lysmd3 Antibody

What is LYSMD3 and what is its biological significance?

LYSMD3 (LysM Domain Containing 3) is a type II membrane protein that functions as a pattern recognition receptor (PRR) for chitin and β-glucan. It is primarily expressed on the surface of human airway epithelial cells and plays a crucial role in recognizing fungal cell wall components . The biological significance of LYSMD3 stems from its involvement in innate immune responses, particularly against fungal pathogens. LYSMD3 mediates the production of inflammatory cytokines such as IL-6 and IL-8 in response to chitin and fungal spores, potentially linking it to allergic disorders like asthma .

What applications are LYSMD3 antibodies commonly used for in research?

LYSMD3 antibodies are utilized in several research applications:

• Western blotting (WB) to detect LYSMD3 expression levels (recommended dilutions: 1:1000-1:5000)

• Immunohistochemistry (IHC) to visualize protein localization in tissues (recommended dilutions: 1:50-1:500)

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

• Co-immunoprecipitation to study protein-protein interactions

• Immunofluorescence to determine subcellular localization

The selection of specific antibodies depends on the experimental requirements, with polyclonal antibodies offering broader epitope recognition compared to monoclonal variants .

What is the expected molecular weight of LYSMD3 in Western blot experiments?

The calculated molecular weight of LYSMD3 is 35 kDa (based on its 306 amino acids) . Consistent with this calculation, experimental observations in Western blot applications typically show a band at approximately 35 kDa . When planning experiments, it's important to include appropriate positive controls (such as Neuro-2a cells or human liver tissue) that have been validated to express LYSMD3 .

What are the optimal sample preparation methods for LYSMD3 detection in different cell types?

For optimal LYSMD3 detection across different cell types:

Epithelial cells (BEAS-2B, A549, primary human bronchial epithelial cells):

• Harvest cells at 80-90% confluence

• Wash cells twice with ice-cold PBS

• For Western blot: Lyse cells directly in RIPA buffer supplemented with protease inhibitors

• For membrane protein enrichment: Use biochemical fractionation with differential centrifugation to separate membrane compartments from cytosolic fractions

• For cell surface LYSMD3: Biotinylate cell-surface proteins prior to lysis and purify with streptavidin beads

Immune cells (macrophages):

• For mouse macrophages, collect cells after appropriate stimulation (e.g., chitin exposure)

• Process similar to epithelial cells with special attention to timing post-stimulation

Regardless of cell type, protein samples should be denatured at 95°C for 5 minutes in Laemmli buffer containing 2-mercaptoethanol before gel loading .

How should immunohistochemistry protocols be optimized for LYSMD3 detection in tissue sections?

For optimal LYSMD3 detection in tissue sections:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

• Antigen retrieval:

  • Preferred method: Heat-induced epitope retrieval with TE buffer pH 9.0

  • Alternative: Citrate buffer (pH 6.0)

  • Heat at 95-100°C for 15-20 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum (matched to secondary antibody host)

  • Apply primary LYSMD3 antibody (1:50-1:500 dilution) and incubate overnight at 4°C

  • Use validated positive control tissues (e.g., human liver)

• Detection and visualization:

  • Apply appropriate HRP-conjugated secondary antibody

  • Develop signal with DAB substrate

  • Counterstain with hematoxylin

  • Mount with permanent mounting medium

For challenging samples, optimize by testing different antibody concentrations and antigen retrieval conditions .

How can LYSMD3 antibodies be used to study the interaction between LYSMD3 and fungal cell wall components?

To investigate LYSMD3-fungal component interactions:

Co-localization studies:

• Culture epithelial cells on glass coverslips

• Expose cells to fluorescently labeled fungi (e.g., Candida albicans) or purified chitin

• Fix cells and immunostain for LYSMD3 using validated antibodies

• Analyze using confocal microscopy to assess LYSMD3 accumulation at sites of fungal contact

Pull-down assays:

• Prepare recombinant LYSMD3 ectodomain (amino acids 1-127)

• Incubate with chitin magnetic beads in appropriate binding buffer (500 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, 0.1% Tween-20, pH 8.0)

• Separate bound and unbound fractions using a magnet

• Analyze fractions by SDS-PAGE and Western blot with anti-LYSMD3 antibody

Binding ELISA:

• Coat plates with chitin oligosaccharides, β-glucan preparations (curdlan, laminarin), or control substrates

• Block plates and add recombinant LYSMD3 at increasing concentrations

• Detect bound LYSMD3 using anti-LYSMD3 antibody and appropriate detection system

• Quantify binding by measuring optical density at 450 nm

These methods can be complemented with competitive inhibition assays using soluble ligands or anti-LYSMD3 antibodies to validate specificity .

What approaches can be used to validate LYSMD3 antibody specificity for research applications?

