SLC15A4 Antibody

What is SLC15A4 and why is it significant in immunological research?

SLC15A4, also known as solute carrier family 15 member 4 (alternatively PHT1, PTR4, hPHT1, or peptide transporter 4), is a transmembrane protein involved in peptide transport across membranes and regulation of immune responses . SLC15A4 has gained significant attention because of its critical role in endosomal toll-like receptor (TLR) signaling pathways, particularly in the TLR7/8-IRF5 innate immune pathway . Studies have demonstrated that SLC15A4 is essential for proper trafficking of TLRs and their ligands to endolysosomes where recognition and signaling are initiated . The protein's role in modulating inflammatory processes highlights its potential relevance in autoimmune disorders, cancer, and chronic inflammatory conditions .

What are the key structural characteristics of SLC15A4 protein?

SLC15A4 exhibits a major facilitator superfamily (MFS) fold structure with 12 transmembrane domains (TMs): TM1-TM6 forming the N-terminal bundle and TM7-TM12 forming the C-terminal bundle . Both N- and C-termini face the cytoplasmic side. The protein contains a lysosomal targeting motif in its N-terminal residues 1-31, which is typically too flexible to be visualized in structural studies . Unlike related transporters SLC15A1 and SLC15A2 that contain extensive extracellular domains with more than ten β strands, SLC15A4 has a relatively small luminal domain between TM9 and TM10 with only two β strands . Interestingly, SLC15A4 can exist in both monomeric and dimeric conformations, with distinct functional implications for each state .

What applications are SLC15A4 antibodies typically used for in research?

SLC15A4 antibodies are versatile research tools employed in multiple experimental applications:

These applications enable researchers to investigate SLC15A4 expression, localization, and function in various experimental contexts .

How should I select an appropriate SLC15A4 antibody for my research?

When selecting an SLC15A4 antibody, several critical factors must be considered:

• Target species compatibility: Ensure the antibody has demonstrated reactivity with your species of interest. Available antibodies show various reactivity profiles including human, mouse, rat, and other species .

• Antibody type: Consider whether a polyclonal or monoclonal antibody better suits your needs. Polyclonal antibodies like the SLC15A4 Polyclonal Antibody (PACO59297) recognize multiple epitopes, potentially offering higher sensitivity but possibly lower specificity than monoclonals .

• Application validation: Verify that the antibody has been validated for your specific application (WB, IHC, IF, etc.) and review any validation images provided .

• Immunogen information: Check the immunogen used to generate the antibody. For example, some SLC15A4 antibodies are raised against recombinant human SLC15A4 protein fragments, such as amino acids 243-317 .

• Purification method: Higher purity antibodies (e.g., >95%, Protein G purified) generally provide better specificity and reduced background .

What protocol is recommended for SLC15A4 immunohistochemistry staining?

For optimal SLC15A4 immunohistochemistry staining, the following protocol has shown success:

• Sample preparation: Use paraffin-embedded tissue sections mounted on appropriate slides.

• Deparaffinization and rehydration: Completely dewax and hydrate sections using standard protocols.

• Antigen retrieval: Perform antigen retrieval under high pressure in citrate buffer (pH 6.0) to expose epitopes potentially masked during fixation .

• Blocking: Block sections with 10% normal goat serum for 30 minutes at room temperature to minimize non-specific binding.

• Primary antibody incubation: Apply the SLC15A4 antibody diluted to 1:200 (or as recommended by manufacturer) and incubate according to optimized conditions.

• Detection: Use an appropriate detection system (e.g., HRP-conjugated secondary antibody and DAB substrate) for visualization.

• Counterstaining: Apply hematoxylin or another suitable counterstain to provide context for SLC15A4 expression.

This protocol has been effectively implemented on systems like the Leica BondTM for staining SLC15A4 in human liver cancer tissues .

What controls should I include when using SLC15A4 antibodies in my experiments?

Rigorous experimental design requires appropriate controls:

These controls are essential for distinguishing genuine SLC15A4 signal from technical artifacts and should be incorporated into every experiment.

