LHFPL5 Antibody

What is LHFPL5 and what is its role in cellular function?

LHFPL5 (LHFPL tetraspan subfamily member 5) is a protein encoded by the LHFPL5 gene in humans. It belongs to the lipoma HMGIC fusion partner (LHFP) gene family, which is a subset of the superfamily of tetraspan transmembrane protein encoding genes . LHFPL5 functions as a critical component of the hair cell's mechanotransduction machinery in the inner ear, where it functionally couples PCDH15 to the transduction channel . Research has demonstrated that LHFPL5 regulates transducer channel conductance and is required for fast channel adaptation . In hair cells, LHFPL5 is responsible for sensory transduction, and mutations in this gene have been associated with hearing loss in humans, particularly in Pakistani populations .

What is the structure of LHFPL5 and how does it relate to its function?

LHFPL5 is a 219 amino acid protein with four transmembrane domains . The protein shares structural homology with claudins, as evidenced by crystal structure analysis . The structure of LHFPL5 includes:

• Four transmembrane helices, with TM1 helices arranged in a V-shape formation when dimerized

• Extracellular β-strands forming a β-sheet structure

• A conserved disulfide bond between Cys68 on β3 and Cys79 on β4

• Another disulfide bond between Cys114 on TM2 and Cys130 on TM3

Structurally, LHFPL5 forms a complex with PCDH15, where two LHFPL5 protomers interact with one another via contacts mediated by TM1 helices, creating a V-shape architecture into which the two central helices of the PCDH15 dimer insert . This structural arrangement is critical for the protein's function in mechanotransduction.

What are the known protein interactions of LHFPL5?

LHFPL5 has been shown to interact with several key proteins in the mechanotransduction complex:

• PCDH15 (Protocadherin-15): LHFPL5 binds strongly to PCDH15, particularly through its N-terminal half, and this interaction is critical for the function of the mechanotransduction machinery .

• TMIE (Transmembrane Inner Ear): LHFPL5, especially its N-terminal half, binds significantly stronger to TMIE compared to its homolog LHFPL3 .

• TMC1 (Transmembrane Channel-like protein 1): LHFPL5 also interacts with TMC1, with this interaction requiring both the N-terminal and C-terminal halves of LHFPL5 for efficient binding .

These protein interactions form part of the sensory mechanotransduction machinery in stereocilia, with LHFPL5 serving as an important structural and functional component of this complex .

How does LHFPL5 contribute to mechanotransduction channel conductance?

LHFPL5 plays a critical role in establishing maximal force transmission to the mechanotransduction channel. In studies with Lhfpl5 −/− hair cells, researchers have observed significantly reduced mechanotransduction (MET) currents. Rescue experiments in outer hair cells (OHCs) demonstrate that only LHFPL5, and not its homologs (LHFPL2, LHFPL3, or LHFPL4), can effectively restore MET currents .

Specifically, when OHCs were injectoporated with LHFPL5, MET currents measured at 1 μm deflection reached 506.1 ± 41.8 pA, compared to only 89.2 ± 15.5 pA for non-transfected cells, and similarly low values for cells transfected with other LHFPL family members (50.7 ± 5.2 pA for LHFPL2, 66.0 ± 16.6 pA for LHFPL3, and 71.7 ± 20.8 pA for LHFPL4) . This demonstrates that LHFPL5 has a unique and specific role in regulating channel conductance that cannot be substituted by other family members.

What are the critical domains of LHFPL5 for protein interactions and function?

Research using chimeric constructs between LHFPL5 and LHFPL3 has identified specific domains critical for LHFPL5's interactions and function:

• N-terminal half: Essential for strong interactions with PCDH15 and TMIE. Chimeric proteins containing the N-terminal half of LHFPL5 (L5-L3) efficiently co-immunoprecipitated with PCDH15 and TMIE, while those with the N-terminal half of LHFPL3 (L3-L5) did not .

• C-terminal half: Important for efficient interactions with TMC1. Both N- and C-terminal parts of LHFPL5 are required for optimal TMC1 binding, as chimeric constructs showed reduced interaction compared to wild-type LHFPL5 .

• N-terminal cytoplasmic domain, TM1, and first extracellular loop: Further studies with more specific chimeras (L5-NcytoTM1Lp1 and L5-NcytoTM1) revealed that these domains are important for TMIE binding but are not sufficient for efficient PCDH15 interaction .

• Functional rescue capacity: The L5-NcytoTM1Lp1 chimera partially rescued MET currents (209.7 ± 35.6 pA at 1 μm deflection) compared to wild-type LHFPL5 (506.1 ± 41.8 pA), while L5-NcytoTM1 showed minimal rescue ability (103.2 ± 12.1 pA) .

What methodology should be used for studying LHFPL5-PCDH15 interactions?

