LGALS4 Human

What is LGALS4 and what protein does it encode?

LGALS4 is a human gene that encodes Galectin-4, a member of the galectin family of beta-galactoside-binding proteins. Galectin-4 is a 36 kDa tandem-repeat galectin comprising 323 amino acids. It contains two homologous carbohydrate recognition domains (CRDs) of approximately 150 amino acids each, connected by a small peptide linker. This structure classifies it as a tandem-repeat galectin, making it intrinsically divalent in its binding capacity . The protein is also known by alternative names including Antigen NY-CO-27, L-36 lactose-binding protein (L36L), and Lactose-binding lectin 4 .

Where is Galectin-4 primarily expressed in the human body?

Galectin-4 is predominantly expressed throughout the gastrointestinal tract, with highest expression in the stomach and intestine . Within the gastrointestinal epithelium, its expression is concentrated in the microvilli, where it can interact with CD3 and bind activated T cells in the lamina propria during intestinal inflammation . While its expression is normally low in tissues outside the digestive system, it can bind to macrophages in the lung, spleen, and kidney . Recent research has also detected Galectin-4 in well-differentiated breast and liver carcinomas, suggesting altered expression patterns in certain cancerous conditions .

What are the fundamental physiological functions of Galectin-4?

Galectin-4 serves multiple physiological functions primarily related to epithelial biology and mucosal immunity:

• Stabilization of lipid rafts, suggesting a role in membrane organization and protein delivery to cells

• Participation in apical trafficking in polarized epithelial cells

• Bactericidal activity against bacteria expressing blood group antigens, contributing to intestinal immunity

• Promotion of intestinal wound healing

• Enhancement of axonal growth and myelination in neurons

• Modulation of cell-cell and cell-matrix interactions through binding to β-galactoside sugars

• Contribution to intestinal homeostasis and epithelial differentiation

Additionally, Galectin-4 can exhibit either pro- or anti-inflammatory activity depending on the experimental model used, indicating context-dependent functional roles .

How does LGALS4 overexpression affect cancer cell proliferation, migration, and invasion?

LGALS4 overexpression has significant inhibitory effects on colorectal cancer (CRC) cells, as demonstrated by multiple experimental approaches:

In proliferation assays using the CCK-8 method, LGALS4 overexpression in LoVo cells reduced viability to approximately 25% compared to control cells after 5 days of culture. Similarly, in HCT-116 cells, viability decreased to approximately 50% of control levels . This indicates a potent anti-proliferative effect of LGALS4.

Regarding migration and invasion, Transwell assays revealed that LGALS4 overexpression substantially decreased both capabilities in CRC cell lines:

• In LoVo cells, invasion capacity decreased 4-fold and migration capacity decreased 2.5-fold compared to controls

• In HCT-116 cells, invasion capacity was reduced by 3-fold and migration capacity by 2-fold compared to controls

These effects likely stem from LGALS4's influence on cell adhesion, cytoskeletal reorganization, or extracellular matrix degradation processes, which are all critical for cell migration and invasion .

What molecular mechanisms explain Galectin-4's role in cell cycle regulation?

Galectin-4 exerts cell cycle regulatory effects primarily by inducing G1 phase arrest through modulation of key cell cycle proteins. Flow cytometric analysis of LGALS4-overexpressing CRC cells revealed:

• In LoVo cells, a ~1.5-fold increase in G1 phase cells and a ~10-fold decrease in S phase cells

• In HCT-116 cells, a ~1.8-fold increase in G1 phase cells and a ~15-fold decrease in S phase cells

At the molecular level, LGALS4 overexpression significantly downregulates the expression of critical cell cycle regulatory proteins:

• CDK1 (cyclin-dependent kinase 1)

• Cyclin B1

• Cyclin A2

These proteins are essential for driving the G1-to-S phase transition. Their downregulation by LGALS4 explains the observed G1 arrest and subsequent inhibition of DNA synthesis. Western blot and RT-qPCR analyses have confirmed these expression changes, providing mechanistic insight into LGALS4's tumor-suppressive properties in CRC cells .

How does LGALS4 influence apoptotic pathways in cancer cells?

LGALS4 significantly promotes apoptosis in colorectal cancer cells through modulation of key apoptosis regulators. Flow cytometry analysis has demonstrated that LGALS4 overexpression increases the apoptosis rate approximately 2.5-fold compared to control cells in both LoVo and HCT-116 cell lines .

