Recombinant Danio rerio Cytosolic sulfotransferase 3 (sult1st3)

What is sult1st3 and what is its function in zebrafish?

Sult1st3, also known as sulfotransferase family 1, cytosolic sulfotransferase 3, is a protein-coding gene in zebrafish (Danio rerio). It was previously identified by the names wu:fj78b11 and zgc:92322 . This enzyme enables sulfotransferase activity and is primarily involved in xenobiotic metabolic processes. Sulfotransferases catalyze the transfer of a sulfonate moiety from the universal sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to various acceptor molecules containing hydroxyl, sulfhydryl, amino, or N-oxide groups .

In zebrafish, sult1st3 is predicted to be active in the cytoplasm, consistent with its classification as a cytosolic sulfotransferase . While specific substrates for sult1st3 have not been fully characterized in the search results, research on related zebrafish sulfotransferases suggests that members of this family may be involved in the metabolism of both endogenous compounds (such as hormones) and xenobiotics (environmental compounds or drugs) .

How is sult1st3 related to human sulfotransferases?

Sult1st3 in zebrafish is orthologous to human SULT1E1 (sulfotransferase family 1E member 1) . This orthology relationship suggests functional similarity between the zebrafish and human enzymes. Human ortholog(s) of this gene have been implicated in several diseases, including breast cancer and endometrial cancer .

Understanding this evolutionary relationship is crucial for researchers using zebrafish as a model organism for human disease studies. The conservation of sulfotransferase functions across species suggests that zebrafish sult1st3 studies may provide insights relevant to human SULT1E1 function in normal physiology and disease states.

What domains and structural features characterize sult1st3?

The sult1st3 protein contains specific functional domains that are characteristic of the sulfotransferase family. According to domain analysis, the protein contains a sulfotransferase domain . The protein lengths of known isoforms (UniProtKB:Q7T2V2 and UniProtKB:Q6DHG7) are both 301 amino acids .

Structurally, like other cytosolic sulfotransferases, sult1st3 likely contains a P-loop containing nucleoside triphosphate hydrolase domain, which is important for binding and orientation of the PAPS cofactor during catalysis . These structural features are critical for the enzyme's function in transferring sulfonate groups to various substrates.

What is the expression pattern of sult1st3 during zebrafish development?

While the search results don't provide specific developmental expression data for sult1st3, studies on related zebrafish sulfotransferases provide a methodological framework for such investigations. For example, SULT1 ST9, SULT3 ST4, and SULT3 ST5 have been analyzed using reverse transcription-polymerase chain reaction (RT-PCR) to examine their expression at different developmental stages during embryogenesis, through larval development, and into maturity .

To determine the developmental expression pattern of sult1st3, researchers could employ similar RT-PCR approaches using stage-specific RNA samples from unfertilized eggs, embryos at various developmental timepoints, larvae, and adult fish (both male and female). The specific primer design for sult1st3 would target its unique sequence regions to ensure specificity .

How can tissue-specific expression of sult1st3 be determined?

Determining tissue-specific expression of sult1st3 would require a combination of molecular and histological techniques. Based on approaches used for other sulfotransferases, researchers could:

• Perform quantitative RT-PCR using RNA isolated from different zebrafish tissues (brain, liver, intestine, gonads, etc.)

• Conduct immunohistochemical analysis using antibodies specific to sult1st3

• Generate transgenic reporter lines with the sult1st3 promoter driving fluorescent protein expression

While studies on human sulfotransferases have shown distinct expression patterns across different tissues (e.g., SULT1A1 and SULT1A3 in various brain regions) , the tissue-specific expression of sult1st3 in zebrafish would need to be determined experimentally. Understanding this distribution is crucial for interpreting the enzyme's physiological roles.

What are the optimal methods for cloning and expressing recombinant sult1st3?

Based on methodologies used for other zebrafish sulfotransferases, researchers can employ the following approach for cloning and expressing recombinant sult1st3:

• Primer Design: Design PCR primers incorporating appropriate restriction sites (e.g., BamHI) based on the sult1st3 sequence .

• Template Preparation: Generate first-strand cDNA by reverse transcription using total RNA isolated from zebrafish tissues where sult1st3 is expressed .

• PCR Amplification: Perform PCR using high-fidelity DNA polymerase (e.g., Ex Taq DNA polymerase) under optimized conditions similar to: 2 min at 94°C for initial denaturation, followed by 20 cycles of 94°C for 35 sec, 60°C for 40 sec, and 72°C for 1 min .

