B4GALT2 Antibody

What is B4GALT2 and what role does it play in cellular functions?

B4GALT2 (Beta-1,4-galactosyltransferase 2) is an enzyme encoded by the B4GALT2 gene in humans. It belongs to a family of seven beta-1,4-galactosyltransferase (beta4GalT) genes that encode type II membrane-bound glycoproteins. These proteins have exclusive specificity for the donor substrate UDP-galactose, transferring galactose in a beta1,4 linkage to acceptor sugars including GlcNAc, Glc, and Xyl .

As a type II membrane protein, B4GALT2 possesses an N-terminal hydrophobic signal sequence that directs the protein to the Golgi apparatus where it remains uncleaved, functioning as a transmembrane anchor. The enzyme specifically synthesizes N-acetyllactosamine in glycolipids and glycoproteins . While its substrate specificity can be affected by alpha-lactalbumin, B4GALT2 is not expressed in lactating mammary tissue .

By sequence similarity, the beta4GalTs form four distinct groups:

• beta4GalT1 and beta4GalT2

• beta4GalT3 and beta4GalT4

• beta4GalT5 and beta4GalT6

• beta4GalT7

Each beta4GalT has a specific function in the biosynthesis of different glycoconjugates and saccharide structures .

What are the common applications for B4GALT2 antibodies in research?

B4GALT2 antibodies are utilized across several experimental applications, with the following being most common:

B4GALT2 antibodies have been successfully validated in multiple tissue and cell types, including human colon cancer tissue, human ovary tissue, PC-3 cells, mouse brain tissue, mouse large intestine tissue, mouse ovary tissue, and mouse skeletal muscle tissue .

What is the molecular profile of B4GALT2?

B4GALT2 has the following molecular characteristics:

• Full Name: UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 2

• Calculated Molecular Weight: 372 amino acids, 42 kDa

• Observed Molecular Weight: 42-45 kDa

• GenBank Accession Number: BC002431

• Gene Symbol: B4GALT2

• Gene ID (NCBI): 8704

• UNIPROT ID: O60909

The protein has several aliases including B4Gal-T2, beta4Gal-T2, Beta-1,4-galactosyltransferase 2, and N-acetyllactosamine synthase, among others .

What are the optimal conditions for Western blot analysis of B4GALT2?

For optimal Western blot (WB) results when working with B4GALT2 antibodies, researchers should consider the following protocol parameters:

• Antibody Dilution: Use a dilution range of 1:1000-1:4000 for polyclonal antibodies such as 20330-1-AP . Alternative antibodies may require 0.04-0.4 μg/mL concentrations .

• Sample Selection: Validated positive controls include:

  • PC-3 cells

  • Mouse brain tissue

  • Mouse large intestine tissue

  • Mouse ovary tissue

  • Mouse skeletal muscle tissue

• Expected Band Size: Look for bands at 42-45 kDa, which corresponds to the observed molecular weight of B4GALT2 .

• Buffer Conditions: Store antibodies in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 for optimal stability .

• Sample-Dependent Optimization: Titration is recommended in each testing system to obtain optimal results, as signal strength can vary between tissue types .

For detailed procedure, manufacturers typically provide specific WB protocols optimized for their B4GALT2 antibodies that should be followed for best results .

How should researchers perform immunohistochemistry with B4GALT2 antibodies?

For successful immunohistochemistry (IHC) with B4GALT2 antibodies, follow these methodological considerations:

• Antibody Dilution: Use a dilution range of 1:20-1:200 for optimal staining with minimal background .

• Antigen Retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Validated Positive Controls:

  • Human colon cancer tissue

  • Human ovary tissue

• Detection System: Choose a detection system appropriate for the host species of the primary antibody (typically rabbit IgG for many B4GALT2 antibodies) .

• Counterstaining: Standard nuclear counterstains like hematoxylin are generally compatible with B4GALT2 antibody staining.

