LGALS7 Human, His

What is LGALS7/galectin-7 and what are its known functions?

Galectin-7 is a β-galactoside-binding protein encoded by the LGALS7 gene located on chromosome 19q13.2. The full-length cDNA of LGALS7 encodes a protein comprising 136 amino acids . Functionally, galectin-7 participates in cell-cell and cell-matrix interactions, cellular proliferation, differentiation, and apoptosis. It plays important roles in epithelial cell development and has been implicated in various pathological processes including cancer progression and inflammatory disorders .

The protein is predominantly expressed in stratified epithelia, particularly in skin tissue, and can be found in both nuclear and cytoplasmic compartments . Its expression pattern is often altered in disease states, making it a potentially valuable biomarker in several conditions.

How is LGALS7 gene expression regulated?

LGALS7 gene expression is regulated through complex mechanisms involving its promoter region. Research has identified specific single nucleotide polymorphisms (SNPs) in the LGALS7 promoter region that can affect expression levels. Two notable SNPs, rs567785577 and rs138945880, located on chromosome 19q13.2, have been identified in the promoter sequence .

Regulation also occurs through endothelin signaling, which has been shown to suppress galectin-7 expression in certain conditions, such as systemic sclerosis . Additionally, studies using transgenic mouse models have demonstrated that the gene can be experimentally manipulated to achieve both overexpression and knockdown, allowing researchers to study the effects of altered LGALS7 expression on various biological processes .

What is the significance of the His-tag in recombinant LGALS7 research?

The histidine tag (His-tag) is a sequence of typically six histidine residues added to the N- or C-terminus of recombinant LGALS7 protein. This modification serves several crucial research purposes:

• Purification: The His-tag enables simple and efficient purification of the recombinant protein using immobilized metal affinity chromatography (IMAC).

• Detection: It facilitates protein detection using commercial anti-His antibodies.

• Protein orientation: When immobilized on surfaces, His-tagged proteins can be oriented in a specific manner.

While the His-tag generally has minimal impact on protein folding and function, researchers should validate that tag addition does not interfere with the specific galectin-7 property being studied, particularly its carbohydrate-binding activity and oligomerization behavior.

How do LGALS7 promoter polymorphisms affect disease susceptibility?

Research has revealed significant associations between LGALS7 promoter polymorphisms and disease susceptibility, particularly in intracerebral hemorrhage (ICH). A two-stage genetic association study identified two ICH susceptibility loci in the LGALS7 promoter region: rs567785577 and rs138945880 .

The study found that the A allele of rs567785577 and the T allele of rs138945880 were associated with a significantly increased risk of ICH, with an unadjusted odds ratio (OR) of 13.5 (95% CI = 2.249-146.5; p = 0.002) . Analysis of homozygous and heterozygous frequencies of these alleles in stroke patients revealed they were in Hardy-Weinberg equilibrium (p > 0.05), but interestingly, these alleles were not observed in homozygous form in healthy control individuals .

This research represents the first effort to genotype the galectin-7 promoter in hemorrhagic stroke patients and suggests that these single loci may serve as genetic risk factors for hemorrhagic stroke. Further studies with larger sample sizes across diverse ethnic populations are needed to fully elucidate the mechanisms underlying these associations .

What protein interactions does galectin-7 participate in and how do they influence cellular pathways?

Galectin-7 engages in numerous protein-protein interactions that mediate its biological effects. Proteomic analyses using iTRAQ (isobaric tags for relative and absolute quantitation) have identified 1,009 differentially expressed proteins associated with galectin-7 expression levels, with 28 of these being well-characterized proteins .

Key interactions include:

These interactions contribute to various cellular pathways involving signal transduction, metabolic processes, and structural organization, ultimately influencing cell proliferation, migration, and response to stimuli.

How does galectin-7 function differ between normal and pathological conditions?

Galectin-7 exhibits context-dependent functions that can vary dramatically between normal and pathological conditions:

In normal tissue:

• Expressed primarily in stratified epithelia, particularly in skin

• Contributes to normal cell differentiation and epithelial development

• Maintains tissue homeostasis through regulation of apoptosis

• Expressed abundantly throughout the epidermis of normal skin

In pathological conditions:

These differential expression patterns and functional roles highlight galectin-7's potential as both a biomarker and therapeutic target in various diseases.

What are the optimal methods for detecting LGALS7/galectin-7 in different sample types?

