HS3ST3B1 Antibody

What biological functions does HS3ST3B1 regulate in normal tissue homeostasis?

HS3ST3B1 (Heparan Sulfate Glucosamine 3-O-Sulfotransferase 3B1) is a key enzyme that catalyzes the transfer of sulfate to the 3-O position of glucosamine residues in heparan sulfate chains. This enzyme plays crucial roles in multiple biological processes including:

• Cell signaling pathway regulation

• Cell adhesion mechanisms

• Extracellular matrix organization

• Developmental processes

The enzyme modifies heparan sulfate proteoglycans (HSPGs) through specific sulfation patterns, which affects interaction with various proteins including growth factors and cytokines. This sulfation is tissue-specific and developmentally regulated, with HS3ST3B1 showing particularly high expression in liver and placenta tissues .

For optimal detection of HS3ST3B1 in Western blotting experiments:

• Protein Extraction:

  • Use RIPA buffer containing protease inhibitors for whole cell lysates

  • For membrane-associated HS3ST3B1, consider membrane fractionation protocols as it is a type II integral membrane protein

• Sample Preparation:

  • Maintain samples at 4°C during preparation to prevent degradation

  • Use reducing conditions (add β-mercaptoethanol to sample buffer)

  • Heat samples at 95°C for 5 minutes before loading

• Electrophoresis Parameters:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Load 20-50 μg of total protein per lane

  • Include positive control samples (e.g., JEG-3 or JAR cell lysates)

• Transfer and Detection:

  • PVDF membranes are recommended over nitrocellulose

  • Blocking with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Primary antibody incubation at 4°C overnight at dilutions of 1:500-1:2000

How can researchers distinguish between HS3ST3B1 and other closely related sulfotransferase isoforms?

Distinguishing between HS3ST3B1 and related sulfotransferases (particularly HS3ST3A1) requires careful experimental design:

• Antibody Selection:

  • Use antibodies targeting non-conserved regions (N-terminal specific antibodies show greater specificity)

  • Validate antibodies using knockout or knockdown controls

  • Consider epitope mapping to ensure specificity

• mRNA Expression Analysis:

  • Design PCR primers spanning unique exon junctions

  • Use isoform-specific probes for in situ hybridization

  • RNA-Seq analysis with isoform-specific mapping

• Functional Differentiation:

  • HS3ST3B1 and HS3ST3A1 sulfate identical disaccharides but may have different expression patterns

  • In salivary gland development, Hs3st3a1 and Hs3st3b1 show overlapping but distinct expression patterns

  • Hs3st3a1 appears to regulate Hs3st3b1 expression in some contexts

• Verification Strategies:

  • Use genetic knockout models (single vs. double knockouts of Hs3st3a1 and Hs3st3b1)

  • Employ mass spectrometry to identify specific sulfation patterns

  • Immunoprecipitation followed by mass spectrometry can confirm antibody specificity

What are the critical controls needed when investigating HS3ST3B1 function through antibody-based techniques?

When studying HS3ST3B1 using antibody-based techniques, incorporate these essential controls:

• Expression Controls:

  • Positive control: Tissues/cells known to express HS3ST3B1 (liver, placenta)

  • Negative control: Tissues/cells with minimal HS3ST3B1 expression

  • HS3ST3B1 overexpression systems (transfected cells)

• Antibody Specificity Controls:

  • Primary antibody omission

  • Isotype-matched control antibodies

  • Pre-absorption with immunizing peptide/protein

  • siRNA/shRNA knockdown of HS3ST3B1

• Genetic Controls:

  • HS3ST3B1 knockout models

  • Compare single (Hs3st3b1 KO) versus double knockout (Hs3st3a1/Hs3st3b1 DKO) phenotypes

• Enzymatic Controls:

  • Treatment with heparinase III to degrade heparan sulfate (relevant for studies examining HS3ST3B1 substrates)

  • Paired samples with and without enzyme treatment can help validate epitope specificity

• Signal Validation:

  • Multiple antibodies targeting different epitopes of HS3ST3B1

  • Orthogonal detection methods (protein vs. mRNA detection)

  • Western blot validation of immunohistochemistry results

How can researchers address potential compensatory mechanisms when studying HS3ST3B1 knockout or knockdown systems?

