B3GNT2 Human

What is B3GNT2 and what is its primary function in human cells?

B3GNT2 is a Golgi-resident glycosyltransferase that catalyzes a key step in the elongation of poly-N-acetyl-lactosamine chains. It specifically transfers N-acetylglucosamine (GlcNAc) to galactose in an N-acetyl-lactosamine repeat via a β1-3 linkage in a divalent metal-dependent manner. B3GNT2 works in alternating fashion with β-1,4-galactosyltransferase (B4GALT) to build the polylactosamine chain: B4GALT adds galactose via β1-4 linkages, while B3GNT2 adds GlcNAc via β1-3 linkages .

How does the structure of B3GNT2 relate to its enzymatic function?

B3GNT2 contains two main domains: a catalytic domain responsible for substrate binding and catalysis, and a novel N-terminal helical domain. The catalytic domain houses the active site where donor (UDP-GlcNAc) and acceptor (typically LacNAc) substrates bind. Crystal structures have revealed that B3GNT2 follows a sequential mechanism for transglycosylation. Key residues involved in metal coordination (D247, H376), substrate binding (K149, D245, Y289, D332), and catalysis (D333, the active site base) are essential for activity. The N-terminal helical domain is hypothesized to stabilize the catalytic domain and may play a role in distinguishing between different glycan acceptors .

How does B3GNT2 differ from other B3GNT family members?

Among the seven identified B3GNTs (B3GNT2-B3GNT8), B3GNT2 demonstrates the strongest polylactosamine synthesizing activity. While B3GNT2, B3GNT3, B3GNT4, and B3GNT8 are all involved in polylactosamine chain elongation, they differ in activity levels and tissue expression. Sequence alignment shows that the active site base Asp333 and other residues involved in substrate recognition are conserved among B3GNT2, B3GNT3, and B3GNT4, but less conserved in B3GNT8. For example, B3GNT8 has Gln instead of Asp245, Phe instead of Tyr289, and Arg instead of His376, which may explain why B3GNT8 is 20-fold less active than B3GNT2 .

What are the established methods for measuring B3GNT2 enzymatic activity?

B3GNT2 activity can be assessed through several approaches:

• In vitro enzymatic assays: Using purified B3GNT2 enzyme with UDP-GlcNAc as donor and various oligosaccharide substrates (LacNAc-PNP or Lactose-PNP). The reaction products can be analyzed using high-performance liquid chromatography (HPLC) or mass spectrometry.

• Cell-based polylactosamine detection: Flow cytometry using LEA lectin (from Lycopersicon esculentum), which specifically binds to polylactosamine structures, can quantify cell surface polylactosamine levels. This approach has been validated in studies comparing wildtype and B3GNT2 knockout cells, with CRISPR-mediated deletion of B3GNT2 resulting in significant decreases in LEA binding .

• Functional rescue experiments: Reconstituting B3GNT2-deficient cells with wildtype or mutant B3GNT2 and measuring restoration of polylactosamine levels can assess the functional significance of specific residues or domains .

What genetic approaches are used to study B3GNT2 function in cellular and animal models?

Several genetic tools have been employed to investigate B3GNT2 function:

• CRISPR-Cas9 gene editing: Used to generate B3GNT2 knockout cell lines, enabling the study of cellular phenotypes in the absence of B3GNT2. This approach has been utilized to demonstrate the essential role of B3GNT2 in polylactosamine synthesis in various cell types, including Jurkat cells .

• B3GNT2 knockout mice: These models have shown dramatic reduction of polylactosamine on glycoproteins in tissues and cells of the immune system, despite the presence of N-glycan core structures. Immunocytes from these mice are hypersensitive and hyperresponsive to stimulation, confirming the inhibitory effect of polylactosamine on excessive immune responses .

• CRISPR activation screens: Genome-scale CRISPR activation screens have identified B3GNT2 as a gene that, when overexpressed, enables cancer cells to evade cytotoxic T cell killing. This approach has revealed B3GNT2 as a potential mediator of immune evasion in cancer .

What advanced structural biology techniques have been applied to study B3GNT2?

