LGALS3 Human, His

What is the molecular structure and functional domains of LGALS3?

LGALS3 is encoded by a gene located on chromosome 14 (locus q21-q22) and is the only chimeric galectin containing a single carbohydrate recognition domain (CRD) linked to a non-lectin domain . The protein consists of approximately 250 amino acids, with the C-terminal region (residues 170-250) containing the CRD responsible for beta-galactoside binding . The N-terminal domain mediates protein-protein interactions and oligomerization. The CRD confers the protein's hemagglutination activity through bivalent carbohydrate binding capabilities .

What key protein interactions have been identified for LGALS3?

LGALS3 interacts with multiple binding partners that affect its biological functions. Key interactions include:

• BARD1: LGALS3 interacts with BARD1's tBRCT domain, as verified through yeast-two-hybrid screens and co-immunoprecipitation assays using HeLa nuclear extracts .

• BRCA1: LGALS3 has been found in complexes with both BARD1 and BRCA1, suggesting its involvement in DNA damage response pathways. Notably, a mono-ubiquitinated form of LGALS3 (Ub-GAL3) is predominantly associated with these complexes .

• Cell surface glycoproteins: Due to its carbohydrate-binding properties, LGALS3 can bind to various cell surface glycoproteins, with each galectin family member preferentially binding to unique subsets of these proteins .

How does LGALS3 participate in DNA damage response pathways?

LGALS3 plays a significant role in early events of DNA damage response (DDR), though its precise mechanism differs from canonical DDR proteins. Experimental evidence shows that:

• LGALS3-silenced cells exhibit increased resistance to various DNA damaging agents including ionizing radiation, etoposide, carboplatin, and mitomycin C .

• Cells lacking LGALS3 show delayed γH2AX foci formation (a marker of DNA damage) after ionizing radiation exposure compared to control cells. Control cells exhibited detectable foci 15 minutes after exposure, while LGALS3-silenced cells only showed foci after 30 minutes .

• While LGALS3 affects early DDR events, it does not significantly alter ATM Ser1981 phosphorylation or CHK2 Thr68 phosphorylation following DNA damage .

• LGALS3-silenced cells demonstrate impaired G2/M cell cycle arrest following ionizing radiation, with a 2-fold higher percentage of mitotic cells compared to control cells, indicating compromised checkpoint functionality .

What methodologies are optimal for studying LGALS3 protein-protein interactions?

Multiple complementary techniques have proven effective for investigating LGALS3 interactions:

• Yeast-two-hybrid screening: Successfully used to identify the interaction between LGALS3 (residues 170-250) and BARD1's tBRCT domain .

• Co-immunoprecipitation (Co-IP): Effective for validating interactions in mammalian cells. Both forward and reverse immunoprecipitations using anti-LGALS3, anti-BARD1, and anti-BRCA1 antibodies have confirmed these interactions in nuclear extracts .

• Tandem affinity purification coupled with mass spectrometry (TAP-MS): This method provides a comprehensive approach to identify LGALS3 interaction partners:

  • Express tagged LGALS3 in HEK293FT cells (pNTAP-GAL3)

  • Lyse cells using NETN buffer (Nonidet P40 0.5% v/v, Tris pH8.0 20 mM, NaCl 50 mM, NaF 50 mM, Na3VO4 100 mM, DTT 1 mM, PMSF 50 μg/mL)

  • Perform tandem affinity purification

  • Resolve purified proteins by SDS-PAGE and stain with Coomassie blue

  • Excise bands, perform in-gel trypsin digestion

  • Analyze digested fragments by nano flow liquid chromatography coupled with mass spectrometry

  • Identify proteins using appropriate software (e.g., Scaffold v.3.2.0, Mascot v.2.2.04)

What are the experimental strategies for studying LGALS3 in cellular stress responses?

