Recombinant Mouse Protein FAM19A4 (Fam19a4)

What is FAM19A4/TAFA4 and what is its structural composition?

FAM19A4 (also known as TAFA4) is a secreted, 12 kDa member of the FAM19/TAFA family of chemokine-like proteins. It is synthesized as a 140 amino acid (aa) precursor in humans, containing a 35 aa signal sequence and a 105 aa mature chain. The mature protein features a distinct pattern of 10 regularly spaced cysteine residues that follow the pattern Cx7CCx13CxCx14Cx11Cx4Cx5Cx10C, where C represents a conserved cysteine residue and x represents any non-cysteine amino acid .

Mouse FAM19A4 protein has 135 amino acids with a molecular mass of 15,018 Da, while human FAM19A4 protein consists of 140 amino acids with a molecular mass of 15,682 Da . The processed mature human FAM19A4 protein (residues 36-140) has a theoretical molecular weight of 11,668.31 Da and an isoelectric point (pI) of 8.89 .

Where is FAM19A4/TAFA4 expressed in biological systems?

Expression of FAM19A4 is predominantly restricted to the central nervous system (CNS). Real-time PCR analysis indicates that FAM19A4 mRNA is expressed at the highest levels in the thalamus . FAM19A expression in the central nervous system is dynamically regulated during development and in the postnatal brain . The protein shows limited expression outside the CNS, making it relatively specific to neural tissues, which is significant for researchers studying neural-specific markers or signaling pathways .

What are the known biological functions of FAM19A4/TAFA4?

FAM19A4/TAFA4 appears to function in several key areas:

• As a neurokine that modulates immune responses in the CNS by functioning as a brain-specific chemokine

• Regulating immune nervous cells in a manner similar to other chemokines

• Potentially controlling axonal sprouting following brain injury

• Enhancing macrophage chemotaxis and phagocytosis in primary human macrophages and resident mouse peritoneal macrophages

• Playing a role in somatosensory functions, particularly in nociception (pain sensation)

Functional studies using both gain- and loss-of-function mouse models have established non-redundant roles for FAM19A4 in regulating physiological processes including locomotor activity, food intake, learning and memory, anxiety- and depressive-like behaviors, social communication, repetitive behaviors, and somatosensory functions .

How can researchers validate the specificity of FAM19A4/TAFA4 antibodies in experimental settings?

Validation of antibodies for FAM19A4/TAFA4 should follow these methodological approaches:

• Flow cytometry validation: As demonstrated with the Mouse Anti-Human TAFA4/FAM19A4 Monoclonal Antibody (Clone 480103), antibodies can be validated in cell lines such as A172 human glioblastoma. The protocol involves:

  • Staining cells with the anti-FAM19A4 antibody versus an isotype control

  • Using appropriate secondary antibodies (e.g., Phycoerythrin-conjugated Anti-Mouse IgG)

  • Fixing cells with paraformaldehyde and permeabilizing with saponin to facilitate intracellular staining

• Cross-reactivity testing: Proper validation requires demonstrating lack of cross-reactivity with related proteins. For example, the anti-human TAFA4/FAM19A4 antibody has been shown to have no cross-reactivity with recombinant human TAFA1, TAFA2, TAFA3, or TAFA5 in direct ELISAs and Western blots .

• Application-specific validation: Researchers should test antibodies in multiple applications including ELISA, Western blotting, and immunohistochemistry to ensure consistent specificity across different experimental contexts .

What considerations are important when designing experiments with recombinant FAM19A4/TAFA4?

When designing experiments with recombinant FAM19A4/TAFA4, researchers should consider:

• Protein source and modifications:

  • Expression system (bacterial vs. eukaryotic) can affect protein folding and post-translational modifications

  • E. coli-derived recombinant FAM19A4 (e.g., Ser35-Arg140 for human) lacks glycosylation but may be sufficient for many applications

  • The presence of tags (His, Myc-His, Fc) can affect protein activity and should be considered when interpreting results

• Storage and stability:

  • Use a manual defrost freezer and avoid repeated freeze-thaw cycles

  • Store at -20 to -70°C for long-term storage (up to 12 months from receipt)

  • Once reconstituted, store at 2-8°C for up to 1 month or at -20 to -70°C for up to 6 months under sterile conditions

• Reconstitution protocols:

  • Typically reconstituted at 200 μg/mL in PBS

  • Carrier-free formulations should be considered when the presence of BSA could interfere with the experimental design

• Experimental controls:

  • Include appropriate positive and negative controls to validate biological activity

  • Consider using both gain- and loss-of-function approaches (recombinant protein addition vs. genetic knockdown)

How does FAM19A4/TAFA4 compare across species, and what implications does this have for translational research?