• Storage Conditions: Store antibody aliquots at -20°C for long-term stability. Aliquoting is unnecessary for -20°C storage for many commercial preparations .

For comprehensive staining protocols, researchers should refer to manufacturer-provided IHC protocols specific to the B4GALT2 antibody being used .

What controls should be included when working with B4GALT2 antibodies?

Proper experimental controls are essential when working with B4GALT2 antibodies to ensure validity and reliability of results:

• Positive Tissue Controls:

  • For WB: PC-3 cells, mouse brain tissue, mouse large intestine tissue, mouse ovary tissue, mouse skeletal muscle tissue

  • For IHC: Human colon cancer tissue, human ovary tissue

• Negative Controls:

  • Isotype controls using non-specific IgG from the same host species

  • Secondary antibody-only controls to assess non-specific binding

  • Tissues known to have low or no B4GALT2 expression

• Specificity Controls:

  • Peptide competition/neutralization assays using the immunogen sequence

  • For the B4GALT2 antibody 20330-1-AP, the immunogen sequence is: "PGVLMGGRY TPPDCTPAQT VAVIIPFRHR EHHLRYWLHY LHPILRRQRL RYGVYVINQH GEDTFNRAKL LNVGFLEALK EDAAYDCFIF SDVDLVPMDD RNLYRCGDQP RHFAIAMD" (60-176 aa encoded by BC002431)

  • For PA5-61580, the immunogen sequence is: "LPPCPDSPPG LVGRLLIEFT SPMPLERVQR ENPGVLMGGR YTPPDCTPAQ TVAVIIPFRH REH"

• Knockdown/Knockout Controls:

  • When possible, include samples with verified B4GALT2 knockdown or knockout to confirm antibody specificity, particularly in functional studies investigating B4GALT2's role in cancer progression

• Cross-Reactivity Assessment:

  • For antibodies claiming multi-species reactivity, validate using samples from each species to confirm expected patterns of reactivity

How is B4GALT2 implicated in cancer biology, particularly in lung adenocarcinoma?

Recent research has revealed significant roles for B4GALT2 in cancer progression, most notably in lung adenocarcinoma (LUAD):

• Prognostic Marker: B4GALT2 has been identified as a central component of the post-translational modification learning signature (PTMLS) with a strong correlation (r=0.82, p<0.05). Elevated B4GALT2 expression consistently predicts poor survival across multiple cohorts (HR=1.62, 95% CI 1.36 to 1.92, log-rank p=3.9e-08) .

• Immune Exclusion Role: B4GALT2 has been implicated in immune exclusion mechanisms in the tumor microenvironment. Analysis revealed distinct immune infiltration patterns associated with B4GALT2 expression, showing negative correlations with immune and ESTIMATE scores but positive associations with tumor purity .

• Relationship with CD8+ T Cells: Clinical correlation studies demonstrated inverse relationships between B4GALT2 levels and CD8A expression (LUAD-TCGA: R=−0.25, p=1.4e-05; LUAD-Atezo: R=−0.2, p=3.2e-06). Multiplex immunofluorescence experiments confirmed spatial co-localization and exclusion relationships between B4GALT2 and CD8+ T cells and CD20+ B cells .

• Functional Studies: Knockdown of B4GALT2 in LUAD cell lines (e.g., A549 and H1299) significantly impaired cell proliferation, validated both in vitro and in vivo. Flow cytometry experiments revealed that B4GALT2 inhibition not only increased the quantity of CD8+ T cells but also enhanced their activity, augmenting antitumor immune responses during anti-PD-1 therapy .

• Immunotherapy Implications: B4GALT2 inhibition showed enhanced efficacy when combined with anti-PD-1 therapy, characterized by a decrease in CD62L+ CD8 T cells and an increase in GZMB+/CD44+/CD69+CD8 T cells, suggesting a potential strategy for improving immunotherapy outcomes in LUAD patients .