To investigate LGALS3's role in cellular stress response, researchers typically employ:

• Gene silencing approaches: Lentiviral-mediated shRNA delivery has been effectively used to silence LGALS3 expression in cell lines:

  • Produce lentiviral particles containing pLKO.1 plasmids encoding shRNAs targeting LGALS3

  • Transduce target cells and select stable cell lines using puromycin

  • Verify knockdown efficiency via western blot analysis

• Cell viability assays following stress induction:

  • Plate cells in 96-well plates (1×10^6 cells/well)

  • Allow attachment for 24 hours

  • Apply stress conditions: irradiation (10-40 Gy) or chemotherapeutic agents (carboplatin: 50-500 μM; etoposide: 10-200 nM; mitomycin C: 50-100 nM)

  • Assess viability using MTT assay (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) after appropriate recovery periods (48-96 hours)

• DNA damage markers analysis:

  • Expose cells to ionizing radiation or DNA-damaging agents

  • Analyze phosphorylation status of ATM (Ser1981), H2AX (Ser139), CHK2 (Thr68) via western blotting or immunofluorescence

  • Quantify γH2AX foci formation at various time points (15 min, 30 min, 1 hr, etc.)

What are the purification strategies for obtaining high-quality recombinant LGALS3-His protein?

Recombinant LGALS3-His protein can be efficiently purified using the following approach:

• Expression system selection: E. coli is commonly used as an expression host for LGALS3-His production due to high yield and relatively straightforward purification procedures .

• Affinity chromatography: The His-tag enables single-step purification using immobilized metal affinity chromatography (IMAC):

  • Lyse bacteria in appropriate buffer containing imidazole (10-20 mM)

  • Load clarified lysate onto Ni-NTA or similar IMAC resin

  • Wash extensively to remove non-specifically bound proteins

  • Elute with increasing imidazole concentrations (250-500 mM)

• Additional purification steps:

  • Size exclusion chromatography to remove aggregates and ensure monomeric protein

  • Endotoxin removal if the protein will be used in cell-based or in vivo experiments

  • Buffer exchange into a physiologically relevant buffer (PBS or similar)

• Quality control assessments:

  • SDS-PAGE to confirm purity

  • Western blot to verify identity

  • Activity assays to confirm carbohydrate binding functionality

  • Endotoxin testing if required for downstream applications

How can LGALS3's role in cardiovascular pathologies be effectively studied?

LGALS3 has been implicated in heart failure and myocardial fibrosis. Research approaches include:

• Genetic association studies:

  • Analyze single-nucleotide polymorphisms (SNPs) in the LGALS3 gene, particularly at positions rs4644 and rs4652, in patients with acute heart failure

  • Correlate genotypes with clinical outcomes and plasma LGALS3 levels

• Biomarker analysis:

  • Measure plasma LGALS3 levels using validated ELISA methods

  • Correlate LGALS3 levels with:

    • Degree of myocardial fibrosis (determined by imaging or biopsy)

    • Clinical outcomes such as major adverse events (MAEs)

    • Disease progression markers

• Histological assessment:

  • Quantify myocardial fibrosis in tissue samples

  • Perform immunohistochemistry to visualize LGALS3 expression and localization

  • Correlate LGALS3 expression with fibrosis severity

Research has demonstrated that plasma LGALS3 levels correlate significantly with the degree of myocardial fibrosis (p < 0.001) and can predict increased risk of major adverse events in acute heart failure patients (p < 0.001) .

How can inconsistent results in LGALS3 localization studies be resolved?

Inconsistencies in LGALS3 localization studies may arise from several factors:

• Cell type-specific expression patterns: Different cell types may express varying levels of LGALS3 and exhibit different subcellular distribution patterns. Always verify localization in your specific cell type of interest.

• Stimulation-dependent translocation: LGALS3 can shuttle between cellular compartments in response to stimuli such as DNA damage. Time-course experiments following stimulation can help capture these dynamic changes.

