Recombinant Mouse UDP-glucuronosyltransferase 1-9 (Ugt1a9)

What is mouse Ugt1a9 and what is its functional role in metabolism?

Mouse Ugt1a9 is a UDP-glucuronosyltransferase enzyme that plays a critical role in the glucuronidation pathway, transforming small lipophilic molecules such as steroids, bilirubin, hormones, and drugs into water-soluble, excretable metabolites. It belongs to the UGT1A family, which forms part of phase II metabolism enzymes. The enzyme is particularly active on phenolic compounds, making it essential for detoxification processes and drug metabolism in mice .

Mouse Ugt1a9, like human UGT1A9, is expressed primarily in liver tissue and is involved in the conjugation of UDP-glucuronic acid to various xenobiotics and endogenous compounds. This conjugation significantly enhances water solubility, facilitating elimination through bile or urine, thereby playing a crucial role in the body's defense against potentially harmful substances.

How is the Ugt1a gene locus organized in mice compared to humans?

The mouse Ugt1a gene locus shares structural similarities with its human counterpart, featuring a complex organization with multiple first exons and common exons. In humans, the UGT1A locus includes thirteen unique alternate first exons followed by four common exons. Four of these alternative first exons are pseudogenes, while the remaining nine can be spliced to the four common exons, resulting in nine distinct proteins with different N-termini but identical C-termini .

Each first exon encodes the substrate binding site and possesses its own promoter region, allowing for tissue-specific and substrate-specific regulation of expression. This complex genetic architecture enables diverse functionality despite structural similarities among family members, a feature conserved between mouse and human UGT1A enzymes.

Which metabolic pathways involve mouse Ugt1a9?

Mouse Ugt1a9 participates in several important metabolic pathways that are crucial for normal physiological function and xenobiotic detoxification. These include:

• Pentose and glucuronate interconversions

• Ascorbate and aldarate metabolism

• Steroid hormone biosynthesis

• Metabolic pathways involving various xenobiotics

• Chemical carcinogenesis pathways

• Retinol metabolism

Through these pathways, Ugt1a9 contributes to homeostasis maintenance and plays a protective role against potential toxins by facilitating their conversion to more easily eliminated forms.

What transgenic mouse models are available for studying Ugt1a9 function?

Transgenic mouse models provide valuable tools for investigating Ugt1a9 function in vivo. Several models have been developed to elucidate the physiological roles and regulatory mechanisms of UGT enzymes:

• PXR knockout mice: These models have demonstrated that prengnane X receptor (PXR) plays a crucial role in regulating Ugt1a9 expression. While wild-type mice show approximately 100% increase in Ugt1a9 mRNA expression after pregnane-16α-carbonitrile (PCN) treatment, this induction is completely abolished in PXR knockout mice .

• UGT humanized mice: These models express human UGT1A genes in place of the mouse orthologs, allowing researchers to study human UGT function in an in vivo context.

• Tissue-specific UGT knockout models: These can help elucidate the tissue-specific roles of Ugt1a9 in drug metabolism and detoxification.

Researchers should select the appropriate model based on their specific experimental questions, considering factors such as the regulatory mechanisms they wish to study and the translational relevance of their research.

How can I optimize expression conditions for recombinant mouse Ugt1a9?

Optimizing expression of recombinant mouse Ugt1a9 requires careful consideration of several factors:

• Expression system selection: Several expression systems have been used successfully for UGT enzymes:

  • Mammalian cell lines (HEK293, CHO)

  • Insect cells (Sf9, High Five)

  • E. coli

  • Cell-free expression systems

  • Wheat germ systems

• Fusion tags: Consider adding fusion tags to facilitate purification and potentially enhance stability:

  • His tag

  • GST tag

  • Avi tag

  • Fc tag

• Co-expression strategies: Co-expression with chaperones or other accessory proteins may improve folding and stability.

• Membrane incorporation: As UGTs are membrane-associated enzymes, proper membrane integration is crucial for activity. When using microsomal preparations, inclusion of alamethicin (12.5 μg/mL) can improve activity by increasing membrane permeability .

• Buffer conditions: Optimize buffer composition, pH, and ionic strength for maximum stability and activity. Standard conditions often include 100 mM Tris-HCl pH 7.4 and 5 mM MgCl₂ .

• Organic solvent limitations: Be aware that addition of more than 5% (v/v) dimethyl sulfoxide, acetonitrile, or ethanol can decrease enzyme activity .

What substrates and assay methods are recommended for measuring mouse Ugt1a9 activity?

For measuring mouse Ugt1a9 activity, several substrates and assay methods can be employed:

• Phenolic compounds: Given Ugt1a9's activity toward phenols, various phenolic substrates can be used .

• Fluorescent substrates: Modified 7-hydroxycoumarin derivatives provide a convenient fluorescence-based assay system. As demonstrated for other UGTs, glucuronidation converts these fluorescent compounds to non-fluorescent glucuronides, allowing real-time monitoring of activity .

