ST6GALNAC5 Human

What is ST6GALNAC5 and what is its primary function?

ST6GALNAC5, also known as ST6 GalNAc alpha-2,6-sialyltransferase V or SIAT7E, is a sialyltransferase enzyme responsible for transferring sialic acid residues to specific gangliosides. Its primary function is catalyzing the conversion of ganglioside GM1b to GD1a by adding a sialic acid in an α2,6-linkage to the GalNAc residue . Gangliosides are acidic glycosphingolipids containing one or more sialic acid residues that play crucial roles in cellular recognition, interaction, adhesion, and signal transduction, particularly in neural tissues . ST6GALNAC5 is part of the larger ST6GalNAc family of sialyltransferases but has distinct substrate specificity and tissue expression patterns.
The enzyme's structure includes a transmembrane domain, stem region, and catalytic domain. Its specificity for ganglioside substrates distinguishes it from other sialyltransferases, making it essential for proper ganglioside composition, particularly in neural tissues where these glycolipids are abundant and functionally significant during development .

Where is ST6GALNAC5 expressed in normal human tissues?

In normal physiological conditions, ST6GALNAC5 expression is predominantly restricted to brain tissues in both humans and mice . This highly tissue-specific expression pattern aligns with the important roles of gangliosides in the nervous system, where they modulate cellular functions especially during developmental stages . The brain-specific expression suggests the involvement of neural-specific transcriptional regulation mechanisms and reflects the specialized function of ST6GALNAC5 in neural development and function.
Immunohistochemical analysis using specific antibodies has confirmed the localization of ST6GALNAC5 protein in brain tissue sections. At the subcellular level, ST6GALNAC5 is typically found in the Golgi apparatus, consistent with its role in glycolipid biosynthesis. When detected in experimental settings using immunofluorescence techniques, ST6GALNAC5 shows specific staining localized to cell membranes and cytoplasm .

What experimental models are optimal for studying ST6GALNAC5 function?

Several cell lines serve as effective models for investigating ST6GALNAC5 function across different research contexts. For prostate cancer research, multiple validated cell line options exist with varying endogenous expression levels: DU145 and C4-2 cells express relatively low levels of ST6GALNAC5, making them suitable for overexpression studies, while 22Rv1 cells exhibit high endogenous expression, providing an ideal system for knockdown experiments . The non-malignant prostate cell line BPH-1 offers a valuable control with low baseline expression .
For breast cancer research focusing on brain metastasis mechanisms, the MDA-MB-231 line has been effectively used to study ST6GALNAC5's role in blood-brain barrier penetration . This cell line can be readily manipulated to alter ST6GALNAC5 expression levels, and its metastatic behavior can be assessed in both in vitro and in vivo models.
When designing experiments, investigators should consider:

• Matching the cell model to the specific research question (basic enzyme function vs. disease mechanism)

• Baseline expression levels of ST6GALNAC5 in candidate cell lines

• The cell line's suitability for the intended genetic manipulation methods

• Whether the cell type appropriately represents the tissue context being studied

What are the current techniques for detecting and measuring ST6GALNAC5 expression and activity?

Comprehensive analysis of ST6GALNAC5 requires assessment at multiple levels: gene expression, protein detection, and enzymatic activity. For gene expression quantification, real-time PCR remains the gold standard, with several validated primer sets available in the literature. When designing qPCR experiments, researchers should target conserved exon regions and include appropriate housekeeping genes for normalization based on the tissue or cell type being studied .
For protein detection, immunological methods are most common. Immunohistochemistry and immunofluorescence can localize ST6GALNAC5 in tissues or cells, as demonstrated in studies using Mouse Anti-Human GalNAc alpha-2,6-sialyltransferase V/ST6GALNAC5 Monoclonal Antibody (such as Clone #719508, detecting the region from Gly30 to Phe336) . Western blotting protocols typically require optimization of lysis conditions to effectively solubilize this transmembrane protein.
Enzymatic activity can be measured through several approaches:

• Phosphatase-coupled assays using fetuin as a substrate

• Radiometric assays tracking transfer of labeled sialic acid to acceptor molecules

• Mass spectrometry to identify and quantify specific ganglioside products
  Researchers should be aware that ST6GALNAC5 activity measurements may be complicated by the presence of other sialyltransferases with overlapping activities, necessitating careful experimental design and appropriate controls.