• Cloning Vector Selection: Clone the PCR product into an appropriate expression vector such as pMAL-c5x or pGEX-2T to generate fusion proteins with maltose-binding protein (MBP) or glutathione S-transferase (GST) tags that facilitate purification .

• Expression System: Transform competent E. coli BL21(DE3) cells with the recombinant plasmid and induce protein expression with IPTG when the culture reaches approximately 0.6 OD at 600 nm .

What purification strategies yield the highest purity and activity of recombinant sult1st3?

For optimal purification of recombinant sult1st3, researchers can implement the following strategy:

• Cell Lysis: After IPTG induction, collect cells by centrifugation and homogenize in ice-cold lysis buffer using a French Press or sonication. Add protease inhibitors (e.g., aprotinin) to prevent degradation .

• Affinity Chromatography: Based on the fusion tag, purify using:

  • For MBP-tagged sult1st3: Use amylose resin and elute with maltose in buffer (50 mM Tris-HCl, pH 8.0)

  • For GST-tagged sult1st3: Use glutathione-Sepharose and either elute with reduced glutathione or cleave the protein directly from the resin using thrombin (in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl₂)

• Quality Control: Verify protein purity using SDS-PAGE and assess activity using appropriate enzymatic assays .

• Storage: Store purified enzyme in buffer containing stabilizers such as DTT (1 mM) with glycerol (10-20%) at -80°C to maintain activity.

This approach has been successful for related zebrafish sulfotransferases and likely would yield active recombinant sult1st3.

How can the substrate specificity of sult1st3 be determined?

To determine the substrate specificity of sult1st3, researchers should employ systematic enzymatic assays testing various potential substrates. Based on approaches used for related sulfotransferases, the following methodology is recommended:

• Prepare standardized enzymatic assay mixtures containing:

  • 50 mM buffer (e.g., MOPS at pH 7.0)

  • 14 μM PAP[³⁵S] (radiolabeled 3'-phosphoadenosine 5'-phosphosulfate)

  • 1 mM DTT

  • 50 μM test substrate

  • Purified recombinant sult1st3

• Test a panel of potential substrates including:

  • Endogenous compounds: steroid hormones (DHEA, pregnenolone, 17β-estradiol, corticosterone, androstene-3,17-dione), thyroid hormones (T₃, T₄)

  • Xenobiotics: phenolic compounds (β-naphthol), aromatic amines (β-naphthylamine), plant phenolics (caffeic acid, gallic acid), pharmaceutical compounds (mestranol, 17α-ethynylestradiol)

• Analyze reaction products using thin-layer chromatography (TLC) with a solvent system containing n-butanol, isopropanol, 88% formic acid, and H₂O in a ratio of 3:1:1:1 (by volume) .

• Quantify sulfated products by autoradiography and scintillation counting .

Based on studies of related sulfotransferases, substrate specificity patterns may emerge - for example, SULT1 family members often show preference for phenolic compounds, while SULT3 family members may exhibit stronger activity toward steroids and other endogenous substrates .

What are the optimal reaction conditions for sult1st3 activity?

Determining optimal reaction conditions requires systematic testing of various parameters:

• pH Optimization:

  • Test activity across a pH range (typically pH 5.0-10.0) using appropriate buffers:

    • pH 5.0-6.5: MES buffer

    • pH 6.5-8.0: MOPS or HEPES buffer

    • pH 8.0-9.0: TAPS or CHES buffer

    • pH 9.0-10.0: CAPS buffer

• Temperature Dependence:

  • Assess activity at temperatures ranging from 25°C to 45°C to determine the temperature optimum

  • Evaluate thermal stability by pre-incubating the enzyme at various temperatures before assaying activity

• Cofactor Requirements:

  • Determine the optimal concentration of PAPS (typically in the range of 1-20 μM)

  • Test the effects of divalent cations (Mg²⁺, Mn²⁺, Ca²⁺) and reducing agents (DTT, β-mercaptoethanol)

• Kinetic Parameters:

  • For identified substrates, determine Km and Vmax values under optimal conditions

  • Calculate catalytic efficiency (kcat/Km) to compare substrate preferences quantitatively

These optimized conditions would be essential for accurate characterization of sult1st3 and comparison with other sulfotransferases.

How can kinetic parameters of sult1st3 be accurately determined?

