FCER1A Human, HEK

What is FCER1A and what is its role in the immune system?

FCER1A (Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide) is the alpha subunit of the high-affinity IgE receptor (FcεRI). This receptor plays a central role in IgE-mediated allergic immune responses. The complete receptor exists as a tetrameric complex composed of one α subunit (FCER1A), one β subunit, and two γ subunits on mast cells and basophils. Alternatively, a trimeric form consisting of one α subunit and two γ subunits is expressed on various cell types including eosinophils and antigen-presenting cells .

FCER1A directly binds IgE with high affinity, while the β and γ chains are responsible for intracellular signal transduction. When allergens bind to receptor-bound IgE, it triggers cell activation and the release of mediators such as histamine, which are responsible for allergic manifestations .

What cell types naturally express FCER1A?

FCER1A is primarily expressed on:

• Mast cells

• Basophils

• Eosinophils (particularly in hypereosinophilic syndrome)

• Langerhans cells

• Dendritic cells

• Monocytes (especially in patients with allergic disorders)

• Platelets

• Bronchial epithelial cells

• Neutrophils (in allergy-induced asthma patients)

What are common challenges when expressing recombinant human FCER1A in HEK cells?

Expressing functional FCER1A in HEK cells presents several challenges:

• Proper folding and post-translational modifications - While FCER1A is naturally glycosylated, recombinant expression may result in different glycosylation patterns that affect function

• Stability issues - Recombinant FCER1A proteins show limited stability at temperatures above 4°C and require careful storage conditions

• Expression efficiency - Optimizing transfection protocols specifically for FCER1A can be challenging due to its complex structure

• Functional verification - Ensuring that the expressed protein maintains proper IgE binding capabilities

Researchers typically overcome these challenges by optimizing expression vectors, culture conditions, and purification strategies specific to FCER1A .

How do genomic variants of FCER1A influence its expression and function in experimental systems?

Genomic variants of FCER1A have been significantly associated with total and allergen-specific IgE levels as well as allergic sensitization. Three strongly correlated FCER1A polymorphisms have been identified that impact both IgE levels and allergic responses .

When designing experimental systems using HEK cells, researchers should consider:

• The specific variant being expressed (wild-type vs. polymorphic)

• The potential impact of these variants on protein expression levels

• Functional differences in IgE binding capacity between variants

• Downstream signaling alterations that may result from structural changes

These genetic variations may influence experimental outcomes, especially in studies comparing different population groups or investigating differential responses to allergic stimuli .

What is the relationship between FCER1A-AS and FCER1A expression, and how might this impact HEK expression systems?

Recent research has identified a natural antisense transcript (NAT) of FCER1A (FCER1A-AS) that plays a positive regulatory role in FCER1A expression. This fully overlapped antisense transcript is co-expressed with the sense transcript (FCER1A-S) in cells expressing FcεRIα .

In HEK expression systems, several considerations emerge:

• Selective knockdown of FCER1A-AS using CRISPR/RfxCas13d (CasRx) leads to marked decreases in both mRNA and protein levels of FCER1A-S

• The FCER1A-AS transcript shows greater stability (half-life of approximately 13 hours) compared to FCER1A-S (half-life of approximately 10 hours)

• FCER1A-AS deficiency results in diminished anaphylactic reactions similar to FCER1A gene knockout effects

When designing expression constructs for HEK cells, researchers should consider whether to include genomic regions that would allow for FCER1A-AS expression, as this could significantly impact the yield and functionality of the expressed FCER1A protein .

How can epistatic effects between FCER1A and other genes be studied using HEK expression systems?

Epistatic effects, particularly between FCER1A and FLG (filaggrin) variants, have been identified as significant in eczema risk. The FCER1A variants rs10489854 and rs2511211 demonstrate synergistic effects on eczema risk after adjustment for FLG effects .

To study these epistatic interactions in HEK cells:

• Co-express FCER1A variants with FLG or other relevant genes in HEK cells

• Utilize model-based multifactor dimensionality reduction (MB-MDR) methods to analyze potential interactions

• Design experiments comparing wild-type and variant forms expressed individually and in combination

• Employ reporter systems to quantify functional outcomes of these interactions

This approach allows researchers to recapitulate and investigate the molecular basis of genetic interactions observed in population studies .

What are the optimal conditions for expressing functional recombinant human FCER1A in HEK cells?

For optimal expression of functional FCER1A in HEK cells, researchers should follow these methodological guidelines:

• Vector selection:

  • Use mammalian expression vectors with strong promoters (CMV/EF1α)

  • Include appropriate signal peptides for membrane localization

  • Consider adding purification tags (His-tag) at the C-terminus to preserve N-terminal IgE binding regions

• Transfection protocol:

  • Use lipid-based transfection reagents for higher efficiency

  • Maintain cell density at 70-80% confluence at transfection

  • Harvest protein 48-72 hours post-transfection

• Culture conditions:

  • Maintain cells in DMEM with 10% FBS at 37°C, 5% CO2

  • Add sodium butyrate (1-5 mM) 24h post-transfection to enhance expression

  • For larger scale production, consider adaptation to suspension culture

• Verification of functional expression:

  • Flow cytometry with fluorescently-labeled IgE

  • Immunoprecipitation with anti-FCER1A antibodies

  • Western blotting for protein detection

What purification strategies yield the highest quality FCER1A protein preparations?

