ST3GAL3 Antibody

What is ST3GAL3 and what cellular functions does it perform?

ST3GAL3 (β-galactoside-α2,3-sialyltransferase-III) is a Golgi-resident type II membrane protein that catalyzes the transfer of sialic acid residues to terminal galactose on glycoproteins and glycolipids, forming α2,3-sialic acid linkages . It predominantly forms the sialyl Lewis a epitope on proteins in humans. ST3GAL3 contains four highly conserved sialylmotifs that constitute part of its active site, and functions as a key enzyme in glycosylation pathways essential for numerous biological processes . The enzyme plays critical roles in neurodevelopment, with mutations linked to intellectual disability and epileptic encephalopathy . Additionally, ST3GAL3 has been implicated in inflammatory responses in conditions like rheumatoid arthritis and in modulating immune evasion in various cancers .

How is ST3GAL3 protein typically detected in research applications?

ST3GAL3 protein can be detected through various complementary techniques:

• Immunofluorescence microscopy: Using ST3GAL3-specific antibodies for subcellular localization studies. Wild-type ST3GAL3 typically shows a perinuclear Golgi signal, while mutant forms may display altered localization patterns (e.g., ER retention) .

• Western blotting: For quantitative assessment of ST3GAL3 protein levels, where expression patterns can be compared between wild-type and variant forms .

• Immunohistochemistry: For detecting ST3GAL3 expression in tissue samples, typically using dilutions around 1:80 of primary antibody followed by biotinylated secondary antibody detection and DAB development .

• Flow cytometry: For analyzing ST3GAL3-dependent glycan patterns on cell surfaces, particularly sialyl Lewis antigens .

The choice of detection method depends on whether researchers are investigating protein levels, subcellular localization, or enzymatic activity outcomes.

What are the optimal sample preparation methods for ST3GAL3 antibody-based experiments?

For effective ST3GAL3 antibody-based experiments, sample preparation should be tailored to the specific application:

For cell culture samples:

• Fix cells with 4% paraformaldehyde for immunofluorescence studies

• For comparing wild-type and mutant ST3GAL3 localization, transfect cells (e.g., murine fibroblast LMTK- cells or HEK-293T) with expression constructs containing the variants of interest

• For protein extraction, use detergent-based lysis buffers that preserve membrane protein integrity while solubilizing Golgi membranes

For tissue samples:

• For immunohistochemistry, perform antigen retrieval after dewaxing in dimethylbenzene and gradient alcohol hydration

• Block endogenous peroxidase activity and non-specific binding sites before primary antibody incubation

• For optimal ST3GAL3 detection in tissues, overnight incubation at 4°C with primary antibody is recommended

For enzymatic activity studies:

• Prepare cell or tissue lysates in buffers that preserve enzymatic activity

• Consider subcellular fractionation to isolate Golgi-enriched fractions for more specific analyses

What are the recommended controls for ST3GAL3 antibody validation?

Robust experimental design for ST3GAL3 antibody studies requires multiple controls:

Positive controls:

• Cell lines with confirmed high ST3GAL3 expression (e.g., AsPC-1, BxPC-3, Capan-2 for pancreatic cancer studies)

• Recombinant ST3GAL3 protein expressed in appropriate systems

Negative controls:

• ST3GAL3 knockdown cell lines generated using validated shRNA constructs (e.g., shST3GAL3_7 and shST3GAL3_10 that showed 77-78% reduction in BxPC-3 cells)

• Tissues or cells from ST3GAL3 knockout models

Specificity controls:

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

• Preabsorption of the antibody with recombinant ST3GAL3 protein

• Validation across multiple detection methods (Western blot, immunofluorescence, IHC)

• Assessment of cross-reactivity with other ST3GAL family members (especially ST3GAL4 and ST3GAL6)

How can researchers effectively design knockdown or knockout experiments to study ST3GAL3 function?

For effective genetic manipulation of ST3GAL3:

shRNA-mediated knockdown:

• Design multiple shRNA constructs targeting different regions of ST3GAL3 mRNA

• Select cell lines with relatively high endogenous ST3GAL3 expression (e.g., BxPC-3, Capan-1, AsPC-1)

• Validate knockdown efficiency at both mRNA (qRT-PCR) and protein levels (Western blot)

• Include scrambled control (SC) shRNA and parental cells as appropriate controls

• Verify that knockdown of ST3GAL3 does not affect expression of other ST3GAL family members, particularly ST3GAL4 and ST3GAL6

CRISPR-Cas9 knockout:

• Design guide RNAs targeting early exons of ST3GAL3

• Screen for complete loss of protein expression rather than just reduced expression

• Validate functional consequences by examining relevant glycan profiles (e.g., sialyl Lewis antigens)

Experimental evaluation:

• Assess phenotypic changes in vitro and in vivo

• For cancer studies, examine both immunocompetent and immunodeficient mouse models to distinguish between intrinsic cellular effects and immune-mediated effects

• Include complementation studies by re-expressing wild-type ST3GAL3 in knockdown/knockout cells to confirm specificity

How do mutations in ST3GAL3 impact its cellular localization and enzymatic activity?