When investigating HS3ST3B1 function through knockout or knockdown approaches, compensatory mechanisms can confound results:

• Comprehensive Isoform Analysis:

  • Monitor expression changes in other HS3ST family members (particularly HS3ST3A1, HS3ST1, and HS3ST6)

  • Research has shown that in Hs3st3b1 KO systems, there is a trend of increased Hs3st1 and Hs3st3a1 expression

  • Use qRT-PCR to quantify changes in related sulfotransferases

• Temporal Considerations:

  • Employ inducible knockout/knockdown systems to avoid developmental compensation

  • Monitor expression changes over time after acute depletion

  • Compare acute versus chronic depletion phenotypes

• Functional Assessment:

  • Analyze sulfation patterns using mass spectrometry or specific antibodies (e.g., HS4C3V)

  • Measure specific downstream effects (e.g., binding of growth factors to heparan sulfate)

  • Perform rescue experiments with HS3ST3B1 re-expression

• Combined Approaches:

  • Use CRISPR/Cas9 knockout with simultaneous knockdown of compensatory isoforms

  • Employ double or triple knockout models for related sulfotransferases

  • Consider pharmacological inhibition combined with genetic approaches

What is the evidence linking HS3ST3B1 to cancer progression, and how can researchers best investigate this connection?

Emerging evidence indicates HS3ST3B1 plays significant roles in cancer biology:

• Cancer-Specific Expression Patterns:

  • Upregulated in non-small cell lung cancer (NSCLC) tissues compared to matched normal tissues

  • Associated with mesenchymal phenotype in cancer cells, suggesting involvement in epithelial-to-mesenchymal transition (EMT)

  • Promotes angiogenesis and proliferation in acute myeloid leukemia (AML)

• Molecular Mechanisms:

  • Induces VEGF expression and shedding in AML cells

  • VEGF-induced activation of ERK and AKT signaling pathways can be attenuated by heparanase inhibitor suramin or VEGFR inhibitor axitinib

  • Knockdown of HS3ST3B1 reverses mesenchymal phenotype in lung cancer cells, upregulating CDH1 and downregulating VIM

• Research Approaches:

  • Tissue microarray analysis comparing cancer vs. normal tissues

  • Cell line models with HS3ST3B1 overexpression or knockdown

  • In vivo xenograft models to assess tumorigenic potential

  • Analysis of correlation between HS3ST3B1 expression and clinical outcomes

• Experimental Design for Cancer Studies:

  • Paired analysis of primary tumors and metastatic lesions

  • Correlation of HS3ST3B1 expression with EMT markers

  • Investigation of specific sulfation patterns in tumor microenvironment

  • Assessment of responses to targeted therapies (e.g., VEGF inhibitors) in relation to HS3ST3B1 status

How does HS3ST3B1 contribute to osteoarthritis progression, and what methodologies best capture this relationship?

Recent research has revealed important connections between HS3ST3B1 and osteoarthritis:

• Molecular Relationship:

  • ALKBH5-mediated m6A demethylation of HS3ST3B1-IT1 (a long non-coding RNA) prevents osteoarthritis progression

  • HS3ST3B1 enhances chondrocyte viability, inhibits chondrocyte apoptosis, and increases extracellular matrix components

  • Direct interaction between HS3ST3B1-IT1 and HS3ST3B1 protein has been confirmed via RNA immunoprecipitation

• Functional Effects:

  • HS3ST3B1 overexpression significantly upregulates COL2A1 and Aggrecan expressions while downregulating MMP13 and ADAMTS-5

  • Affects apoptosis-related proteins: upregulates Bcl-2 and downregulates Bax, cleaved Caspase-9, cleaved Caspase-3, and cleaved PARP

  • HS3ST3B1 knockdown has opposite effects, promoting chondrocyte apoptosis

• Research Methodologies:

  • Human primary chondrocyte cultures with HS3ST3B1 manipulation

  • Analysis of HS3ST3B1 expression in OA cartilage vs. normal tissue

  • Assessment of extracellular matrix components and degradative enzymes

  • Evaluation of cell viability and apoptosis following HS3ST3B1 modulation

• Experimental Approaches:

  • Overexpression systems using HS3ST3B1 expression plasmids

  • siRNA-mediated knockdown (multiple independent siRNAs recommended)

  • Cell viability assays (CCK-8) and flow cytometry for apoptosis analysis

  • Western blot for apoptosis markers and ECM components

What is the significance of HS3ST3B1 in neurodegenerative disorders, and how can researchers investigate this relationship?