These findings establish B4GALT2 as both a novel prognostic marker and a potential therapeutic target in LUAD treatment strategies, particularly in the context of immunotherapy optimization .

What genetic variants of B4GALT2 have clinical significance?

Research has identified specific B4GALT2 genetic variants with clinical significance, particularly in the context of platelet reactivity and drug response:

• c.909C>T Variant (rs1061781):

  • This synonymous variant (p.Ile303=) in B4GALT2 showed significant association with platelet response after Bonferroni correction (p<0.003) .

  • In multivariate analysis, B4GALT2 c.909C>T remained an independent genetic predictor of on-treatment platelet reactivity (p=0.03) along with CYP2C19 loss-of-function alleles (p=0.01) .

  • This association remained significant even after inclusion of relevant clinical variables in the regression model (adjusted R²=0.09) .

• c.366G>C Variant (rs1859728):

  • This variant was also tested in association studies with platelet reactivity .

  • In univariate analysis, it showed association with platelet response, but did not remain significant in the multivariate model .

• Functional Implications:

  • The exact mechanism by which these B4GALT2 variants influence platelet function remains to be fully elucidated.

  • The variants may affect glycosylation processes critical for platelet membrane protein function and drug-receptor interactions.

  • These variants could potentially serve as biomarkers for predicting response to antiplatelet therapies in cardiovascular patients .

This genetic evidence suggests that B4GALT2 plays an important role in platelet function, potentially through its glycosylation activity, and certain genetic variants may influence therapeutic outcomes in patients receiving antiplatelet therapy .

What are the challenges and considerations in B4GALT2 knockdown experiments?

When designing and conducting B4GALT2 knockdown experiments, researchers should consider several challenges and methodological aspects:

• Target Validation:

  • Validate knockdown efficiency using multiple methods (Western blot, qPCR) to confirm reduction in both protein and mRNA levels .

  • Use appropriate B4GALT2 antibodies with validated specificity for Western blot confirmation (recommended dilution 1:1000-1:4000) .

• Model System Selection:

  • Cell line selection is critical: A549 and H1299 lung adenocarcinoma cell lines have been successfully used in B4GALT2 knockdown studies .

  • For in vivo experiments, consider mouse models with human tumor xenografts to study B4GALT2's role in tumor growth and immune interactions .

• Immunological Assessment:

  • When studying B4GALT2's role in immune regulation, include comprehensive immune profiling using flow cytometry to assess changes in immune cell populations after knockdown .

  • Key markers to analyze include CD8+ T cell quantities and activation markers (CD44+, CD69+, GZMB+) .

• Functional Readouts:

  • Cell proliferation assays are essential to determine the oncogenic properties of B4GALT2 .

  • When combined with immunotherapy (e.g., anti-PD-1), assess both tumor growth and immune infiltration to comprehensively evaluate therapeutic potential .

• Mechanism Investigation:

  • Consider both immune-modulatory effects and glycosylation-related mechanisms when interpreting B4GALT2 knockdown results .

  • The dual role of B4GALT2 (glycosylation function and immune modulation) presents a complex experimental scenario requiring careful controls and multifaceted analysis .

• Off-target Effects:

  • Use multiple knockdown methods (e.g., different siRNA/shRNA sequences) to mitigate potential off-target effects.

  • Include rescue experiments with wild-type B4GALT2 expression to confirm phenotype specificity.

Research investigating B4GALT2 in cancer models should consider both its enzymatic function in glycosylation and its apparent role in immune regulation, requiring comprehensive experimental design and analysis .

How do post-translational modifications related to B4GALT2 affect cancer immunotherapy response?

The relationship between B4GALT2-mediated post-translational modifications and cancer immunotherapy response represents an emerging area of research with significant clinical implications:

• PTMLS Framework: B4GALT2 has been identified as a key component of the post-translational modification learning signature (PTMLS), with a strong correlation coefficient of r=0.82 (p<0.05). This signature effectively predicts immunotherapy response across various cancer types .

