Recombinant Human UDP-GalNAc:beta-1,3-N-acetylgalactosaminyltransferase 1 (B3GALNT1), partial

What is the genomic organization and expression pattern of B3GALNT1?

B3GALNT1 is located on chromosome 3q26.1 and spans positions 161,083,883 to 161,105,349 on the negative strand. The gene contains 10 exons with the protein coding sequence historically considered to be contained within a single exon . B3GALNT1 belongs to the beta-1,3-galactosyltransferase (beta3GalT) gene family, which encodes type II membrane-bound glycoproteins .

Expression analysis reveals interesting tissue-specific patterns. Unlike some related genes, B3GALNT1 shows differential expression patterns in peripheral blood versus bone marrow. This tissue-specific regulation suggests complex transcriptional control mechanisms that may involve enhancer elements and tissue-specific transcription factors .

When investigating expression patterns, researchers should consider:

• RT-PCR analysis of different tissue samples

• RNA-seq for quantitative expression profiling

• Western blot confirmation of protein-level expression

• Immunohistochemistry for spatial distribution in tissues

What is the enzymatic function and specificity of B3GALNT1?

B3GALNT1 functions as a UDP-GalNAc:beta-1,3-N-acetylgalactosaminyltransferase that catalyzes the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc onto globotriaosylceramide (Gb3) to form globoside (Gb4) . This enzymatic activity is critical for the biosynthesis of P blood group antigens.

Unlike other beta3GalT family members, B3GALNT1 does not use N-acetylglucosamine as an acceptor sugar . The enzyme belongs to the glycosyltransferase 31 family (EC 2.4.1.79) and is characterized as a metal-dependent transferase .

For experimental validation of enzymatic activity, researchers should:

• Express recombinant enzyme in appropriate mammalian expression systems

• Purify using affinity chromatography (typically His-tagged constructs)

• Perform activity assays using UDP-GalNAc as donor and globotriaosylceramide as acceptor

• Analyze products using HPLC/MS techniques to confirm correct glycan structure formation

How do structural features of the catalytic domain influence B3GALNT1 function?

While the specific crystal structure of B3GALNT1 hasn't been fully characterized, insights can be drawn from related glycosyltransferases. These enzymes typically adopt a GT-A fold characterized by a central eight-stranded β-sheet flanked by α-helices, as observed in the UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase structure .

For B3GALNT1 and related enzymes, disulfide bonds are critical for structural integrity and catalytic function. Mutagenesis studies of conserved cysteine residues in related enzymes demonstrate that these disulfides are essential for proper folding and activity .

Of particular interest is the unusual conformation that the GalNAc moiety can adopt in the binding pocket, which differs from the canonical "tucked under" conformation observed for UDP-Gal in related enzymes . This flexibility in substrate conformation may be important for the dual specificity observed in some related glycosyltransferases.

For structural studies, researchers should consider:

What approaches can be used to study the conformational dynamics of UDP-GalNAc binding to B3GALNT1?

Studying conformational dynamics requires a combination of structural and biophysical techniques. Research on related glycosyltransferases provides valuable methodological approaches :

• Synthetic UDP-GalNAc derivatives: Modifications at the C5 position of the GalNAc moiety can create derivatives with altered binding properties that are more amenable to crystallization. Compounds like 5-(5-formylthien-2-yl)-UDP-α-d-GalNAc have been successfully used in structural studies of related enzymes .

• NMR spectroscopy: This technique can capture the dynamic nature of sugar conformations in the binding pocket. For B3GALNT1, deuterium labeling of specific positions in UDP-GalNAc would allow tracking of conformational changes during binding.

• Enzyme kinetics with modified substrates: Comparing kinetic parameters (KM, kcat) for different UDP-GalNAc derivatives can provide insights into how substrate conformation affects catalysis. This approach has revealed that seemingly similar derivatives can exhibit vastly different kinetic properties .

