LGALS10 Human

What is the molecular structure of human LGALS10 and how does it differ from other galectins?

Human LGALS10 (Galectin-10) belongs to the prototype galectin subfamily but exhibits a novel dimerization structure that distinguishes it from other prototype galectins such as Galectin-1, -2, and -7. Crystallographic studies at 1.55–2.00 Å resolution reveal that LGALS10 forms a unique dimer with a distinctive global shape . In the LGALS10 dimer, Glu33 from one subunit modifies the carbohydrate-binding site of the other subunit, which essentially inhibits disaccharide binding . This structural arrangement is not observed in other prototype galectins and contributes to LGALS10's unique functional properties.

Human LGALS10 is 142 amino acids in length with one Galectin domain (amino acids 6-138) containing two dimerization motifs (amino acids 6-10 and 131-135) . Two molecular weight isoforms of 15 and 14 kDa have been described, though these variants have not been fully characterized . Importantly, unlike other widely conserved galectins, there is no known structural rodent counterpart to human LGALS10, which creates challenges for preclinical research .

Which cell types express LGALS10 and how can researchers detect its expression?

LGALS10 has a remarkably restricted expression pattern. It is predominantly and abundantly expressed in human eosinophils, where it is one of the most abundant cytoplasmic proteins . Besides eosinophils, LGALS10 is also expressed in basophils and CD25+ regulatory T cells (Tregs) . Some myeloid leukemia cells have also been found to express LGALS10, but otherwise, its expression is highly cell-type specific .

For detection, researchers can employ several methodologies:

• Western blot analysis: Using specific antibodies such as Goat Anti-Human Galectin-10 Antigen Affinity-purified Polyclonal Antibody, LGALS10 can be detected at approximately 16 kDa under reducing conditions .

• Flow cytometry: Intracellular staining of cells fixed and permeabilized with appropriate buffers allows detection of LGALS10 in specific cell populations. This is particularly useful for Treg analysis when combined with surface markers like CD25 .

• PCR detection: Quantitative real-time PCR can be performed using specific primers:

  • Forward: 5′-GCGACCACTTGCCTGTTTCT-3′

  • Reverse: 5′-CATGACCACACGACGACCA-3′

• Immunohistochemistry: LGALS10 can be detected in tissue sections using specific antibodies (typically at 1:100 dilution) with DAB staining and hematoxylin counterstaining .

What is the relationship between LGALS10 and Charcot-Leyden crystals?

LGALS10 forms Charcot-Leyden crystals (CLCs) in vivo, which are characteristic bipyramidal crystals observed in various eosinophilic diseases . These crystals have long been recognized as hallmarks of eosinophilic inflammation in tissues and body fluids. The formation of CLCs is linked to the release of LGALS10 through a specific cell death program called extracellular trap cell death (ETosis) .

During ETosis, eosinophils rapidly disintegrate their plasma membranes, releasing the majority of cytoplasmic LGALS10, which can subsequently form CLCs in the extracellular environment . Therefore, the presence of CLCs in tissue samples or body fluids indicates not only eosinophilic inflammation but specifically suggests active ETosis of eosinophils at the site of inflammation.

Current research indicates that LGALS10/CLCs are not merely passive markers of eosinophilic inflammation but may play functional roles in immunity and disease pathogenesis . This represents an important shift in understanding, as these structures were previously viewed as simply diagnostic indicators.

How does LGALS10 function as a prognostic biomarker in ovarian cancer and what mechanisms underlie this role?

The underlying mechanisms for LGALS10's favorable prognostic impact may relate to:

• Differential expression patterns: Studies have shown that LGALS10 mRNA and protein expression are decreased in ovarian cancer cells compared to normal ovarian cells (P<0.05) . This suggests that loss of LGALS10 may contribute to cancer development or progression.

• Subtype specificity: LGALS10 predicts a longer OS specifically in females with serous ovarian cancer across all stages, suggesting that its function may interact with the unique biology of this histological subtype .