Obtaining high-quality FCER1A preparations requires careful purification strategies:

• Initial preparation:

  • Harvest cells in PBS containing protease inhibitors

  • Lyse cells using mild detergents (0.5-1% NP-40 or Triton X-100)

  • Centrifuge at 14,000 × g for 15 minutes to remove cellular debris

• Purification steps:

  • For His-tagged constructs: Ni-NTA affinity chromatography

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for higher purity

• Buffer optimization:

  • Maintain in PBS with 50mM Arginine to improve stability

  • Add 0.05% NaN3 as preservative for long-term storage

  • Consider adding carrier proteins (0.1% HSA or BSA) for long-term storage

• Storage recommendations:

  • Aliquot to avoid freeze-thaw cycles

  • Store at -20°C for long-term stability

  • Protein remains stable at 4°C for approximately one week

How can researchers verify the functional integrity of recombinant FCER1A expressed in HEK cells?

Verification of functional integrity requires multiple analytical approaches:

• Binding affinity assessment:

  • Surface Plasmon Resonance (SPR) to determine IgE binding kinetics

  • ELISA-based binding assays with purified IgE

  • Flow cytometry with fluorescently-labeled IgE for cell-surface expressed FCER1A

• Structural analysis:

  • Circular dichroism (CD) spectroscopy to verify proper folding

  • Limited proteolysis to assess domain organization

  • Mass spectrometry to confirm post-translational modifications

• Functional assays:

  • Calcium flux assays in cells expressing the complete receptor complex

  • Degranulation assays in reconstituted systems

  • Downstream signaling assessment via phosphorylation studies

• Quality control metrics:

  • Purity assessment by SDS-PAGE (>90% purity recommended)

  • Endotoxin testing for preparations intended for immunological studies

  • Glycosylation analysis by lectin blotting or mass spectrometry

How do FCER1A expression levels in HEK cells compare with natural expression in immune cells?

When comparing FCER1A expression between HEK systems and natural immune cells:

• Expression level differences:

  • HEK cells typically achieve higher expression levels due to strong promoters

  • Natural expression in mast cells and basophils is tightly regulated and responsive to cytokines like IL-3

  • Quantitative PCR and western blotting should be used to compare expression levels

• Functional considerations:

  • HEK cells lack endogenous FcεRIβ and FcεRIγ subunits needed for complete receptor assembly

  • Co-expression systems may be needed to reconstitute full receptor functionality

  • IL-3 induces both FCER1A-S and FCER1A-AS in natural systems, which may not occur in standard HEK expression

• Comparative analysis approaches:

  • Flow cytometric quantification using standardized beads

  • Western blot densitometry against known standards

  • qPCR with appropriate reference genes for transcript quantification

What are common issues when studying FCER1A genetic variants and how can they be addressed?

Researchers frequently encounter these challenges when studying FCER1A variants:

• Haplotype complexity:

  • Multiple FCER1A variants often occur in linkage disequilibrium

  • Solution: Express individual variants and combinations to dissect effects

  • Use site-directed mutagenesis to create specific variants in expression constructs

• Functional characterization difficulties:

  • Subtle functional differences may be difficult to detect

  • Solution: Develop highly sensitive assays specific to FCER1A function

  • Employ multiple methodologies to validate observations

• Cell-type specific effects:

  • Variants may show different impacts in different cellular contexts

  • Solution: Compare expression in HEK cells with immune cell lines

  • Consider co-expression of cell-type specific factors that might influence variant effects

How can researchers effectively target both FCER1A and its antisense transcript in experimental designs?

To effectively study the relationship between FCER1A and its antisense transcript:

• Selective targeting approaches:

  • Use CRISPR/RfxCas13d (CasRx) for selective RNA knockdown of FCER1A-AS

  • Design strand-specific primers for reverse transcription to distinguish sense and antisense transcripts

  • Employ strand-specific RNAi approaches with carefully designed sequences

• Expression construct considerations:

  • Include genomic regions that contain both sense and antisense promoters

  • Design modular constructs that allow selective expression of each transcript

  • Use bidirectional promoters to mimic natural co-expression

• Analytical techniques:

  • Strand-specific RT-PCR covering different exons and UTR regions

  • RNA stability assays to compare half-lives of sense and antisense transcripts

  • RNA-protein interaction studies to elucidate regulatory mechanisms

How might FCER1A research in HEK systems contribute to therapeutic developments for allergic diseases?

FCER1A research using HEK expression systems offers several promising therapeutic avenues:

• Drug discovery applications:

  • High-throughput screening platforms using FCER1A-expressing HEK cells

  • Structure-based drug design targeting the IgE binding interface

  • Development of decoy receptors or receptor antagonists

• Therapeutic antibody development:

  • Expression of FCER1A fragments for immunization and antibody production

  • Functional screening of therapeutic antibodies that block IgE-FCER1A interaction

  • Engineering modified FCER1A variants with enhanced or altered functions

• Genetic therapy approaches:

  • Testing CRISPR-based therapies targeting specific FCER1A variants

  • Antisense oligonucleotide development targeting FCER1A-AS for regulation

  • Gene editing validation in HEK systems before advancing to primary cells

What role might the FCER1A-AS transcript play in developing novel regulatory approaches to allergic diseases?

The discovery of FCER1A-AS opens new regulatory possibilities:

• Antisense-based therapeutic strategies:

  • Developing modulators of FCER1A-AS to indirectly control FCER1A expression

  • Testing antisense oligonucleotides that stabilize or destabilize FCER1A-AS

  • Creating synthetic antisense transcripts with enhanced regulatory properties

• Diagnostic applications:

  • Expression analysis of both FCER1A-S and FCER1A-AS as biomarkers

  • Correlation studies between antisense transcript levels and disease severity

  • Personalized medicine approaches based on sense/antisense ratio

• Experimental models:

  • Development of transgenic models with selective FCER1A-AS deficiency

  • In vitro disease models using HEK cells with manipulated FCER1A-AS expression

  • Investigation of cell-specific regulation of antisense transcript expression