The research by Jørgensen et al. demonstrated that UDP-GalNAc can adopt a catalytically productive conformation different from the "tucked under" conformation, suggesting that B3GALNT1 might accommodate different substrate conformations during catalysis .

How do mutations in B3GALNT1 affect blood group phenotypes and reproductive health?

Inactivating mutations in B3GALNT1 give rise to the rare Pk phenotype, which lacks P and PX2 antigens . The P antigen is carried by globoside (Gb4), an abundant glycosphingolipid in the red blood cell membrane, and its absence leads to the production of naturally occurring anti-P antibodies.

A striking clinical case involved two Thai sisters with a homozygous nonsense mutation (c.420T>G) in B3GALNT1, which introduces a premature stop codon (p.Tyr140Ter) . This mutation completely abolished enzyme activity and resulted in:

• The rare Pk blood group phenotype

• Production of anti-P and anti-PX2 antibodies

• Multiple spontaneous abortions (8 and 11 miscarriages, respectively)

The mechanism linking B3GALNT1 mutations to reproductive failure involves the cytotoxic attack of anti-P antibodies on the globoside-rich fetal portion of the placenta . Screening of 384 Thai donors indicated an allele frequency of 0.13% for this mutation .

For clinical research, the following approaches are recommended:

• Hemagglutination and flow cytometry for phenotyping

• Sequencing of B3GALNT1 coding regions in cases of unexplained recurrent miscarriage

• Development of allele-specific PCR for rapid genotyping of known mutations

• Counseling and management strategies for affected individuals

What is the relationship between B3GALNT1 and the P1PK blood group system?

B3GALNT1 plays a crucial role in the synthesis of antigens in the P1PK blood group system. The relationship between the P and Pk antigens has been clarified through transcriptional and genetic studies .

The P1PK blood group system involves three key antigens:

• Pk: The precursor structure, present in all individuals except those with rare inactivating mutations

• P: Synthesized by B3GALNT1 adding GalNAc to the Pk structure

• P1: A related antigen with variable expression

A previously unrecognized polymorphic A4GALT exon was found to be linked to the P1/P2 polymorphism, establishing the long-suspected connection between P1 and Pk antigens . The P2 allele was shown to lower A4GALT transcript levels and reduce both P1 and Pk antigen expression .

Researchers investigating blood group polymorphisms should:

• Use both serological and molecular methods for comprehensive phenotyping

• Consider the impact of regulatory region variations on gene expression

• Investigate potential linkage disequilibrium between related blood group genes

• Understand population-specific allele frequencies when designing genetic studies

How is B3GALNT1 implicated in cancer pathways and what experimental evidence supports this connection?

B3GALNT1 has been identified as a potential therapy target gene for non-small cell lung cancer (NSCLC) based on integrated analysis of gene expression and promoter methylation patterns . Principal component analysis-based unsupervised feature extraction identified B3GALNT1 as one of eleven critical genes potentially mediating NSCLC metastasis.

Disease association analysis using the Gendoo server revealed specific cancer connections:

The methodology for identifying these associations involved:

• Integrated analysis of gene expression and promoter methylation patterns of aggressive NSCLC cell lines

• Principal component analysis (PCA) to identify genes with differential patterns

• Selection of genes showing correlation between expression and methylation

• Validation through disease association databases

Researchers established B3GALNT1 as a candidate for targeted inhibition strategies, suggesting that compounds that inhibit its gene expression could have therapeutic potential in NSCLC .

What experimental approaches should be used to validate B3GALNT1 as a therapeutic target?

