LGALSL Human

What is LGALSL protein and what is its role in human cells?

LGALSL (Lectin galactoside-binding-like protein) is a 172-amino acid protein that belongs to the galectin family. Also known as Galectin-Related Protein (GRP) or HSPC159, LGALSL contains one galectin domain . The human LGALSL gene is located on chromosome 2 .

While typical galectins are soluble β-galactoside-binding animal lectins that modulate cell-to-cell adhesion, cell-to-extracellular matrix interactions, and participate in tumor progression, pre-mRNA splicing, and apoptosis, LGALSL appears to have diverged functionally . Unlike other family members, LGALSL does not bind carbohydrates or lactose since the critical residues required for binding are not conserved .

The specific cellular functions of LGALSL remain to be fully characterized, but its evolutionary conservation suggests important biological roles. Researchers should consider investigating protein interaction networks to elucidate LGALSL's specific functions, as the protein may have evolved novel molecular interactions distinct from other galectin family members.

How is LGALSL structurally related to other galectin family proteins?

LGALSL shares structural homology with other galectin family members through its galectin domain but exhibits key differences that likely account for its unique functional properties:

• LGALSL contains a single galectin domain similar to prototype galectins

• Unlike other galectins, LGALSL lacks conservation of critical residues required for lactose and carbohydrate binding

• This structural divergence suggests LGALSL has evolved specialized functions distinct from classical galectin roles

From an evolutionary perspective, LGALSL is part of the galectin family expansion in vertebrates. Syntenic analyses reveal that in most jawed vertebrates (Gnathostomata), LGALS8 is associated with LGALSL, and LGALSL2 (also called LGALSLA) is associated with LGALS3 on one chromosomal segment . During mammalian evolution, lineage-specific rearrangements occurred, resulting in LGALS8 and LGALSL being localized on different chromosomes, while LGALSL2 has been lost in mammals .

Comparative structural analysis between LGALSL and other galectins can provide insights into its functional specialization and evolutionary history.

What are the key structural features of LGALSL protein?

LGALSL protein consists of 172 amino acids containing one galectin domain with several distinctive structural features:

For structural studies, recombinant LGALSL can be produced as a single, non-glycosylated polypeptide chain . The Human Protein Atlas provides in-house generated structures predicted using the AlphaFold source code , which can guide experimental approaches.

When expressed recombinantly, LGALSL is typically formulated in a solution containing 20mM Tris-HCl buffer (pH 8.0), 0.1M NaCl, 20% glycerol, and 1mM DTT . This formulation maintains protein stability for structural and functional investigations.

To fully understand LGALSL's structure-function relationship, researchers should consider comparative structural analysis with other galectins, particularly examining differences in binding sites and surface properties.

What methods are most effective for analyzing LGALSL expression patterns across different tissues?

To comprehensively analyze LGALSL expression patterns, researchers should employ multiple complementary approaches:

• Transcriptomic Analysis:

  • RNA-seq data from resources like The Human Protein Atlas to identify tissues with high, medium, or low expression

  • Single-cell RNA-seq to determine cell type-specific expression patterns

  • Quantitative RT-PCR with tissue-specific cDNA panels for targeted validation

• Protein Detection:

  • Immunohistochemistry using validated antibodies to confirm protein-level expression in tissues

  • Western blotting to quantify relative protein levels across tissue samples

  • ELISA for quantitative measurement in serum, plasma, and tissue lysates

• Promoter Analysis:

  • Identifying regulatory elements controlling tissue-specific expression

  • Reporter assays to validate promoter activity in different cell types

• Epigenetic Profiling:

  • Analysis of DNA methylation and histone modifications at the LGALSL locus across tissues

  • Correlation of epigenetic status with expression levels

When interpreting expression data, researchers should consider that LGALSL may exhibit cell type-specific or context-dependent expression patterns that could provide clues to its specialized functions.

What are the recommended methods for detecting LGALSL protein in biological samples?

