Recombinant Human Protein FAM19A2 protein (FAM19A2) (Active)

What is FAM19A2/TAFA2 protein and what are its key structural features?

FAM19A2 (Family with Sequence Similarity 19 Member A2), also known as TAFA2, is a secreted 11 kDa member of the FAM19/TAFA family of chemokine-like proteins. It is synthesized as a 131 amino acid precursor containing a 30 amino acid signal sequence and a 101 amino acid mature chain . A defining characteristic of FAM19A2 is its 10 regularly spaced cysteine residues that follow the pattern CX...7CCX13CXCX14CX11CX4CX5CX10C, where C represents a conserved cysteine residue and X represents any non-cysteine amino acid . This cysteine pattern is conserved across TAFA family members (with the exception of TAFA5) and is thought to be critical for the protein's tertiary structure and function. Human TAFA2 shares 97% amino acid identity with mouse TAFA2, indicating high evolutionary conservation .

What is the tissue expression pattern of endogenous FAM19A2?

FAM19A2 exhibits a predominantly central nervous system (CNS)-restricted expression pattern. While FAM19A2 expression can be detected in peripheral tissues including colon, heart, lung, spleen, kidney, and thymus, its expression in the CNS is 50- to 1000-fold higher than in these other tissues . Within the CNS, FAM19A2 shows regional specificity, with highest expression observed in the occipital and frontal cortex (3- to 10-fold higher than other cortical regions) and the medulla . This distinctive expression pattern suggests specialized neural functions for FAM19A2 that likely differ from its potential roles in peripheral tissues. Expression analysis using tissue-specific RT-PCR or immunohistochemistry is recommended to confirm expression in tissues of interest for experimental design.

What are the proposed biological functions of FAM19A2?

While the full spectrum of FAM19A2 biological functions remains to be determined, several hypotheses have emerged based on structural similarities and experimental data:

• As a brain-specific chemokine modulating immune responses in the CNS, potentially working in concert with other chemokines to optimize recruitment and activity of immune cells

• As a novel class of neurokines functioning as regulators of immune nervous cells

• As a controller of axonal sprouting following brain injury

• As a regulator of skeletal (stromal) stem cell migration through activation of Rac1-p38 signaling

• As a factor enhancing neurite outgrowth, as demonstrated in rat embryonic cortical neurons

• As a potential regulator of insulin sensitivity pathways, as suggested by genome-wide association studies that identified FAM19A2 as a novel insulin sensitivity locus

Research approaches combining recombinant protein application with specific pathway inhibitors can help elucidate which of these proposed functions predominate in different cellular contexts.

What methodological considerations should be addressed when using recombinant FAM19A2 in neuronal culture systems?

When utilizing recombinant FAM19A2 in neuronal culture experiments, several methodological considerations must be addressed:

• Protein Immobilization: Evidence suggests that immobilized FAM19A2 effectively enhances neurite outgrowth. For example, rHuTAFA2 immobilized at 624 μg/ml on 96-well plates significantly enhances neurite outgrowth of E16-E18 rat embryonic cortical neurons . Consider immobilizing the protein rather than simply adding it to culture medium for neuronal experiments.

• Embryonic Stage Selection: For developmental neurobiology studies, the embryonic stage of neuronal isolation appears critical. E16-E18 rat embryonic cortical neurons have been successfully used in FAM19A2 studies . Earlier or later developmental stages may respond differently.

• Dosage Optimization: Titration experiments are essential as different neuronal populations may exhibit varying dose-response relationships to FAM19A2. Begin with concentrations in the range reported in the literature (e.g., 624 μg/ml for immobilized protein) and adjust based on preliminary results.

• Signaling Pathway Analysis: Include appropriate inhibitors of proposed downstream pathways (particularly Rac1-p38 signaling components) to confirm mechanistic hypotheses .

• Species Considerations: Although human FAM19A2 shares 97% amino acid identity with mouse FAM19A2 , species-specific responses cannot be ruled out. Validation using species-matched systems is recommended for critical experiments.

