FCGR3A Human, Sf9

What is FCGR3A and what is its molecular structure?

FCGR3A is a receptor for the Fc portion of immunoglobulin G (IgG) involved in the removal of antigen-antibody complexes from circulation and antibody-dependent cellular mediated cytotoxicity (ADCC). It plays a critical role in immune responses including antibody-dependent enhancement of virus infections . The recombinant FCGR3A Human produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 200 amino acids (specifically amino acids 18-208) with a molecular mass of 22.8kDa . On SDS-PAGE, it typically appears at approximately 28-40kDa due to glycosylation patterns. The protein is commonly produced with a 6 amino acid His tag at the C-terminus to facilitate purification through proprietary chromatographic techniques . When designing experiments with this recombinant protein, researchers should account for potential functional differences caused by the expression system's glycosylation patterns and the presence of the His tag.

How does FCGR3A differ from related Fc receptors?

FCGR3A (CD16a) shares high sequence similarity with FCGR3B (CD16b), but they have distinct cellular expression patterns and membrane anchoring mechanisms. FCGR3A is expressed primarily on natural killer (NK) cells as an integral membrane glycoprotein anchored through a transmembrane peptide, while FCGR3B is expressed on polymorphonuclear neutrophils (PMN) with a phosphatidylinositol (PI) linkage . This difference in anchoring impacts signal transduction capabilities, as FCGR3A can effectively transmit activation signals through its transmembrane domain. Within the broader Fc receptor family, FCGR3A is classified as a low-affinity receptor for IgG, distinguishing it from high-affinity receptors like FCGR1 (CD64). When designing experiments to study FCGR3A, researchers must use specific primers and antibodies that can distinguish between these closely related receptors, especially FCGR3A and FCGR3B, to ensure accurate results.

What are the key polymorphisms in FCGR3A and how do they affect function?

The FCGR3A gene contains numerous polymorphisms with potential functional significance. A comprehensive analysis revealed 234 polymorphisms within the gene, with 69% occurring in intron regions, 16% in UTR regions, and 15% in exon regions. Only 16% of all polymorphisms have a minor allele frequency (MAF) greater than 0.01 . The most extensively studied polymorphism is V158F (rs396991), which affects binding affinity to IgG. In a study of 76 individuals, the V158F polymorphism distribution was 38% homozygous T/T, 7% homozygous G/G, and 55% heterozygous . The V158 variant (G allele) demonstrates higher binding affinity for IgG1 and IgG3 compared to the F158 variant (T allele), affecting NK cell activation thresholds and ADCC potency. This polymorphism has clinical significance, as seen in rheumatoid arthritis patients where FCGR3A rs396991-TT genotype carriers showed higher response to tocilizumab, while the G allele was associated with better response to rituximab .

How can FCGR3A polymorphisms be detected in research settings?

Researchers have developed multiple methods for detecting FCGR3A polymorphisms, addressing the challenges posed by the high homology between FCGR3A and FCGR3B. A comprehensive approach combines FCGR3A gene-specific amplification followed by sequencing. Both Sanger sequencing and next-generation sequencing methods like MinION (Nanopore Technologies) have been successfully employed . When implementing these methods, researchers should design primers that specifically target FCGR3A to avoid cross-amplification of FCGR3B. PCR-RFLP (Restriction Fragment Length Polymorphism) has traditionally been used to detect specific variants like V158F, but more comprehensive approaches are needed to capture the full range of polymorphisms. A pilot study comparing Sanger sequencing and MinION sequencing of 14 DNA samples showed good concordance between the methods, with both approaches detecting 13 SNPs listed in the 1000 Genome Project and 10 additional SNPs not previously listed . This highlights the importance of using comprehensive sequencing approaches rather than targeted genotyping of known variants.

How does FCGR3A expression correlate with immune infiltration in cancer?