Several validated methods are available for detecting and quantifying LGALSL protein in biological samples:

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • Commercial ELISA kits are available for quantitative measurement of Human LGALSL in serum, plasma, and other biological samples

  • Typical detection range: Dependent on kit specifications (check manufacturer guidelines)

  • Sample types: Serum, plasma, cell culture supernatants, tissue lysates

• Western Blotting:

  • Recommended primary antibody dilution: 1:1000-1:2000 (optimized based on antibody source)

  • Sample preparation: Protein extraction using RIPA or NP-40 buffer with protease inhibitors

  • Detection: Anti-LGALSL antibodies followed by species-appropriate secondary antibodies

• Immunohistochemistry/Immunofluorescence:

  • Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Recommended antibody dilution: 1:100-1:500 for tissue sections

  • Controls: Include LGALSL-expressing and non-expressing tissues

• Mass Spectrometry:

  • Sample preparation: Tryptic digestion followed by LC-MS/MS analysis

  • Target peptides: Select unique peptides from the LGALSL sequence for targeted assays

  • Quantification: Label-free or isotope-labeled internal standards

When selecting detection methods, consider the research question, required sensitivity, sample availability, and whether qualitative or quantitative data is needed. Validation of antibody specificity is crucial for accurate results, especially given structural similarities between galectin family members.

How has LGALSL evolved across vertebrate species, and what does this tell us about its function?

Evolutionary analysis of LGALSL provides valuable insights into its functional significance. Syntenic studies reveal a complex evolutionary history with important phylogenetic patterns:

• Chromosomal Organization:

  • In most jawed vertebrates (Gnathostomata), LGALS8 is associated with LGALSL, and LGALSL2 (LGALSLA) is associated with LGALS3 on one chromosomal segment

  • During mammalian evolution, lineage-specific rearrangements occurred, resulting in LGALS8 and LGALSL being localized on different chromosomes

  • LGALSL2 has been lost during mammalian evolution

• Methodological Approach for Evolutionary Analysis:

  • Sequence collection: Gather LGALSL homologs from diverse vertebrate species using BLAST searches

  • Multiple sequence alignment: Use MUSCLE or MAFFT algorithms with manual refinement

  • Phylogenetic reconstruction: Maximum likelihood or Bayesian methods with appropriate substitution models

  • Synteny analysis: Compare genomic context across species using genome browsers and synteny databases

  • Selection analysis: Calculate dN/dS ratios across protein regions to identify selection patterns

• Functional Implications:

  • The evolutionary conservation of LGALSL suggests essential biological functions despite loss of carbohydrate-binding capability

  • Lineage-specific patterns may indicate adaptive specialization in different vertebrate groups

  • Conserved domains likely represent functionally important regions

The association of LGALSL with LGALS8 throughout most of vertebrate evolution, followed by chromosomal separation in mammals, suggests potential functional relationships between these proteins that may have diverged in mammals. This evolutionary pattern provides a foundation for hypotheses about LGALSL's specialized functions.

What experimental approaches can reveal regulatory mechanisms controlling LGALSL expression?

Understanding LGALSL's regulatory mechanisms requires a multi-faceted approach combining computational analysis with experimental validation:

• Promoter Characterization:

  • In silico analysis: Identify potential transcription factor binding sites using tools like JASPAR, TRANSFAC

  • Promoter reporter assays: Generate luciferase constructs with progressively truncated LGALSL promoter regions

  • Mutational analysis: Site-directed mutagenesis of predicted regulatory elements

  • ChIP assays: Identify transcription factors binding to the LGALSL promoter in vivo

• Epigenetic Regulation:

  • DNA methylation analysis: Bisulfite sequencing of the LGALSL promoter region

  • Histone modification profiling: ChIP-seq for active (H3K4me3, H3K27ac) and repressive (H3K27me3, H3K9me3) marks

  • Chromatin accessibility: ATAC-seq to identify open chromatin regions near LGALSL

  • Experimental validation: Treatment with epigenetic modifiers (HDAC inhibitors, DNA methyltransferase inhibitors)

• Post-transcriptional Regulation:

  • miRNA binding site prediction: Computational identification of miRNA target sites in LGALSL mRNA

  • 3'UTR reporter assays: Validate miRNA regulation using luciferase constructs containing LGALSL 3'UTR

  • RNA-binding protein analysis: RIP-seq to identify proteins interacting with LGALSL mRNA

  • mRNA stability assays: Actinomycin D chase experiments to measure LGALSL mRNA half-life

• Response to Stimuli:

  • Treatment with cytokines, growth factors, and stress inducers to identify conditions affecting LGALSL expression

  • Time-course analysis to determine expression dynamics

  • Cell type-specific responses to identify context-dependent regulation

By integrating these approaches, researchers can construct a comprehensive model of LGALSL regulation across different cellular contexts and physiological conditions.