How should researchers approach FAM19A2 functional studies in relation to immune regulation in the CNS?

Given FAM19A2's proposed role in immune regulation within the CNS, a multifaceted experimental approach is recommended:

• Co-culture Systems: Establish neuron-microglia or neuron-astrocyte co-culture systems treated with recombinant FAM19A2 to evaluate cell-specific responses and intercellular communication effects.

• Inflammatory Challenge Models: Pre-treat cultures with FAM19A2 before applying inflammatory stimuli (LPS, TNF-α, IL-1β) to assess its potential immunomodulatory effects.

• Receptor Identification: Employ receptor-capturing techniques (such as chemical crosslinking coupled with mass spectrometry) to identify potential FAM19A2 receptors on neural and immune cells.

• Cytokine/Chemokine Profiling: Analyze secretome changes following FAM19A2 treatment using multiplex assays to identify downstream inflammatory mediators.

• In vivo Models: Consider stereotactic injection of recombinant FAM19A2 in animal models of neuroinflammation (EAE, stroke, traumatic brain injury) with subsequent analysis of immune cell infiltration, activation states, and inflammatory markers.

The high CNS-specific expression of FAM19A2 (50-1000 fold higher than in peripheral tissues) suggests specialized neuroimmune functions that warrant careful experimental isolation from its potential peripheral effects.

What experimental approaches can clarify the role of FAM19A2 in insulin sensitivity?

Genome-wide association studies have identified FAM19A2 as a novel insulin sensitivity locus , suggesting metabolic functions beyond its neural roles. To investigate this connection, researchers should consider:

• Tissue-Specific Expression Analysis: Quantify FAM19A2 expression in metabolically active tissues (liver, adipose, muscle, pancreas) under normal and diabetic conditions using qPCR and immunohistochemistry.

• Glucose Uptake Assays: Measure 2-deoxyglucose uptake in skeletal muscle, adipocyte, and hepatocyte cultures treated with recombinant FAM19A2 to assess direct effects on insulin-stimulated glucose disposal.

• Insulin Signaling Pathway Analysis: Evaluate phosphorylation of insulin receptor, IRS1/2, Akt, and AS160 in response to FAM19A2 treatment with and without insulin co-stimulation.

• Conditional Knockout Models: Generate tissue-specific FAM19A2 knockout mice (particularly in brain, liver, and muscle) and characterize their metabolic phenotypes including glucose tolerance tests, insulin tolerance tests, and hyperinsulinemic-euglycemic clamps.

• Human Genetic Correlation Studies: Analyze correlation between FAM19A2 variants (particularly rs10506418) and direct measures of insulin sensitivity (M-value from euglycemic clamps or steady-state plasma glucose from insulin suppression tests) in metabolic cohorts.

The association between FAM19A2 and insulin sensitivity raises intriguing questions about potential neuroendocrine mechanisms connecting CNS signaling with peripheral metabolism that warrant detailed investigation.

What are the recommended methods for validating FAM19A2 activity in biological assays?

When validating recombinant FAM19A2 activity, researchers should implement these methodological approaches:

• Neurite Outgrowth Assay: The ability to enhance neurite outgrowth in E16-E18 rat embryonic cortical neurons when immobilized at 624 μg/ml serves as a functional validation of bioactive FAM19A2 . Quantify neurite length, branching, and complexity using automated image analysis software.

• Migration Assays: Assess FAM19A2's effect on cell migration using Boyden chamber or wound healing assays with skeletal (stromal) stem cells, monitoring Rac1-p38 signaling activation as a mechanistic readout .

• Protein Quality Control: Verify protein integrity using SDS-PAGE (>95% purity) and HPLC analysis, confirming the expected molecular weight of approximately 11.2 kDa . Mass spectrometry can provide additional confirmation of protein identity and post-translational modifications.

• Endotoxin Testing: As an immune-modulatory protein, ensuring endotoxin-free preparations (<0.1 EU/μg) is critical to avoid confounding inflammatory responses in biological assays.