How does SLC15A4 interact with TASL in endolysosomal TLR signaling pathways?

The interaction between SLC15A4 and TASL represents a critical regulatory mechanism in endolysosomal TLR signaling. Recent cryo-EM structural studies have revealed key insights into this interaction:

SLC15A4 undergoes a conformational change from an outward-facing (lysosomal lumen-exposed) state to an inward-facing state during TASL recruitment . This conformational shift creates a binding pocket that accommodates the N-terminal helix of TASL. The interaction occurs with a precise 1:1 stoichiometry between SLC15A4 and TASL .

The N-terminal sequences of TASL, particularly residues 1-30, are crucial for this binding interaction. Multiple constructs retaining these N-terminal sequences (TASL(1–30), TASL(1–20), TASL(1–215), TASL(1–20&207–301)) have demonstrated the ability to bind SLC15A4 .

This interaction forms the molecular basis for the SLC15A4-TASL complex that functions as a signaling module in the TLR7/8-IRF5 pathway, which has significant implications for autoimmune disease pathogenesis and potential therapeutic interventions .

How does SLC15A4 deficiency affect different immune cell populations?

SLC15A4 deficiency manifests differently across immune cell types, revealing its diverse roles in immune function:

In plasmacytoid dendritic cells (pDCs):

• Failed acquisition of activation markers

• Defective NF-κB translocation

• Impaired inflammatory cytokine production

• Disrupted IRF5 activation

In B cells:

• Normal proliferation and activation marker expression

• Selective defects in endosomal TLR responses

• Preserved responses to non-TLR stimuli

These differential effects highlight the cell type-specific functions of SLC15A4. Interestingly, some studies using the CAL-1 pDC cell line reported that SLC15A4 deficiency affected IRF5 activation but not MAPK and NF-κB pathways, suggesting complex regulatory mechanisms that may vary by experimental system . These differences might be attributed to unique signaling pathways in different cell types or experimental conditions.

What is the significance of SLC15A4 in autoimmune disease research?

SLC15A4 has emerged as a key focus in autoimmune disease research based on several compelling findings:

• Lupus pathogenesis: Recognition of self-nucleic acids by endosomal TLRs is central to lupus-like autoimmunity in mouse models, and SLC15A4 is required for this recognition .

• Therapeutic potential: The critical role of SLC15A4 in autoimmune pathways supports ongoing efforts to identify pharmacologic inhibitors as potential treatments for lupus and other inflammatory disorders .

• Structural insights: Recent cryo-EM structures of SLC15A4-TASL complexes provide a structural basis for drug discovery targeting SLC15A4- or TASL-related human autoimmune diseases .

• Type I interferon pathway: SLC15A4 is involved in the type I interferon (IFN-I) pathway, which is dysregulated in many autoimmune conditions .

This evidence positions SLC15A4 as both a key mechanistic player and potential therapeutic target in autoimmune disease pathways.

What factors affect SLC15A4 antibody specificity and how can I optimize it?

Optimizing SLC15A4 antibody specificity requires addressing several potential factors:

• Antibody selection: Polyclonal antibodies recognize multiple epitopes and may exhibit higher background compared to monoclonals. The SLC15A4 Polyclonal Antibody (PACO59297) has been validated for specificity with human samples, but optimization may be required for different experimental systems .

• Blocking conditions: Insufficient blocking can lead to non-specific binding. The recommended protocol suggests using 10% normal goat serum for blocking in IHC applications .

• Antibody dilution: Using the recommended dilution ranges (ELISA:1:2000-1:10000, IHC:1:200-1:500, IF:1:50-1:200) is crucial for optimizing signal-to-noise ratio .

• Purification quality: Higher purity antibodies (e.g., >95% Protein G purified) generally show improved specificity. The storage conditions (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative) help maintain antibody integrity .

• Cross-reactivity testing: Test for potential cross-reactivity with related transporters (e.g., other SLC15 family members) in your experimental system.

