SULT1C4 Human

What is SULT1C4 and what is its primary function?

SULT1C4 is a member of the cytosolic sulfotransferase family that utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as a sulfonate donor to catalyze the sulfate conjugation of various phenolic compounds . This enzyme plays a significant role in phase II metabolism of xenobiotics and endogenous compounds by adding sulfate groups, which typically increases their water solubility and facilitates excretion. SULT1C4 has notable activity toward a diverse range of substrates, displaying particularly high sulfonation capacity toward xenobiotics including various drugs (e.g., acetaminophen), environmental chemicals (e.g., bisphenol A), and procarcinogens (e.g., hydroxymethyl furans) . While it can sulfonate estrogenic compounds, dietary flavonoids (phytoestrogens) and environmental estrogens like bisphenol A are better substrates than 17beta-estradiol . Additionally, SULT1C4 mediates the sulfation of anticancer drugs doxorubicin and its analog epirubicin .

Where is SULT1C4 expressed in human tissues?

SULT1C4 exhibits a distinctive developmental expression pattern in human tissues. Studies using targeted quantitative proteomics of liver cytosolic fractions have demonstrated that SULT1C4 expression is highest in prenatal and infant liver, showing a developmental stage-specific pattern . This expression pattern differs markedly from other SULT family members - while SULT1A1 expression remains high throughout development, SULT1A2, 1B1, and 2A1 expression is highest in infant and/or adult liver . Research models frequently use Caco-2 cells (intestinal model) and HepaRG cells (liver model) for SULT1C4 studies, as these cell lines express the enzyme at detectable levels, with Caco-2 cells showing particularly high expression . Interestingly, there exists a clear discordance between SULT1C4 mRNA and protein levels in human liver specimens, with mRNA being abundant in prenatal liver while protein levels remain very low .

What is the genomic location and structure of the SULT1C4 gene?

The human SULT1C4 gene is located on chromosome 2q12.3, spanning positions 108,377,911–108,388,989 of the genome . The full-length SULT1C4 transcript variant 1 (TV1) contains seven exons and encodes a protein of 302 amino acids . Multiple transcript variants have been identified, including a variant lacking exons 3 and 4 (TV2, NM_001321770), two noncoding RNA variants (TV3, NR_135776 and TV4, NR_135779), and a predicted transcript variant (TVX1, XM_017003807) . An additional noncoding RNA variant consisting of two exons and a retained intron is also indexed in the Ensembl database (ENST00000494122.1) . The 5'-end of the SULT1C4 transcript varies across cell types, with 5'-RACE analysis in Caco-2 and HepaRG cells revealing multiple transcription start sites ranging from 345-690 nucleotides upstream of the translation start codon .

What methodologies are recommended for expressing and purifying SULT1C4 protein?

For efficient expression and purification of SULT1C4 protein, researchers have successfully employed bacterial expression systems using Escherichia coli. The purification process typically involves a multi-step approach to achieve high purity and yield. According to published protocols, the following methodology has proven effective: bacterial expression followed by cell lysis, DEAE-sepharose chromatography, and size exclusion chromatography . Using this approach, SULT1C4 can be purified approximately 115-fold compared to cytosol, with a specific activity of 26.0 nmol of 1-naphthol sulfated per min per milligram protein and a yield of 34.5% . Pure SULT1C4 typically elutes from DEAE-sepharose with approximately 150–200 mM NaCl .

For recombinant protein production, commercial sources provide human SULT1C4 expressed in E. coli with >95% purity, suitable for SDS-PAGE and mass spectrometry applications . For research requiring tagged proteins, expression vectors containing DDK-tagged SULT1C4 can be prepared using pKK233-2-SULT1C4 as template with appropriate primer pairs and PCR conditions (95°C for 2 minutes; 20 cycles of 95°C for 20 seconds, 64°C for 20 seconds, and 72°C for 30 seconds; and 72°C for 3 minutes) . Transfection into HEK293T cells using Lipofectamine 2000 has been successfully employed for mammalian expression .

How can the enzymatic activity of SULT1C4 be measured and characterized?

Multiple methodologies are available for measuring SULT1C4 enzymatic activity, with the choice depending on the specific research question. For kinetic studies of PAPS binding, intrinsic fluorescence spectroscopy provides a sensitive approach. This method involves measuring the fluorescence of purified SULT1C4 (typically at 200 nM) at room temperature with continuous stirring using a spectrofluorometer, with excitation at 282 nm and emission at 342 nm . PAPS is titrated into the reaction mixture in increasing concentrations, allowing determination of binding parameters . Due to the adenosine ring's absorption of the protein's emission at high PAPS concentrations, parallel experiments with AMP as a blank control are recommended .