FCGR3A expression shows significant correlation with immune cell infiltration across multiple cancer types. Analysis from the TIMER database revealed that FCGR3A expression is associated with tumor purity in 28 cancer types and shows correlation with various immune cell populations . In low-grade glioma (LGG), FCGR3A expression is positively correlated with infiltrating levels of:

• B cells

• CD8+ T cells

• CD4+ T cells

• Macrophages

• Neutrophils

• Dendritic cells (DCs)

These correlations suggest FCGR3A plays a vital role in the recruitment or retention of immune cells within the tumor microenvironment. When investigating this phenomenon, researchers should employ multiple methodologies including single-cell RNA sequencing to distinguish cell-specific expression patterns, multiplex immunohistochemistry to visualize spatial relationships, and functional assays to determine the mechanistic links between FCGR3A expression and immune cell behavior. Experimental designs should account for tumor heterogeneity and include appropriate controls for tissue-specific expression patterns. The strong correlation between FCGR3A and immune infiltration highlights its potential as both a prognostic biomarker and a target for immunotherapeutic approaches.

What is the prognostic value of FCGR3A in cancer and how should it be evaluated?

To properly evaluate FCGR3A's prognostic value, researchers should:

The relationship between FCGR3A expression, immune infiltration, and clinical outcomes suggests complex interactions within the tumor microenvironment that warrant further mechanistic investigation.

How do FCGR3A polymorphisms affect response to monoclonal antibody therapies?

FCGR3A polymorphisms significantly impact response to monoclonal antibody therapies through alteration of binding affinity and subsequent effector functions. The V158F polymorphism (rs396991) has been particularly well-studied in this context. In rheumatoid arthritis patients, treatment outcomes show genotype-specific patterns:

For tocilizumab treatment:

• Patients carrying the FCGR3A rs396991-TT genotype showed higher EULAR response (OR, 5.075; 95%CI, 1.20-21.33; P = .027) at 12 months

• Patients who were biologic DMARD-naïve at treatment initiation demonstrated better outcomes, suggesting interaction between prior treatment history and genetic factors

For rituximab treatment:

• The FCGR3A rs396991-G allele was associated with improved low disease activity rate (OR, 4.904; 95%CI, 0.84-28.48; P = .077) and greater improvement in DAS28 (B = -1.083; 95%CI, -1.98 to -0.18; P = .021) at 18 months

• The FCGR2A rs1801274-TT genotype also showed better response, indicating the importance of considering multiple Fc receptor polymorphisms together

When investigating these effects, researchers should employ prospective study designs with adequate sample sizes to account for polymorphism frequencies, include comprehensive genotyping of multiple Fc receptor genes, and measure outcomes at multiple timepoints using standardized response criteria. Functional validation through in vitro ADCC assays using cells from donors with different genotypes can provide mechanistic insights into the clinical observations.

What methodological considerations are important when producing FCGR3A Human in Sf9 cells?

Producing functional FCGR3A Human in Sf9 cells requires careful attention to several methodological aspects:

• Construct design:

  • Include amino acids 18-208 of the native sequence to maintain proper protein folding

  • Consider the impact of tag placement (N- vs C-terminal) on receptor function

  • Optimize codon usage for Sf9 expression system efficiency

• Expression conditions:

  • Determine optimal multiplicity of infection (MOI) through titration experiments

  • Evaluate expression at different time points post-infection (typically 48-72 hours)

  • Consider temperature adjustments during expression phase

• Purification strategy:

  • Implement multi-step chromatography beginning with affinity purification via the His-tag

  • Follow with ion exchange and/or size exclusion chromatography for higher purity

  • Monitor glycosylation heterogeneity throughout purification process

• Quality control:

  • Verify molecular weight by SDS-PAGE (expected 28-40kDa due to glycosylation)

  • Confirm identity by mass spectrometry or Western blotting

  • Assess functional activity through binding assays with IgG

  • Evaluate glycosylation patterns if relevant to intended application

Researchers should note that Sf9-expressed proteins have insect-type glycosylation, which differs from mammalian patterns and may affect certain interactions. For applications where mammalian glycosylation is critical, alternative expression systems might be more appropriate. Documentation of purification yields, purity assessments, and functional validation are essential for reproducible research using FCGR3A Human, Sf9.

How does FCGR3A interact with other components of the immune system in disease pathogenesis?