How can ST6GALNAC5 be genetically manipulated for functional studies?

Effective genetic manipulation of ST6GALNAC5 can be achieved through several complementary approaches depending on the experimental goals. For overexpression studies, plasmid vectors expressing tagged versions (such as Flag-tagged ST6GALNAC5) have been successfully employed in multiple cell lines . These constructs can be delivered via lipid-based transfection in most adherent cell lines or electroporation in harder-to-transfect cells.
For loss-of-function studies, RNA interference provides a reliable approach. Using multiple siRNAs targeting different regions of ST6GALNAC5 helps control for off-target effects, as demonstrated in 22Rv1 cell experiments where two independent siRNAs produced consistent phenotypes . For stable knockdown, shRNA expression vectors or CRISPR-Cas9 gene editing can be employed.
Rescue experiments, combining knockdown of endogenous expression with reintroduction of wild-type or mutant forms, offer particularly robust evidence of specificity. This approach was effectively used to demonstrate that ST6GALNAC5 is a downstream target of GATA2 in cancer cell invasion, where ectopic expression of ST6GALNAC5 in GATA2-depleted DU145 cells significantly reversed the compromised invasiveness phenotype .
When implementing these strategies, verification of the genetic manipulation through both RNA and protein expression analysis is essential to confirm effectiveness.

What are the key considerations for developing ST6GALNAC5-specific antibodies?

Developing effective antibodies against ST6GALNAC5 presents several technical challenges that researchers must address. As a type II membrane protein anchored to the Golgi membrane, ST6GALNAC5's topology affects epitope accessibility, particularly in intact cells. The catalytic domain faces the Golgi lumen, which can complicate antibody binding in non-permeabilized samples .
The high sequence similarity between ST6GALNAC5 and other members of the ST6GalNAc family (six members total) necessitates careful epitope selection to ensure specificity. Researchers should target unique regions that are not conserved across the family to minimize cross-reactivity. The region from Gly30 to Phe336 has been successfully used as an immunogen for monoclonal antibody development, as seen with antibody clone #719508 .
Validation strategies should include:

• Testing on cells with confirmed high and low/absent ST6GALNAC5 expression

• Verification with knockdown/knockout cells as negative controls

• Confirmation using multiple detection methods (Western blot, immunofluorescence)

• Cross-reactivity testing against other ST6GalNAc family members
  Application-specific optimization is critical, as antibodies that work well for immunofluorescence may not perform adequately for Western blotting or immunoprecipitation due to differences in epitope presentation under various experimental conditions.

How is ST6GALNAC5 implicated in cancer progression and metastasis?

ST6GALNAC5 plays significant roles in cancer progression, particularly in promoting metastasis through multiple mechanisms. In breast cancer, ST6GALNAC5 overexpression enhances GD1α ganglioside expression on cell surfaces, reducing adhesion to the blood-brain barrier and facilitating brain-specific metastasis . This specialized role is particularly significant given that ST6GALNAC5 is normally expressed predominantly in brain tissue, suggesting that cancer cells may exploit this brain-specific glycosylation pathway to colonize the brain microenvironment.
In prostate cancer, ST6GALNAC5 serves as an adverse prognostic biomarker with mechanistic importance. Functional studies have demonstrated that ST6GALNAC5 overexpression significantly enhances invasion in multiple prostate cancer cell lines, including DU145 and C4-2, increasing invasiveness by approximately 2-fold compared to control cells . Conversely, ST6GALNAC5 knockdown in 22Rv1 prostate cancer cells not only reduces proliferation but also significantly decreases invasive capacity .
The prognostic significance of ST6GALNAC5 is evident from survival analyses across multiple cancer datasets. In prostate cancer, high ST6GALNAC5 expression correlates with poor disease-free survival in both TCGA-PRAD dataset (HR = 1.943, p = 0.0017) and GSE21032 dataset (HR = 1.954, p = 0.0490) . ROC curve analysis demonstrates ST6GALNAC5's superior ability to predict disease-free survival at both 3-year (AUC = 0.880) and 5-year (AUC = 0.846) timepoints compared to other ST6GALNAC family members .