• Immune modulation: Given LGALS10's expression in regulatory T cells and its role in eosinophil function, it may influence tumor immunity, potentially enhancing anti-tumor immune responses when present at higher levels.

Researchers investigating LGALS10 in ovarian cancer should consider multivariate analysis to determine if its prognostic value is independent of established clinical factors. Additionally, functional studies examining how LGALS10 affects ovarian cancer cell proliferation, migration, and immune evasion would provide mechanistic insights.

What are the current hypotheses regarding LGALS10's unique saccharide binding properties and how can they be experimentally tested?

LGALS10 exhibits atypical saccharide binding properties compared to other galectins. Structural and biochemical studies have revealed several unique features:

• Inhibition of disaccharide binding: In the LGALS10 dimer, Glu33 from one subunit modifies the carbohydrate-binding site of another, inhibiting disaccharide binding .

• Interaction with small hydroxylated molecules: Despite limited disaccharide binding, LGALS10 can interact with glycerol and potentially other small hydroxylated molecules. His53 appears to be crucial for this binding .

• Negative regulation by Trp72: Surprisingly, alanine substitution of the conserved Trp72 (which is crucial for saccharide binding in other galectins) actually enhances erythrocyte agglutination, suggesting that Trp72 negatively regulates LGALS10 ligand binding .

To experimentally test hypotheses regarding LGALS10's binding properties, researchers could:

• Site-directed mutagenesis: Create mutants targeting key residues (His53, Trp72, Glu33) to assess their impact on binding properties.

• Glycan array screening: Utilize glycan arrays to comprehensively profile LGALS10's binding specificity to various glycans and identify preferred ligands.

• Surface plasmon resonance (SPR): Determine binding kinetics and affinities for potential ligands, comparing wild-type LGALS10 with mutant variants.

• X-ray crystallography: Solve co-crystal structures with various ligands to visualize binding modes at atomic resolution.

• Cellular assays: Assess how mutations affect LGALS10's functional properties in cell-based systems, such as agglutination assays or cell adhesion studies.

These approaches would provide insights into the structural basis of LGALS10's unique binding properties and potentially identify physiologically relevant ligands.

How does extracellular trap cell death (ETosis) regulate LGALS10 release and what methodologies can detect this process?

The release of LGALS10 from eosinophils primarily occurs through extracellular trap cell death (ETosis) rather than conventional secretory processes like piecemeal degranulation or exocytosis . During ETosis, eosinophils rapidly disintegrate their plasma membranes, releasing the majority of cytoplasmic LGALS10 into the extracellular environment .

To study and detect ETosis-mediated LGALS10 release, researchers can employ several methodologies:

• Immunofluorescence microscopy: Visualization of extracellular DNA fibers (using DNA stains) co-localized with LGALS10 immunostaining provides direct evidence of ETosis. The characteristic web-like structures of extracellular traps containing LGALS10 can be quantified.

• Flow cytometry: Detection of cells undergoing ETosis through the combination of vital dyes (indicating membrane integrity loss), DNA stains, and LGALS10 antibodies.

• ELISA for soluble LGALS10: Quantification of LGALS10 levels in cell supernatants or biological fluids (serum, bronchoalveolar lavage fluid) as a surrogate marker for ETosis.

• Live cell imaging: Real-time visualization of the ETosis process using fluorescently labeled LGALS10 or antibodies against LGALS10.

• Electron microscopy: Ultrastructural analysis of eosinophils undergoing ETosis and LGALS10 release.

Understanding the regulatory mechanisms of ETosis-mediated LGALS10 release is crucial, as elevated LGALS10 levels in serum and tissue suggest a high degree of eosinophil ETosis, which may serve as a biomarker for disease activity and treatment effectiveness in various eosinophilic conditions .

What are the optimized protocols for detecting LGALS10 in different sample types?

