Recombinant Human Protein FAM19A3 (FAM19A3)

Basic Research Questions

• What is FAM19A3 and where is it primarily expressed?

  FAM19A3, also called TAFA3, is a member of the FAM19/TAFA family of small secreted proteins that are predominantly expressed in the central and peripheral nervous systems . Human TAFA3 is 133 amino acids in length with a predicted molecular weight of approximately 14-15 kDa . It belongs to a family of five conserved chemokine-like proteins (FAM19A1-5 or TAFA1-5) that function as neurokines . In the brain, FAM19A3 shows the highest expression in the parietal cortex and basal ganglia . It's also notably expressed in the hypophysial pars tuberalis, which serves as an important interface between neuroendocrine brain centers .

• How is FAM19A3 structurally related to other protein families?

  FAM19A3 displays chemokine-like signatures and appears to be distantly related to CCL3/MIP-1 alpha as they share a common CC motif . The FAM19A family consists of five homologous proteins that are approximately 12-15 kDa in size, and all contain conserved structural motifs typical of secreted signaling molecules . Unlike other FAM19A family members, FAM19A3 expression is not significantly affected by metabolic states such as fasting and refeeding, but instead shows regulation by circadian rhythms, particularly in the hypophysial pars tuberalis .

• How is FAM19A3 expression regulated in the brain?

  FAM19A3 expression demonstrates several unique regulatory patterns:

  • Circadian regulation: In the hypophysial pars tuberalis, FAM19A3 transcript levels oscillate with low expression at mid-day and high expression at midnight . This oscillation is lost in mice lacking the melatonin receptor subtype 1 (MT1), suggesting melatonin-dependent regulation .

  • Hypoxia response: FAM19A3 is significantly upregulated in response to hypoxia caused by focal cerebral ischemia, particularly in the infarcted mouse brain regions .

  • Cell-type specificity: FAM19A3 expression and secretion are promoted by M2 stimuli in microglia, indicating that it might function as an M2-type gene .

  Unlike other FAM19A family members whose expression changes with nutritional status, FAM19A3 expression remains relatively stable during fasting and refeeding cycles .

Experimental Methodologies and Applications

• How can recombinant FAM19A3 protein be used in experimental settings?

  Recombinant FAM19A3 has been used in several experimental contexts:

  • Neurite outgrowth assays: Recombinant Human TAFA3/FAM19A3, when immobilized at 4 μg/mL on a 96-well plate, can significantly enhance neurite outgrowth .

  • Microglia polarization studies: Recombinant FAM19A3 has been used to promote M2 polarization and inhibit M1 polarization of microglia in vitro .

  • In vivo cerebral ischemia models: Intraperitoneal injection of recombinant FAM19A3 attenuates cerebral ischemia and neuronal cell death and promotes microglial polarization toward the anti-inflammatory M2 type in brain ischemia models .

  When designing experiments with recombinant FAM19A3, researchers should consider protein concentration, buffer composition, and the appropriate controls based on the specific experimental context.

• What are the specifications and handling recommendations for recombinant human FAM19A3 protein?

  Based on commercial recombinant human FAM19A3 protein specifications:

  • Source: E. coli-derived human TAFA3/FAM19A3 protein spanning Ala31-Arg133 .

  • Formulation: Typically lyophilized from a 0.2 μm filtered solution in PBS .

  • Reconstitution: Recommended to reconstitute at 400 μg/mL in sterile PBS .

  • Storage and stability: Store at recommended temperature and avoid repeated freeze-thaw cycles .

  • Carrier-free option: Available without bovine serum albumin (BSA) for applications where BSA might interfere .

  For experimental use, it's important to note that recombinant FAM19A3 has been shown to form disulfide-bonded complexes with neurexins during biosynthesis , which may impact its behavior in certain experimental systems.

• How can FAM19A3 knockout models be generated and what considerations should be taken?

  While the search results don't provide specific methods for generating FAM19A3 knockout models, they mention that:

  • FAM19A3 knockout mice have been generated and show phenotypes including impaired response to social novelty, deficit in social communication, increased repetitive behaviors, and elevated anxiety .

  Based on general practices in the field, considerations for generating FAM19A3 knockout models include:

  • Gene targeting approach: CRISPR/Cas9 system or traditional homologous recombination in embryonic stem cells.

  • Verification of knockout: Confirming the absence of FAM19A3 at both mRNA and protein levels.

  • Control for compensatory mechanisms: Other FAM19A family members might compensate for the loss of FAM19A3, potentially masking some phenotypes.

  • Background strain considerations: The genetic background of mice can influence phenotypic outcomes of gene deletion.

  • Sex-specific effects: As seen with FAM19A1 knockout mice, which show sexually dimorphic phenotypes , monitoring for sex-specific effects in FAM19A3 knockout models is advisable.

• What techniques are recommended for detecting FAM19A3 expression in tissue samples?

  Based on the search results and general practices in protein detection:

  • RNA-level detection:

    • RT-PCR or qPCR for mRNA expression analysis

    • RNA sequencing for transcriptome-wide analysis

    • Single-cell RNA sequencing for cell-type-specific expression patterns

  • Protein-level detection:

    • Western blotting with specific antibodies

    • Immunohistochemistry or immunofluorescence for tissue localization

    • ELISA for quantitative analysis in tissue lysates or biological fluids

  When studying FAM19A3 expression in the brain, it's important to consider its circadian regulation, particularly in structures like the hypophysial pars tuberalis , and to collect samples at consistent times to account for this variation.

Future Research Directions

• What are the key unresolved questions about FAM19A3 function?

  Several important aspects of FAM19A3 biology remain to be fully elucidated:

  • Receptor identification: While FAM19A proteins appear to bind to different classes of cell surface receptors (e.g., GPCRs and neurexins) , the specific receptors mediating FAM19A3's effects on microglia polarization and other cellular functions need further characterization.

  • Downstream signaling pathways: The molecular mechanisms by which FAM19A3 promotes M2 polarization and inhibits M1 polarization of microglia are not fully understood .

  • Developmental roles: How FAM19A3 expression and function change during brain development requires further investigation.

  • Disease implications: The potential involvement of FAM19A3 in neurological disorders beyond cerebral ischemia, particularly in conditions with altered microglial function or neuroinflammation, warrants exploration.

  • Therapeutic potential: The efficacy, safety, and delivery methods for using FAM19A3 as a potential therapeutic agent for neuroinflammatory conditions need further research.

• How might FAM19A3 contribute to neurological disease pathogenesis or therapy?

  Based on its known functions, FAM19A3 may have implications for several neurological conditions:

  • Stroke and cerebral ischemia: Recombinant FAM19A3 attenuates cerebral ischemia in the MCAO mouse model by promoting anti-inflammatory M2 polarization of microglia and macrophages .

  • Neurodevelopmental disorders: FAM19A3 knockout mice exhibit impaired social communication and increased repetitive behaviors , which are features reminiscent of autism spectrum disorders.

  • Anxiety disorders: FAM19A3 deficiency leads to elevated anxiety in mouse models , suggesting a potential role in anxiety regulation.

  • Circadian rhythm disorders: Given its regulation by melatonin and circadian rhythms , FAM19A3 might be involved in disorders associated with disrupted circadian function.

  The development of FAM19A3-based therapies for these conditions would require further research into its mechanism of action, dosing, delivery methods, and potential side effects.