ST3GAL3 mutations have distinct effects on protein localization and function:

Effect on cellular localization:

• Wild-type ST3GAL3 localizes predominantly to the perinuclear Golgi apparatus

• The p.Ala13Asp mutation, located in the transmembrane domain (TMD), causes significant ER retention with rare Golgi localization

• The p.Asp370Tyr mutation, located in the catalytic domain (CD), shows complete ER retention with no detectable Golgi localization

• The p.Y220* nonsense variant results in a truncated protein that, surprisingly, can still partially localize to the Golgi apparatus despite being enzymatically inactive

Impact on enzymatic activity:

These structure-function relationships highlight that both proper Golgi localization and intact catalytic domains are essential for ST3GAL3 function. Notably, even when some mutant forms reach the Golgi, they may still lack enzymatic activity, suggesting multiple mechanisms by which mutations can impair ST3GAL3 function.

What is the role of ST3GAL3 in cancer progression and how can antibodies be used to study this relationship?

ST3GAL3 plays complex roles in cancer biology that can be investigated using antibody-based approaches:

ST3GAL3 in cancer progression:

• In pancreatic ductal adenocarcinoma, ST3GAL3 knockdown reduces migratory and invasive phenotypes and impairs E-selectin-dependent adhesion

• In ovarian cancer, elevated ST3GAL3 correlates with poor prognosis in high-grade serous carcinoma, and its knockdown inhibits tumor growth in immunocompetent mouse models

• In prostate cancer, the related enzyme ST3GAL1 negatively correlates with androgen signaling and promotes immune evasion through sialoglycan synthesis

Antibody-based research approaches:

• Expression profiling: Use ST3GAL3 antibodies for IHC analysis of tumor tissue microarrays to correlate expression with clinical outcomes

• Functional studies: Combine ST3GAL3 knockdown with antibody detection of downstream glycan products (e.g., sialyl Lewis antigens) by flow cytometry

• Mechanistic investigations: Use antibodies to study how ST3GAL3 manipulation affects signaling pathways (e.g., Notch/p21/CDK signaling in cervical cancer)

• Immune contexture analysis: Multiplex immunofluorescence using ST3GAL3 antibodies alongside immune cell markers to study tumor microenvironment remodeling

These approaches can reveal how ST3GAL3-mediated sialylation modulates tumor cell behavior and immune cell interactions, potentially identifying new therapeutic targets.

How does ST3GAL3 activity influence immune responses in disease contexts?

ST3GAL3-mediated sialylation has significant immunomodulatory effects across various disease contexts:

In cancer immunology:

• ST3GAL3 knockdown enhances antitumor immunity by increasing functional CD8+ T cell infiltration into tumors

• ST3GAL3 manipulation reprograms tumor-associated macrophages from immunosuppressive M2-like to tumor-suppressive M1-like phenotypes

• The sialoglycans produced by ST3GAL3 can engage Siglec immunoreceptors (like Siglec-7 and Siglec-9), creating immunosuppressive signals that prevent immune clearance of cancer cells

In inflammatory conditions:

• ST3GAL3 promotes inflammatory responses in fibroblast-like synoviocytes in rheumatoid arthritis

• Its activity may modulate Toll-like receptor (TLR) pathway signaling, influencing innate immune responses

Research methodologies:

• Co-culture systems combining ST3GAL3-manipulated cells with immune cells (e.g., bone marrow-derived macrophages) to study cellular crosstalk

• Transwell experiments to distinguish between contact-dependent and soluble factor-mediated effects

• Flow cytometric analysis of immune cell phenotypes following exposure to cells with altered ST3GAL3 expression

• Assessment of cytokine/chemokine production profiles, particularly focusing on molecules like CXCL10 that mediate immune cell recruitment

These findings highlight ST3GAL3's role as a potential immunomodulatory target, with implications for both cancer immunotherapy and inflammatory disease treatment strategies.

What are the common challenges in distinguishing ST3GAL3 from other sialyltransferase family members?