• Immunotherapy Prediction: High PTMLS scores (associated with high B4GALT2 expression) correlate with lower immune activity and a "cold tumor" phenotype, generally predicting poorer responses to immunotherapy. This pattern was validated across 12 immunotherapy cohorts spanning multiple cancer types (n=1201) .

• Mechanism of Immune Regulation:

  • B4GALT2's glycosylation function may modify surface receptors and ligands on both tumor and immune cells.

  • Elevated B4GALT2 expression is associated with immune exclusion, characterized by reduced CD8+ T cell infiltration and function .

  • Flow cytometry analysis revealed that B4GALT2 inhibition decreased CD62L+ CD8 T cells (naive phenotype) and increased GZMB+/CD44+/CD69+CD8 T cells (activated phenotype), enhancing antitumor immunity .

• Therapeutic Implications:

  • B4GALT2 knockdown experiments demonstrated enhanced efficacy of anti-PD-1 immunotherapy in animal models .

  • This suggests B4GALT2 inhibition may represent a strategy to convert "cold" tumors to "hot" tumors, potentially sensitizing previously resistant patients to immunotherapy .

  • The combined approach of B4GALT2 targeting with immunotherapy represents a promising therapeutic strategy .

• Biomarker Potential: B4GALT2 expression levels could serve as biomarkers for patient stratification in immunotherapy trials, identifying patients who might benefit from combination strategies targeting both B4GALT2 and immune checkpoints .

The link between B4GALT2-mediated glycosylation and immunotherapy response opens new avenues for improving cancer treatment, with potential applications across multiple tumor types beyond lung adenocarcinoma .

What are the storage and handling recommendations for B4GALT2 antibodies?

Proper storage and handling of B4GALT2 antibodies is crucial for maintaining their functionality and specificity:

Following these handling guidelines will help ensure optimal antibody performance and reproducible experimental results when working with B4GALT2 antibodies .

How should researchers troubleshoot non-specific binding with B4GALT2 antibodies?

When encountering non-specific binding issues with B4GALT2 antibodies, researchers should systematically address the problem using the following approaches:

• Optimize Antibody Dilution:

  • Start with manufacturer's recommended dilution ranges (e.g., 1:1000-1:4000 for WB; 1:20-1:200 for IHC) .

  • Perform a dilution series to identify optimal concentration balancing specific signal and background.

• Blocking Optimization:

  • Evaluate different blocking agents (BSA, normal serum, commercial blocking buffers).

  • Increase blocking time or concentration if background remains high.

  • Ensure blocking agent is from a species different from the primary and secondary antibody sources.

• Antigen Retrieval Adjustments (for IHC):

  • Try both recommended antigen retrieval methods: TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative) .

  • Adjust retrieval time and temperature if needed.

• Washing Procedure Enhancements:

  • Increase number and duration of wash steps.

  • Use fresh buffers with appropriate detergent concentration.

  • Ensure complete buffer removal between different antibody applications.

• Secondary Antibody Considerations:

  • Ensure secondary antibody is appropriate for the host species of the B4GALT2 antibody.

  • Pre-adsorb secondary antibody against tissue lysates if cross-reactivity is suspected.

  • Run secondary-only controls to identify potential direct binding issues.

• Sample Preparation Factors:

  • Ensure proper tissue fixation and processing.

  • For Western blots, optimize protein extraction and denaturation conditions.

  • Consider using fresh tissue samples if applicable.

• Validation Controls:

  • Use peptide competition assays with the immunogen sequence to confirm specificity.

  • Include known positive controls (e.g., PC-3 cells, mouse brain tissue for WB; human colon cancer tissue for IHC) .

  • Consider B4GALT2 knockdown samples as negative controls where available.

By systematically addressing these factors, researchers can significantly improve specificity and reduce non-specific binding when working with B4GALT2 antibodies.

What are emerging applications of B4GALT2 in cancer research and immunotherapy?