What is the relationship between ST6GALNAC5 mutations and coronary artery disease?

Genetic studies have established a compelling link between ST6GALNAC5 mutations and coronary artery disease (CAD). Through genetic linkage analysis and exome sequencing in an Iranian pedigree with CAD, researchers identified a p.Val99Met mutation in ST6GALNAC5 as the likely causative variant . This mutation affects a highly conserved amino acid, was absent in 800 controls, and was predicted to damage protein function through in silico analysis .
Further sequencing of ST6GALNAC5 in 160 Iranian patients revealed a candidate causative stop-loss mutation in two additional patients with CAD . Functional characterization demonstrated that both the p.Val99Met and stop-loss mutations caused increased sialyltransferase activity, suggesting that dysregulation of sialic acid metabolism contributes to CAD pathogenesis .
The ST6GALNAC5 gene is positioned within genomic loci previously linked to CAD-associated parameters, providing additional support for its role in cardiovascular disease . While hypercholesterolemia was a prominent feature in the studied family, clinical and genetic data suggested that this condition was not directly caused by the ST6GALNAC5 mutation but may interact with the altered sialyltransferase activity .
These findings align with the substantial literature suggesting a relationship between sialyltransferase activity, sialic acid levels, and coronary disease pathogenesis, potentially through effects on endothelial function, inflammatory responses, and lipid metabolism.

What signaling pathways and molecular interactions does ST6GALNAC5 influence?

ST6GALNAC5 participates in multiple signaling networks through its effects on cell surface glycosylation. Gene Set Enrichment Analysis (GSEA) has identified associations between ST6GALNAC5 expression and several key signaling pathways in prostate cancer, including Hedgehog signaling, neurotrophin signaling, ERBB signaling, and SNARE interactions . These diverse pathways suggest that ST6GALNAC5-mediated glycosylation affects multiple cellular processes relevant to both normal development and disease progression.
Gene Ontology enrichment analysis of ST6GALNAC5-associated genes further revealed significant associations with cell junction components and biological processes related to cell communication and cell-cell signaling . KEGG pathway analysis identified the neuroactive ligand-receptor interaction pathway as particularly relevant to ST6GALNAC5 function, consistent with its predominant expression in neural tissues .
At the protein interaction level, ST6GALNAC5 networks with other glycosylation-related enzymes, including ST3GAL1, ST3GAL2, and ST8SIA5, suggesting coordinated regulation of cell surface glycan patterns . ST6GALNAC5 also interacts with HBEGF protein, which has been implicated in cancer cell metastasis, providing another mechanistic link to its role in cancer progression .
In functional contexts, ST6GALNAC5 has been reported to enhance anchorage-independent growth by activating the HGF/MET signaling pathway and is required for epithelial-to-mesenchymal transition in cancer cells . These molecular interactions collectively explain how ST6GALNAC5 influences diverse cellular phenotypes across different biological contexts.

How is ST6GALNAC5 regulated at the transcriptional level?

ST6GALNAC5 transcription is controlled through multiple regulatory mechanisms, with GATA2 emerging as a key upstream regulator. Bioinformatic analysis identified a putative GATA2 binding site within intron 1 of the ST6GALNAC5 gene, and this interaction has been experimentally verified through multiple approaches . Luciferase reporter assays demonstrated that GATA2 increases ST6GALNAC5 transcriptional activity in a dose-dependent manner, while mutation of the binding site significantly reduces this response .
Chromatin immunoprecipitation (ChIP) experiments have confirmed direct binding of GATA2 to a specific region (+801 to +885) containing the predicted GATA2 binding motif (+843 to +847) within ST6GALNAC5 intron 1 . Functional validation showed that GATA2 knockdown significantly downregulates ST6GALNAC5 expression at both mRNA and protein levels in prostate cancer cells .
The brain-specific expression pattern of ST6GALNAC5 under normal conditions suggests additional tissue-specific regulatory mechanisms involving neural-specific transcription factors. This restricted expression is disrupted in pathological states such as cancer, where aberrant expression appears in non-neuronal tissues including breast and prostate tumors .
Interestingly, ST6GALNAC5 shows different expression patterns across cancer types – upregulated in some cancers while downregulated in others like glioma . This differential regulation suggests complex context-dependent control mechanisms that respond to tissue-specific factors and disease-related signaling changes.