Detecting LGALS10 requires different approaches depending on the sample type. Here are optimized protocols for various applications:

Western Blot Analysis for Cell/Tissue Lysates

• Sample preparation:

  • For cell lines (e.g., HL-60): Lyse cells in RIPA buffer with protease inhibitors

  • For tissue samples: Homogenize in tissue lysis buffer with protease inhibitors

• Electrophoresis conditions:

  • Use 12-15% SDS-PAGE gels

  • Load 20-30 μg protein per lane

  • Run under reducing conditions

• Transfer and detection:

  • Transfer to PVDF membrane

  • Block with 5% non-fat milk in TBST

  • Probe with anti-LGALS10 antibody (1 μg/mL)

  • Use appropriate HRP-conjugated secondary antibody

  • Develop using ECL detection system

• Expected result: A specific band at approximately 16 kDa

Flow Cytometry for Intracellular LGALS10

• Cell preparation:

  • Isolate cells (PBMCs, eosinophils, or cultured cells)

  • Surface stain for relevant markers (e.g., CD25 for Tregs)

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • Permeabilize with a suitable buffer (e.g., FlowX FoxP3/Transcription Factor Fixation & Perm Buffer)

• Staining:

  • Incubate with anti-LGALS10 antibody (optimal concentration determined empirically)

  • Add fluorophore-conjugated secondary antibody

  • Wash and analyze by flow cytometry

• Controls: Include isotype control antibody to assess non-specific binding

Immunohistochemistry for Tissue Sections

• Tissue processing:

  • Fix tissue in formalin and embed in paraffin

  • Section at 4-5 μm thickness

• Antigen retrieval:

  • Deparaffinize and rehydrate sections

  • Perform heat-induced epitope retrieval (citrate buffer pH 6.0)

• Staining procedure:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with serum

  • Incubate with anti-LGALS10 antibody (1:100 dilution) at 4°C overnight

  • Apply secondary antibody

  • Develop with DAB and counterstain with hematoxylin

• Scoring system: Semi-quantitative assessment based on staining intensity and percentage of positive cells

How can researchers quantify LGALS10 expression at mRNA and protein levels for comparative studies?

Accurate quantification of LGALS10 expression is essential for comparative studies. Here are methodological approaches for both mRNA and protein quantification:

mRNA Quantification

• Real-time quantitative PCR (RT-qPCR):

  • Extract total RNA using standard methods (TRIzol or column-based)

  • Synthesize cDNA using reverse transcriptase

  • Perform qPCR with LGALS10-specific primers:

    • Forward: 5′-GCGACCACTTGCCTGTTTCT-3′

    • Reverse: 5′-CATGACCACACGACGACCA-3′

  • Use GAPDH as reference gene:

    • Forward: 5′-GACTCATGACCACAGTCCATGC-3′

    • Reverse: 5′-CAGGTCAGGTCCACCACTGA-3′

  • Calculate relative expression using the 2^(-ΔΔCt) method

• Digital droplet PCR (ddPCR):

  • For absolute quantification without standard curves

  • Particularly useful for low-abundance transcript detection

  • Use the same primers as for RT-qPCR

• RNA-Seq:

  • For transcriptome-wide analysis with LGALS10 expression as part of the dataset

  • Allows identification of correlations with other genes and potential regulatory networks

Protein Quantification

• Western blot with densitometry:

  • Perform western blot as described in section 3.1.1

  • Use β-actin or GAPDH as loading control

  • Quantify band intensity using image analysis software

  • Express results as relative density normalized to loading control

• ELISA:

  • For quantification in serum, plasma, or cell culture supernatants

  • Either commercial kits or laboratory-developed assays

  • Standard curve using recombinant LGALS10 for absolute quantification

• Mass spectrometry-based proteomics:

  • For global proteome analysis with LGALS10 quantification

  • Label-free or isotope-labeled approaches

  • Provides absolute quantification and identification of post-translational modifications

Comparative Analysis Guidelines

• Standardization: Use the same methodology across all samples being compared

• Technical replicates: Minimum of three replicates to assess variability

• Biological replicates: Analyze multiple independent samples

• Statistical analysis: Appropriate tests (t-test, ANOVA) with multiple testing correction