X-ray crystallography has been the principal method for elucidating B3GNT2 structure. Five crystal structures of the luminal domain of human B3GNT2 have been determined at resolutions ranging from 1.85 to 2.35 Å, capturing the enzyme in different states:

• Unliganded (apo) state (2.35 Å)

• Donor substrate-bound state with UDP-GlcNAc (2.26 Å)

• Acceptor substrate-bound state with LacNAc (2.19 Å)

• Product-bound state with UDP (1.85 Å)

• Dual product-bound state with UDP and trisaccharide GlcNAc β1-3Gal β1-4GlcNAc (2.20 Å)

These structures have provided critical insights into the catalytic mechanism, substrate recognition, and the role of specific residues in enzyme function. The structures have guided the design of B3GNT2 inhibitors through structure-based drug design approaches .

How does B3GNT2-mediated polylactosamine synthesis affect immune cell function?

B3GNT2-synthesized polylactosamine chains play a crucial role in modulating immune responses:

• Suppression of excessive immune responses: Long polylactosamine chains on bi-, tri-, and tetraantennary N-glycans (as well as O-glycans) have been demonstrated to suppress excessive immune responses. The B3GNT2 gene is widely expressed in mice with high-level expression detected in the immune system .

• Regulation of immunocyte sensitivity: B3GNT2 knockout mice studies have shown that immunocytes with diminished surface polylactosamine on N-glycan are hypersensitive and hyperresponsive to stimulation. This indicates that polylactosamine has an inhibitory effect on excessive immune responses, with B3GNT2 playing a crucial role as the major polylactosamine synthase .

• T cell-tumor cell interactions: B3GNT2 has been identified as targeting more than 10 ligands and receptors to disrupt interactions between tumor and T cells, reducing T cell activation. This mechanism contributes to cancer cells' ability to evade immune surveillance .

What is the relationship between B3GNT2 and autoimmune diseases?

Genome-wide association studies have revealed connections between B3GNT2 and several autoimmune conditions:

• Rheumatoid arthritis: Single nucleotide polymorphisms (SNPs) reducing B3GNT2 expression are associated with rheumatoid arthritis in Japanese populations .

• Ankylosing spondylitis: SNPs affecting B3GNT2 expression have been linked to ankylosing spondylitis in people of European descent .

• Psoriasis: B3GNT2-related genetic variations have been associated with psoriasis in European populations .

These associations align with the functional studies showing that reduced polylactosamine synthesis due to diminished B3GNT2 activity can lead to hyperresponsive immune cells, potentially contributing to autoimmune pathology .

How do changes in B3GNT2 expression affect immune surveillance in cancer?

B3GNT2 has emerged as a significant factor in cancer immune evasion:

• CRISPR activation screen identification: Genome-scale CRISPR activation screens have identified B3GNT2 as one of four top candidate genes (along with CD274/PD-L1, MCL1, and JUNB) that, when overexpressed, confer resistance to cytotoxic T cell killing in diverse cancer cell types and mouse xenografts .

• Mechanism of immune evasion: B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets multiple ligands and receptors to disrupt interactions between tumor and T cells, reducing T cell activation. This suggests that B3GNT2-mediated glycosylation alters the tumor cell surface in ways that impair recognition or killing by immune cells .

• Therapeutic implications: Inhibition of B3GNT2 has been shown to sensitize tumor models to T cell cytotoxicity, highlighting its potential as a target for cancer immunotherapy .

What mutations in B3GNT2 have been identified in human diseases?

Several mutations with functional consequences have been identified:

• Colorectal cancer mutations: Three significant mutations have been identified in colorectal cancer tissues and patient-derived cell lines:

  • R6X: Generates a soluble form of B3GNT2 through an alternative translation start

  • P186T: Located in the β2-α2 loop, though its mechanism of action is not clear from structural studies

  • D247H: Located in the active site, severely impacts proper metal coordination and catalysis as demonstrated in activity assays

These mutations significantly alter B3GNT2 subcellular location or impair enzymatic activity and consequently enhance the migratory potential of cancer cells .

How are alterations in B3GNT2 expression linked to cancer progression?

Changes in B3GNT2 expression have been observed in various cancers:

How do B3GNT2 inhibitors affect disease models, particularly in immune-related conditions?