• Dose-Response Curves: Establish complete dose-response relationships in each biological system, as effective concentrations may vary significantly between different cell types and assay formats.

Implementing these validation approaches ensures that observed biological effects can be attributed to specific FAM19A2 activity rather than contaminants or degradation products.

How should researchers interpret conflicting data between FAM19A2's neural and metabolic functions?

The emerging dual role of FAM19A2 in both neural and metabolic regulation presents potential challenges in data interpretation. To address seemingly conflicting observations:

• Tissue-Specific Isoform Analysis: Investigate whether alternative splicing produces tissue-specific FAM19A2 isoforms with distinct functions in neural versus metabolic tissues. RNA-seq and isoform-specific qPCR can identify such variants.

• Receptor Diversity Hypothesis: Consider the possibility that FAM19A2 interacts with different receptors in neural versus peripheral tissues. Receptor capture experiments in different cell types can test this hypothesis.

• Concentration-Dependent Effects: Establish complete dose-response curves in different systems, as FAM19A2 may exhibit different biological activities at different concentrations relevant to its 50-1000 fold expression difference between CNS and peripheral tissues .

• Pathway Integration Analysis: Use systems biology approaches to map how FAM19A2 signaling pathways in neurons might overlap with insulin signaling networks, potentially explaining the GWAS associations .

• In vivo Models with Tissue-Specific Manipulation: Develop conditional knockout or overexpression models with tissue-specific FAM19A2 manipulation to dissect neural versus peripheral effects on metabolism.

The identification of FAM19A2 as an insulin sensitivity locus despite its predominant CNS expression suggests complex neuroendocrine connections that may require integrated experimental paradigms spanning neuroscience and metabolism research.

What experimental controls are essential when investigating FAM19A2 in stem cell migration studies?

When investigating FAM19A2's reported role in skeletal (stromal) stem cell migration , the following experimental controls are critical:

• Heat-Inactivated FAM19A2: Include heat-denatured protein preparations to confirm that observed migration effects depend on the native protein structure rather than contaminants.

• Related Family Members: Include other FAM19/TAFA family proteins (especially the closely related TAFA1, TAFA3, and TAFA4) to determine the specificity of FAM19A2's effects on stem cell migration.

• Signaling Pathway Inhibitors: Include specific inhibitors of the Rac1-p38 pathway (e.g., NSC23766 for Rac1, SB203580 for p38 MAPK) to confirm the proposed mechanism .

• Checkerboard Analysis: Perform checkerboard assays (varying FAM19A2 concentrations in upper and lower chambers) to distinguish between chemotactic (directional) and chemokinetic (random motility) effects.

• Stem Cell Source and Passage Control: Standardize the source, isolation method, and passage number of skeletal stem cells, as responsiveness to migration signals can vary with these parameters.

• Time-Course Analysis: Collect migration data at multiple time points to distinguish between effects on migration rate versus ultimate migration capacity.

These controls help establish whether FAM19A2's effect on stem cell migration represents a direct chemotactic activity consistent with its proposed role as a chemokine-like molecule or an indirect effect on cellular motility machinery.

What are the key considerations for storing and handling recombinant FAM19A2 to maintain biological activity?

Proper storage and handling of recombinant FAM19A2 is critical to maintain its biological activity for research applications:

• Storage Conditions: Store lyophilized FAM19A2 desiccated at -20°C . Once reconstituted, aliquot the protein to minimize freeze-thaw cycles and store at -80°C for long-term storage or at -20°C for short-term use.

• Reconstitution Buffer: Typically use sterile PBS or similar physiological buffer. For specific applications requiring higher concentrations, consider including 0.1% BSA as a carrier protein to prevent adhesion to tubes and loss of active protein.

• Avoiding Aggregation: The 10 cysteine residues in FAM19A2 may contribute to aggregation through disulfide bond formation. Consider including a mild reducing agent (0.1mM DTT) during storage, but not in final working solutions for cell-based assays.

