Recombinant Human Allergin-1 (MILR1), partial

What is Allergin-1 and what are its synonyms in scientific literature?

Allergin-1, also known as mast cell immunoglobulin-like receptor 1 (MILR1), has several synonyms in the scientific literature including Allergy inhibitory receptor 1, Mast cell antigen 32 (MCA-32), Mast cell Ag-32, and in mouse models, Gm885 and Mca32. It functions as an immunoglobulin-like receptor that plays an inhibitory role in the degranulation of mast cells, negatively regulating IgE-mediated mast cell activation and suppressing type I immediate hypersensitivity reactions .

What are the key structural characteristics of recombinant Allergin-1?

Recombinant Allergin-1 typically contains an extracellular domain with immunoglobulin-like features. In mouse models, this domain spans approximately amino acids 34-150. When produced recombinantly, the protein often includes tags for purification and detection purposes, such as N-terminal His-tags and C-terminal Myc-tags. The mouse version has a molecular weight of approximately 20.1kDa when produced as a partial recombinant protein . Human Allergin-1 exists in multiple isoforms, with Allergin-1S1 being the predominant isoform expressed on human primary mast cells .

How are the different isoforms of human Allergin-1 distributed in tissues?

Human Allergin-1 exists in multiple isoforms, with Allergin-1S1 being predominantly expressed on human primary mast cells in both bronchoalveolar lavage (BAL) fluid and nasal scratching specimens. This has been confirmed through seven-color flow cytometry methods specifically developed for assessing expression on small numbers of human primary mast cells . The expression pattern of Allergin-1 is not limited to mast cells, as it has also been detected on other myeloid cells including monocytes, granulocytes, and dendritic cells, suggesting a broader role in immune regulation beyond mast cell functions .

How does Allergin-1 inhibit mast cell activation at the molecular level?

Allergin-1 functions as an inhibitory receptor that suppresses IgE-mediated activation of mast cells. At the molecular level, the inhibition occurs when Allergin-1 is co-ligated with FcεRIα (the high-affinity IgE receptor). This co-ligation triggers inhibitory signaling pathways that counteract the activating signals initiated by FcεRI. In experimental settings, researchers have demonstrated this inhibitory function by using TNP-specific IgE and TNP-conjugated anti-Allergin-1 antibodies to artificially co-ligate FcεRI and Allergin-1 .

The inhibitory effect can be measured by monitoring cell surface CD107a expression, which serves as a marker for mast cell degranulation. When Allergin-1 is co-ligated with FcεRI, there is a significant decrease in the population of CD107a-positive mast cells compared to when mast cells are activated through FcεRI alone .

What genetic polymorphisms of MILR1 are associated with allergic conditions?

Genetic studies have identified several polymorphisms in the MILR1 gene, including rs6504230, c.−170_−166delAGGAA, rs8071835, rs143526766, and rs12936887, as well as two rare missense variants (Val273Ala and Leu311Val). Among these, the C allele of rs6504230 has been shown to have protective effects against atopy (P=0.002) .

Functional analyses using luciferase reporter assays have demonstrated that the C allele of rs6504230 is associated with increased expression of MILR1. This finding is consistent with expression quantitative trait loci (eQTL) analysis using human leukocytes . The increased expression of MILR1 associated with this polymorphism aligns with the known inhibitory function of Allergin-1 in suppressing IgE-mediated responses, providing a mechanistic explanation for the protective effect against atopy observed in population studies.

How do mouse and human Allergin-1 compare in function and structure?

Mouse and human Allergin-1 share significant structural and functional similarities. Database searches have identified mouse Allergin-1 containing a single Ig-like domain in the extracellular portion with approximately 50% amino acid identity with human Allergin-1S1, suggesting evolutionary conservation of this receptor .

Functionally, both mouse and human Allergin-1 suppress IgE-mediated mast cell activation. In mouse models, Allergin-1 expression on mast cells has been shown to suppress the development of IgE- and mast cell-dependent systemic and cutaneous anaphylaxis . Similarly, human Allergin-1S1 inhibits FcεRI-mediated activation in primary mast cells, indicating a conserved functional role in regulating allergic responses across species .

What are the optimal methods for assessing Allergin-1 expression on human primary mast cells?