Researchers can employ several validated methods for detecting galectin-7 in various sample types:

• Western Blot:

  • Effective for protein lysates from tissues and cell lines

  • PVDF membranes probed with anti-galectin-7 antibodies (e.g., Mouse Anti-Human Galectin-7 Monoclonal Antibody) at 2 μg/mL concentration

  • Detects a specific band at approximately 14 kDa under reducing conditions

  • Recommended protocol: Use Immunoblot Buffer Group 1 for optimal results

• Immunocytochemistry/Immunofluorescence:

  • Suitable for fixed cell lines (e.g., HEK001 human epidermal keratinocyte cell line)

  • Use 5 μg/mL of anti-galectin-7 antibody for 3 hours at room temperature

  • Counterstain with DAPI to visualize nuclei

  • Can detect both cytoplasmic and nuclear localization

• Immunohistochemistry:

  • Optimal for paraffin-embedded tissue sections

  • Use 5 μg/mL of anti-galectin-7 antibody for 1 hour at room temperature

  • Visualize using DAB (brown) with hematoxylin counterstain (blue)

  • VisUCyte HRP Polymer Detection Reagents recommended for signal amplification

• ELISA:

  • Appropriate for quantifying galectin-7 in serum or other biological fluids

  • Particularly useful for studies correlating serum levels with disease manifestations

For optimal results, researchers should determine the ideal antibody dilutions for each application through titration experiments.

How can LGALS7 expression be manipulated in experimental models?

Several validated approaches exist for manipulating LGALS7 expression in experimental models:

• Transgenic animal models:

  • Overexpression models: Generate by microinjection of pcDNA3.1-LGALS7 plasmid vectors into fertilized oocytes

  • Knockdown models: Create using pSilencer-LGALS7 plasmid vectors containing shRNA constructs

  • Maintain transgenic lines by backcrossing with appropriate mouse strains (e.g., BALB/c)

  • Verify genotypes using PCR amplification of tail snip DNA with transgene-specific primers

• Cell culture models:

  • Transient transfection: Use lipid-based transfection reagents with expression vectors containing the LGALS7 cDNA sequence

  • Stable transfection: Select transfected cells with appropriate antibiotics to establish stable cell lines

  • siRNA/shRNA approaches: Knockdown LGALS7 expression using RNA interference technology

  • CRISPR/Cas9 gene editing: For precise modification of the LGALS7 gene or its regulatory elements

• Verification of expression changes:

  • Confirm altered expression at the mRNA level using RT-qPCR

  • Validate protein level changes using Western blot or immunofluorescence

  • Assess functional consequences through appropriate biological assays

These methodologies enable researchers to study the consequences of altered galectin-7 expression in various biological contexts.

What are the best approaches for studying galectin-7 protein interactions?

Several complementary approaches can be employed to study galectin-7 protein interactions:

• Proteomic analysis using iTRAQ:

  • Effective for identifying differentially expressed proteins associated with galectin-7 expression

  • Requires HPLC separation of peptides using gradient elution

  • Process samples in triplicate with a requirement that peptides be identified at least twice

  • Can reveal hundreds of potential interaction partners (1,009 differentially expressed proteins identified in one study)

• Co-immunoprecipitation (Co-IP):

  • Pull down galectin-7 using specific antibodies and identify binding partners

  • Can be performed in reverse to confirm interactions

  • Western blot analysis of precipitated complexes confirms specific interactions

• Proximity ligation assay (PLA):

  • Detects protein interactions in situ with high sensitivity and specificity

  • Particularly useful for confirming interactions in tissue samples

• Functional validation approaches:

  • Assess biological relevance of interactions through knockdown/overexpression studies

  • Examine co-localization using immunofluorescence microscopy

  • Analyze effects on downstream signaling pathways

• Bioinformatic analysis of interaction networks:

  • Use the STRING database to detect functional interactions between identified proteins

  • Apply gene ontology (GO) analysis to categorize interactions into biological processes, cellular components, or molecular functions

  • Pathway enrichment analysis using KEGG or Reactome databases helps identify key biological pathways affected

These methods can be combined to provide a comprehensive understanding of galectin-7's interactome and its functional implications.

What is the significance of galectin-7 in cancer research and potential therapeutic applications?