What is the optimal protocol for expressing and purifying LGALSL protein for structural studies?

Based on available information, the following optimized protocol for LGALSL expression and purification is recommended:

• Expression System Selection:

  • E. coli is a suitable host for LGALSL production as a non-glycosylated polypeptide

  • Recommended strain: BL21(DE3) for high-level expression

  • Alternative systems: Consider insect cells (Sf9, Hi5) if E. coli yields insoluble protein

• Expression Construct Design:

  • Vector: pET series with T7 promoter for high expression

  • Fusion tag: N-terminal 6xHis-tag (the search results mention a 23-amino acid His-tag construct)

  • Cleavage site: TEV protease recognition sequence between tag and LGALSL

  • Codon optimization: Adjust codons for expression host

• Expression Conditions:

  • Culture medium: LB or TB media supplemented with appropriate antibiotics

  • Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

  • Temperature: Test both standard (37°C) and reduced temperature (16-25°C) induction

  • Duration: 3-4 hours (37°C) or overnight (16-25°C)

• Purification Strategy:

  • Cell lysis: Sonication or high-pressure homogenization in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, protease inhibitors)

  • Initial capture: Ni-NTA affinity chromatography

  • Intermediate purification: Ion exchange chromatography (Q or SP Sepharose)

  • Polishing step: Size exclusion chromatography

  • Purity assessment: SDS-PAGE (target >90% purity)

• Buffer Optimization:

  • Final formulation: 20 mM Tris-HCl buffer (pH 8.0), 0.1 M NaCl, 20% glycerol, and 1 mM DTT

  • Concentration: 0.5-1.0 mg/ml

  • Storage: Aliquot and store at -80°C, avoid repeated freeze-thaw cycles

• Quality Control:

  • Mass spectrometry to confirm protein identity

  • Dynamic light scattering to assess homogeneity

  • Circular dichroism to verify proper folding

  • Thermal shift assay to evaluate stability

This protocol should yield high-quality LGALSL protein suitable for crystallization trials, NMR studies, or other structural biology applications. The predicted AlphaFold structure available from the Human Protein Atlas can guide experimental design and structural analysis.

What experimental strategies can elucidate LGALSL's potential roles in disease pathways?

To investigate LGALSL's involvement in disease processes, researchers should implement a comprehensive experimental strategy:

• Expression Profiling in Disease States:

  • Analyze LGALSL expression in publicly available datasets (TCGA, GEO) across different diseases

  • Compare expression levels between normal and pathological tissues using qPCR, Western blotting, and immunohistochemistry

  • Correlate expression with disease progression, staging, and patient outcomes

• Genetic Association Studies:

  • Examine LGALSL genetic variants in relevant disease cohorts

  • Perform case-control association studies for identified variants

  • Analyze functional consequences of disease-associated variants

• Cellular Models:

  • Generate LGALSL knockout, knockdown, and overexpression cell lines

  • Assess effects on:

    • Cell proliferation, migration, and invasion

    • Apoptosis and cell death pathways

    • Inflammatory responses

    • Response to stress conditions

• Mechanistic Studies:

  • Identify LGALSL interaction partners in disease-relevant cell types

  • Map associated signaling pathways using phosphoproteomics

  • Evaluate effects on gene expression using RNA-seq

  • Investigate subcellular localization changes in disease states

• In Vivo Models:

  • Generate LGALSL knockout or conditional knockout mouse models

  • Challenge with disease-inducing conditions

  • Evaluate tissue-specific phenotypes

  • Perform rescue experiments with wild-type or mutant LGALSL

• Therapeutic Targeting Assessment:

  • Develop tools to modulate LGALSL function (small molecules, peptides, antibodies)

  • Test effects in cellular and animal disease models

  • Evaluate potential as biomarker or therapeutic target

Given LGALSL's relationship to the galectin family, which participates in tumor progression, immune responses, and cell-cell interactions , focusing on cancer, inflammatory conditions, and developmental disorders may be particularly productive research directions.