The optimal method for assessing Allergin-1 expression on human primary mast cells involves a seven-color flow cytometry approach, particularly when working with limited biological samples such as bronchoalveolar lavage (BAL) fluid or nasal scratching specimens (NSS) .

This methodology includes:

• Sample preparation: Process BAL fluid or NSS to preserve cell viability

• Antibody staining: Use a panel of markers including:

  • Anti-CD45 to identify leukocytes

  • Anti-c-kit (CD117) and anti-FcεRIα to identify mast cells

  • Specific anti-Allergin-1 monoclonal antibodies (e.g., EX32 for Allergin-1S1 and EX29 for Allergin-1S2)

• Flow cytometric analysis: Gate on CD45+c-kit+FcεRIα+ cells to identify mast cells, then analyze Allergin-1 expression

This method allows for the identification of specific Allergin-1 isoforms expressed on very small numbers of primary mast cells at the single-cell level, which is particularly valuable given the typically low frequency of mast cells in these samples .

How can researchers assess the inhibitory function of Allergin-1 on mast cells?

Researchers can assess the inhibitory function of Allergin-1 on mast cells using a multi-color flow cytometry-based activation assay, which is particularly valuable when working with limited primary cell numbers. The protocol involves the following steps:

• Incubate mast cells with allergen-specific IgE (e.g., TNP-specific mouse IgE)

• Stimulate cells with:

  • Control condition: TNP-conjugated F(ab')2 fragment of control immunoglobulin

  • Test condition: TNP-conjugated anti-Allergin-1 antibody (e.g., EX33)

• Detect mast cell activation by measuring cell surface CD107a expression, a marker of degranulation

• Analyze using multi-color flow cytometry to identify the percentage of activated (CD107a+) mast cells

This method has several advantages over conventional ELISA-based assays for histamine, cytokine, or β-hexosaminidase release, as it can be applied to extremely small numbers of primary uncultured mast cells, such as those found in BAL fluid .

What purification strategies yield high-quality recombinant Allergin-1 for functional studies?

Based on established protocols for recombinant proteins similar to Allergin-1, the following purification strategy can be recommended:

• Expression system: E. coli is commonly used for expressing recombinant Allergin-1 with appropriate tags (N-terminal His-tag and C-terminal Myc-tag)

• Purification steps:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) using the His-tag

  • Intermediate purification: Ion exchange chromatography to separate charged variants

  • Polishing: Size exclusion chromatography to remove aggregates and ensure homogeneity

• Quality control:

  • Purity assessment: SDS-PAGE (target >85% purity)

  • Western blot: Confirm identity using anti-tag antibodies

  • Functional validation: Binding assays to confirm interaction with target cells

This strategy typically yields recombinant protein with >85% purity as assessed by SDS-PAGE, which is suitable for most research applications including cell-based assays and biochemical studies .

How might recombinant Allergin-1 be utilized in therapeutic approaches for allergic diseases?

Recombinant Allergin-1 presents several potential therapeutic applications for allergic diseases based on its natural inhibitory function on mast cell activation:

• Direct application: Recombinant Allergin-1 could potentially be engineered as a therapeutic agent to engage endogenous inhibitory pathways on mast cells, thereby suppressing IgE-mediated allergic responses.

• Combinatorial approaches: Following the model of allergen-toxin fusion proteins developed for house dust mite allergens (Der p 1), Allergin-1 could be utilized in the design of fusion proteins that specifically target and modulate the activity of allergic effector cells .

• Genetic modulation: The identification of MILR1 promoter polymorphisms that affect expression levels suggests that genetic or epigenetic approaches to upregulate Allergin-1 expression might provide protection against atopic conditions .

The therapeutic potential is supported by mouse studies showing that Allergin-1 expression on mast cells suppresses the development of IgE- and mast cell-dependent systemic and cutaneous anaphylaxis, indicating that enhancing Allergin-1 signaling could potentially mitigate allergic reactions in humans .

What are the challenges in translating Allergin-1 research into clinical applications?

Several challenges must be addressed to translate Allergin-1 research into clinical applications:

• Complex expression pattern: While Allergin-1 inhibits mast cell activation, it is also expressed on other myeloid cells including monocytes, granulocytes, and dendritic cells. This broad expression profile complicates the development of targeted therapies, as interventions affecting Allergin-1 might have effects beyond mast cells .