At the molecular level, LGALS4 modulates several critical pro- and anti-apoptotic factors:

These expression changes were confirmed through both RT-qPCR and western blot analyses . The upregulation of pro-apoptotic factors (BAX, CASP3, CASP9) combined with downregulation of anti-apoptotic BCL2 creates a cellular environment that favors apoptosis, explaining LGALS4's pro-apoptotic effect in CRC cells.

What experimental methods are most effective for detecting Galectin-4 in research samples?

Several validated methods have proven effective for detecting Galectin-4 in various research contexts:

• Western Blot Analysis:

  • Successfully detects Galectin-4 at approximately 36 kDa under reducing conditions

  • Effective concentration: 2 μg/mL of Anti-Human Galectin-4 Monoclonal Antibody (e.g., MAB1227)

  • Optimal for detecting Galectin-4 in tissue lysates and cell lines (e.g., COLO 205, T84, HT-29)

  • Best performed using PVDF membranes and appropriate immunoblot buffers

• Immunohistochemistry:

  • Effectively detects Galectin-4 in colon tissue

  • Optimal dilution: 1:500 in PBS

  • Specifically identifies crypt epithelial cells at lower dilutions

  • Requires mouse antibody-specific IHC kit for optimal staining

• Immunofluorescence/Immunocytochemistry:

  • Effective for cancer cell samples

  • Optimal dilution: 1:1000 in PBS

  • Provides clear detection of Galectin-4 protein in cellular contexts

• Flow Cytometry:

  • Highly sensitive for detecting internal Galectin-4

  • Optimal dilution: 1:100 in fixing buffer

  • Particularly useful for examining apoptosis and cell cycle effects in cancer cells

• RT-qPCR:

  • Effective for measuring LGALS4 mRNA expression

  • Has been successfully used to compare expression levels between normal cells (e.g., NCM460) and CRC cells (e.g., LoVo, HCT-116, SW480)

Each method offers specific advantages depending on the research question and sample type.

How does LGALS4 relate to metabolic reprogramming in cancer cells?

LGALS4 appears to play a significant role in cancer cell metabolism, particularly in relation to glycolysis. Recent research has revealed a complex relationship between LGALS4 expression and metabolic adaptations in colorectal cancer:

• LGALS4-overexpressing CRC cells exhibit increased survival under glucose deprivation conditions

• These cells show enhanced tolerance to glycolytic inhibition compared to control cells

• Flow cytometry analysis confirms that LGALS4 significantly reduces apoptosis induced by glucose deprivation

Interestingly, these findings suggest that while LGALS4 generally acts as a tumor suppressor by inhibiting proliferation and promoting apoptosis under normal conditions, it may paradoxically enhance cancer cell survival under metabolic stress. This metabolic reprogramming may represent an adaptation mechanism that allows tumor cells to survive in nutrient-poor microenvironments.

The downregulation of LGALS4 observed in various cancers may be related to metabolic reprogramming that facilitates rapid proliferation and immune evasion . These findings highlight the complex and context-dependent roles of LGALS4 in cancer biology and suggest that therapeutic strategies targeting aerobic glycolysis may represent promising approaches for colorectal cancer treatment.

What is the clinical significance of Galectin-4 expression in various cancer types?

Galectin-4 expression has been linked to clinical outcomes in multiple cancer types, with emerging evidence suggesting both diagnostic and prognostic value:

Colorectal Cancer (CRC):

• LGALS4 is strongly underexpressed in CRC compared to normal colonic tissue

• Functions as a potential tumor suppressor, with overexpression inducing cell cycle arrest, reducing migration, and sensitizing cells to apoptosis

• Interacts with Wnt signaling proteins and downregulates Wnt target genes, potentially explaining its tumor-suppressive effects

• Elevated levels of circulating Galectin-4 correlate with disease progression, suggesting utility as a follow-up marker post-surgery

Other Cancer Types:
Galectin-4 has been detected in various cancers and may be involved in the development and progression of:

• Pancreatic carcinoma

• Hepatocellular carcinoma

• Breast carcinoma

• Gastric cancer

• Lung cancer

Prognostic Implications:

• Surface profiling of CRC cells and tumor-infiltrating lymphocytes from surgical samples reveals that Galectin-4 expression patterns align with prognostic categories

• May serve as part of minimal antigenic panels that predict disease relapse and patient survival

• In ovarian cancer, LGALS4 expression has been correlated with prognosis, though detailed findings were limited in the search results

These findings collectively suggest that LGALS4 could serve as both a therapeutic target and a prognostic marker in cancer management, highlighting its importance in clinical cancer research.

What cell lines and experimental models are most appropriate for studying LGALS4 function?