FCGR3A functions within a complex network of immune components, with interactions that influence disease development and progression. Key interactions include:

• Coordination with other Fc receptors:

  • The balance between activating (FCGR3A, FCGR2A) and inhibitory (FCGR2B) receptors determines cellular activation thresholds

  • Combined polymorphism analysis of FCGR2A and FCGR3A provides better predictive value for treatment response than single-gene analysis

  • Co-expression patterns vary across immune cell subsets, creating cell-specific response profiles

• Integration with complement system:

  • Synergistic activity between FCGR3A-mediated and complement-mediated immune complex clearance

  • Defects in either system can lead to immune complex deposition and inflammation

• Cytokine and chemokine networks:

  • FCGR3A engagement influences cytokine production by NK cells and other immune cells

  • Cytokine feedback loops can modulate FCGR3A expression and function

In disease contexts, particularly autoimmune disorders like systemic lupus erythematosus, mutations in FCGR3A contribute to pathogenesis through altered immune complex handling, dysregulated ADCC, and abnormal cytokine production . When studying these interactions, researchers should employ systems biology approaches combining transcriptomics, proteomics, and functional assays to map the complex relationship networks. Methodologically, it's important to use multiparameter flow cytometry, proximity ligation assays, and carefully designed in vivo models that represent the complexity of human immune system interactions.

What are the emerging applications of FCGR3A research in precision medicine?

FCGR3A research is increasingly important for precision medicine approaches, particularly in immunotherapy and autoimmune disease treatment. Key applications include:

• Pharmacogenetic stratification for antibody therapies:

  • Genotyping patients for FCGR3A V158F polymorphism before initiating therapy

  • Stratifying patients based on predicted ADCC efficiency

  • Adjusting dosing regimens based on FCGR3A-dependent drug clearance rates

• Biomarker development:

  • Using FCGR3A expression as a prognostic biomarker in cancers like LGG

  • Monitoring NK cell FCGR3A expression levels during immunotherapy

  • Evaluating polymorphism status as predictive biomarkers for treatment selection

• Therapeutic antibody engineering:

  • Designing Fc modifications to enhance binding to specific FCGR3A variants

  • Creating antibodies with selective engagement of activating or inhibitory Fc receptors

  • Developing bispecific antibodies that co-engage FCGR3A and tumor antigens

• Diagnostic applications:

  • Assessing FCGR3A polymorphisms as risk factors for autoimmune diseases

  • Evaluating FCGR3A expression in tumor biopsies for patient stratification

  • Developing companion diagnostics for antibody therapeutics

When implementing these applications, researchers must consider the polygenic nature of treatment responses, with combined analysis of multiple Fc receptor genes often providing better predictive value than FCGR3A alone . Methodologically, researchers should develop standardized genotyping assays, establish clear clinical endpoints, and design trials with adequate power to detect genotype-specific effects. The integration of FCGR3A research into precision medicine approaches has potential to significantly improve treatment outcomes across multiple disease areas.

What are the future directions for FCGR3A research?

Future FCGR3A research should focus on several promising directions that build upon current knowledge while addressing existing gaps:

• Comprehensive genetic analysis:

  • Whole gene sequencing to identify rare variants beyond commonly studied polymorphisms

  • Haplotype analysis across the Fc receptor gene cluster to understand combined genetic effects

  • Functional validation of newly identified variants using CRISPR-engineered cell lines

• Structural biology approaches:

  • Cryo-EM structures of FCGR3A in complex with various antibody Fc regions

  • Molecular dynamics simulations to understand how polymorphisms affect binding interfaces

  • Structure-based design of modified antibodies with optimized FCGR3A engagement

• Single-cell analysis technologies:

  • Single-cell RNA sequencing to map FCGR3A expression across immune cell subsets

  • Spatial transcriptomics to understand FCGR3A in tissue microenvironments

  • Multi-parameter protein analysis to correlate FCGR3A with other immune receptors

• Translational applications:

  • Development of standardized FCGR3A genotyping assays for clinical implementation

  • Large-scale clinical studies correlating FCGR3A variants with treatment outcomes

  • Integration of FCGR3A data into comprehensive predictive models for immunotherapy response