B3GNT2 inhibitors have shown promising effects in disease models:

• Cancer immunotherapy: Inhibition of B3GNT2 has been shown to sensitize tumor models to T cell cytotoxicity, suggesting potential applications in enhancing cancer immunotherapy responses .

• Imidazolone-based B3GNT2 inhibitors: Novel inhibitors using imidazolone as an amide bioisostere have been developed. These compounds alleviate torsional strain of the amide bond on binding to B3GNT2 and improve potency, isoform selectivity, and certain physicochemical and pharmacokinetic properties .

• Mechanism of action: B3GNT2 inhibitors function by interfering with the enzyme's ability to catalyze the transfer of N-acetylglucosamine to nascent glycan structures, thereby reducing polylactosamine synthesis and potentially normalizing hyperactive immune responses in disease settings .

What are the key structural features of the B3GNT2 active site?

The B3GNT2 active site has several critical features revealed by crystallographic studies:

• Metal coordination site: Coordinates divalent metal ions (likely Mn²⁺) through residues D247 and H376. This metal coordination is essential for catalysis, as mutations in these residues (D247A, H376Q, H376L, H376E) abolish enzyme activity .

• Donor substrate (UDP-GlcNAc) binding site: Key residues involved include K149, D245, and Y289. Mutations in these residues (K149A, D245A, Y289F) significantly impair enzyme activity .

• Acceptor substrate binding site: A279 is positioned less than 4 Å from the acetyl group of the GlcNAc of the acceptor substrate. Mutation of A279 to either Val or Leu creates steric hindrance with GlcNAc and impairs activity, while A279G maintains partial activity .

• Catalytic base: D333 serves as the active site base. The D333N mutation abolishes enzyme activity in cell assays .

How does the helical domain of B3GNT2 contribute to its function?

The N-terminal helical domain of B3GNT2 has unique functional implications:

• Structural stabilization: The helical domain is hypothesized to stabilize the catalytic domain, maintaining optimal conformation for enzyme activity .

• Substrate selectivity: This domain may play a crucial role in distinguishing among different glycan acceptors, contributing to the substrate specificity of B3GNT2 .

• Novel structural feature: Comparison with other glycosyltransferases, such as mouse Fringe, reveals that this helical domain is a distinctive feature of B3GNTs. This domain may represent an evolutionary adaptation that enhances functional specificity .

What are the mechanistic insights from B3GNT2 crystal structures in different ligand-bound states?

Crystal structures of B3GNT2 in various states have provided significant mechanistic insights:

• Sequential reaction mechanism: Kinetic studies combined with structural analysis show that the B3GNT2 transglycosylation reaction follows a sequential mechanism rather than a ping-pong mechanism. This means both donor and acceptor substrates must bind before the reaction can proceed .

• Conformational changes: Comparison of structures in different states reveals conformational changes associated with substrate binding and product release, providing insights into the dynamic aspects of catalysis .

• Structure-guided inhibitor design: The structures have enabled rational design of B3GNT2 inhibitors, particularly informing the development of imidazolone-based inhibitors that improve potency and selectivity by alleviating torsional strain in binding .

How is B3GNT2 being targeted in cancer immunotherapy approaches?

B3GNT2 is emerging as a promising target in cancer immunotherapy:

• Enhancing T cell-mediated cytotoxicity: Inhibition of B3GNT2 has been shown to sensitize tumor models to T cell cytotoxicity, suggesting that B3GNT2 inhibitors could potentially enhance the efficacy of existing T cell-based immunotherapies .

• Combination with immune checkpoint inhibitors: Given that B3GNT2 and PD-L1 (CD274) were both identified as top candidates in immune evasion screens, there may be potential for combining B3GNT2 inhibitors with checkpoint inhibitors like anti-PD-1/PD-L1 antibodies .

• Targeting glycan-dependent immune evasion: B3GNT2 targets multiple ligands and receptors involved in tumor-T cell interactions. Inhibiting B3GNT2 could potentially restore these interactions and enhance immunosurveillance .

What experimental models best reflect the role of B3GNT2 in human disease?