Based on the research literature, the following experimental models have proven effective for studying LGALS4 function:

Cell Lines:

• Colorectal cancer (CRC) cell lines: LoVo and HCT-116 cells have shown significantly lower LGALS4 expression compared to normal cells, making them ideal for overexpression studies

• Additional CRC cell lines: SW480, COLO 205, T84, and HT-29 have also been successfully used in LGALS4 research

• Normal colon cell line: NCM460 serves as an appropriate control, exhibiting higher baseline LGALS4 expression than CRC cell lines

Experimental Approaches:

• Gene overexpression models: Transfection with LGALS4 overexpression vectors has successfully demonstrated functional effects in LoVo and HCT-116 cells

• Protein detection models: Human colon tissue and CRC cell lines have proven effective for protein detection using western blot, immunohistochemistry, and immunofluorescence techniques

• Functional assays:

  • Cell viability: CCK-8 assay over 5-day periods effectively demonstrates anti-proliferative effects

  • Migration/invasion: Transwell assays clearly show LGALS4's impact on these processes

  • Cell cycle analysis: Flow cytometry with appropriate cell cycle staining provides robust data

  • Apoptosis assessment: Flow cytometry with annexin V/PI staining effectively quantifies apoptotic effects

When designing experiments, researchers should consider that LGALS4 effects may be context-dependent, as indicated by its varied roles in inflammation depending on the mouse model used .

How can researchers effectively measure changes in glycolysis associated with LGALS4 expression?

To effectively investigate LGALS4's impact on glycolysis in cancer cells, researchers can employ several complementary approaches:

• Glucose Deprivation Assays:

  • Culture cells in glucose-limited medium and compare survival rates between LGALS4-overexpressing and control cells

  • Measure apoptosis rates under glucose deprivation using flow cytometry with annexin V/PI staining

• Glycolytic Inhibition Studies:

  • Treat cells with glycolytic inhibitors (e.g., 2-deoxyglucose) at various concentrations

  • Compare cell viability, apoptosis rates, and metabolic activity between LGALS4-modified and control cells

• Metabolic Flux Analysis:

  • Measure extracellular acidification rate (ECAR) using a Seahorse XF analyzer to quantify glycolytic activity

  • Compare oxygen consumption rate (OCR) to assess mitochondrial respiration

  • Calculate glycolytic capacity and glycolytic reserve

• Expression Analysis of Glycolytic Enzymes:

  • Use RT-qPCR and western blotting to measure expression of key glycolytic enzymes (e.g., HK2, PKM2, LDHA)

  • Compare expression patterns between LGALS4-overexpressing and control cells

• Glucose Uptake Assays:

  • Use fluorescent glucose analogs (e.g., 2-NBDG) to measure glucose uptake rates

  • Compare uptake between LGALS4-modified and control cells using flow cytometry

• Lactate Production Measurement:

  • Quantify lactate levels in culture medium using commercially available lactate assay kits

  • Monitor changes in lactate production over time in response to LGALS4 modulation

These methodological approaches can provide comprehensive insights into how LGALS4 affects glycolytic pathways and metabolic reprogramming in cancer cells, which appears to be a significant aspect of its biological function .

What techniques are most effective for studying LGALS4's interactions with cell cycle regulatory proteins?

To effectively investigate LGALS4's interactions with cell cycle regulatory proteins, researchers should consider the following complementary techniques that have yielded important insights:

• Flow Cytometric Cell Cycle Analysis:

  • Method: Stain fixed cells with propidium iodide or other DNA-binding dyes

  • Analysis: Compare distribution of cells in G1, S, and G2/M phases between LGALS4-overexpressing and control cells

  • Findings: This approach has successfully demonstrated that LGALS4 overexpression increases G1 phase cells (~1.5-fold in LoVo cells; ~1.8-fold in HCT-116 cells) and decreases S phase cells (~10-fold in LoVo cells; ~15-fold in HCT-116 cells)

• Western Blot Analysis of Cell Cycle Proteins:

  • Target proteins: CDK1, Cyclin B1, and Cyclin A2

  • Method: Use appropriate antibodies to detect protein expression levels

  • Controls: Include loading controls (e.g., β-actin, GAPDH)

  • Analysis: Perform densitometric analysis for quantification

  • Findings: This technique has confirmed that LGALS4 overexpression significantly decreases the expression of these key cell cycle regulatory proteins

• RT-qPCR for mRNA Expression Analysis:

  • Target genes: Genes encoding cell cycle regulatory proteins

  • Method: Extract RNA, perform reverse transcription, and conduct qPCR

  • Analysis: Use the 2^(-ΔΔCT) method to calculate relative expression levels

  • Controls: Include appropriate housekeeping genes for normalization

  • Findings: This approach complements protein analysis by confirming transcriptional changes