How does LGALSL differ functionally from other galectin family members despite structural similarities?

LGALSL exhibits significant functional divergence from typical galectin family members despite containing a galectin domain. Understanding these differences requires systematic comparative analysis:

• Structural Comparison:

  • LGALSL contains a galectin domain but lacks conserved residues required for β-galactoside binding

  • AlphaFold predicted structures available from the Human Protein Atlas can be compared with experimental structures of other galectins

  • Differences in binding pocket architecture and surface properties likely explain functional divergence

• Methodological Approach for Functional Comparison:

  • Glycan Array Analysis:

    • Test LGALSL against comprehensive glycan arrays

    • Compare binding profiles with other galectin family members

    • Identify any unique binding preferences

  • Protein-Protein Interaction Profiling:

    • Perform comparative IP-MS analyses of LGALSL vs. other galectins

    • Identify unique and shared interacting partners

    • Map interaction interfaces through mutagenesis

  • Domain Swap Experiments:

    • Generate chimeric proteins exchanging domains between LGALSL and classical galectins

    • Test functionality of chimeras to map functional regions

    • Identify critical residues through site-directed mutagenesis

  • Subcellular Localization:

    • Compare localization patterns with other galectin family members

    • Identify unique trafficking or compartmentalization

• Functional Assays:

  • Comparative analysis of effects on:

    • Cell adhesion and migration

    • Apoptosis induction

    • Immune response modulation

    • RNA processing

• Evolutionary Context:

  • Reconstruct the evolutionary trajectory of functional divergence

  • Identify key mutations that led to loss of carbohydrate binding

  • Determine if LGALSL gained new functions during evolution

Understanding LGALSL's unique functional properties will provide insights into protein family evolution and may reveal novel cellular mechanisms distinct from classical galectin functions.

What are the most effective approaches for identifying LGALSL binding partners?

Identifying LGALSL's interactome requires multiple complementary approaches to capture both stable and transient interactions:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Protocol Overview:

    • Express N-terminally His-tagged LGALSL in relevant cell lines

    • Perform pulldown with immobilized metal affinity chromatography

    • Analyze bound proteins by LC-MS/MS

    • Filter against appropriate controls (untransfected cells, unrelated bait proteins)

  • Variations:

    • SILAC labeling for quantitative comparison

    • Crosslinking to stabilize weak interactions

    • Tandem affinity purification for higher specificity

• Proximity-Based Labeling:

  • BioID Method:

    • Generate LGALSL-BioID2 fusion construct

    • Express in target cells and supply biotin

    • Purify biotinylated proteins and identify by MS

    • Map proximity network around LGALSL

  • APEX2 Method:

    • Express LGALSL-APEX2 fusion

    • Treat cells with biotin-phenol and H₂O₂ for rapid labeling

    • Identify biotinylated proteins to map microenvironment

• Protein Microarray Screening:

  • Screen purified LGALSL against:

    • Commercial protein microarrays

    • Custom arrays of candidate interactors

    • Arrays representing specific pathway components

• Biophysical Interaction Analysis:

  • Surface Plasmon Resonance (SPR):

    • Immobilize purified LGALSL on sensor chip

    • Flow potential binding partners over surface

    • Measure binding kinetics and affinities

  • Isothermal Titration Calorimetry (ITC):

    • Directly measure thermodynamic parameters of interactions

    • Determine binding stoichiometry

• In Silico Approaches:

  • Structure-based docking to predict potential binding partners

  • Network analysis using interaction databases

  • Leverage the interaction network visualization tools from the Human Protein Atlas

• Validation Strategies:

  • Co-immunoprecipitation with endogenous proteins

  • Fluorescence resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • Colocalization by immunofluorescence microscopy

By implementing multiple complementary approaches, researchers can build a comprehensive interactome map to guide functional studies and pathway analysis.

What cellular pathways might involve LGALSL and how can these be experimentally mapped?