Several experimental models have provided valuable insights into B3GNT2 function in disease contexts:

• B3GNT2 knockout mice: These models have demonstrated the importance of B3GNT2-synthesized polylactosamine in regulating immune cell function, with knockout mice showing hypersensitive and hyperresponsive immunocytes .

• Cell line models with CRISPR-mediated B3GNT2 deletion: Systems like B3GNT2-deficient Jurkat cells have been instrumental in studying polylactosamine synthesis and evaluating the functional significance of specific B3GNT2 residues through reconstitution experiments .

• Human cancer xenograft models: These have been used to demonstrate that B3GNT2 overexpression confers resistance to T cell-mediated killing in vivo, validating findings from in vitro screens .

• Point mutation models: Cell lines expressing specific B3GNT2 mutations identified in colorectal cancer (R6X, P186T, D247H) have helped elucidate how these mutations enhance cancer cell migration .

How do B3GNT2 inhibitors compare with other glycosyltransferase inhibitors in terms of specificity and efficacy?

B3GNT2 inhibitors have distinct characteristics compared to other glycosyltransferase inhibitors:

• Novel chemical scaffolds: Recent developments include imidazolone-based inhibitors that function as amide bioisosteres. These compounds alleviate torsional strain of the amide bond on binding to B3GNT2, improving potency and isoform selectivity compared to traditional inhibitor scaffolds .

• Structure-guided design advantages: The availability of high-resolution crystal structures of B3GNT2 in multiple states has enabled rational, structure-based drug design, potentially leading to more specific inhibitors compared to glycosyltransferases where such structural information is lacking .

• Targeting immune regulation: Unlike many glycosyltransferase inhibitors that broadly affect glycan synthesis, B3GNT2 inhibitors specifically target polylactosamine synthesis with direct implications for immune regulation, potentially offering more precise modulation of immune responses in disease settings .

What are the key unresolved questions in B3GNT2 biology?

Several important questions remain to be addressed:

• Specific glycoprotein targets: While B3GNT2 is known to synthesize polylactosamine chains on N-glycans, the specific glycoprotein targets that mediate its effects on immune regulation and disease processes are not fully characterized .

• Tissue-specific roles: Although B3GNT2 is widely expressed with high-level expression in the immune system, its functions in other tissues and cell types require further investigation .

• Regulatory mechanisms: The factors controlling B3GNT2 expression and activity in normal and disease states are not completely understood, particularly how its expression is dysregulated in cancer .

• Interaction with other glycosylation pathways: How B3GNT2-mediated polylactosamine synthesis interacts with or is influenced by other glycosylation pathways remains to be elucidated .

How might multi-omics approaches advance our understanding of B3GNT2 function?

Integrative multi-omics approaches could provide comprehensive insights:

• Glycoproteomics: Identifying the specific glycoproteins modified by B3GNT2 and how these modifications change in disease states could reveal new molecular mechanisms .

• Transcriptomics and epigenomics: Understanding the regulatory mechanisms controlling B3GNT2 expression in different tissues and disease contexts could identify upstream targets for modulating its activity .

• Structural glycomics: Detailed analysis of glycan structures influenced by B3GNT2 activity across different cell types and disease models could provide insights into functional specificity .

• Systems biology integration: Combining these approaches with proteomic, metabolomic, and functional genomic data could reveal network-level effects of B3GNT2 perturbation and identify potential compensatory mechanisms .

What potential clinical applications of B3GNT2 research are most promising?

Several clinical applications show particular promise:

• Cancer immunotherapy: B3GNT2 inhibitors could potentially enhance the efficacy of existing immunotherapies by preventing cancer cells from evading T cell recognition and killing .

• Autoimmune disease treatment: Given the role of B3GNT2 in regulating immune cell responses, targeted modulation of its activity might help normalize hyperactive immune responses in autoimmune conditions like rheumatoid arthritis, ankylosing spondylitis, and psoriasis .

• Diagnostic biomarkers: Changes in B3GNT2 expression or polylactosamine levels could serve as diagnostic or prognostic biomarkers in cancer, particularly colorectal cancer where specific mutations have been identified .

• Precision medicine approaches: Understanding the relationship between specific B3GNT2 genetic variants and disease phenotypes could enable more personalized therapeutic strategies for patients with autoimmune conditions or cancer .