To study LHFPL5-PCDH15 interactions, researchers have successfully employed the following methodological approaches:

• Co-immunoprecipitation: Expressing FLAG-tagged LHFPL5 (or variants) together with PCDH15 in HEK293 cells, followed by immunoprecipitation with anti-FLAG antibodies. The PCDH15-CD2 splice variant has been used as it is a component of tip links . Typically, cell extracts are prepared ~36 hours after transfection, and protein complexes are isolated and resolved on SDS-PAGE gels.

• Cryo-electron microscopy: This technique has been used to determine the structure of the PCDH15-LHFPL5 complex, revealing how the complex is composed of PCDH15 and LHFPL5 subunit pairs related by a 2-fold axis .

• Rigid-body fitting: Computational approaches using crystal structures of related proteins (such as claudin) have been employed to model the LHFPL5 structure and its interaction with PCDH15 .

• Cross-correlation analysis: Final refinement using phenix.real_space_refine with secondary structure restraints, resulting in a final cross correlation of 0.837 (0.875 for backbone atoms) .

What controls should be included in immunoprecipitation experiments with LHFPL5?

When conducting immunoprecipitation experiments with LHFPL5, researchers should include the following controls:

• Input analysis: Analyze the input amount of epitope-tagged proteins prior to immunoprecipitation to confirm expression levels .

• Total co-precipitating protein analysis: Assess the total amount of co-precipitating proteins to normalize pull-down efficiency .

• Negative controls: Include non-relevant proteins or empty vectors to control for non-specific binding.

• Homolog comparisons: Using related proteins such as LHFPL3 provides valuable comparative data on binding specificity, as demonstrated in studies comparing LHFPL5 with LHFPL3 .

• Chimeric constructs: Employing chimeric proteins (such as L5-L3 and L3-L5) can serve as controls while also providing insight into domain-specific functions .

For quantification of results, studies have successfully compared the relative binding efficiency of different LHFPL constructs to partners like PCDH15, TMIE, and TMC1 .

What considerations should researchers take when selecting anti-LHFPL5 antibodies?

When selecting anti-LHFPL5 antibodies for research, consider the following factors:

• Validated applications: Ensure the antibody has been validated for your specific application. For example, some antibodies are validated for Western blot (0.04-0.4 μg/mL), immunohistochemistry (1:50-1:200), and ELISA .

• Species reactivity: Verify that the antibody reacts with your species of interest. Available antibodies have been validated for human, mouse, and rat samples .

• Immunogen sequence: Check the immunogen sequence used to generate the antibody. For example, one commercial antibody uses the sequence "FVLGYRQDKLLPDDYKADGTEEV" , which may influence epitope recognition.

• Enhanced validation: Look for antibodies that have undergone enhanced validation, such as recombinant expression validation .

• Detected tissues: Consider which tissues the antibody has been tested in. For instance, some antibodies have demonstrated positive Western blot results in mouse and rat epididymis tissue .

• Storage and handling: Follow recommended storage conditions (-20°C) and shipping requirements (wet ice) to maintain antibody integrity .

What experimental approach is recommended for studying LHFPL5 function in hair cells?

For studying LHFPL5 function in hair cells, researchers have successfully employed a methodological approach combining:

• Injectoporation: OHCs are injectoporated at P3 to express LHFPL5 proteins along with GCaMP3 to identify transfected cells by GFP fluorescence .

• Patch clamp recording: MET currents are recorded after 1 day in vitro (DIV) by patching on the cell body of OHCs .

• Stereocilia deflection: Stepwise deflection of stereocilia from −400 to 1,000 nm using a stiff glass probe allows for measurement of MET currents in response to mechanical stimulation .

• Rescue experiments: Comparing MET currents between non-transfected cells and cells expressing wild-type or chimeric LHFPL5 constructs provides valuable insights into structure-function relationships .

This experimental approach has proven effective in demonstrating that LHFPL5, but not its homologs, can rescue MET currents in Lhfpl5−/− hair cells, and in identifying critical domains required for LHFPL5 function .

How should epitope tagging be considered when studying LHFPL5 function?

When designing experiments involving epitope-tagged LHFPL5, researchers should be aware that epitope tags can affect protein function. Research has shown that epitope tags slightly affected LHFPL5 function by reducing peak currents . Therefore:

• For immunolocalization studies: Epitope-tagged versions of LHFPL5 are suitable and have been successfully used to visualize protein localization in stereocilia .

• For functional analysis: Non-epitope-tagged versions are recommended, as rescue experiments with proteins lacking an epitope tag showed better preservation of function .

• For biochemical studies: FLAG-tagged LHFPL5 has been successfully used for co-immunoprecipitation experiments with PCDH15, TMIE, and TMC1 .