How can I distinguish between different conformational states of SLC15A4?

Distinguishing between the outward-facing and inward-facing conformational states of SLC15A4 presents a technical challenge but is crucial for understanding its function in TASL recruitment and signaling:

• Structural analysis: Cryo-EM analyses have successfully resolved structures of human SLC15A4 in both the outward-facing apo state (both monomeric and dimeric forms) and the inward-facing TASL-bound state .

• Conformation-specific reagents: The development of conformation-specific antibodies or nanobodies that preferentially recognize specific SLC15A4 states would be valuable tools, though these are not yet widely available.

• Functional readouts: Since the conformational change is associated with TASL binding, assays measuring TASL recruitment can serve as indirect indicators of SLC15A4 conformational state.

• Mutational analysis: Introduction of mutations that stabilize specific conformations based on the resolved structures could provide experimental tools to study each state independently.

The transition between these conformational states represents a regulatory mechanism for SLC15A4 function in immune signaling pathways .

How should I interpret inconsistent results across different studies of SLC15A4 function?

The search results reveal some inconsistencies in reported SLC15A4 functions across studies, which may be explained by:

• Cell type differences: Divergent results between B cells and pDCs suggest cell type-specific roles for SLC15A4 .

• Experimental systems: Studies using primary cells versus cell lines (e.g., CAL-1 pDC line) may yield different results due to the complexity of signaling networks and compensatory mechanisms .

• Pathway specificity: SLC15A4 deficiency may affect some signaling pathways (e.g., IRF5 activation) but not others (MAPK, NF-κB) depending on the experimental context .

• Methodological differences: Variations in knockout strategies, stimulation conditions, and readout assays can contribute to apparently contradictory results.

• Redundancy with related transporters: Potential functional overlap between SLC15A4 and related transporters like SLC15A3 may complicate interpretation, though recent evidence suggests non-redundancy in their functions .

When interpreting SLC15A4 literature, researchers should carefully consider these factors and directly compare different cell types or model systems under identical experimental conditions where possible.

What are promising approaches for targeting SLC15A4 therapeutically?

Based on current understanding of SLC15A4 structure and function, several therapeutic targeting strategies show promise:

• Small molecule inhibitors: The recently solved cryo-EM structures of SLC15A4 provide a foundation for structure-guided drug discovery . Small molecules that stabilize the outward-facing conformation or occupy the TASL binding pocket could inhibit SLC15A4-mediated signaling.

• Peptide-based inhibitors: Design of peptides that mimic TASL's N-terminal region but lack signaling capability could competitively inhibit the SLC15A4-TASL interaction.

• Antibody-based approaches: Development of antibodies that specifically recognize and block functionally important domains of SLC15A4 could provide selective inhibition.

• RNA-based therapeutics: siRNA or antisense oligonucleotides targeting SLC15A4 expression could downregulate its levels in specific tissues.

These approaches are supported by the understanding that SLC15A4 is required for endosomal TLR recognition events that contribute to lupus-like autoimmunity .

What techniques are emerging for studying SLC15A4 localization and trafficking?

Advanced techniques for investigating SLC15A4 localization and trafficking include:

• Live-cell imaging: Fluorescently tagged SLC15A4 constructs allow real-time visualization of trafficking and localization in response to stimuli.

• Super-resolution microscopy: Techniques like STORM, PALM, or STED provide nanoscale resolution of SLC15A4 localization in relation to endolysosomal compartments.

• Correlative light and electron microscopy (CLEM): Combines fluorescence localization with ultrastructural context to precisely define SLC15A4's subcellular environment.

• Proximity labeling approaches: BioID or APEX2 fused to SLC15A4 can identify proximal proteins in different trafficking states.

• Organelle isolation techniques: Improved methods for isolating and analyzing endolysosomal compartments enable biochemical characterization of SLC15A4 in its native environment.

These approaches can help elucidate how SLC15A4 contributes to proper trafficking of TLRs and their ligands to endolysosomes , a process critical for immune signaling.