FAM19A4/TAFA4 shows significant conservation across species, with human and mouse variants exhibiting 90% amino acid identity . This high degree of conservation has important implications:

• Cross-species experimental design:

  • The high homology suggests functional conservation, potentially allowing mouse models to provide insights relevant to human biology

  • Studies in mouse models have established roles in somatosensory functions that may translate to human conditions

• Structural differences:

  • Human FAM19A4: 140 amino acids, 15,682 Da

  • Mouse FAM19A4: 135 amino acids, 15,018 Da

  • These slight differences may affect antibody recognition and should be considered when selecting reagents

• Functional conservation assessment:

  • Functional studies in mice have revealed roles in modulating mechanical and chemical hypersensitivity following inflammation and nerve injury

  • Knockout mouse models show enhanced mechanical and chemical hypersensitivity, suggesting conserved roles in pain modulation that may be relevant to human pain disorders

What methods are available for detecting FAM19A4/TAFA4 expression in tissue samples?

Several methodological approaches are available for detecting FAM19A4/TAFA4 expression:

• Flow cytometry:

  • Intracellular staining using validated antibodies at 0.25-1 μg/10^6 cells

  • Requires fixation with paraformaldehyde and permeabilization with saponin

  • Can be applied to cell lines such as A172 human glioblastoma

• Immunohistochemistry:

  • Can be used to detect FAM19A4 protein in tissue sections

  • Enables co-localization studies with other neural markers

  • Has been applied to study FAM19A4 expression in the thalamus and other CNS regions

• Real-time PCR:

  • For mRNA expression analysis

  • Has been used to demonstrate restricted expression in the CNS

  • Allows quantitative assessment of expression levels across different brain regions

• Methylation analysis:

  • While not detecting the protein directly, DNA methylation of the FAM19A4 gene has been developed as a biomarker in certain contexts

  • Uses quantitative methylation-specific PCR (qMSP) targeting FAM19A4 loci

How can recombinant FAM19A4/TAFA4 be used in neuroinflammation studies?

Recombinant FAM19A4/TAFA4 can be strategically employed in neuroinflammation studies through several methodological approaches:

• Macrophage function studies:

  • Application of recombinant FAM19A4 to primary human macrophages or resident mouse peritoneal macrophages

  • Assessment of phagocytosis enhancement and reactive oxygen species production

  • Evaluation of chemotaxis in response to FAM19A4 stimulation

• Pain modulation studies:

  • Intrathecal delivery of recombinant FAM19A4 to reverse carrageenan-induced mechanical hypersensitivity

  • Assessment of pain alleviation in complete Freund adjuvant (CFA)-inflamed paws

  • Comparison between FAM19A4 knockout and wild-type mice to establish the role in pain modulation

• Neuroprotection assessment:

  • Evaluation of FAM19A4's role in attenuating cerebral ischemia and neuronal cell death

  • Study of microglial polarization toward anti-inflammatory M2 phenotype in brain ischemia models

  • Comparison with related family members such as FAM19A3, which has demonstrated neuroprotective effects

What cell types and models are appropriate for studying FAM19A4/TAFA4 function?

Based on current research, the following cell types and models are appropriate for FAM19A4/TAFA4 studies:

• Cell types:

  • A172 human glioblastoma cell line (validated for antibody detection)

  • Primary human macrophages (demonstrated response to FAM19A4 stimulation)

  • Resident mouse peritoneal macrophages (enhanced phagocytosis in response to FAM19A4)

  • Neural stem cells (FAM19A family members regulate neural stem cell fate)

• Animal models:

  • FAM19A4 knockout mice (show enhanced mechanical and chemical hypersensitivity)

  • Mouse inflammation models (carrageenan-induced, CFA-induced)

  • Nerve injury models for studying pain modulation

  • Brain ischemia models for studying neuroprotective effects

• In vitro systems:

  • Hippocampal cultures (FAM19A family members affect synaptic transmission)

  • Neurospheres (for studying neural stem cell proliferation and differentiation)

What are the emerging roles of FAM19A4/TAFA4 in neuroimmune signaling?