• Isoform specificity: Human Allergin-1 exists in multiple isoforms with potentially different functions. Therapeutic approaches would need to consider isoform-specific effects, particularly focusing on Allergin-1S1 as the predominant isoform on primary mast cells .

• Delivery system development: Efficient delivery of recombinant proteins or genetic interventions to target tissues represents a significant challenge, particularly for accessing mast cells in tissues.

• Safety considerations: As Allergin-1 plays a role in immune regulation, modulating its function could potentially affect normal immune responses beyond allergic reactions.

To develop Allergin-1 as a molecular therapeutic target, the role of Allergin-1 on myeloid cells as well as mast cells in both allergic and non-allergic diseases needs further clarification .

How do genetic variations in MILR1 influence susceptibility to atopic conditions?

Genetic variations in MILR1, particularly the promoter polymorphism rs6504230, significantly influence susceptibility to atopic conditions. The C allele of rs6504230 has been identified as having protective effects against atopy (P=0.002) . This protection operates through a molecular mechanism involving increased expression of MILR1, as demonstrated through luciferase reporter assays using the promoter region of MILR1 .

These findings align with the functional role of Allergin-1 in suppressing FcεRI-mediated activation of mast cells. The increased expression of MILR1 associated with the C allele likely enhances the inhibitory capacity against mast cell degranulation, thereby reducing the likelihood of developing atopic sensitization to common allergens .

This genetic evidence complements functional studies and provides an important link between natural genetic variation and allergic disease susceptibility, highlighting MILR1 as a potential therapeutic target for allergic diseases.

What are the key methodological considerations when working with primary human mast cells to study Allergin-1?

Working with primary human mast cells to study Allergin-1 presents several methodological challenges that researchers must address:

• Sample acquisition and cell rarity:

  • Primary mast cells are rare in biological samples (BAL fluid, nasal scrapings)

  • Develop protocols optimized for small cell numbers

  • Consider ethical approvals for obtaining human samples

• Cell identification and isolation:

  • Use multi-parameter flow cytometry with markers CD45+c-kit+FcεRIα+

  • Employ gentle isolation techniques to preserve viability and function

• Functional assays:

  • Traditional biochemical assays (β-hexosaminidase, histamine release) require large cell numbers

  • Flow cytometry-based assays using CD107a as a degranulation marker offer single-cell resolution for small samples

  • Microscopy-based approaches may provide spatial information about receptor distribution

• Isoform specificity:

  • Use isoform-specific antibodies (e.g., EX32 for Allergin-1S1 and EX29 for Allergin-1S2)

  • Consider differential expression patterns when interpreting results

• Validation approaches:

  • Compare cultured mast cells (easier to obtain) with primary cells

  • Consider species differences when translating findings between mouse models and human studies

These methodological considerations are crucial for generating reliable data when studying Allergin-1 on primary human mast cells, particularly given the limited availability of these cells and their sensitivity to manipulation .

How do the mechanisms of Allergin-1 inhibition compare with other inhibitory receptors on mast cells?

The inhibitory mechanism of Allergin-1 can be compared with other mast cell inhibitory receptors across several dimensions:

Unlike some other inhibitory receptors with well-characterized ligands, the endogenous ligand for Allergin-1 remains to be identified, representing an important area for future research. Understanding the unique and shared features of these inhibitory receptors will be crucial for developing targeted therapeutic approaches that modulate specific aspects of mast cell function in allergic diseases .

What are the most promising future research directions for Allergin-1?

The most promising future research directions for Allergin-1 include:

• Ligand identification: Discovering the endogenous ligand(s) for Allergin-1 would provide crucial insights into its physiological role and potential for therapeutic targeting.

• Isoform-specific functions: Further characterization of the functional differences between Allergin-1 isoforms could reveal specialized roles in different tissues or disease contexts.

• Signaling pathway elucidation: Detailed mapping of the signaling pathways downstream of Allergin-1 activation would identify potential points for therapeutic intervention.

• Genetic association studies: Expanding on the known association between MILR1 polymorphisms and atopy to explore connections with other allergic or inflammatory conditions.

• Therapeutic development: Design and testing of recombinant proteins or small molecules that can enhance Allergin-1 inhibitory function for potential treatment of allergic conditions.