• Clinical drug substrates: Mycophenolic acid and propofol have been used as substrates for human UGT1A9 and may also work for the mouse ortholog .

• Assay setup recommendations:

  • Buffer: 100 mM Tris-HCl pH 7.4, 5 mM MgCl₂

  • UDPGA concentration: 0.5 mM

  • Substrate concentration range: 0-15 μM (for kinetic studies)

  • For microsomal preparations: include alamethicin (12.5 μg/mL)

  • Controls: samples without substrate, without UDPGA, or without enzyme

• Detection methods:

  • Fluorescence spectroscopy (excitation/emission wavelengths depend on substrate)

  • HPLC or LC-MS/MS for non-fluorescent substrates

  • For 7-hydroxycoumarin derivatives, monitor fluorescence decrease at 390 nm excitation and 460 nm emission

How is mouse Ugt1a9 regulated at the transcriptional level?

Mouse Ugt1a9 is subject to complex transcriptional regulation through multiple mechanisms:

• Nuclear receptors: The Pregnane X Receptor (PXR) plays a crucial role in regulating Ugt1a9. Studies with PXR knockout mice have shown that PXR activation by pregnane-16α-carbonitrile (PCN) increases Ugt1a9 mRNA expression by approximately 100% in wild-type mice, while this induction is completely abolished in PXR knockout mice .

• Promoter structure: Each first exon of the Ugt1a gene complex has its own promoter, allowing for specific regulation of individual UGT isoforms including Ugt1a9 .

• Other transcription factors: In addition to PXR, other nuclear receptors, the aryl hydrocarbon receptor (AhR), and nuclear factor erythroid 2-related factor 2 (Nrf2) may also regulate Ugt1a9 expression .

• Tissue-specific regulation: Different regulatory mechanisms may operate in different tissues, contributing to tissue-specific expression patterns of Ugt1a9.

Understanding these regulatory mechanisms is crucial for interpreting experimental results and designing interventions to modulate Ugt1a9 activity.

What is known about polymorphisms in Ugt1a9 and their impact on enzyme function?

While most polymorphism studies have focused on human UGT1A9, these findings provide valuable insights for mouse research:

In human UGT1A9, several promoter polymorphisms significantly affect protein expression and enzymatic activity:

• Expression variability: Human UGT1A9 expression can vary by up to 17-fold in liver microsomes, with significant correlation to specific SNPs .

• Key promoter SNPs: Position -275, -331/-440, -665, and -2152 SNPs are significantly associated with altered UGT1A9 protein levels .

• Functional consequences: Livers with -275 and -2152 variants show significantly higher glucuronidating activities toward substrates like mycophenolic acid and propofol, indicating an "extensive glucuronidator" phenotype .

• T10 polymorphism: The -109 to -98 T10 polymorphism, previously reported to increase reporter gene expression in HepG2 cells, was not linked to changes in UGT1A9 protein levels in human liver microsomes .

When working with mouse models, researchers should be aware that strain differences might reflect similar polymorphic variations, potentially affecting experimental outcomes and interpretation.

How can I design experiments to investigate the role of mouse Ugt1a9 in drug metabolism?

Designing rigorous experiments to investigate mouse Ugt1a9's role in drug metabolism requires a multifaceted approach:

• In vitro enzyme kinetics:

  • Use recombinant mouse Ugt1a9 or liver microsomes

  • Include proper controls (no enzyme, no substrate, no UDPGA)

  • Determine kinetic parameters (Km, Vmax) for various substrates

  • Compare wild-type versus genetically modified enzyme variants

• Inhibition studies:

  • Use selective inhibitors to distinguish between UGT isoforms

  • Employ competitive and non-competitive inhibition analysis

  • Consider substrate-dependent inhibition patterns

• Comparative studies:

  • Compare mouse Ugt1a9 with human UGT1A9 to evaluate species differences

  • Compare activity across different mouse strains to account for genetic variability

  • Evaluate tissue-specific differences (liver vs. intestine vs. kidney)

• In vivo studies:

  • Utilize wild-type and Ugt1a9-knockout or transgenic mice

  • Administer substrate drugs and monitor pharmacokinetics

  • Analyze metabolite profiles in plasma, urine, and bile

  • Consider the impact of inducing agents like PXR activators

• Data analysis recommendations:

  • Plot enzyme kinetics using non-linear regression

  • Calculate clearance rates and extrapolate to in vivo scenarios

  • Apply physiologically-based pharmacokinetic modeling when appropriate

What methods can differentiate between Ugt1a9 and other UGT isoforms?

Differentiating between Ugt1a9 and other UGT isoforms is crucial for accurate experimental interpretation:

• Selective substrates: While finding completely selective substrates is challenging, certain compounds show preferential metabolism by specific UGT isoforms. For example, while 7-hydroxycoumarin is primarily glucuronidated by UGT1A6, certain C3-substituted 7-hydroxycoumarin derivatives show high selectivity for specific UGTs .

