Protein Functional Analysis: How to Conduct Research?

Abstract

Eosinophil peroxidase (EPX) is a heme-containing oxidoreductase stored in the granules of eosinophils. It plays a central role in the host's immune defense and is closely related to various inflammatory and allergic diseases. This article provides a detailed overview of the biological functions of EPX, emphasizing its role in pathogen clearance, inflammation regulation, and disease pathogenesis. In addition, this article discusses the research methods of EPX, including recombinant expression, enzymatic analysis, and genetic methods. It aims to lay a foundation for new researchers entering this field and propose future directions for in-depth research on the role of EPX in health and disease.

1. Introduction

Eosinophils are multifunctional granulocytes that are mainly involved in the pathophysiology of defense against parasitic infections and allergic diseases. Among the effector molecules secreted by eosinophils, eosinophil peroxidase (EPX) stands out for its powerful oxidative capacity and wide range of biological effects.

EPX catalyzes hydrogen peroxide-mediated oxidation of halides and pseudohalides to generate reactive oxygen species (ROS), which kill pathogens. However, these oxidants can also cause tissue damage and chronic inflammation. Therefore, EPX is both a protector and a potential invader in the immune system, making it a research subject of great interest in the biomedical field. Therefore, understanding the function of EPX and developing reliable research methods are crucial to advance therapeutic strategies for eosinophilic diseases.

2. Biological functions of EPX proteins

2.1 Antimicrobial activity

EPX plays a major role in the host defense system. The enzyme uses hydrogen peroxide (H₂O₂) produced by eosinophil NADPH oxidase to catalyze the halogenation of the following substrates:

Bromide (Br⁻) → hypobromous acid (HOBr)

Thiocyanate (SCN⁻) → hypothiocyanic acid (HOSCN)

These oxidants are cytotoxic to bacteria, fungi, and helminths, destroying membrane integrity and inactivating microbial proteins. This mechanism is particularly important in infections involving multicellular parasites, as eosinophils are activated and degranulate, releasing high concentrations of EPX.

2.2 Inflammatory regulation

In addition to direct cytotoxicity, EPX is also involved in shaping the inflammatory environment like typical oxidative chemokines and cytokines, changing their activity and signaling. It can also induce epithelial cell stress response and trigger the production of more inflammatory mediators. In this way, more leukocytes are recruited to enhance the immune response. These mechanisms highlight the role of EPX as an inflammatory amplifier, especially in chronic diseases such as asthma.

2.3 Role in allergic reactions

In allergic diseases, eosinophils are usually the main infiltrating immune cells, and EPX plays a central role in their effector function. The released EPX can damage respiratory or intestinal epithelial cells, thereby destroying tight junctions and impairing barrier function. It can also promote Th2-biased immune responses and maintain allergic inflammation by activating mast cells and basophils through ROS. In addition, elevated levels of EPX in sputum, bronchoalveolar lavage fluid, or tissues are associated with the severity of asthma, eosinophilic esophagitis, and atopic dermatitis.

2.4 Potential role of autoimmunity

Recent evidence suggests that EPX-mediated oxidation may lead to the formation of new antigens by modifying host proteins. This may trigger an autoimmune response, although its direct causal relationship remains to be fully confirmed.

3. Clinical significance of EPX

3.1 EPX as a biomarker

Because EPX is specific for eosinophils, it is a good biomarker of eosinophil activation:

Clinical uses include monitoring asthma, allergic inflammation, and eosinophilic gastrointestinal diseases.

Quantitative assays (detection of EPX in sputum or plasma by ELISA) can provide a non-invasive diagnostic tool.

3.2 Role of EPX in Disease Pathogenesis

In diseases such as hypereosinophilic syndrome (HES) or eosinophilic granulomatosis with polyangiitis (EGPA), EPX can cause end-organ damage, especially in the heart and lungs.

In cancer research, some studies have shown that eosinophils and EPX may affect tumor immunity, although this area is still under active investigation.

4. EPX Research Methods

EPX research involves molecular biology and functional immunology. Common methods are as follows:

4.1 Protein expression and purification

Recombinant EPX is usually expressed using the following methods:

Mammalian cells (e.g. HEK293, CHO) for expression similar to native glycosylation. Baculovirus-insect cell system (e.g. Sf9) for expression of complex, secreted proteins. E. coli expression system for higher yields. In addition, His-tagged or Fc-tagged constructs facilitate purification.

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4.2 Activity Assays

The peroxidase activity of EPX can be quantified using the following substrates: Amplex Red (fluorescence), TMB, ABTS (colorimetric), bromide for native substrate assays, or SCN⁻, which allow determination of enzyme kinetics (Km, Vmax) and comparison of wild-type and mutant proteins.

4.3 Functional models in vitro and in vivo 

Cell culture models: co-culture eosinophils or epithelial cells with EPX to study oxidative damage.

Mouse asthma models: allergen sensitization can induce eosinophilia; measure EPX levels to understand its relationship with disease severity.

Knock-in/knock-out studies: gain insight into the effects of EPX on specific pathologies.

4.4 Genetic manipulation and EPX knockout models

CRISPR/Cas9 technology can generate EPX knockout (KO) cell lines or mice. These cell lines or mice can be used to:

Study immune responses in the absence of EPX

Validate the role of EPX in disease models

Assess compensatory mechanisms of other peroxidases (e.g., MPO)

4.5 Omics and bioinformatics approaches

Transcriptomics (RNA sequencing): measure EPX gene expression under different stimulation conditions.

Proteomics: detect EPX and its oxidative targets in biological samples.

Pathway analysis: revealing the position of EPX in the immune signaling network.

5. Experimental challenges and considerations

EPX research faces the following challenges:

Protein instability: EPX is sensitive to oxidation and denaturation.

Glycosylation heterogeneity: affects activity and immunogenicity.

Assay interference: other peroxidases (e.g., MPO, LPO) may interfere with the measurement results.

Species differences: mouse EPX and human EPX do not function exactly the same.

Ethical sources: human eosinophils require invasive sampling (e.g., bronchoalveolar lavage).

Appropriate controls and validation steps are essential to ensure specificity and reproducibility.

6. Conclusion and outlook

EPX is a multifunctional immune effector whose role is not limited to killing microorganisms but may also modulate inflammation, allergic reactions, and even autoimmunity. Depending on the specific situation, it can act as both an enzyme that kills pathogens and a mediator of tissue damage.

Given its dual nature, EPX is both a therapeutic target and a biomarker. Advances in recombinant protein production, gene editing, and high-throughput omics technologies are accelerating our understanding of EPX biology.

Future Directions

• Development of EPX inhibitors as novel anti-inflammatory drugs
• EPX imaging agents for disease monitoring
• Integrating single-cell transcriptomics to dissect EPX functions at the cellular level
• Further elucidating the role of EPX in tumor immunology and autoimmunity
• Understanding EPX at the molecular, cellular, and systemic levels is critical for developing precision therapies for eosinophil-related diseases.