While specific LGALSL-associated pathways remain to be fully characterized, several experimental strategies can systematically map its functional networks:

• Global Pathway Analysis Following LGALSL Perturbation:

  • Transcriptomic Profiling:

    • Perform RNA-seq after LGALSL knockdown/knockout or overexpression

    • Identify differentially expressed genes using DESeq2 or similar tools

    • Conduct pathway enrichment analysis using GSEA, KEGG, or Reactome databases

    • Validate key targets by qRT-PCR and Western blotting

  • Proteomics and Post-Translational Modifications:

    • TMT or iTRAQ-based quantitative proteomics

    • Phosphoproteomics to identify altered signaling pathways

    • Glycoproteomics to investigate potential effects on protein glycosylation

• Targeted Pathway Investigation:

  • Based on galectin family functions , examine LGALSL's potential involvement in:

    • Cell adhesion and extracellular matrix interactions

    • Apoptosis and cell survival pathways

    • Immune regulation and inflammation

    • RNA processing and pre-mRNA splicing

    • Cancer-related signaling pathways

• Experimental Mapping Approaches:

  • Pathway Reporter Assays:

    • Screen LGALSL effects on luciferase reporters for major signaling pathways (NF-κB, MAPK, Wnt, etc.)

    • Identify pathways modulated by LGALSL expression levels

  • Protein-Protein Interaction Network Analysis:

    • Map LGALSL interactors to known pathways

    • Identify hub proteins connecting LGALSL to specific pathways

    • Validate functional relevance through targeted perturbations

  • Live Cell Imaging:

    • Monitor pathway activation using fluorescent reporters

    • Track LGALSL localization during pathway activation

    • Measure dynamic protein interactions using FRET biosensors

• Integrative Analysis:

  • Combine multi-omics data (transcriptomics, proteomics, interactomics)

  • Build network models of LGALSL-associated pathways

  • Identify potential feedback mechanisms and pathway crosstalk

Given LGALSL's divergence from typical galectin functions, researchers should remain open to discovering novel pathway associations beyond those established for other family members.

What are the most rigorous approaches for validating LGALSL knockout or knockdown models?

Comprehensive validation of LGALSL genetic perturbation models is essential for ensuring experimental reliability. The following multi-layered approach is recommended:

• Genomic Validation:

  • For CRISPR-Cas9 Knockout:

    • PCR amplification and sequencing of the targeted region

    • Analysis of indel patterns and predicted protein consequences

    • Verification of frameshift or nonsense mutations

    • Whole-genome sequencing to assess off-target effects

  • For RNAi Knockdown:

    • Use multiple independent siRNA/shRNA sequences targeting different regions

    • Include non-targeting controls and rescue controls

    • Evaluate potential off-targets using bioinformatic prediction tools

• Transcript Validation:

  • Quantitative Analysis:

    • RT-qPCR with primers spanning multiple exons

    • Digital droplet PCR for precise quantification

    • Northern blotting for size verification

  • Qualitative Analysis:

    • RNA-seq to verify transcript reduction and assess compensatory changes

    • 5' and 3' RACE to identify potential alternative transcripts

    • Examination of potential splice variants that might escape targeting

• Protein Validation:

  • Detection Methods:

    • Western blotting using antibodies targeting different epitopes

    • ELISA for quantitative assessment

    • Immunofluorescence to confirm reduced protein expression in situ

    • Mass spectrometry-based targeted proteomics (SRM/MRM)

  • Validation Controls:

    • Include positive controls (known LGALSL-expressing cells)

    • Include negative controls (cells naturally lacking LGALSL)

    • Compare multiple antibodies to ensure specificity

• Functional Validation:

  • Phenotypic Reversal:

    • Rescue experiments by reintroducing wild-type LGALSL

    • Use of inducible expression systems for temporal control

    • Complementation with domain mutants to map functional regions

  • Comparative Analysis:

    • Parallel validation in multiple cell types or model systems

    • Correlation of phenotype severity with knockdown/knockout efficiency

    • Comparison with related family members (other galectins) to assess specificity

By implementing this comprehensive validation strategy, researchers can establish reliable model systems for investigating LGALSL function while minimizing the risk of misinterpreting results due to off-target effects or incomplete gene inactivation.