Galectin-7 has emerged as an important molecule in cancer research with dual roles as either tumor suppressor or promoter depending on the cancer type:

In ovarian cancer:

• Increases invasive behavior by inducing MMP-9 expression

• Enhances cell motility, contributing to metastatic potential

• May represent a potential therapeutic target to reduce tumor aggressiveness

In head and neck squamous cell carcinoma (HNSCC):

Potential therapeutic approaches targeting galectin-7:

• Small molecule inhibitors that block carbohydrate recognition domains

• Neutralizing antibodies that interfere with galectin-7 function

• Gene silencing strategies to reduce galectin-7 expression in tumors where it promotes progression

• Development of galectin-7-based biomarkers for early detection and treatment monitoring

Research in this field continues to evolve, with pre-clinical studies exploring these various approaches. The context-dependent nature of galectin-7 in different cancer types necessitates careful consideration when developing therapeutic strategies.

How do LGALS7 genetic variations contribute to stroke risk and what are the mechanistic implications?

The relationship between LGALS7 genetic variations and stroke risk represents an emerging area of research with significant clinical implications:

A genetic association study identified two critical SNPs in the LGALS7 promoter region (rs567785577 and rs138945880) significantly associated with increased risk of intracerebral hemorrhage (ICH):

• The A allele of rs567785577 and T allele of rs138945880 conferred an unadjusted odds ratio of 13.5 (95% CI = 2.249-146.5; p = 0.002)

• These SNPs are located on chromosome 19q13.2

• Traditional vascular disease risk factors (hypertension, diabetes, lipid disorders) were more common in stroke patients but did not account for the genetic association

Mechanistic implications:

• Altered galectin-7 expression may affect vascular integrity through:

  • Modulation of blood-brain barrier permeability via interaction with proteins like alpha-1-acid glycoprotein 2, which adds negative charges to the matrix component of the blood-brain barrier

  • Influence on functions of synthetic and contractile smooth muscle cells within cerebral vessel walls

  • Effects on transport proteins such as monocarboxylate transporter MCT1, affecting nutrient delivery

• Cerebrovascular accumulation of β-amyloid may disrupt blood flow within the brain, adversely affecting:

  • Vascular blood supply

  • Astrocyte function

  • Vascular endothelium

  • Associated nutrient transporters

These findings suggest potential for:

• Development of genetic screening tools to identify high-risk individuals

• Novel therapeutic targets focusing on galectin-7-associated pathways

• Personalized prevention strategies for hemorrhagic stroke

Further studies with expanded case numbers and diverse ethnic populations are needed to fully elucidate the mechanisms underlying these associations .

What is the role of galectin-7 in skin disorders and autoimmune conditions?

Galectin-7 plays significant roles in skin homeostasis and pathology, particularly in conditions like systemic sclerosis (SSc):

In normal skin:

• Abundantly expressed throughout the epidermis

• Contributes to normal keratinocyte differentiation and epidermal homeostasis

In systemic sclerosis (SSc):

• Remarkably downregulated in the basal and suprabasal layers of lesional epidermis of involved skin

• Patients with diffuse pigmentation and esophageal dysfunction show significantly decreased serum galectin-7 levels compared to those without these symptoms

• Suppression appears to be stimulated by autocrine endothelin signaling in SSc keratinocytes

Mechanistic implications:

• Altered galectin-7 levels may contribute to the fibrotic process characteristic of SSc

• Decreased galectin-7 may influence keratinocyte function and epithelial-mesenchymal interactions

• Changes in galectin-7 expression might affect immune cell function and inflammatory responses

• The protein may serve as a biomarker for specific SSc manifestations

Potential research and therapeutic directions:

• Development of serum galectin-7 as a biomarker for SSc progression and specific organ involvement

• Investigation of therapeutic approaches to restore normal galectin-7 levels in affected tissues

• Exploration of the relationship between endothelin signaling inhibitors and galectin-7 expression

• Further research into galectin-7's role in other autoimmune and inflammatory skin conditions

Understanding these mechanisms may lead to novel diagnostic and therapeutic approaches for SSc and potentially other skin and autoimmune disorders.

What are the optimal conditions for purification and storage of recombinant LGALS7-His?