• Co-Immunoprecipitation (Co-IP):

  • Purpose: Identify direct physical interactions between LGALS4 and cell cycle proteins

  • Method: Precipitate LGALS4 using specific antibodies and probe for co-precipitated cell cycle proteins

  • Controls: Include IgG controls and input samples

• Chromatin Immunoprecipitation (ChIP):

  • Purpose: Determine if LGALS4 influences transcription factor binding to promoters of cell cycle genes

  • Method: Precipitate chromatin with antibodies against transcription factors and quantify bound DNA by qPCR

• Immunofluorescence Microscopy:

  • Purpose: Visualize co-localization of LGALS4 with cell cycle proteins

  • Method: Double staining with antibodies against LGALS4 and cell cycle proteins

  • Analysis: Calculate Pearson's correlation coefficient to quantify co-localization

By combining these techniques, researchers can comprehensively characterize how LGALS4 influences cell cycle regulation, which appears to be a key mechanism underlying its tumor-suppressive effects in colorectal cancer .

What are the most promising therapeutic applications of LGALS4 research?

Based on current understanding of LGALS4 biology, several promising therapeutic directions emerge:

• LGALS4 as a Target for Cancer Therapy:

  • Development of approaches to restore LGALS4 expression in colorectal cancers where it is downregulated

  • Gene therapy approaches to deliver functional LGALS4 to tumor cells

  • Small molecule compounds that mimic LGALS4 activity or induce its expression

• Combination Therapies:

  • LGALS4-based interventions combined with conventional chemotherapies

  • Research indicates LGALS4 overexpression enhances 5-fluorouracil (5-FU)-induced apoptosis in CRC cells, suggesting synergistic potential

  • Combination with glycolytic inhibitors, given LGALS4's influence on metabolic reprogramming

• Diagnostic and Prognostic Applications:

  • Development of LGALS4-based biomarkers for cancer detection and monitoring

  • Elevated circulating Galectin-4 levels correlate with CRC progression, suggesting utility as a follow-up marker post-surgery

  • Potential for LGALS4 to complement existing biomarkers (e.g., CEA/CA19-9) in enhancing CRC monitoring

• Targeting Metabolic Vulnerabilities:

  • Therapeutic approaches targeting aerobic glycolysis in LGALS4-low tumors

  • Exploitation of the relationship between LGALS4 expression and glucose metabolism

• Inflammatory Bowel Disease Therapies:

  • Development of interventions based on LGALS4's role in intestinal inflammation and wound healing

  • Potential applications in promoting mucosal healing in inflammatory conditions

These therapeutic directions reflect LGALS4's multifaceted roles in cancer biology, metabolism, and mucosal immunity, with particular promise in colorectal cancer where its tumor-suppressive effects are well-documented.

What experimental challenges must be addressed in LGALS4 research?

Researchers investigating LGALS4 face several experimental challenges that must be addressed for optimal results:

• Context-Dependent Functions:

  • LGALS4 can exhibit either pro- or anti-inflammatory activity depending on the experimental model

  • Research designs must account for this variability by carefully selecting appropriate models and controls

  • The paradoxical role of LGALS4 in both promoting apoptosis under normal conditions but increasing survival under glucose deprivation requires careful experimental design

• Tissue-Specific Expression Patterns:

  • LGALS4 expression is primarily restricted to the gastrointestinal tract under normal conditions

  • Researchers using non-GI tissues or cell lines must consider baseline expression levels

  • Detection methods may need optimization for tissues with naturally low expression

• Post-Translational Modifications:

  • The functional activity of Galectin-4 may be influenced by post-translational modifications

  • Research should incorporate methods to detect and characterize these modifications

  • The impact of PTMs on antibody recognition should be considered when selecting detection reagents

• Carbohydrate Recognition Domain (CRD) Specificity:

  • Galectin-4 has two distinct CRDs with different binding specificities

  • Experiments investigating ligand interactions must consider which CRD is involved

  • Design of inhibitors or mimetics must account for this dual specificity

• In Vivo Translation:

  • Bridging the gap between in vitro findings and in vivo relevance

  • Development of appropriate animal models that recapitulate human LGALS4 expression and function

  • Need for careful validation of findings across multiple experimental systems

• Technical Considerations:

  • Optimal antibody dilutions vary by application (1:500 for IHC, 1:1000 for ICC/IF, 1:100 for flow cytometry)

  • Cell-specific optimization may be necessary (e.g., different transfection efficiencies for LGALS4 overexpression)

  • Careful selection of control cell lines with appropriate baseline LGALS4 expression