutations in mouse models, researchers should consider:

• Mutation classification and frequency:

  • ENO1 alterations occur in 1.8% of samples studied in cancer contexts

  • Mutations include inframe mutations, missense mutations, truncating mutations, fusion, amplification, and deep deletion

• Statistical analysis approaches:

  • One-way ANOVA with specific thresholds (|log 2FC| ≥ 1.00, P < 0.01) for differential expression analysis

  • Survival curve analysis to assess prognostic significance of ENO1 mutations

  • Comparison between altered and unaltered groups (195 vs. 10,772 samples in cited study)

• Bioinformatic analysis:

  • Utilization of databases like GTEx Portal for normal tissue expression

  • GEPIA database for analyzing TCGA data with appropriate P-values, fold changes, and ranks

  • Construction of survival curves and violin plots for visualizing results

Methodological approach: A comprehensive strategy combining in silico analysis of existing datasets with experimental validation in appropriate mouse models is recommended to confirm the functional consequences of specific ENO1 mutations.

How do anti-ENO1 antibodies affect mouse models of autoimmune disease?

The current understanding of anti-ENO1 antibodies' effects in mouse models includes:

• Immunogenicity of ENO1: When mice are injected with ENO1, they develop a strong IgG response to mouse ENO1, confirming its immunogenicity .

• Potential protective role: There is speculation that anti-ENO1 antibodies might have a protective role in autoimmune conditions, though mechanisms remain to be fully elucidated .

• No impact on ACPA production: Pre-immunization with ENO1 has no impact on anti-citrullinated protein antibody (ACPA) production, with titers comparable between treated and control groups .

• Measurement methods: Anti-ENO1 antibody titers are typically measured using ELISA techniques with purified polyclonal antibodies from immunized mice as standards .

Methodological approach: Researchers studying anti-ENO1 antibodies should employ passive transfer experiments, epitope mapping, and functional assays to determine their specific effects on disease progression and immune cell function.

What are the optimal dosing protocols for ENO1 administration in mouse models?

Based on experimental evidence, the following dosing parameters should be considered:

Methodological approach: Researchers designing ENO1 administration protocols should start with 100 μg dose delivered intraperitoneally, with careful consideration of timing relative to disease induction. Dose-response studies including doses above and below 100 μg are recommended to determine optimal therapeutic windows for specific disease models.

How should researchers control for LPS contamination when working with recombinant ENO1 in mouse studies?

LPS contamination is a critical consideration when working with recombinant proteins produced in E. coli:

• LPS removal techniques:

  • Affinity chromatography using specialized resins (e.g., Endotrap)

  • The study reported removal of 99.5% of LPS without loss of ENO1 protein

• Quantification of residual LPS:

  • Use sensitive commercial kits (e.g., Qcl chromogenic LAL kit)

  • Ensure LPS levels are below detection limits (<1 EU/mL)

• Essential control groups:

  • Include groups receiving the same buffer used for ENO1 solubilization

  • Include groups receiving purified LPS at concentrations equivalent to any residual contamination

  • Consider groups receiving heat-inactivated ENO1 to distinguish between enzymatic activity and other protein properties

Methodological approach: Implementation of rigorous LPS removal, quantification, and appropriate control groups is essential to confidently attribute observed effects to ENO1 rather than LPS contamination, particularly in immunological studies.

What are the best methods for assessing ENO1-mediated effects on histological parameters in mouse models?

For comprehensive histological assessment of ENO1 effects:

• Tissue processing protocol:

  • Dissect both hind and anterior paws

  • Fix tissues in 10% phosphate-buffered formaldehyde for 48 hours

  • Process for histological examination with appropriate staining

• Multi-parameter scoring system:

  Parameter	What to Assess
  Inflammatory score	Inflammatory infiltrates
  Synovitis score	Synovial tissue hypertrophy
  Cartilage destruction	Cartilage resorption
  Bone destruction	Bone erosion

• Key features to document:

  • Synovial tissue hypertrophy

  • Newly formed cartilage

  • Fibrin deposits

  • Cartilage resorption

• Analysis approach:

  • Use appropriate statistical tests (t-tests were used in the cited study)

  • Calculate correlations between histological findings and clinical data (Spearman's rank correlation coefficient)

Methodological approach: Implement this comprehensive histological assessment approach to thoroughly evaluate ENO1-mediated effects on joint pathology, ensuring assessment of both inflammatory and tissue remodeling parameters.

How can researchers effectively measure anti-ENO1 antibody responses in mouse serum?

For accurate quantification of anti-ENO1 antibodies:

• ELISA development:

  • Coat plates with 1 μg/well of recombinant ENO1

  • Block with appropriate buffer (PBS1X, Tween 0.05%, SVF 5%)

  • Incubate 50 μL of mouse serum for 1 hour

  • Use mouse anti-IgG HRP-conjugated antibodies for detection

  • Develop with TMB substrate

• Standard preparation:

  • Immunize BALB/C mice with ENO1 to generate polyclonal antibodies

  • Purify anti-ENO1 antibodies from these immunized mice

  • Use these purified antibodies as standards

• Time-course measurements:

  • Collect serum at multiple time points (day 0, 15, 30, 45, 60)

  • Store serum at -80°C until analysis

Methodological approach: This standardized ELISA method allows for reliable quantification of anti-ENO1 antibodies, essential for monitoring immune responses in various experimental settings involving ENO1 administration or immunization.

What techniques are recommended for analyzing ENO1 mutation effects in mouse cancer models?

For comprehensive analysis of ENO1 mutation effects:

• Mutation characterization methods:

  • Classify ENO1 alterations into specific categories: inframe mutation, missense mutation, truncating mutation, fusion, amplification, and deep deletion

  • Determine mutation frequency across different sample cohorts

• Expression analysis approach:

  • Compare ENO1 expression levels between tumors with and without ENO1 mutations

  • Use one-way ANOVA with specific thresholds (|log 2FC| ≥ 1.00, P < 0.01)

• Survival analysis techniques:

  • Generate Kaplan-Meier survival curves to assess prognostic significance

  • Compare outcomes between altered and unaltered groups

• Immune infiltration assessment:

  • Analyze relationship between ENO1 mutation status and levels of tumor-infiltrating immune cells

  • This provides insights into how ENO1 mutations influence the tumor immune microenvironment

Methodological approach: A multi-dimensional analysis combining genomic characterization, expression profiling, and functional assessment of tumor-immune interactions is recommended for comprehensive evaluation of ENO1 mutation effects in cancer contexts.