Recent research has expanded our understanding of FAM19A4/TAFA4's role in neuroimmune signaling:

• Microglial regulation:

  • FAM19A family members (particularly FAM19A3) promote microglial polarization toward anti-inflammatory M2 phenotype

  • This suggests FAM19A4 may have similar immunomodulatory effects in the CNS

• Receptor interactions:

  • FAM19A proteins appear to bind to different classes of cell surface receptors

  • These include G-protein coupled receptors (GPCRs) and neurexins

  • The specific receptors for FAM19A4 are still being characterized

• Pain modulation mechanisms:

  • Intrathecal delivery of FAM19A4 can reverse inflammation-induced mechanical hypersensitivity

  • FAM19A4 knockout mice show enhanced sensitivity to painful stimuli

  • These findings indicate a key role in modulating nociceptive signaling

What cellular signaling pathways are affected by FAM19A4/TAFA4?

While specific signaling pathways for FAM19A4 are still being fully characterized, research on the FAM19A family provides insights:

• GPCR signaling:

  • FAM19A1 binds to G-protein-coupled receptor 1 (GPR1)

  • This engagement activates the ROCK/ERK1 pathway (suppressing cell proliferation)

  • It also activates the ROCK/STAT3 pathway (promoting neuronal differentiation)

  • Similar GPCR-mediated signaling may apply to FAM19A4

• Neurexin-mediated pathways:

  • FAM19A1 binding to neurexins reduces O-glycosylation and heparan sulfate modifications

  • This affects inhibitory synaptic transmission in hippocampal cultures

  • FAM19A4 may have similar effects on neuronal cell adhesion molecules

• Inflammatory signaling:

  • In macrophages, FAM19A4 enhances phagocytosis and reactive oxygen species production

  • The specific intracellular signaling mechanisms mediating these effects are areas of ongoing research

What are the optimal storage and handling conditions for recombinant FAM19A4/TAFA4 protein?

For optimal stability and activity of recombinant FAM19A4/TAFA4 protein, researchers should follow these protocols:

• Long-term storage:

  • Use a manual defrost freezer to avoid temperature fluctuations

  • Store at -20 to -70°C for up to 12 months from receipt date

  • Avoid repeated freeze-thaw cycles which can degrade protein structure and activity

• Reconstitution protocols:

  • Lyophilized protein is typically reconstituted at 200 μg/mL in PBS

  • After reconstitution, the protein can be stored at 2-8°C for up to 1 month under sterile conditions

  • For longer storage after reconstitution, aliquot and store at -20 to -70°C for up to 6 months

• Working solution preparation:

  • Carrier-free preparations are recommended when the presence of BSA could interfere with experimental outcomes

  • Standard preparations typically contain BSA as a carrier protein to enhance stability and shelf-life

How can researchers evaluate the functional activity of recombinant FAM19A4/TAFA4?

To evaluate the functional activity of recombinant FAM19A4/TAFA4, researchers can employ several methodological approaches:

• Macrophage functional assays:

  • Phagocytosis assays using fluorescently-labeled particles or bacteria

  • Chemotaxis assays using Boyden chambers or similar migration assays

  • Reactive oxygen species (ROS) production measurement using fluorescent probes

• Pain modulation models:

  • Measure mechanical pain thresholds using von Frey filaments before and after FAM19A4 administration

  • Assess thermal pain sensitivity using hot plate or tail flick tests

  • Compare responses in inflammatory pain models (carrageenan, CFA) with and without FAM19A4 treatment

• Receptor binding studies:

  • While specific receptors for FAM19A4 are still being characterized, binding assays can be developed based on knowledge from other FAM19A family members

  • Competition binding assays with known FAM19A ligands may help identify receptor interactions

What are the methodological considerations for comparing mouse and human FAM19A4/TAFA4 in translational studies?

When conducting translational studies comparing mouse and human FAM19A4/TAFA4, researchers should consider:

• Sequence homology and structural differences:

  • The 90% amino acid identity between mouse and human FAM19A4 suggests functional conservation

  • Differences in protein size (mouse: 135 aa, 15,018 Da; human: 140 aa, 15,682 Da) may affect antibody recognition and functional properties

• Species-specific antibody selection:

  • Antibodies should be validated for cross-reactivity between species if used in comparative studies

  • Some antibodies may be species-specific and not suitable for cross-species comparisons

• Experimental readouts:

  • Behavioral assays in mouse models should be carefully correlated with human clinical parameters

  • Cell-based assays should compare equivalent cell types from both species when possible

  • Biomarker development should account for potential species differences in signaling pathways and receptor distribution

By addressing these methodological considerations, researchers can design more robust translational studies that effectively bridge findings between mouse models and human applications in FAM19A4/TAFA4 research.