What factors influence ST6GALNAC5 activity post-translationally?

While the search results don't provide comprehensive information about post-translational modifications of ST6GALNAC5 specifically, several mechanisms likely regulate its activity based on what is known about structurally similar glycosyltransferases.
As a type II membrane protein residing in the Golgi apparatus, ST6GALNAC5 likely undergoes N-linked glycosylation, which may be critical for proper protein folding, stability, and Golgi localization. The evidence that the functional protein spans from Gly30 to Phe336 suggests potential N-terminal processing during protein maturation .
The p.Val99Met mutation identified in CAD patients demonstrates how single amino acid changes can significantly affect enzyme function, in this case increasing sialyltransferase activity . Similarly, the stop-loss mutation found in other CAD patients would extend the C-terminus of the protein, potentially altering its processing, localization, or interaction with regulatory factors .
ST6GALNAC5 activity may also be regulated through:

• Phosphorylation events that respond to cellular signaling cascades

• Protein-protein interactions within the Golgi compartment

• Changes in substrate availability due to altered expression of upstream glycosyltransferases

• Regulation of protein trafficking between cellular compartments
  Understanding these post-translational regulatory mechanisms could provide additional targets for modulating ST6GALNAC5 activity in therapeutic contexts.

What are the most promising diagnostic applications for ST6GALNAC5 measurements?

ST6GALNAC5 shows significant potential as a diagnostic and prognostic biomarker, particularly in cancer settings. In prostate cancer, ST6GALNAC5 expression demonstrates remarkable discriminative ability for predicting disease-free survival, with ROC curve analysis showing AUC scores of 0.880 for 3-year and 0.846 for 5-year survival prediction . This superior predictive power compared to other ST6GALNAC family members highlights its potential utility in clinical risk stratification.
For implementing ST6GALNAC5 as a diagnostic marker, several methodological approaches could be effective:

• Tissue-based RNA expression analysis in tumor biopsies

• Immunohistochemical detection using validated antibodies

• Measurement of ST6GALNAC5-specific ganglioside products in tissue or liquid biopsies

• Analysis of ST6GALNAC5 mutations through targeted sequencing in cardiovascular disease patients
  The detection of specific ST6GALNAC5 mutations (such as p.Val99Met) could also serve as a genetic risk marker for coronary artery disease in appropriate populations . Combined with traditional risk factors, ST6GALNAC5 status could enhance predictive algorithms for both cancer progression and cardiovascular disease risk.
  Development of standardized assays with established clinical thresholds will be necessary before ST6GALNAC5 can be implemented in routine clinical practice. Multi-center validation studies comparing its performance against existing biomarkers would provide the evidence base needed for clinical adoption.

How might ST6GALNAC5 be targeted therapeutically?

The involvement of ST6GALNAC5 in multiple disease processes suggests several potential therapeutic targeting strategies. Direct enzyme inhibition represents the most straightforward approach, with small molecules designed to bind the catalytic site and block sialyltransferase activity. Such inhibitors could be particularly valuable in contexts where increased enzyme activity contributes to pathology, as seen with the CAD-associated mutations .
For cancer applications, disrupting the GATA2-ST6GALNAC5 regulatory axis offers another promising approach. Since GATA2 directly controls ST6GALNAC5 expression through binding to its intron 1 region, small molecules or oligonucleotides that interfere with this interaction could downregulate ST6GALNAC5 expression specifically . The rescue experiments showing that ST6GALNAC5 can reverse the anti-invasive effects of GATA2 depletion provide proof-of-concept for this therapeutic strategy .
Additional therapeutic avenues include:

• Antisense oligonucleotides or siRNAs targeting ST6GALNAC5 mRNA

• Antibody-drug conjugates targeting cancer cells with aberrant ST6GALNAC5 expression

• Metabolic glycoengineering to alter sialic acid incorporation into gangliosides

• Combination therapies targeting both ST6GALNAC5 and interacting signaling pathways
  For brain metastasis prevention, inhibiting ST6GALNAC5-mediated changes in cell surface gangliosides could reduce cancer cell penetration across the blood-brain barrier, potentially limiting this devastating complication of systemic malignancies .