For robust determination of sult1st3 kinetic parameters, researchers should:

• Perform Initial Velocity Studies:

  • Ensure reactions are measured in the linear range of enzyme activity

  • Use substrate concentrations spanning at least 0.2-5 times the Km value (typically a range from 0.1 μM to 100 μM for most SULT substrates)

  • Include proper controls without substrate or enzyme

• Data Analysis Methods:

  • Apply appropriate enzyme kinetic models:

    • Michaelis-Menten equation for simple kinetics

    • Hill equation if cooperativity is observed

    • Competitive, non-competitive, or uncompetitive inhibition models when examining inhibitors

• Considerations for Substrate Inhibition:

  • Many sulfotransferases exhibit substrate inhibition at higher concentrations

  • Test a wide range of substrate concentrations to detect potential substrate inhibition

  • Apply modified kinetic equations accounting for substrate inhibition when appropriate

• Reporting Standards:

  • Report Km (μM), Vmax (nmol/min/mg), kcat (min⁻¹), and catalytic efficiency (kcat/Km)

  • Include standard errors and experimental conditions in all reports

  • Present results in tabular format for comparison with other SULT enzymes

Example table format for reporting kinetic parameters:

This systematic approach would yield reliable kinetic parameters for comparison with other sulfotransferases.

How can gene editing tools be used to study sult1st3 function in vivo?

Modern gene editing approaches offer powerful tools to investigate sult1st3 function in zebrafish:

• CRISPR/Cas9 Knockout Strategies:

  • Design sgRNAs targeting exonic regions of sult1st3

  • Generate complete knockouts or specific domain mutations

  • Analyze phenotypic consequences across development

  • Examine changes in metabolite profiles through targeted metabolomics

• Conditional Knockdown Approaches:

  • Employ morpholino oligonucleotides for transient developmental knockdown

  • Use inducible promoter systems for temporal control of sult1st3 expression

  • Create tissue-specific knockout lines using Cre-lox systems

• Overexpression and Rescue Studies:

  • Generate transgenic lines overexpressing wild-type or mutant sult1st3

  • Perform rescue experiments in knockout backgrounds

  • Examine dosage effects through titrated expression systems

• Phenotypic Analysis:

  • Compare developmental milestones and morphology

  • Analyze xenobiotic sensitivity in wild-type versus mutant fish

  • Assess substrate metabolism through analytical chemistry approaches

  • Examine gene expression changes through RNA-seq to identify downstream pathways

These approaches would help elucidate the physiological and developmental roles of sult1st3 in zebrafish, potentially providing insights relevant to human SULT1E1 function.

What is the role of sult1st3 in xenobiotic metabolism and detoxification?

Based on information about related sulfotransferases, sult1st3 likely plays an important role in xenobiotic metabolism. To investigate this role specifically:

• Comparative Toxicity Assays:

  • Expose sult1st3 knockout and wild-type zebrafish to various xenobiotics

  • Determine LC50 values and time-dependent toxicity profiles

  • Analyze behavioral responses to sub-lethal toxicant exposure

• Metabolite Profiling:

  • Use liquid chromatography-mass spectrometry (LC-MS) to identify and quantify sulfated metabolites

  • Compare metabolite profiles between wild-type and sult1st3-deficient fish

  • Trace the fate of labeled xenobiotics to identify sult1st3-dependent metabolic pathways

• Cellular Localization Studies:

  • Examine tissue-specific expression patterns in response to xenobiotic exposure

  • Determine subcellular localization through fractionation and immunohistochemistry

  • Analyze potential translocation or expression changes upon toxicant exposure

• Pathway Interaction Analysis:

  • Investigate potential interactions with other detoxification enzymes (cytochrome P450s, UDP-glucuronosyltransferases)

  • Examine compensatory changes in other SULT family members in sult1st3 knockouts

  • Identify potential synergistic or antagonistic relationships with other detoxification pathways

Since xenobiotic metabolism is a key function of sulfotransferases, understanding sult1st3's specific role would have implications for environmental toxicology and pharmacology research.

How does sult1st3 activity influence hormone signaling in zebrafish?

Given that human SULT1E1 (ortholog of zebrafish sult1st3) is known to be involved in estrogen metabolism, sult1st3 may play a role in hormone signaling in zebrafish. To investigate this:

• Hormone Metabolism Assays:

  • Test sult1st3 activity toward various steroid hormones in vitro

  • Quantify sulfated hormone metabolites in wild-type versus sult1st3 knockout fish

  • Examine hormone-dependent developmental processes in mutant versus wild-type fish

• Reproductive and Developmental Effects:

  • Analyze gonadal development in sult1st3-deficient fish

  • Assess fertility, fecundity, and reproductive behaviors

  • Examine sexually dimorphic traits and secondary sex characteristics

• Endocrine Disruption Sensitivity:

  • Test sensitivity to endocrine-disrupting chemicals in wild-type versus sult1st3 knockout fish

  • Examine potential protective effects of sult1st3 against estrogen-mimicking compounds

  • Investigate potential biomarker applications for environmental estrogen exposure

• Gene Expression Analysis:

  • Perform transcriptome analysis of hormone-responsive tissues in wild-type versus mutant fish

  • Examine expression changes in hormone receptor genes and hormone-responsive genes

  • Investigate feedback mechanisms between hormone levels and sult1st3 expression

Understanding the role of sult1st3 in hormone metabolism could provide insights into endocrine regulation and potential implications for reproductive and developmental biology.