• Antibody specificity issues: Different antibodies may recognize different epitopes or forms of LGALS3:

  • Use multiple validated antibodies targeting different regions

  • Include appropriate controls (LGALS3-silenced cells)

  • Consider using tagged LGALS3 constructs for verification

• Post-translational modifications: Modified forms of LGALS3, such as the mono-ubiquitinated form (Ub-GAL3), may show different localization patterns. Using antibodies specific to modified forms or western blot analysis to distinguish these forms can clarify results .

What considerations should be made when interpreting LGALS3 knockdown phenotypes?

When analyzing LGALS3 knockdown experiments:

• Verify knockdown efficiency: Assess protein levels via western blot to confirm significant reduction. The research shows that effective LGALS3 silencing should result in nearly undetectable protein levels in whole cell lysates .

• Consider compensatory mechanisms: Other galectin family members may compensate for LGALS3 loss. Measuring expression levels of related galectins following LGALS3 knockdown can help identify compensatory upregulation.

• Evaluate off-target effects: Include appropriate controls (scrambled shRNA) and consider rescue experiments with shRNA-resistant LGALS3 constructs to confirm phenotype specificity.

• Context-dependent functions: LGALS3 may exhibit different functions depending on:

  • Cell type (cancer vs. normal cells)

  • Stress conditions (type and intensity of DNA damage)

  • Protein interaction partners present in the specific cellular context

• Interpretation of seemingly paradoxical results: For example, LGALS3-silenced cells show increased resistance to DNA damage despite delayed DDR response, suggesting complex roles in cell fate decisions following DNA damage .

What are the emerging areas of investigation for LGALS3 in disease pathways?

Current research has opened several promising avenues for future LGALS3 investigations:

• Therapeutic targeting in cancer:

  • The findings that LGALS3 silencing confers resistance to DNA-damaging agents suggests potential for combination therapies targeting LGALS3 alongside conventional chemotherapy or radiation .

  • Further research should explore whether LGALS3 inhibition could protect normal tissues while sensitizing cancer cells to treatment.

• Biomarker development in cardiovascular disease:

  • Building on the correlation between LGALS3, myocardial fibrosis, and clinical outcomes, developing standardized LGALS3 assays for risk stratification in heart failure patients .

  • Investigating whether genetic polymorphisms in LGALS3 could predict treatment response in personalized medicine approaches.

• Non-classical secretion mechanisms:

  • Elucidating the precise pathways by which LGALS3 is secreted despite lacking a signal peptide .

  • Understanding how these secretion mechanisms are regulated in normal and disease states.

• Post-translational modifications:

  • Further characterizing the mono-ubiquitinated form of LGALS3 (Ub-GAL3) and its specific functions .

  • Investigating other potential modifications (phosphorylation, acetylation, etc.) and their impact on LGALS3 activity.

What technological advances may facilitate deeper understanding of LGALS3 biology?

Emerging technologies likely to advance LGALS3 research include:

• CRISPR/Cas9 gene editing:

  • Creating precise LGALS3 knockout or knockin cell lines and animal models

  • Introducing specific mutations to study structure-function relationships

  • Developing conditional knockout systems to study temporal aspects of LGALS3 function

• Proximity labeling proteomics (BioID, APEX):

  • Identifying transient or weak interaction partners of LGALS3 that may be missed by conventional co-IP approaches

  • Mapping compartment-specific interactomes in different cellular locations

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize LGALS3 localization at nanoscale resolution

  • Live-cell imaging with fluorescently tagged LGALS3 to monitor dynamic changes in localization and interactions

• Single-cell analyses:

  • Single-cell RNA-seq to uncover cell type-specific expression patterns and responses

  • Single-cell proteomics to identify heterogeneity in LGALS3 protein levels and modifications

These technological advances will enable researchers to address fundamental questions about LGALS3 biology and potentially develop novel therapeutic strategies targeting this multifunctional protein.