Purification and storage of recombinant LGALS7-His requires careful attention to maintain protein integrity and activity:

Purification protocol:

• Expression system: E. coli-derived recombinant human Galectin-7 (Ser2-Phe136) is commonly used

• Immobilized Metal Affinity Chromatography (IMAC):

  • Use Ni-NTA or Co2+ resins for optimal His-tag binding

  • Apply imidazole gradient elution (typically 20-250 mM)

  • Buffer composition: 50 mM sodium phosphate, 300 mM NaCl, pH 7.4 with appropriate imidazole concentrations

• Additional purification steps:

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for higher purity if needed

Storage conditions:

• Short-term storage (1-2 weeks):

  • 4°C in sterile buffer containing mild preservatives

  • Buffer composition: PBS with 0.1% sodium azide

• Long-term storage:

  • -20°C or -80°C in aliquots to avoid freeze-thaw cycles

  • Addition of stabilizers: 20% glycerol or 5% trehalose

  • Avoid repeated freeze-thaw cycles which can lead to protein denaturation

Quality control:

• SDS-PAGE to confirm purity (should show a band at approximately 14 kDa)

• Western blot with anti-His or anti-galectin-7 antibodies

• Mass spectrometry to confirm protein identity

• Functional assays to verify carbohydrate-binding activity

Reconstitution after lyophilization:

• Use sterile water or appropriate buffer

• Gentle mixing rather than vortexing to avoid protein denaturation

• Allow complete reconstitution before use in experiments

Following these guidelines will ensure optimal protein quality for downstream applications.

How can researchers validate the functional activity of recombinant LGALS7-His?

Validating the functional activity of recombinant LGALS7-His is crucial for ensuring meaningful experimental results:

Carbohydrate binding assays:

• Solid-phase binding assays:

  • Coat plates with β-galactoside-containing glycoproteins or glycolipids

  • Measure binding of recombinant LGALS7-His using anti-His antibodies

  • Include appropriate controls (e.g., binding in the presence of lactose as a competitive inhibitor)

• Hemagglutination assay:

  • Use trypsinized rabbit erythrocytes or glutaraldehyde-fixed human erythrocytes

  • Determine the minimum concentration required for visible agglutination

  • Compare with a reference standard of known activity

Cellular functional assays:

• Apoptosis induction:

  • Treat appropriate cell lines (e.g., keratinocytes or epithelial cancer cells) with recombinant LGALS7-His

  • Measure apoptosis using Annexin V/PI staining and flow cytometry

  • Compare with untreated controls and positive apoptosis inducers

• Cell adhesion modulation:

  • Assess effects on cell-matrix or cell-cell adhesion

  • Compare with wild-type galectin-7 to ensure the His-tag doesn't interfere with function

Structural validation:

• Circular dichroism (CD) spectroscopy:

  • Confirm proper protein folding

  • Compare spectrum with native galectin-7

• Thermal shift assay:

  • Assess protein stability

  • Compare melting temperature with reference standards

Interaction validation:

• Pull-down assays to confirm binding to known galectin-7 interaction partners

• Surface plasmon resonance (SPR) to quantify binding kinetics

These complementary approaches provide comprehensive validation of recombinant LGALS7-His functionality, ensuring that experimental observations truly reflect the biological activities of galectin-7.

What are the key considerations when using LGALS7-His in different experimental systems?

When using LGALS7-His across various experimental systems, researchers should consider several critical factors:

In cell culture systems:

• Protein internalization:

  • Galectin-7 can be internalized by cells, affecting experimental interpretation

  • Monitor cellular uptake using fluorescently labeled protein

  • Consider timing in experiments as internalization may change over time

• Endotoxin contamination:

  • E. coli-derived recombinant proteins may contain endotoxins

  • Use endotoxin removal methods during purification

  • Test final preparations using LAL assay

  • Endotoxin contamination can confound results, particularly in immune cell experiments

• Concentration considerations:

  • Use dose-response experiments to determine optimal concentrations

  • Different cell types may have varying sensitivity to galectin-7

In animal models:

• Route of administration:

  • Consider bioavailability and tissue distribution

  • His-tagged proteins may have altered pharmacokinetics

  • Validate tissue penetration using immunohistochemistry

• Immunogenicity:

  • The His-tag may increase protein immunogenicity

  • Monitor for immune responses in long-term studies

  • Consider using tag-free protein for extended in vivo studies

In protein interaction studies:

• Tag interference:

  • The His-tag may interfere with some protein-protein interactions

  • Include controls with tag-free protein when possible

  • Consider using C-terminal vs. N-terminal tags depending on the known functional domains

• Oligomerization effects:

  • Galectin-7 can form dimers

  • Verify that His-tagging doesn't alter oligomerization behavior

  • Use size exclusion chromatography to assess oligomeric state

In all systems:

• Controls:

  • Include tag-only controls to distinguish tag effects from galectin-7 effects

  • Use heat-inactivated protein as a negative control

  • Include wild-type galectin-7 when possible for comparison

• Storage stability:

  • Verify protein activity after storage using functional assays

  • Avoid repeated freeze-thaw cycles