How can structural biology approaches enhance understanding of sult1st3 function?

Advanced structural biology techniques can provide detailed insights into sult1st3 function:

• Protein Crystallography:

  • Express, purify, and crystallize recombinant sult1st3

  • Determine high-resolution crystal structures of apo-enzyme and enzyme-substrate complexes

  • Identify key catalytic residues and substrate binding sites

  • Compare structural features with human SULT1E1 to understand evolutionary conservation

• Molecular Dynamics Simulations:

  • Model substrate binding and catalytic mechanisms

  • Simulate conformational changes during catalysis

  • Predict effects of mutations on protein stability and function

  • Investigate water networks and proton transfer pathways

• Structure-Based Drug Design:

  • Use structural information to design selective inhibitors or substrate analogs

  • Develop tools to modulate sult1st3 activity in vivo

  • Create structure-activity relationship (SAR) models for substrate specificity

• Site-Directed Mutagenesis Validation:

  • Design mutations of predicted key residues based on structural data

  • Express and characterize mutant proteins to validate structural hypotheses

  • Correlate structure-function relationships through activity assays

These approaches would provide molecular-level understanding of sult1st3 catalysis and substrate recognition, potentially enabling applications in drug development and toxicology.

What are the potential implications of sult1st3 polymorphisms in zebrafish populations?

Investigating polymorphisms in sult1st3 could reveal important insights into natural variation and functional consequences:

• Population Genetics Approaches:

  • Sequence sult1st3 from diverse wild zebrafish populations

  • Identify common variants and their frequencies

  • Examine evidence for selective pressures through population genetic analyses

• Functional Characterization of Variants:

  • Express and purify variant forms of sult1st3

  • Compare enzymatic activities and substrate specificities

  • Analyze thermal stability and other biophysical properties

• Phenotypic Consequences:

  • Generate zebrafish lines expressing different natural variants

  • Compare xenobiotic sensitivity and metabolism

  • Examine potential differences in hormone regulation and developmental processes

• Comparative Analysis with Human SULT1E1 Polymorphisms:

  • Relate zebrafish sult1st3 variants to known human SULT1E1 polymorphisms

  • Assess potential as a model for human variation

  • Examine conservation of functionally significant variants

This research area could provide insights into natural adaptation mechanisms and potentially model human polymorphism effects seen in SULT1E1, where variations have been linked to altered drug metabolism and potentially disease risk .

How can systems biology approaches integrate sult1st3 function into broader metabolic networks?

Systems biology offers tools to understand sult1st3 in the context of broader biological networks:

• Multi-Omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and sult1st3 knockout fish

  • Identify metabolic pathways affected by sult1st3 deficiency

  • Construct network models of sult1st3-dependent processes

• Pathway Analysis:

  • Map sult1st3 substrates and products onto known metabolic pathways

  • Identify regulatory interactions with other detoxification systems

  • Examine potential feedback mechanisms and compensatory responses

• Mathematical Modeling:

  • Develop kinetic models of sult1st3-dependent metabolic pathways

  • Simulate effects of altered enzyme activity on metabolite concentrations

  • Predict emergent system properties through in silico experiments

• Physiological Integration:

  • Connect molecular-level findings to tissue, organ, and whole-organism phenotypes

  • Investigate developmental stage-specific roles in different tissues

  • Examine potential interactions with environmental factors

How can zebrafish sult1st3 research inform human SULT1E1 studies?