• Validation: Confirm key findings using an alternative method

Cellular Models

• Human eosinophilic cell lines:

  • HL-60 cells differentiated toward eosinophilic lineage

  • AML14.3D10 cells (eosinophilic cell line)

  • Primary human eosinophils isolated from peripheral blood

• LGALS10 overexpression/knockdown systems:

  • Lentiviral transduction for stable expression in various cell types

  • siRNA or CRISPR-Cas9 for gene silencing in cells that naturally express LGALS10

  • Inducible expression systems for temporal control

• 3D culture systems:

  • Organoids from tissues affected by eosinophilic diseases

  • Co-culture systems with eosinophils and target tissue cells

Ex Vivo Models

• Human tissue explants:

  • Short-term culture of tissue biopsies from patients with eosinophilic diseases

  • Treatment with recombinant LGALS10 or neutralizing antibodies

• Precision-cut tissue slices:

  • Maintain 3D architecture and cellular diversity

  • Useful for studying LGALS10 in tissue context

In Vivo Approaches

• Humanized mouse models:

  • Mice engrafted with human immune cells including eosinophils

  • Allows study of human LGALS10 in a living organism

• Transgenic expression models:

  • Generate mice expressing human LGALS10 under tissue-specific promoters

  • Study effects of human LGALS10 in specific compartments

• Non-human primates:

  • Potential for studying LGALS10 in species with closer homology to humans

  • Limited by ethical and practical considerations

Computational Approaches

• Structure-based functional prediction:

  • Use solved crystal structures to predict functional interactions

  • Molecular dynamics simulations to study protein behavior

• Systems biology:

  • Integrate -omics data to predict LGALS10 functions in different contexts

  • Network analysis to identify functional associations

Each model system has advantages and limitations, and researchers should select the appropriate model based on their specific research questions and available resources.

How can LGALS10 be utilized as a biomarker for eosinophilic diseases, and what are the optimal detection methods in clinical samples?

LGALS10 has emerged as a promising biomarker for various eosinophilic diseases, including asthma, eosinophilic esophagitis, rhinitis, sinusitis, atopic dermatitis, and eosinophilic granulomatosis with polyangiitis . Its utility spans disease activity assessment, diagnosis, and monitoring treatment effectiveness.

Clinical Applications of LGALS10 as a Biomarker

Optimal Detection Methods for Clinical Samples

• Serum/Plasma LGALS10:

  • ELISA-based quantification

  • Reference ranges need to be established for different patient populations

  • Sample handling critical: immediate processing or storage at -80°C

• Tissue LGALS10 Detection:

  • Immunohistochemistry for formalin-fixed paraffin-embedded (FFPE) tissue

  • Dual staining with eosinophil markers (e.g., major basic protein)

  • Digital pathology for quantitative assessment

• Charcot-Leyden Crystal Detection:

  • Microscopic evaluation of respiratory samples (sputum, nasal lavage)

  • Wright-Giemsa or H&E staining

  • Confirmation with LGALS10 immunostaining

• Non-invasive Sampling:

  • Induced sputum for respiratory diseases

  • Nasal lavage fluid for upper airway disorders

  • Tears for ocular allergic disorders

Analytical Considerations for Clinical Implementation

• Pre-analytical factors:

  • Standardize collection procedures

  • Define acceptable time intervals between collection and processing

  • Establish stability data for different storage conditions

• Analytical validation:

  • Precision (intra- and inter-assay variability <10%)

  • Accuracy (recovery 90-110%)

  • Linearity within the clinical measurement range

  • Detection limits appropriate for disease states

• Clinical validation:

  • Establish reference intervals in healthy individuals

  • Determine decision thresholds for specific clinical applications

  • Assess confounding factors (medications, comorbidities)

What is the prognostic significance of LGALS10 expression in various cancers and how does it compare to established biomarkers?

Research has begun to elucidate the prognostic significance of LGALS10 in various cancers, with the most substantive evidence in ovarian cancer. Understanding its performance relative to established biomarkers is essential for potential clinical implementation.