• Freeze-Thaw Stability: FAM19A2's activity in neurite outgrowth assays may be particularly sensitive to freeze-thaw cycles. Validate protein activity after storage using functional assays.

• Working Concentration Preparation: When diluting stock solutions to working concentrations, use buffers containing carrier protein (0.1-0.5% BSA) and prepare fresh dilutions for each experiment.

These handling considerations are particularly important given FAM19A2's complex cysteine-rich structure and its potential for conformation-dependent receptor interactions.

How can researchers address the challenge of detecting endogenous FAM19A2 expression in different tissues?

Detection of endogenous FAM19A2 presents challenges due to its varying expression levels across tissues (50-1000 fold higher in CNS than peripheral tissues) :

• RNA Detection Methods:

  • Use highly sensitive qRT-PCR with validated primers spanning exon junctions

  • For low-expressing tissues, consider nested PCR approaches or digital droplet PCR

  • RNA-scope in situ hybridization can provide cellular resolution with higher sensitivity than conventional ISH

• Protein Detection Methods:

  • Validate antibodies using overexpression systems and knockout controls

  • Employ tissue enrichment techniques (e.g., subcellular fractionation) before Western blotting

  • Consider using mass spectrometry-based approaches for unambiguous detection in complex samples

• Reference Standards:

  • Include positive control tissues (occipital cortex or frontal cortex) where FAM19A2 expression is highest

  • Generate calibration curves using recombinant FAM19A2 for absolute quantification

• Cell Type Resolution:

  • Single-cell RNA-seq can identify specific cell populations expressing FAM19A2

  • Flow cytometry with intracellular staining can quantify protein levels in mixed cell populations

These approaches can help overcome the technical challenges of detecting FAM19A2 across diverse tissues with widely varying expression levels.

What strategies can address potential reproducibility issues in FAM19A2 functional studies?

Ensuring reproducibility in FAM19A2 functional studies requires addressing several potential variables:

• Protein Source Standardization:

  • Document the expression system used (E. coli-derived human FAM19A2 is common)

  • Specify the exact amino acid sequence (typically Ala31-His131)

  • Confirm batch-to-batch consistency using SDS-PAGE, HPLC, and activity assays

• Activity Benchmarking:

  • Establish standard curves for neurite outgrowth enhancement or stem cell migration

  • Create internal reference standards to normalize between experiments

• Cell System Standardization:

  • For neuronal assays, standardize embryonic age (E16-E18) and cortical region

  • For stem cell studies, document source, isolation protocol, and passage number

  • Consider using established cell lines for initial studies before moving to primary cells

• Protocol Documentation:

  • Record specific immobilization methods for neurite outgrowth assays (concentration, coating time, temperature)

  • Document medium composition, including serum lot numbers when relevant

  • Specify imaging and quantification parameters for morphological endpoints

• Biological Replicates:

  • Include both technical replicates (multiple wells) and biological replicates (different cell preparations)

  • Calculate appropriate sample sizes based on preliminary data variability

  • Consider multi-laboratory validation for key findings

Addressing these factors systematically can significantly improve reproducibility in FAM19A2 functional studies across different research groups.

How does the identification of FAM19A2 as an insulin sensitivity locus change our understanding of its biological functions?

The identification of FAM19A2 as a novel insulin sensitivity locus through genome-wide association studies represents a paradigm shift in our understanding of this predominantly CNS-expressed protein:

• Neuroendocrine Axis Hypothesis: This finding suggests FAM19A2 may function within a neuroendocrine axis connecting brain signaling with peripheral metabolic regulation. This expands its proposed functions beyond local neural activities to include systemic metabolic control.

• Mechanistic Possibilities: Several mechanisms could explain this connection:

  • FAM19A2 might influence hypothalamic circuits controlling peripheral insulin sensitivity

  • Low-level FAM19A2 expression in peripheral tissues might directly modulate insulin signaling

  • FAM19A2 could regulate the production or secretion of other factors that affect insulin sensitivity

• Clinical Implications: The association with insulin sensitivity suggests potential relevance to metabolic disorders, expanding FAM19A2's clinical significance beyond neurological conditions.