As sult1st3 is orthologous to human SULT1E1, research in zebrafish can provide valuable insights for human applications:

• Comparative Functional Analysis:

  • Systematically compare substrate specificities between zebrafish sult1st3 and human SULT1E1

  • Identify conserved regulatory mechanisms and expression patterns

  • Assess conservation of protein-protein interactions and metabolic pathways

• Disease Modeling:

  • Create zebrafish models mimicking human SULT1E1 mutations associated with disease

  • Investigate developmental and physiological consequences

  • Test potential therapeutic interventions in zebrafish before moving to mammalian models

• Drug Metabolism Studies:

  • Use zebrafish as a model to predict human drug metabolism patterns

  • Investigate the role of SULT1E1/sult1st3 in metabolizing therapeutic compounds

  • Examine potential drug-drug interactions mediated by sulfotransferase activity

• Environmental Toxicology Applications:

  • Develop zebrafish-based assays for environmental toxicants affecting sulfotransferase activity

  • Validate findings through comparative studies with human cells and tissues

  • Create biomarkers for xenobiotic exposure relevant to human health

Given that human SULT1E1 has been implicated in breast cancer and endometrial cancer , understanding the fundamental biology of its zebrafish ortholog could potentially contribute to cancer research and therapeutic development.

What methodological challenges exist in studying recombinant sult1st3 and how can they be overcome?

Researchers face several challenges when working with recombinant sulfotransferases, including:

• Protein Solubility and Stability Issues:

  • Challenge: Recombinant SULTs may form inclusion bodies or show limited solubility

  • Solution: Optimize expression conditions (temperature, IPTG concentration), use solubility-enhancing fusion tags (MBP, SUMO), and include stabilizing agents (glycerol, reducing agents) in purification buffers

• Assay Sensitivity and Specificity:

  • Challenge: Detecting sulfotransferase activity can be technically demanding

  • Solution: Employ multiple complementary assay methods (radiochemical assays with PAP[35S], HPLC-based methods, coupled enzyme assays) and include appropriate controls

• Substrate Availability and Handling:

  • Challenge: Many potential substrates have poor solubility or stability

  • Solution: Develop appropriate solubilization methods (use of compatible co-solvents like DMSO at <1% final concentration), prepare fresh substrate solutions, and establish stability profiles

• Interpretation of Kinetic Data:

  • Challenge: Complex kinetic behaviors including substrate inhibition

  • Solution: Use appropriate kinetic models, test wide concentration ranges, and employ global data fitting approaches for complex mechanisms

These methodological considerations are crucial for generating reliable data on sult1st3 function and comparing results across different studies.

What emerging technologies could advance sult1st3 research?

Several cutting-edge technologies hold promise for advancing sult1st3 research:

• CRISPR-Based Approaches:

  • Base editing for introducing specific point mutations

  • Prime editing for precise genomic modifications

  • CRISPRi/CRISPRa for temporally controlled gene expression modulation

  • CRISPR screening to identify genetic interactions

• Advanced Imaging Techniques:

  • Live cell imaging of fluorescently tagged sult1st3

  • Super-resolution microscopy to examine subcellular localization

  • Correlative light and electron microscopy (CLEM) for structural context

  • Whole-organism imaging of substrate metabolism in transparent zebrafish larvae

• Single-Cell Technologies:

  • Single-cell RNA-seq to examine cell-type specific expression patterns

  • Spatial transcriptomics to map expression in tissue context

  • Single-cell proteomics to examine protein-level regulation

  • Integration of multi-modal single-cell data for comprehensive understanding

• Computational Approaches:

  • Machine learning for substrate prediction

  • Quantum mechanics/molecular mechanics (QM/MM) simulations of reaction mechanisms

  • Network pharmacology to predict drug interactions

  • Virtual screening for novel substrates or inhibitors

These technologies could overcome current limitations and provide unprecedented insights into sult1st3 biology and function.

How can contradictory findings in sulfotransferase research be reconciled?

Researchers sometimes encounter contradictory results in sulfotransferase studies. Strategies to address these include:

• Standardization of Experimental Protocols:

  • Develop consensus methods for expression, purification, and activity assays

  • Create standard reference materials (purified enzymes, substrate sets)

  • Establish reporting standards for experimental conditions and kinetic parameters

• Systematic Meta-Analysis:

  • Compare methodologies across contradictory studies

  • Identify variables that might explain discrepancies (pH, temperature, buffer composition)

  • Re-analyze raw data when available using consistent analytical approaches

• Collaborative Validation Studies:

  • Conduct multi-laboratory validation studies

  • Test identical enzyme preparations across different assay platforms

  • Develop proficiency testing programs for sulfotransferase assays

• Consideration of Biological Variables:

  • Investigate effects of post-translational modifications

  • Examine contextual factors (presence of cofactors, inhibitors)

  • Consider developmental and physiological state differences

  • Account for genetic background effects in in vivo studies

Through these approaches, apparent contradictions may be resolved, leading to more robust and reproducible findings in the field.