For substrate specificity studies, various analytical approaches can be employed. Activity toward specific substrates can be measured using radiometric assays with 35S-PAPS or HPLC methods to detect sulfated products. Using these approaches, SULT1C4 has been shown to display sulfating activity toward multiple substrates, including hyperoside, with the capability to determine kinetic parameters for comparison with other SULT enzymes . When analyzing multiple SULT1C4 allozymes, differential sulfating activities toward substrates like hyperoside can reveal the functional impact of genetic polymorphisms .

How do you explain the discordance between SULT1C4 mRNA and protein levels in human liver?

The pronounced discordance between SULT1C4 mRNA and protein levels presents an intriguing research question. While RT-qPCR and RNA-seq analyses indicate that SULT1C4 mRNA is abundant in prenatal liver, targeted quantitative proteomics reveals very low SULT1C4 protein levels in the same tissues . This discrepancy suggests complex post-transcriptional regulation mechanisms affecting SULT1C4 expression.

Several hypotheses may explain this phenomenon:

• Alternative Transcript Variants: The expression of multiple SULT1C4 transcript variants, including those that do not encode functional protein (noncoding RNAs TV3 and TV4), could contribute to high mRNA measurements without corresponding protein production . Research approaches to investigate this include RT-PCR with transcript variant-specific primers spanning unique exon-exon junctions, cloning and sequencing of amplified products, and quantitative analysis using synthetic standards .

• Translational Regulation: Post-transcriptional mechanisms may inhibit efficient translation of SULT1C4 mRNA. Investigating this aspect requires analysis of polysome association, mRNA secondary structure, or regulatory RNA-binding proteins.

• Protein Stability: Rapid degradation of SULT1C4 protein could result in low steady-state levels despite high mRNA expression. Pulse-chase experiments and proteasome inhibition studies would help evaluate this hypothesis.

Research characterizing the specific SULT1C4 transcript variants expressed in different tissues and developmental stages has revealed important insights. Methodology for such studies includes 5'-RACE to determine transcription start sites, RT-PCR with primers spanning different exon junctions, cloning and sequencing of PCR products, and quantitative analysis using transcript variant-specific primers .

What approaches are used for molecular modeling and structural analysis of SULT1C4?

A comprehensive molecular modeling workflow for SULT1C4 includes:

• Structure Preparation: Using the crystal structure (PDB 2GWH) as a template, homology modeling software like MOE can be used to complete missing residues .

• Energy Minimization: The complete structure should be energy minimized using appropriate force fields such as Amber99 .

• Protonation State Assignment: Protonation is performed using functions like MOE 3D Protonate set to physiological conditions (pH 7.4, 150 mM NaCl) .

• Structure Validation: Quality assessment using validation servers such as NIH Structure Analysis and Verification Server (NIH-SAVES), with a score of 90% or higher considered acceptable .

• Substrate Docking and Binding Analysis: Molecular docking of various substrates and analysis of binding interactions to understand substrate specificity.

This comprehensive structural analysis approach provides a foundation for understanding enzyme-substrate interactions and the impact of genetic polymorphisms on SULT1C4 function.

How do genetic polymorphisms affect SULT1C4 activity?

Genetic polymorphisms significantly impact SULT1C4 enzymatic activity. Research has demonstrated that different SULT1C4 allozymes display differential sulfating activities toward substrates like hyperoside, clearly revealing the functional consequences of genetic variations . This finding has important implications for understanding individual differences in xenobiotic metabolism and drug response.

To investigate the effects of polymorphisms, researchers have employed the following methodological approaches:

• Allozyme Preparation: Generation of expression constructs for different SULT1C4 variants through site-directed mutagenesis or gene synthesis.

• Activity Assays: Comparative analysis of enzymatic activity using standardized substrates across different allozymes.

• Kinetic Analysis: Determination of kinetic parameters (Km, Vmax) to quantify the impact of polymorphisms on enzyme efficiency.

• Structure-Function Correlation: Mapping polymorphisms onto the protein structure to understand mechanistic bases for altered activity.

This research provides a robust biochemical foundation for further studies on the functional impact of SULT1C4 genetic polymorphisms and their potential role in variability in drug metabolism and toxicity .

What is the role of SULT1C4 in xenobiotic metabolism and toxicology?

SULT1C4 plays a significant role in xenobiotic metabolism through its capability to sulfonate a wide range of compounds. Several studies have demonstrated that SULT1C4 possesses the highest sulfonation capacity among SULT1C enzymes toward various xenobiotics, including drugs (acetaminophen), environmental chemicals (bisphenol A), and procarcinogens (hydroxymethyl furans) . This broad substrate specificity makes SULT1C4 an important enzyme in phase II metabolism pathways that typically convert compounds to more water-soluble forms for excretion.

For toxicological research, SULT1C4's activity toward environmental estrogens and procarcinogens is particularly significant. The enzyme mediates the sulfation of doxorubicin and epirubicin, two antitumor anthracyclines used in cancer treatment . Additionally, SULT1C4 catalyzes the sulfation of dietary flavonoids and environmental estrogens with higher efficiency than endogenous estrogens like 17β-estradiol . This activity pattern suggests SULT1C4 may play a specialized role in detoxification of environmental compounds rather than regulation of endogenous hormones.