• Enzyme kinetics: Different UGT isoforms often exhibit distinct kinetic properties with the same substrate. Characterizing Km and Vmax values can help identify the contributing isoforms.

• Recombinant enzymes: Use purified recombinant Ugt1a9 as a positive control and compare with other recombinant UGT isoforms.

• Tissue expression patterns: Leverage knowledge of differential tissue expression. For instance, UGT1A10 is expressed in intestine but not liver, while UGT1A9 is expressed in both tissues .

• Molecular techniques:

  • siRNA knockdown in cell lines

  • Antibody-based detection (if isoform-specific antibodies are available)

  • RT-PCR with isoform-specific primers

• Transgenic models: Knockout or humanized mouse models for specific UGT isoforms provide definitive tools for assigning metabolic functions.

How do species differences between mouse and human UGT1A9 affect translational research?

Understanding species differences is crucial for translating findings from mouse models to humans:

When designing translational studies, these species differences should be carefully considered, and when possible, humanized mouse models may provide more directly translatable results.

What factors contribute to variability in Ugt1a9 activity measurements?

Several factors can contribute to variability in Ugt1a9 activity measurements:

• Genetic factors:

  • Strain differences in mice

  • Polymorphisms affecting enzyme expression or activity

• Experimental conditions:

  • Buffer composition and pH

  • Presence of detergents or organic solvents (>5% can decrease activity)

  • UDPGA concentration

  • Alamethicin concentration in microsomal preparations

  • Incubation time and temperature

• Enzyme source:

  • Recombinant enzyme vs. tissue microsomes

  • Expression system used for recombinant enzyme production

  • Storage conditions and freeze-thaw cycles

• Protein concentration:

  • Linearity of activity with protein concentration should be verified

  • Both too low and too high protein concentrations can lead to misleading results

• Detection methods:

  • Sensitivity and specificity of analytical methods

  • Matrix effects in complex biological samples

To minimize variability, researchers should standardize protocols, include appropriate controls, and perform detailed method validation before conducting critical experiments.

How should I interpret kinetic data from Ugt1a9 enzymatic assays?

Proper interpretation of kinetic data from Ugt1a9 enzymatic assays requires consideration of several factors:

What are emerging technologies for studying Ugt1a9 structure-function relationships?

Several emerging technologies are advancing our understanding of Ugt1a9 structure-function relationships:

• Homology modeling and molecular docking: Computational approaches can predict how substrates interact with Ugt1a9, guiding the design of selective substrates and inhibitors. This approach has been successfully used for UGT1A10 to design selective fluorescent substrates .

• Site-directed mutagenesis: Creating specific mutations, such as the H210M mutation in UGT1A10, can provide insights into critical residues for substrate binding and catalysis .

• Cryo-electron microscopy: This technique offers the potential to determine high-resolution structures of membrane-bound UGTs, overcoming challenges associated with crystallography.

• Hydrogen-deuterium exchange mass spectrometry: This method can identify flexible regions and conformational changes upon substrate binding.

• CRISPR/Cas9 genome editing: Creating precise genetic modifications in cell lines or animals to study structure-function relationships in physiologically relevant contexts.

• Fluorescent probes: Development of isoform-selective fluorescent substrates and probes enables real-time monitoring of enzyme activity in complex systems .

These technologies, often used in combination, are advancing our understanding of how UGT structure determines substrate specificity and catalytic efficiency.

How can recombinant mouse Ugt1a9 be applied in drug discovery and toxicology research?

Recombinant mouse Ugt1a9 offers valuable applications in drug discovery and toxicology research:

• Drug metabolism profiling:

  • Early assessment of metabolic stability

  • Identification of metabolic soft spots in drug candidates

  • Comparison between species to predict human metabolism

• Drug-drug interaction prediction:

  • Identifying compounds that inhibit or induce Ugt1a9

  • Assessing risk of metabolic interactions with co-administered drugs

  • Evaluating the impact of Ugt1a9 polymorphisms on drug response

• Toxicology applications:

  • Studying detoxification of environmental chemicals

  • Investigating metabolism of potential carcinogens

  • Evaluating the role of glucuronidation in toxicity modulation

• Biomarker development:

  • Using Ugt1a9 activity as a biomarker for liver function

  • Measuring Ugt1a9 activity to monitor drug-induced enzyme induction

  • Correlating Ugt1a9 genotypes with metabolic phenotypes

• Translational research:

  • Comparing mouse and human UGT1A9 to improve prediction of human drug metabolism

  • Using transgenic models to evaluate the impact of human UGT1A9 polymorphisms

• Experimental design considerations:

  • Use recombinant enzyme for initial screening

  • Confirm findings with liver microsomes

  • Validate in vivo using appropriate animal models

  • Consider species differences when extrapolating to humans

By incorporating recombinant mouse Ugt1a9 in these applications, researchers can gain valuable insights into drug metabolism and toxicity while reducing reliance on animal testing through improved in vitro-in vivo correlations.