Emerging evidence suggests potential roles for heparan sulfate sulfotransferases in neurodegenerative conditions:

• Connection to Alzheimer's Disease (AD):

  • While HS3ST1 (rather than HS3ST3B1) has been more directly linked to AD as a genetic risk locus ,

  • 3-O-sulfated heparan sulfate domains show increased presence in AD brain samples

  • Specific 3-O-sulfated HS may enhance tau internalization and spread of tau pathology

• Research Approaches:

  • Analysis of HS3ST3B1 expression in brain regions affected by neurodegeneration

  • Examination of sulfation patterns in different tauopathies using LC-MS/MS methods

  • Comparison of HS3ST3B1 levels across neurodegenerative disorders (AD, FTLD_tau, LBD)

  • Investigation of interactions between 3-O-sulfated HS domains and tau protein

• Experimental Design:

  • Brain tissue analysis from different neurodegenerative conditions

  • In vitro models of tau internalization and propagation

  • Analysis of heparan sulfate composition in different brain regions

  • Correlation of HS3ST3B1 activity with disease progression markers

• Methodological Considerations:

  • LC-MS/MS for specific sulfation pattern analysis

  • Use of synthetic tetradecasaccharides with specific 3-O-sulfated domains

  • Tau internalization assays with competitive inhibition using synthetic HS oligosaccharides

  • Comparison of single gene knockouts versus combinatorial approaches

How can researchers accurately assess HS3ST3B1 enzyme activity rather than just its expression?

Measuring HS3ST3B1 enzyme activity provides more functional information than expression analysis alone:

• In Vitro Sulfotransferase Assays:

  • Radiometric assays using [35S]PAPS (3'-phosphoadenosine 5'-phosphosulfate) as the sulfate donor

  • Incubation of recombinant HS3ST3B1 or cell/tissue extracts with defined HS substrates

  • Measurement of transferred [35S] to substrate

• Mass Spectrometry Analysis:

  • LC-MS/MS to identify specific 3-O-sulfated disaccharides or oligosaccharides

  • Comparison of sulfation patterns in samples with and without HS3ST3B1 manipulation

  • Targeted analysis of characteristic 3-O-sulfated epitopes

• Functional Binding Assays:

  • Assessment of binding between 3-O-sulfated HS and known interacting proteins

  • Competitive inhibition assays with synthetic 3-O-sulfated oligosaccharides

  • Surface plasmon resonance (SPR) to measure binding kinetics

• Cell-Based Activity Reporters:

  • FRET-based sensors designed to detect 3-O-sulfation

  • Reporter systems with readouts triggered by specific 3-O-sulfated HS interactions

  • Use of epitope-specific antibodies (e.g., HS4C3V) that recognize 3-O-sulfated domains

What strategies can researchers employ to investigate the intracellular trafficking and localization of HS3ST3B1?

Understanding HS3ST3B1 localization is crucial for comprehending its biological functions:

• Fluorescent Protein Fusions:

  • Generate HS3ST3B1-GFP or HS3ST3B1-mCherry fusion constructs

  • Validate functionality of tagged proteins through activity assays

  • Live-cell imaging to track trafficking and localization

• Immunolocalization Approaches:

  • Co-staining with organelle markers (ER, Golgi, endosomes)

  • Super-resolution microscopy for detailed subcellular localization

  • Electron microscopy with immunogold labeling for ultrastructural analysis

• Biochemical Fractionation:

  • Subcellular fractionation followed by Western blotting

  • Density gradient centrifugation to separate organelles

  • Enzyme activity assays in different cellular fractions

• Trafficking Studies:

  • Photoactivatable or photoconvertible fusion proteins

  • RUSH (Retention Using Selective Hooks) system for synchronized trafficking

  • BioID or APEX proximity labeling to identify proteins in close proximity to HS3ST3B1

What methodological approaches can resolve contradictory findings regarding HS3ST3B1 expression or function in different experimental systems?