Researchers face several challenges when specifically targeting ST3GAL3 among related sialyltransferases:

Structural similarity challenges:

• The ST3GAL family contains multiple members (ST3GAL1-6) with similar catalytic domains and conserved sialylmotifs

• ST3GAL3, ST3GAL4, and ST3GAL6 can have overlapping substrate preferences and partially redundant functions

Expression pattern considerations:

• Cell-type specific expression patterns exist; for example, ST3GAL3 expression is extremely low in lymphoblastoid cell lines

• In many cell types, ST3GAL3 expression is 4-20 fold lower than ST3GAL4 expression

Strategies for specificity:

• Validate antibody specificity using recombinant proteins and knockout/knockdown models

• Perform careful RNA analysis (qRT-PCR) to determine relative expression levels of all ST3GAL family members in your experimental system

• When manipulating ST3GAL3 expression, verify that other ST3GAL family members remain unaffected

• Consider functional redundancy - loss of ST3GAL3 activity may be partially compensated by other family members (e.g., in the synthesis of sialyl-Lewis a epitope)

How can researchers effectively measure ST3GAL3 enzymatic activity in experimental settings?

Methods for measuring ST3GAL3 enzymatic activity include:

In vitro enzymatic assays:

• Use recombinant or immunoprecipitated ST3GAL3 protein

• Supply appropriate acceptor substrates (lactotetraosylceramide shows strong preference as acceptor; gangliotetraosylceramide is also efficiently utilized)

• Monitor the transfer of radioactively or fluorescently labeled CMP-sialic acid to these acceptors

• Conduct kinetic studies with varying substrate concentrations to determine enzyme parameters (Km, Vmax)

Cellular sialylation assessment:

• Flow cytometric analysis using lectins or antibodies that recognize ST3GAL3-dependent glycan structures

• Monitor changes in sialyl Lewis antigens (sLea, sLex) expression following ST3GAL3 manipulation

• Consider the potential redundancy with other sialyltransferases - some glycan structures may still be synthesized despite ST3GAL3 knockdown

Specialized techniques:

• Mass spectrometry analysis of glycan profiles before and after ST3GAL3 manipulation

• HPLC separation of released glycans followed by sialic acid-specific detection

• Biomarker measurements (e.g., CA19.9 antigen in serum, which contains the sialyl-Lewis a epitope)

These methodologies allow researchers to directly assess the functional consequences of ST3GAL3 manipulation or mutation.

What experimental approaches can help resolve contradictory findings regarding ST3GAL3 function in different disease models?

Contradictory findings about ST3GAL3 function can arise from context-dependent effects. Resolving these contradictions requires:

Comprehensive experimental design:

• Test hypotheses across multiple cell lines representing different disease subtypes or tissues

• Compare results in 2D culture, 3D organoids, and in vivo models to account for microenvironmental influences

• Examine both immunocompetent and immunodeficient animal models to distinguish between cell-autonomous and immune-mediated effects

Mechanistic dissection:

• Perform detailed pathway analysis to determine context-specific signaling networks affected by ST3GAL3

• Consider how ST3GAL3 activity may differentially impact various substrate proteins or lipids in different cellular contexts

• Investigate the complex interplay between ST3GAL3 and other glycosyltransferases in the glycosylation pathway

Translational validation:

• Correlate experimental findings with patient-derived samples and clinical data

• Utilize tissue microarrays with proper scoring systems to evaluate ST3GAL3 expression across large patient cohorts

• Apply systems biology approaches to integrate genomic, transcriptomic, and glycomic data

Technical considerations:

• Account for differences in antibody sensitivity and specificity across studies

• Standardize experimental conditions including culture medium, which can affect glycosylation patterns

• Consider the impact of genetic background and epigenetic factors on ST3GAL3 function

By implementing these approaches, researchers can better understand the seemingly contradictory roles of ST3GAL3 in different disease contexts and develop more targeted therapeutic strategies.

How might ST3GAL3 antibodies be used in developing novel therapeutic approaches for neurodevelopmental disorders?

Given ST3GAL3's critical role in neurodevelopment, antibody-based research could advance therapeutic approaches for associated disorders:

Diagnostic applications:

• Develop antibodies that specifically recognize mutant forms of ST3GAL3 for early detection in patient samples

• Create antibody-based assays to measure ST3GAL3-dependent glycan profiles as biomarkers for disease severity or progression

• Use antibodies to identify cellular populations with altered ST3GAL3 expression in brain tissue samples

Therapeutic target validation:

• Employ antibody-mediated labeling to track the subcellular fate of mislocalized ST3GAL3 mutants (e.g., p.Ala13Asp, p.Asp370Tyr)

• Identify potential chaperone targets that could rescue proper Golgi localization of mutant ST3GAL3

• Map the binding partners of ST3GAL3 in neural cells to understand downstream pathways affected by mutations

Mechanistic insights:

• Investigate how ST3GAL3 deficiency affects ganglioside composition in neural tissues, particularly "minor gangliosides" that may be critical for brain function

• Elucidate the specific glycan structures regulated by ST3GAL3 that are essential for neuronal development and function

• Examine how ST3GAL3 mutations affect neural cell morphology, synaptic connections, and circuit formation

These research directions could potentially lead to novel therapeutic strategies aimed at compensating for ST3GAL3 deficiency or restoring proper glycan profiles in patients with neurodevelopmental disorders.