Research methodologies for investigating SULT1C4's role in xenobiotic metabolism include in vitro sulfation assays with purified enzyme or cellular systems expressing SULT1C4, metabolite identification using mass spectrometry, and correlation of enzymatic activity with toxicological endpoints.

How does the developmental expression pattern of SULT1C4 affect xenobiotic metabolism in the developing fetus?

The distinctive developmental expression pattern of SULT1C4, with highest levels in prenatal and infant liver, suggests it plays a specialized role in fetal and neonatal xenobiotic metabolism . This temporal expression pattern has important implications for understanding drug metabolism and environmental chemical detoxification during critical developmental periods.

Despite high mRNA expression in prenatal liver, the observed low SULT1C4 protein levels present a paradox that complicates the assessment of its functional significance in fetal metabolism . Research approaches to address this question include:

Understanding SULT1C4's developmental expression has significant implications for pediatric pharmacology and risk assessment of environmental exposures during pregnancy and early childhood, potentially influencing susceptibility to adverse effects during these vulnerable periods.

How can researchers overcome challenges in detecting and quantifying SULT1C4 protein in human tissues?

The discordance between SULT1C4 mRNA and protein levels presents significant technical challenges for researchers. Several methodological approaches can be employed to improve detection and quantification:

• Targeted Proteomics: Development of sensitive and specific mass spectrometry-based methods for SULT1C4 quantification, such as multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM), can enhance detection sensitivity .

• Enrichment Strategies: Immunoprecipitation or affinity purification techniques prior to analysis can concentrate SULT1C4 protein from complex samples.

• Antibody Selection and Validation: Rigorous validation of antibodies for specificity against SULT1C4, considering potential cross-reactivity with other SULT family members.

• Translation Assessment: Polysome profiling to determine whether SULT1C4 mRNA is efficiently loaded onto ribosomes, providing insights into translational efficiency.

• Protein Stability Analysis: Investigation of post-translational modifications or protein degradation pathways that might affect SULT1C4 stability and detectability.

• Cell-Based Models: Development of cellular models with controlled expression of SULT1C4 for mechanistic studies of protein regulation.

These approaches can help overcome the technical challenges in SULT1C4 research and provide more accurate assessment of its expression and function in various tissues and developmental stages.

What are the optimal experimental systems for studying SULT1C4 function?

Selecting appropriate experimental systems is crucial for meaningful SULT1C4 research. Based on the literature, several systems have been successfully employed:

• Recombinant Protein Systems: Purified recombinant SULT1C4 expressed in E. coli provides a clean system for enzymatic characterization, kinetic studies, and substrate specificity analysis . This approach has been used to achieve >95% purity suitable for various applications .

• Cell Culture Models:

  • HepaRG cells serve as a liver model that expresses detectable levels of SULT1C4

  • Caco-2 cells provide an intestinal model with high SULT1C4 expression

  • HEK293T cells are suitable for transient expression of SULT1C4 variants for comparative studies

• Human Tissue Samples: Liver specimens from different developmental stages (prenatal, infant, adult) provide context for physiologically relevant expression analysis .

• Transcript Variant Expression Systems: Development of expression constructs for specific SULT1C4 transcript variants enables investigation of their relative expression and functional significance .

Each system offers distinct advantages depending on the research question. Recombinant systems provide controlled conditions for biochemical characterization, while cell models offer insights into physiological regulation and processing. Human tissue samples provide the most relevant context but present challenges in terms of variability and limited availability.

What are the most promising areas for future SULT1C4 research?

Based on current knowledge and gaps identified in the literature, several promising research directions emerge:

• Resolving the mRNA-Protein Discordance: Further investigation into the mechanisms underlying the discrepancy between high SULT1C4 mRNA expression and low protein levels in prenatal liver would provide fundamental insights into post-transcriptional regulation .

• Functional Significance of Transcript Variants: Comprehensive characterization of the biological roles and relative contributions of different SULT1C4 transcript variants, including potential regulatory functions of noncoding RNA variants .

• Role in Developmental Toxicology: Exploration of SULT1C4's contribution to detoxification processes during prenatal and early postnatal development, and its impact on susceptibility to xenobiotics during these critical periods .

• Pharmacogenomic Implications: Expanded investigation of SULT1C4 genetic polymorphisms and their impact on drug metabolism, focusing on pediatric pharmacology and personalized medicine applications .

• Substrate Specificity Determinants: Structural analysis and mutagenesis studies to identify key residues determining SULT1C4's distinctive substrate preferences, particularly its high activity toward environmental chemicals compared to endogenous compounds .

• Regulatory Network Analysis: Identification of transcription factors and signaling pathways controlling SULT1C4 expression during development and in response to environmental stimuli.

These research directions would significantly advance understanding of SULT1C4 biology and its role in human health and disease, particularly in the context of developmental pharmacology and toxicology.