To validate B3GALNT1 as a therapeutic target, researchers should employ a multi-faceted approach:

• Functional validation in cell models:

  • CRISPR/Cas9 knockout studies to assess the impact on cancer cell phenotypes

  • siRNA/shRNA knockdown with rescue experiments using wild-type and mutant constructs

  • Overexpression studies to determine if increased expression promotes oncogenic properties

• Enzymatic inhibition strategies:

  • Development of specific B3GALNT1 inhibitors based on UDP-GalNAc derivatives

  • Structure-activity relationship studies to optimize inhibitor potency and selectivity

  • Cell-based assays to evaluate the impact of enzymatic inhibition on cancer cell behavior

• Glycosphingolipid profiling:

  • Mass spectrometry-based analysis of glycosphingolipid patterns in normal vs. cancer cells

  • Correlation of glycosphingolipid profiles with B3GALNT1 expression levels

  • Functional studies to determine how altered glycosphingolipid patterns affect cell signaling

• Validation in animal models:

  • Generation of conditional B3GALNT1 knockout mice to study tumor development and progression

  • Xenograft models with B3GALNT1-manipulated cancer cell lines

  • Testing of B3GALNT1 inhibitors in appropriate animal models of cancer

The identification of B3GALNT1 as a potential therapy target gene provides a starting point for developing novel therapeutic strategies for NSCLC and potentially other cancers .

What mechanisms control the tissue-specific expression of B3GALNT1?

The transcriptional regulation of B3GALNT1 involves complex mechanisms that result in tissue-specific expression patterns. Studies of related blood group glycosyltransferase genes provide insights into potential regulatory mechanisms .

Key aspects of transcriptional regulation include:

• Allele-specific regulatory elements: Sequence analysis of up- and downstream regions of B3GALNT1 may reveal allele-specific motifs that influence transcription, similar to what has been observed for ABO alleles .

• Tissue-specific transcription factors: Different transcription factor binding patterns likely contribute to the differential expression observed between peripheral blood and bone marrow samples.

• Epigenetic regulation: Promoter methylation patterns have been shown to correlate with B3GALNT1 expression in cancer contexts, suggesting epigenetic control . Integrated analysis of gene expression and promoter methylation patterns has revealed significant correlations for B3GALNT1 in the context of cancer.

• Enhancer regions: Similar to other glycosyltransferase genes, B3GALNT1 expression may be regulated by enhancer elements. Studies of ABO have shown that minisatellite elements in the enhancer region can affect transcription, although the correlation is not always straightforward .

Research approaches should include:

• ChIP-seq for identifying transcription factor binding sites

• ATAC-seq for chromatin accessibility analysis

• Bisulfite sequencing for methylation profiling

• Reporter gene assays to validate enhancer/promoter function

How do epigenetic modifications impact B3GALNT1 expression in different disease contexts?

Epigenetic modifications play a crucial role in regulating B3GALNT1 expression, particularly in disease states. Research on NSCLC has demonstrated a relationship between promoter methylation and gene expression for B3GALNT1 .

In cancer contexts, principal component analysis (PCA) of gene expression and promoter methylation data has identified B3GALNT1 as a gene with coordinated changes in both parameters . This suggests that altered methylation patterns may drive changes in B3GALNT1 expression during cancer progression.

Methodological approaches for studying epigenetic regulation include:

• Genome-wide methylation analysis:

  • Illumina methylation arrays for broad coverage

  • Targeted bisulfite sequencing for detailed promoter analysis

  • Single-cell methylation analysis for heterogeneity assessment

• Chromatin modification profiling:

  • ChIP-seq for histone modifications associated with active/repressed chromatin

  • CUT&RUN or CUT&Tag for more efficient profiling

  • HiChIP for integrating 3D genome organization with epigenetic marks

• Functional validation:

  • DNA methyltransferase inhibitors to reverse hypermethylation

  • CRISPR-based epigenetic editing to target specific regions

  • Reporter assays with methylated vs. unmethylated constructs

• Clinical correlation:

  • Integration of methylation data with patient outcomes

  • Identification of methylation signatures as biomarkers

  • Development of epigenetic therapies targeting B3GALNT1 regulation

Understanding the epigenetic regulation of B3GALNT1 could provide opportunities for developing novel diagnostic markers and therapeutic approaches in cancer and other diseases.