To ensure LYSMD3 antibody specificity:

Genetic validation:

• Generate LYSMD3 knockout cell lines using CRISPR-Cas9 (as demonstrated with sgRNAs targeting LYSMD3)

• Alternatively, use siRNA knockdown to reduce LYSMD3 expression

• Compare antibody signal between wildtype and knockout/knockdown samples

• Absence of signal in knockout cells confirms specificity

Peptide competition:

• Pre-incubate LYSMD3 antibody with excess immunizing peptide/recombinant protein

• In parallel, use untreated antibody as control

• Apply both antibody preparations to identical samples

• Specific signal should be blocked by peptide competition

Cross-reactivity assessment:

• Test antibody against recombinant proteins from all LYSMD family members (LYSMD1, LYSMD2, LYSMD3, LYSMD4)

• Perform Western blots on cells with confirmed expression of different LYSMD proteins

• Confirm signal specificity for LYSMD3 versus other family members

Immunoprecipitation-mass spectrometry:

• Perform immunoprecipitation with LYSMD3 antibody

• Analyze precipitated proteins by mass spectrometry

• Confirm LYSMD3 as the predominant protein identified

This comprehensive validation ensures reliable results in subsequent experiments .

How should experiments be designed to study LYSMD3's role in innate immune responses?

For investigating LYSMD3's immune function:

Cellular models:

• Human bronchial epithelial cells (BEAS-2B)

• Alveolar type II epithelial cells (A549)

• Primary human bronchial epithelial cells (NHBE)

• IL-33-producing human bronchial epithelial cells (HBE33)

• Mouse macrophages for comparative studies

Experimental approaches:

• Loss-of-function studies:

  • siRNA knockdown (multiple non-overlapping siRNAs recommended)

  • CRISPR-Cas9 knockout (using two different sgRNAs targeting LYSMD3)

  • Validate knockdown/knockout by Western blot with anti-LYSMD3 antibody

• Stimulation protocols:

  • Chitin oligosaccharides (DP7) at 10-50 μg/ml

  • Chitin particles (1-5 μg/ml)

  • Alternaria spores or extract

  • Control stimuli: TLR3 ligand (Poly I:C), TLR5 ligand (Flagellin), peptidoglycan

• Functional readouts:

  • Cytokine production (IL-6, IL-8) by ELISA

  • IL-33 release from HBE33 cells

  • Cell viability monitoring (MTT assay, LDH release)

• Complementation:

  • Rescue experiments with recombinant LYSMD3

Include both positive controls (known PRR ligands) and negative controls (unrelated stimuli) to confirm specificity of LYSMD3-mediated responses .

What are common challenges when using LYSMD3 antibodies and how can they be addressed?

Common challenges and solutions:

When encountering discrepancies between studies (such as Golgi vs. plasma membrane localization) , employ complementary approaches like cell fractionation, surface biotinylation, and immunofluorescence with markers for different cellular compartments .

How can researchers distinguish between human and mouse LYSMD3 in comparative studies?

For species-specific LYSMD3 detection:

• Sequence comparison:

  • Analyze amino acid sequence conservation between human and mouse LYSMD3

  • Identify regions of divergence that might affect antibody recognition

• Antibody selection:

  • Choose antibodies raised against species-specific epitopes

  • Verify cross-reactivity claims with experimental validation

  • For polyclonal antibodies, confirm reactivity through Western blotting of samples from both species

• Experimental validation:

  • Test antibody specificity against recombinant human and mouse LYSMD3

  • Include appropriate positive controls from both species

  • For Western blots, look for slight differences in migration pattern due to species differences

• Species-specific assays:

  • Design primers that amplify species-specific regions for qRT-PCR validation

  • Use species-matched cell lines as controls (human BEAS-2B vs. mouse embryonic fibroblasts)

• Complementary approaches:

  • Use epitope-tagged constructs for overexpression studies

  • Consider species-specific CRISPR knockouts as negative controls

This approach ensures reliable species discrimination in comparative studies of LYSMD3 function .

How can researchers reconcile conflicting findings about LYSMD3 subcellular localization?

The literature contains apparent contradictions regarding LYSMD3 localization. He et al. (2021) demonstrated LYSMD3 expression on the plasma membrane of epithelial cells , while other studies reported Golgi localization . To resolve these discrepancies:

Integrated experimental approach:

• Fractionation studies:

  • Perform careful biochemical fractionation to separate plasma membrane, Golgi, and other compartments

  • Use established markers for each compartment (Na⁺/K⁺-ATPase for plasma membrane, GM130 for Golgi)

  • Analyze LYSMD3 distribution by Western blot across fractions

• Cell surface biotinylation:

  • Biotinylate cell surface proteins using membrane-impermeable reagents

  • Purify biotinylated proteins with streptavidin

  • Analyze for LYSMD3 presence by Western blot

• Microscopy with multiple markers:

  • Perform co-localization studies with plasma membrane and Golgi markers

  • Use super-resolution microscopy for improved spatial resolution

  • Analyze under both basal and stimulated conditions (e.g., fungal exposure)

• Dynamic trafficking studies:

  • Track LYSMD3 movement using fluorescently tagged constructs

  • Investigate whether stimulation with chitin or fungi alters localization

  • Consider that LYSMD3 might shuttle between compartments

• Context-specific expression:

  • Examine whether localization differs by cell type or activation state

  • Consider that LYSMD3 might reside primarily in the Golgi but translocate to the surface upon stimulation

This comprehensive approach recognizes that both localizations may be correct under different conditions or represent different pools of the protein .