• Research Direction Impact: This finding encourages integration of neuroscience and metabolism research approaches when studying FAM19A2, potentially revealing novel brain-periphery communication pathways.

• Therapeutic Target Evaluation: FAM19A2 might represent a novel CNS-originating target for insulin resistance and type 2 diabetes, a perspective not previously considered for this protein family.

This genetic evidence linking FAM19A2 to insulin sensitivity highlights the importance of considering both central and peripheral functions when designing comprehensive research programs to elucidate its biological roles.

What emerging technologies might advance our understanding of FAM19A2 signaling mechanisms?

Several cutting-edge technologies could significantly advance our understanding of FAM19A2 signaling:

• CRISPR-Based Approaches:

  • CRISPR activation/inhibition (CRISPRa/CRISPRi) systems can provide temporal control over FAM19A2 expression

  • CRISPR screens targeting potential receptor candidates could identify FAM19A2 binding partners

  • Base editing or prime editing can introduce specific variants identified in GWAS studies to investigate their functional consequences

• Advanced Imaging Techniques:

  • Super-resolution microscopy combined with fluorescently labeled FAM19A2 can visualize receptor binding and internalization dynamics

  • Lattice light-sheet microscopy enables long-term live imaging of FAM19A2-induced cellular responses with minimal phototoxicity

  • Expansion microscopy can provide nanoscale resolution of FAM19A2 localization in complex neural tissues

• Single-Cell Multi-Omics:

  • Integrated single-cell transcriptomics and proteomics can identify cell populations responsive to FAM19A2

  • Spatial transcriptomics can map FAM19A2 expression and downstream responses within intact tissue architecture

  • Single-cell ATAC-seq can reveal chromatin accessibility changes following FAM19A2 signaling

• Protein Structure Technologies:

  • AlphaFold2 and similar AI-based structure prediction tools can generate high-confidence models of FAM19A2-receptor interactions

  • Cryo-EM approaches could resolve the structure of FAM19A2 bound to its receptor(s)

  • Hydrogen-deuterium exchange mass spectrometry can map dynamic conformational changes during ligand binding

These technologies could resolve current knowledge gaps regarding FAM19A2's receptors, signaling mechanisms, and tissue-specific functions.

How might FAM19A2 research contribute to emerging therapeutic strategies for metabolic or neurological disorders?

FAM19A2's unique characteristics position it as a potential therapeutic target with several promising applications:

• Metabolic Disorder Applications:

  • The identification of FAM19A2 as an insulin sensitivity locus suggests potential applications in type 2 diabetes and insulin resistance

  • FAM19A2-based therapeutics might offer a novel CNS-driven approach to improving peripheral insulin sensitivity

  • Personalized medicine approaches could target specific FAM19A2 variants identified in metabolic GWAS studies

• Neurological Applications:

  • FAM19A2's ability to enhance neurite outgrowth suggests potential applications in neuroregeneration after injury or stroke

  • Its proposed role in axonal sprouting could be leveraged for neuroplasticity-promoting therapies

  • As a potential neuroimmune modulator, FAM19A2-based approaches might benefit neuroinflammatory conditions

• Delivery Strategies:

  • Blood-brain barrier (BBB) penetration will be critical for CNS-targeted applications, suggesting the need for specialized delivery vehicles

  • For peripheral applications, modified FAM19A2 variants with extended half-life might be required

  • Cell-specific targeting could be achieved through fusion proteins or nanoparticle delivery systems

• Therapeutic Modalities:

  • Recombinant protein administration (systemic or CNS-directed)

  • Gene therapy approaches to modulate endogenous FAM19A2 expression

  • Small molecule mimetics or modulators targeting FAM19A2 signaling pathways

  • Biologics targeting FAM19A2 receptors once identified

The dual role of FAM19A2 in neural and metabolic functions presents unique opportunities for developing therapies that could address the interconnection between neurological and metabolic disorders, an increasingly important frontier in medical research.