The evolving understanding of B4GALT2's role in cancer biology suggests several promising research directions:

• Combinatorial Immunotherapy Approaches:

  • Investigating synergistic effects between B4GALT2 inhibition and established checkpoint inhibitors (anti-PD-1, anti-CTLA-4) .

  • Exploring triple combination therapies adding targeted agents to B4GALT2 inhibition and immunotherapy.

• Biomarker Development:

  • Validating B4GALT2 expression as a predictive biomarker for immunotherapy response across multiple cancer types beyond LUAD .

  • Developing clinical assays for B4GALT2 assessment in patient samples to guide treatment decisions.

• Mechanistic Investigations:

  • Elucidating the precise glycosylation-related mechanisms through which B4GALT2 influences immune cell function and tumor progression .

  • Identifying specific glycoprotein targets modified by B4GALT2 that mediate immune exclusion.

• Therapeutic Development:

  • Designing specific small molecule inhibitors or biologics targeting B4GALT2 enzymatic activity.

  • Exploring RNA interference or CRISPR-based approaches for B4GALT2 inhibition in clinical applications.

• Expanding Cancer Indications:

  • Investigating B4GALT2's role in other "cold" tumor types beyond lung adenocarcinoma.

  • Determining cancer-specific patterns of B4GALT2 expression and function across diverse tumor types.

• Immune Microenvironment Dynamics:

  • Characterizing temporal changes in immune cell populations and function following B4GALT2 modulation.

  • Developing spatial transcriptomics and proteomics approaches to map B4GALT2 influence on the tumor immune microenvironment .

• Clinical Translation:

  • Establishing multicenter clinical validation of PTMLS and B4GALT2 as predictive markers for immunotherapy response .

  • Designing early-phase clinical trials combining B4GALT2 targeting with existing immunotherapies.

These research directions hold potential to transform B4GALT2 from a novel biomarker to a therapeutic target with significant impact on cancer immunotherapy outcomes .

How does B4GALT2's function in glycosylation impact broader cellular processes?

The glycosylation function of B4GALT2 has far-reaching implications beyond its direct enzymatic activity, affecting multiple cellular processes:

• Protein Structure and Function Modification:

  • B4GALT2 transfers galactose in a beta1,4 linkage to acceptor sugars (GlcNAc, Glc, and Xyl), creating N-acetyllactosamine in glycolipids and glycoproteins .

  • These modifications can alter protein folding, stability, and functional properties of cell surface and secreted proteins.

• Cell Signaling Pathway Modulation:

  • Glycosylation of receptor tyrosine kinases and other signaling proteins can modulate their activation, clustering, and downstream signaling.

  • B4GALT2-mediated modifications may influence cancer-related signaling networks, potentially explaining its association with tumor progression .

• Cell-Cell and Cell-Matrix Interactions:

  • Altered glycosylation patterns impact adhesion molecule function and extracellular matrix interactions.

  • In cancer contexts, these changes may facilitate invasion, metastasis, and immune evasion .

• Immune Recognition Mechanisms:

  • Glycan structures serve as recognition elements for immune cells through lectins and other pattern recognition receptors.

  • B4GALT2-dependent glycosylation may create patterns that inhibit anti-tumor immune responses, explaining the observed immune exclusion phenotype .

• Drug Response Modulation:

  • Glycosylation can affect drug binding, uptake, and metabolism, potentially explaining B4GALT2 variants' influence on clopidogrel response .

  • Modified glycans on therapeutic targets may alter binding of targeted therapies and antibody-based drugs.

• Cross-talk with Other Post-translational Modifications:

  • Glycosylation can influence or be influenced by other modifications like phosphorylation, ubiquitination, and acetylation.

  • This creates complex regulatory networks impacting protein fate and function.

• Therapeutic Targeting Considerations:

  • Understanding B4GALT2's broader impact informs approaches to targeting its activity.

  • Potential for developing glycomimetics or engineered glycans to modulate B4GALT2-dependent processes.