When faced with contradictory findings regarding HS3ST3B1, researchers should consider:

• Methodological Standardization:

  • Define consistent antibody validation criteria

  • Standardize detection methods across studies

  • Establish uniform sample preparation protocols

• Context-Dependent Expression:

  • Assess tissue-specific and developmental regulation

  • Compare expression in cell lines versus primary tissues

  • Examine regulation by microenvironmental factors

• Isoform-Specific Analysis:

  • Distinguish between HS3ST3B1 and closely related family members

  • Employ isoform-specific detection methods

  • Consider potential coordinated regulation of multiple isoforms

• Functional Redundancy Assessment:

  • Combinatorial knockdown/knockout approaches

  • Activity-based assays rather than expression-only studies

  • Analysis of substrate specificity in different systems

• Resolution Approaches:

  • Multi-center collaborative studies with standardized protocols

  • Meta-analysis of existing datasets with careful consideration of methodology

  • Development of more specific detection tools and activity assays

How can single-cell approaches advance our understanding of HS3ST3B1 function in heterogeneous tissues?

Single-cell technologies offer powerful new approaches to study HS3ST3B1:

• Single-Cell RNA Sequencing:

  • Map HS3ST3B1 expression across cell types within tissues

  • Identify co-expression patterns with other HS biosynthetic enzymes

  • Discover cell type-specific regulatory networks

  • Research has shown cell-specific expression in salivary gland development, with HS3ST3B1 found in myoepithelial cells and duct cells

• Single-Cell Proteomics:

  • Quantify HS3ST3B1 protein abundance at single-cell resolution

  • Correlate with expression of other sulfotransferases

  • Analyze cell-specific post-translational modifications

• Spatial Transcriptomics:

  • Map HS3ST3B1 expression within tissue architecture

  • Correlate expression with tissue microenvironments

  • Link expression patterns to functional domains in tissues

• Functional Single-Cell Studies:

  • CRISPR screens with single-cell readouts

  • Clonal analysis of HS3ST3B1 knockout/knockin cells

  • Single-cell activity sensors for 3-O-sulfotransferase function

What are the most promising approaches for targeting HS3ST3B1 or its modified substrates therapeutically?

Emerging therapeutic strategies targeting HS3ST3B1 include:

• Small Molecule Modulators:

  • Develop selective inhibitors of HS3ST3B1 enzymatic activity

  • Screen compound libraries for molecules that alter 3-O-sulfation patterns

  • Consider PAPS (3'-phosphoadenosine 5'-phosphosulfate) analogs as competitive inhibitors

• Genetic Modulation Strategies:

  • CRISPR-based approaches for precise gene editing

  • Antisense oligonucleotides targeting HS3ST3B1 mRNA

  • Viral vector-mediated gene therapy for conditions requiring increased HS3ST3B1

• Substrate-Directed Approaches:

  • Synthetic 3-O-sulfated oligosaccharides as competitive inhibitors

  • Designer heparan sulfate mimetics with specific sulfation patterns

  • Antibodies or peptides targeting 3-O-sulfated HS domains

• Disease-Specific Applications:

  • For cancer: Combined targeting of HS3ST3B1 and VEGF signaling

  • For osteoarthritis: Enhancement of HS3ST3B1 function to promote chondrocyte survival

  • For neurodegeneration: Inhibition of 3-O-sulfated HS domains that promote tau propagation

How can computational approaches enhance our understanding of HS3ST3B1 substrate specificity and biological functions?

Computational methods offer powerful tools for studying HS3ST3B1:

• Structural Modeling:

  • Homology modeling of HS3ST3B1 active site

  • Molecular docking of substrate oligosaccharides

  • Molecular dynamics simulations of enzyme-substrate complexes

• Bioinformatic Analysis:

  • Phylogenetic analysis of HS3ST family members across species

  • Identification of conserved structural features

  • Network analysis of HS3ST3B1 interactions with other proteins

• Machine Learning Applications:

  • Prediction of substrate specificity based on oligosaccharide sequences

  • Integration of multi-omics data to predict HS3ST3B1 functions

  • Pattern recognition in sulfation motifs across different tissues

• Systems Biology Approaches:

  • Pathway analysis incorporating HS3ST3B1 and its substrates

  • Tissue-specific interaction networks

  • Prediction of compensatory mechanisms in knockout systems