LGALS10 as a Prognostic Marker in Ovarian Cancer

*Exact hazard ratios not provided in the source material

Methodological Approaches for Prognostic Studies

• Retrospective cohort analysis:

  • Tissue microarray analysis of archival samples

  • Correlation with long-term clinical outcomes

  • Multivariate regression models adjusting for clinicopathological factors

• Prospective validation:

  • Include LGALS10 measurement in clinical trials

  • Pre-specified analysis of prognostic value

  • Time-dependent receiver operating characteristic (ROC) analysis

• Integration with molecular profiling:

  • Combine with genomic signatures

  • Pathway analysis to understand biological context

  • Machine learning approaches for integrated biomarker models

What technical challenges exist in standardizing LGALS10 detection for clinical applications?

The translation of LGALS10 detection from research to clinical applications faces several technical challenges that must be addressed to ensure reliability and reproducibility:

Pre-analytical Variables

• Sample collection and processing:

  • Blood collection tubes (EDTA vs. heparin vs. serum)

  • Processing delays affect LGALS10 stability

  • Cell lysis during processing may release intracellular LGALS10

• Tissue handling:

  • Fixation protocols affect epitope availability

  • Cold ischemia time impacts protein integrity

  • Decalcification procedures for bone marrow samples

Analytical Standardization

• Antibody variability:

  • Different commercial antibodies show variable specificity and sensitivity

  • Lot-to-lot variability affects quantitative measurements

  • Need for reference standards and calibrators

• Assay platforms:

  • ELISA vs. multiplex vs. automated immunoassay platforms

  • Need for platform-specific reference ranges

  • Inter-laboratory harmonization

• Cut-off determination:

  • Disease-specific thresholds required

  • Population-specific reference ranges

  • Statistical approaches for optimal cut-point selection

Post-analytical Considerations

• Result interpretation:

  • Context-specific interpretation (disease type, treatment status)

  • Integration with other laboratory and clinical findings

  • Longitudinal monitoring protocols

• Quality assurance:

  • Internal quality control materials

  • External quality assessment programs

  • Proficiency testing

Proposed Solutions for Standardization

• Reference materials development:

  • Purified recombinant LGALS10 as primary calibrator

  • Stabilized cell lysates as secondary standards

  • Certified reference materials through international collaboration

• Assay standardization initiatives:

  • International working groups for method harmonization

  • Standard operating procedures for pre-analytical variables

  • Clinical Laboratory Standards Institute (CLSI) guidelines development

• Digital pathology approaches:

  • Automated image analysis algorithms for tissue LGALS10 quantification

  • Machine learning for standardized interpretation

  • Virtual microscopy for centralized expert review

What unexplored therapeutic applications might exist for targeting LGALS10 in inflammatory and malignant conditions?

Given the emerging understanding of LGALS10's functions, several potential therapeutic applications warrant exploration:

Targeting LGALS10 in Eosinophilic Diseases

• Anti-LGALS10 antibodies:

  • Neutralization of extracellular LGALS10

  • Prevention of Charcot-Leyden crystal formation

  • Disruption of eosinophil-epithelial interactions

• Small molecule inhibitors:

  • Target the unique LGALS10 carbohydrate-binding site

  • Exploit the distinct Glu33-mediated binding inhibition

  • Design based on His53 interaction with small hydroxylated molecules

• ETosis modulation:

  • Inhibit pathways leading to LGALS10 release via ETosis

  • Targeted degradation of extracellular LGALS10

  • Enhancement of Charcot-Leyden crystal resolution

Cancer Therapeutic Approaches

• LGALS10 induction therapy:

  • Based on favorable prognostic impact in ovarian cancer

  • Epigenetic modifiers to restore LGALS10 expression

  • Differentiation therapy to enhance LGALS10 in cancer cells

• Immune modulation:

  • Targeting LGALS10 in regulatory T cells

  • Enhancing anti-tumor immunity

  • Combination with immune checkpoint inhibitors

• Biomarker-guided therapy:

  • LGALS10 expression as predictor of response

  • Treatment stratification based on LGALS10 levels

  • Monitoring therapy effectiveness through LGALS10 dynamics

Novel Delivery Approaches

• Targeted nanoparticles:

  • Eosinophil-targeted delivery of LGALS10 modulators

  • Tumor-specific delivery systems

  • Inhalational formulations for respiratory applications

• Gene therapy:

  • CRISPR-based modulation of LGALS10 expression

  • AAV-mediated delivery to specific tissues

  • mRNA therapeutics for transient LGALS10 modulation

How might advanced genomic and proteomic approaches enhance our understanding of LGALS10 regulation and function?

Advanced -omics technologies offer powerful approaches to deepen our understanding of LGALS10:

Genomic Approaches

• Single-cell RNA sequencing:

  • Cell-specific expression patterns in health and disease

  • Identification of previously unknown LGALS10-expressing cell populations

  • Trajectory analysis for developmental regulation

• Epigenetic profiling:

  • DNA methylation analysis of the LGALS10 promoter

  • Histone modification patterns affecting expression

  • Chromatin accessibility studies (ATAC-seq)

• Genome-wide association studies (GWAS):

  • Identification of genetic variants influencing LGALS10 expression

  • Population-specific differences in regulation

  • Disease associations with LGALS10 polymorphisms

Proteomic Approaches

• Interactome analysis:

  • Proximity labeling techniques (BioID, APEX) to identify LGALS10 protein partners

  • Affinity purification-mass spectrometry

  • Protein-protein interaction networks in different cellular contexts

• Post-translational modifications:

  • Comprehensive PTM mapping (phosphorylation, glycosylation, etc.)

  • Functional consequences of modifications

  • PTM dynamics during cellular activation and disease states

• Spatial proteomics:

  • Subcellular localization in different cell types

  • Translocation during activation and ETosis

  • Imaging mass cytometry for tissue context

Integrative Multi-omics

• Data integration approaches:

  • Combined genomic, transcriptomic, and proteomic analysis

  • Network biology to identify regulatory hubs

  • Causal inference methods for mechanistic insights

• Temporal dynamics:

  • Time-course experiments during cell activation

  • Disease progression analysis

  • Treatment response monitoring

• Computational modeling:

  • Machine learning for pattern recognition

  • Pathway enrichment analysis

  • Systems biology approaches to predict functional impacts

What structural biology techniques could further elucidate the unique properties of LGALS10 and inform drug design?

Despite important insights from the crystal structure of LGALS10, several structural biology techniques could provide additional critical information:

Advanced Structural Approaches

• Cryo-electron microscopy (Cryo-EM):

  • Visualization of LGALS10 in different conformational states

  • LGALS10 interactions with binding partners

  • Structure of Charcot-Leyden crystals at near-atomic resolution

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Solution structure dynamics

  • Ligand binding studies

  • Protein-protein interaction interfaces

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Conformational dynamics in solution

  • Effects of ligand binding on protein flexibility

  • Allosteric regulation mechanisms

Computational Structural Biology

• Molecular dynamics simulations:

  • Conformational sampling of LGALS10

  • Binding pocket dynamics

  • Effects of mutations on structure and function

• Virtual screening and docking:

  • Identification of novel LGALS10 binders

  • Structure-based drug design

  • Exploitation of unique binding properties

• Quantum mechanics/molecular mechanics (QM/MM):

  • Detailed analysis of binding site interactions

  • Reaction mechanisms

  • Electronic properties relevant to function

Structural Biology of LGALS10 Complexes

• Co-crystallization studies:

  • LGALS10 with physiological ligands

  • Complexes with binding partners

  • Drug candidates targeting LGALS10

• Small-angle X-ray scattering (SAXS):

  • Low-resolution shape of LGALS10 complexes in solution

  • Oligomerization states

  • Conformational ensembles

• Cross-linking mass spectrometry:

  • Mapping interaction surfaces

  • Validation of computational models

  • Analysis of multi-protein complexes