How can LYSMD3 antibodies be used to investigate contradictions between studies on LYSMD3's role in immune response?

There are contrasting findings about LYSMD3's immune functions. While He et al. (2021) demonstrated its role in chitin recognition and inflammatory responses , Liu et al. (2018) found no evidence for LYSMD3's role during mammalian immune response in their models . To address these contradictions:

Evidence reconciliation strategy:

• Model-specific investigation:

  • Compare immune responses in different cell types using the same LYSMD3 antibodies

  • Use antibodies to track LYSMD3 expression in different tissues and under various stimulation conditions

  • Assess LYSMD3 expression in tissues relevant to each study (lung epithelium vs. other tissues)

• Stimulus-specific responses:

  • Use LYSMD3 antibodies to monitor protein levels after exposure to:

    • Chitin oligosaccharides and particles

    • Fungal pathogens (Candida, Alternaria)

    • Bacterial components

    • Other PRR ligands

  • Determine if LYSMD3 expression or localization changes with specific stimuli

• Signaling pathway analysis:

  • Combine LYSMD3 antibodies with phospho-specific antibodies to track activation of downstream pathways

  • Compare signaling cascades under conditions used in different studies

  • Identify potential stimulus-specific or context-dependent signaling

• Genetic background effects:

  • Use LYSMD3 antibodies to compare expression levels across different mouse strains or human donors

  • Investigate potential compensatory mechanisms in knockout models

  • Examine expression of other LYSMD family members in knockout contexts

• Temporal dynamics:

  • Track LYSMD3 expression and localization over time after stimulation

  • Determine optimal timepoints for different readouts and stimuli

This systematic approach may reconcile apparently contradictory findings by identifying context-specific roles for LYSMD3 in immune responses .

How can LYSMD3 antibodies be utilized to explore the relationship between LYSMD3 and allergic disorders such as asthma?

Given LYSMD3's role in sensing chitin (a common allergen component) and its expression in airway epithelium, researchers can use LYSMD3 antibodies to explore its connection to allergic disorders through:

• Clinical sample analysis:

  • Compare LYSMD3 expression levels in bronchial biopsies from asthmatic vs. healthy subjects

  • Correlate expression with disease severity and fungal sensitization

  • Examine LYSMD3 localization in airway epithelium from patients with allergic diseases

• Mechanistic studies:

  • Investigate LYSMD3 interactions with other allergic pathway components

  • Track LYSMD3-dependent release of epithelial alarmins (IL-33) using antibody-based blocking

  • Analyze LYSMD3-dependent gene expression changes in response to allergens

• Mouse models of allergic inflammation:

  • Use antibodies to track LYSMD3 expression in experimental asthma models

  • Correlate LYSMD3 levels with inflammation severity

  • Compare LYSMD3 knockout mice responses to allergen challenges

• Therapeutic targeting assessment:

  • Develop and test blocking antibodies against LYSMD3

  • Evaluate their potential to reduce fungal-induced allergic responses

  • Monitor LYSMD3 engagement and downstream effects

This research could establish LYSMD3 as a clinically relevant target for allergic diseases and reveal new intervention strategies .

What is the relationship between LYSMD3 and other LYSMD family members, and how can antibody-based approaches help distinguish their functions?

The LYSMD protein family includes four members (LYSMD1-4) with conserved LysM domains but potentially distinct functions. Antibody-based approaches can help distinguish their roles:

• Expression pattern comparison:

  • Use specific antibodies against each LYSMD family member

  • Compare tissue distribution and subcellular localization

  • Correlate expression with functional specialization

• Functional redundancy assessment:

  • In LYSMD3 knockout systems, monitor other family members' expression

  • Investigate compensatory upregulation

  • Use antibodies to pull down protein complexes and identify shared binding partners

• Domain-specific functions:

  • Generate antibodies targeting specific domains of LYSMD proteins

  • Use domain-blocking antibodies to dissect functional contributions

  • Compare binding properties of LysM domains across family members

• Evolutionary conservation analysis:

  • Apply antibodies recognizing conserved epitopes across species

  • Track evolutionary relationships between LYSMD proteins

  • Compare functions from invertebrates to mammals

• Interactome mapping:

  • Use antibodies for immunoprecipitation coupled with mass spectrometry

  • Identify unique and shared interaction partners

  • Create comprehensive protein-protein interaction networks

Recent research indicates that different LYSMD proteins may perform specialized functions, with LYSMD1/2 promoting activation of Rab32-family GTPases for lysosome-related organelle biogenesis , while LYSMD3 functions as a pattern recognition receptor . Understanding these functional distinctions could reveal specialized roles for each family member .