ST3GAL4 Antibody

What is ST3GAL4 and what is its primary function in cells?

ST3GAL4 (ST3 beta-galactoside alpha-2,3-sialyltransferase 4) is a glycosyltransferase belonging to the glycosyltransferase 29 family. It catalyzes the transfer of sialic acid (N-acetyl-neuraminic acid; Neu5Ac) from the nucleotide sugar donor CMP-Neu5Ac onto acceptor galactose-terminated glycoconjugates through an alpha2-3 linkage .

The enzyme plays several important biological roles:

• Sialylation of plasma proteins like von Willebrand factor

• Biosynthesis of sialyl Lewis X epitopes on both O- and N-glycans

• Sialylation of gangliosides in glycosphingolipid biosynthesis

• Contribution to immune cell adhesion and migration through sialylation of receptors

ST3GAL4 has a molecular weight of approximately 38 kDa, though it is often observed at 50-55 kDa on Western blots due to post-translational modifications, particularly glycosylation .

Where is ST3GAL4 localized within cells and which tissues express it most highly?

Subcellular localization:

• Golgi apparatus, specifically the Golgi stack membrane as a single-pass type II membrane protein

• Also found as a secreted form in body fluids

Tissue expression profile:

• Highly expressed in adult placenta, heart, and kidney

• Also abundant in ovary and testes

• Expression levels vary across different hematopoietic cells, with notable upregulation in several subtypes of acute myeloid leukemia (AML) compared to normal hematopoietic stem cells

What are the validated applications for ST3GAL4 antibodies in research?

Based on published literature and commercial validation data, ST3GAL4 antibodies have been successfully used in the following applications:

Most commercially available antibodies have been validated in human samples, with some cross-reactivity to mouse and rat specimens .

What are the recommended dilutions and protocols for using ST3GAL4 antibodies?

Recommended dilutions by application:

Important methodological considerations:

• For IHC applications, following dewaxing and hydration, antigen retrieval mediated by high pressure in a citrate buffer (pH 6.0) has been reported to be effective

• Section blocking with 10% normal goat serum for 30 minutes at room temperature is recommended before primary antibody incubation

• Primary antibody (in 1% BSA) should be incubated at 4°C overnight for optimal results

How does ST3GAL4 contribute to immune evasion in acute myeloid leukemia?

Recent research has revealed that ST3GAL4 plays a critical role in immune evasion in AML through the following mechanism:

• Siglec-9 ligand generation: ST3GAL4 drives the synthesis of sialylated N-glycans that function as ligands for Siglec-9, an inhibitory receptor expressed on immune cells, particularly macrophages and dendritic cells .

• Experimental evidence: CRISPR-Cas9 knockout of ST3GAL4 in AML cell lines:

  • Dramatically reduced Siglec-9 ligand expression

  • Enhanced sensitivity to phagocytosis by macrophages

  • Promoted increased macrophage activation (higher CD86 expression)

  • Stimulated increased TNF-α secretion by macrophages

• Molecular specificity: Mass spectrometry analysis of cell-surface glycosylation in ST3GAL4 KO cells revealed:

  • Dramatic decrease in sialylated N-linked glycans

  • Changes in sialylation observed in 23 out of 77 detected N-linked glycans

  • Both fucosylated and non-fucosylated N-linked structures were affected

  • Bi-, tri-, and tetra-antennary structures showed comparable decreases in sialylation

This research suggests that ST3GAL4 represents a potential target for immunotherapy in AML, as its inhibition could enhance anti-leukemic immune responses.

What evidence exists for upregulation of ST3GAL4 in malignancies?

Analysis of ST3GAL4 expression across different cancer types has revealed significant findings:

• AML-specific upregulation: The BloodSpot database analysis showed marked upregulation of ST3GAL4 expression in several subtypes of AML compared to terminal monocytes or hematopoietic stem cells .

• Cell line data: DepMap analysis showed that AML cell lines exhibit much higher expression of ST3GAL4 than cell lines derived from other forms of blood cancer .

• Clinical significance: Elevated expression of ST3GAL4 has been associated with worse survival in a large cohort of AML patients .

• Genetic associations: In clinical datasets, ST3GAL4 upregulation is particularly pronounced in AML with MLL gene translocations, suggesting specific genetic pathways controlling its expression .

• Cross-tissue expression: Variants associated with transferrin glycosylation have been associated with ST3GAL4 expression in liver and whole blood, suggesting broader regulatory networks across tissues .

How can researchers functionally validate the impact of ST3GAL4 inhibition?

To assess the functional impact of ST3GAL4 inhibition or knockout, researchers can employ several methodological approaches:

• Phagocytosis assays:

  • Label ST3GAL4 WT or KO cells with cell-permeable dye (e.g., CellTrace Far Red)

  • Co-culture with Siglec-9-expressing macrophages

  • Quantify phagocytosis by measuring fluorescence increase in CD11b+ cells

  • Include controls with Siglec-9 blocking antibody and sialidase treatment

• Macrophage activation assessment:

  • Co-culture ST3GAL4 WT or KO cells with macrophages

  • Measure expression of activation markers (e.g., CD86) by flow cytometry

  • Analyze secretion of inflammatory cytokines (e.g., TNF-α) in supernatants using multiplexed assays like Luminex

• Glycan profile analysis:

  • Isolate N-linked glycans from cell membranes

  • Perform mass spectrometry analysis (e.g., MALDI-MS) to profile glycan structures

  • Quantify changes in sialylation patterns and specific glycan structures

• Validation in primary cells:

  • For non-genetically tractable cells (e.g., primary patient samples), use sialidase enzyme treatment as a surrogate for genetic manipulation

  • Confirm phenotypic changes are consistent with genetic knockout results

What molecular weight should be detected when using ST3GAL4 antibodies?

When working with ST3GAL4 antibodies, researchers should be aware of the following:

• Calculated molecular weight: 38 kDa based on the 333 amino acid sequence

• Observed molecular weight: Typically 36-38 kDa for the unmodified protein, but often observed at 50-55 kDa due to post-translational modifications, particularly glycosylation

• Multiple isoforms: Up to 7 different isoforms have been reported for ST3GAL4, which may appear as multiple bands on Western blots

• Splice variants: Multiple splice variants exist, which may require validation in specific experimental contexts

What are effective validation strategies for confirming ST3GAL4 antibody specificity?

To ensure the specificity of ST3GAL4 antibodies, researchers should consider these validation approaches:

• Genetic validation:

  • CRISPR-Cas9 knockout of ST3GAL4 as a negative control

  • Rescue experiments with exogenous ST3GAL4 cDNA expression in knockout cells

• Expression analysis:

  • Test the antibody in cell lines with known expression levels of ST3GAL4

  • Confirmed positive cell lines include HCT 116, HeLa, HepG2, and MCF-7 cells

• Multiple antibody approach:

  • Use multiple antibodies targeting different epitopes of ST3GAL4

  • Compare detection patterns across different applications

• Functional validation:

  • Correlate antibody detection with functional readouts (e.g., Siglec-9 binding)

  • Biochemical assays measuring sialyltransferase activity

What are common challenges when using ST3GAL4 antibodies in experimental applications?

Researchers may encounter several challenges when working with ST3GAL4 antibodies:

• Post-translational modifications:

  • ST3GAL4 undergoes extensive glycosylation that can affect antibody binding

  • Consider deglycosylation treatments if inconsistent results are observed

• Isoform specificity:

  • With up to 7 different isoforms reported, antibodies may not detect all variants

  • Check epitope locations against known isoform sequences

• Subcellular localization:

  • ST3GAL4 exists in both membrane-bound (Golgi) and secreted forms

  • Different extraction methods may be required depending on the experimental question

• Tissue-specific expression:

  • Expression levels vary significantly across tissues

  • Include appropriate positive controls when examining new tissue types

• Antigen retrieval in IHC:

  • Different protocols recommend different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Optimization may be required for specific tissue types

What are promising therapeutic implications of targeting ST3GAL4?

Based on recent research findings, several therapeutic approaches targeting ST3GAL4 show promise:

• Immunotherapy enhancement:

  • ST3GAL4 inhibition could enhance immune cell recognition and clearance of AML cells

  • Combining ST3GAL4 inhibitors with existing immunotherapies might improve efficacy

• Dual-targeting approach:

  • ST3GAL4 drives both Siglec-9 ligand and E-selectin ligand synthesis

  • Targeting ST3GAL4 could disrupt multiple survival pathways simultaneously

• Safety profile:

  • ST3GAL4 knockout mice are viable with only minor deficiencies in platelet function

  • This suggests ST3GAL4 inhibitors could potentially have manageable side effect profiles

• Patient stratification:

  • Genetic signatures based on ST3GAL4 and related glycosylation enzymes could identify patients likely to benefit from targeted therapies

  • This may be particularly relevant in genetically heterogeneous cancers like AML

What experimental models are most appropriate for studying ST3GAL4 function?

Several experimental models have proven valuable for studying ST3GAL4:

What are unexplored areas of ST3GAL4 biology that warrant further investigation?

Despite significant advances, several aspects of ST3GAL4 biology remain to be fully explored:

• Protein-specific glycosylation:

  • While ST3GAL4 broadly affects N-linked glycans, specific "professional ligands" may exist

  • Interactomics and glycoproteomics approaches could identify key protein carriers

• Genetic regulation:

  • The mechanisms controlling ST3GAL4 upregulation in AML, particularly in MLL-rearranged cases, remain unclear

  • Transcriptional and epigenetic regulation studies are needed

• Pathway interactions:

  • Interactions between ST3GAL4 and other glycosylation enzymes in creating complex glycan patterns

  • Cross-talk between glycosylation and other post-translational modifications

• Non-cancer functions:

  • While extensively studied in cancer, the physiological roles of ST3GAL4 in normal development and immunity deserve further investigation

  • Potential roles in other biological processes beyond known functions

• Therapeutic targeting:

  • Development of specific small molecule inhibitors of ST3GAL4

  • Strategies to modify glycosylation patterns in a tissue-specific manner