Recombinant Dog UDP-glucuronosyltransferase 2B31 (UGT2B31)

What is UGT2B31 and what is its primary function in dogs?

UGT2B31 is a canine UDP-glucuronosyltransferase belonging to the 2B subfamily that plays a crucial role in drug detoxification in dogs. This enzyme catalyzes glucuronidation, which represents a major route of clearance for a diverse set of both drug and endogenous substrates . The enzyme functions by conjugating glucuronic acid to various substrates, increasing their water solubility and facilitating their elimination from the body. Structurally, UGT2B31 shows high sequence identity (75-76%) with several human UGT enzymes, particularly UGT2B15 .

What is the substrate specificity profile of recombinant canine UGT2B31?

Recombinant canine UGT2B31 displays a substrate specificity profile similar to human UGT2B7 and rat UGT2B1. The enzyme catalyzes the glucuronidation of several compound classes including:

• Phenolic compounds

• Opioids (notably morphine)

• Carboxylic acid-containing drugs

Specifically with morphine, UGT2B31 exclusively forms morphine-3-glucuronide with a Km value of approximately 1300 μM, which is remarkably similar to the Km observed with human UGT2B7 for the same reaction . This selective regioselectivity is an important characteristic that distinguishes its catalytic behavior.

What are the recommended expression systems for producing recombinant canine UGT2B31?

Based on successful previous studies, the following expression systems are recommended for recombinant canine UGT2B31:

• V79 cell expression system: This has been validated for functional UGT2B31 expression and is the system used in the original characterization studies . The V79 cells provide appropriate post-translational modifications and membrane integration for the enzyme.

• Bacterial expression systems: While not explicitly mentioned in the search results for UGT2B31, these systems can be used for high-yield production, though they may require refolding protocols to achieve functional enzyme.

• Insect cell systems: These can be particularly useful when studying membrane-bound enzymes like UGTs, as they provide eukaryotic processing capabilities.

The methodological approach involves:

• Isolation of the gene from a dog cDNA library

• Cloning into an appropriate expression vector

• Transfection/transformation of the host system

• Verification of expression (Western blot)

• Activity assays to confirm functional expression

How can researchers determine enzyme kinetic parameters for UGT2B31?

To determine enzyme kinetic parameters for UGT2B31, researchers should follow this methodological approach:

• Prepare recombinant enzyme: Express UGT2B31 in an appropriate system (e.g., V79 cells) and prepare microsomes containing the recombinant enzyme .

• Substrate concentration gradient: Set up reactions with varying concentrations of the substrate of interest (e.g., morphine) while maintaining constant enzyme concentration.

• Assay conditions: Include UDPGA (UDP-glucuronic acid) as the co-substrate and maintain appropriate buffer conditions (typically pH 7.4-7.5).

• Analytical quantification: Utilize LC-MS/MS or HPLC methods to quantify the formation of glucuronide conjugates.

• Data analysis: Plot the reaction velocity versus substrate concentration and fit to appropriate enzyme kinetic models (Michaelis-Menten, Hill, or substrate inhibition models).

For example, when characterizing UGT2B31 with morphine, a Km value of approximately 1300 μM was determined, similar to that observed with human UGT2B7 . This parameter provides important information about the enzyme's affinity for the substrate.

What tissue distribution pattern does UGT2B31 exhibit in dogs?

UGT2B31 shows a distinctive tissue expression pattern, with the highest expression observed in the liver compared to other tissues . This is consistent with its role in xenobiotic metabolism and detoxification processes. The pronounced hepatic expression suggests that the enzyme contributes significantly to first-pass metabolism of drugs and other xenobiotics.

For researchers studying tissue distribution, recommended methodologies include:

• RT-qPCR analysis: For quantifying UGT2B31 mRNA expression across different tissues

• Western blotting: To confirm protein expression using UGT2B31-specific antibodies

• Immunohistochemistry: For spatial localization within tissue sections

• Activity assays: Using tissue-specific microsomes to correlate expression with functional activity

Understanding this tissue distribution pattern is crucial when designing studies to evaluate drug metabolism in dogs or when using canine models for pharmacological research.

How can researchers assess the functional activity of recombinant UGT2B31?

To assess the functional activity of recombinant UGT2B31, researchers should implement a combination of approaches:

• Microsomal incubation assays: Prepare microsomes from cells expressing recombinant UGT2B31 and incubate with:

  • Known substrates (e.g., morphine, phenolic compounds, carboxylic acid-containing drugs)

  • UDPGA as co-substrate

  • Appropriate buffer systems and conditions (pH 7.4, 37°C)

• Control comparisons: Include appropriate controls:

  • Microsomes from non-transfected cells

  • Incubations without UDPGA

  • Incubations with known UGT inhibitors

• Analytical detection methods:

  • LC-MS/MS for quantitative detection of glucuronide metabolites

  • HPLC-UV for compounds with appropriate chromophores

  • Radiometric assays using radiolabeled substrates for high sensitivity

• Comparative analysis: Compare activity profiles with:

  • Dog liver microsomes (to assess physiological relevance)

  • Human UGT2B7 (for cross-species comparison)

For example, functional analysis of UGT2B31 has demonstrated its ability to form morphine-3-glucuronide with kinetic parameters similar to human UGT2B7, confirming both its catalytic activity and its relevance as a comparative model for human drug metabolism .

What role does UGT2B31 play in drug metabolism and pharmacokinetics in dogs?

UGT2B31 plays a crucial role in drug detoxification in dogs through the following mechanisms:

• Phase II metabolism: As a UDP-glucuronosyltransferase, UGT2B31 catalyzes the conjugation of glucuronic acid to various xenobiotics, enhancing their water solubility and facilitating excretion .

• Substrate range: The enzyme's ability to glucuronidate phenols, opioids, and carboxylic acid-containing drugs indicates its broad contribution to the metabolism of diverse drug classes .

• Species-specific clearance patterns: While dogs generally show high clearance of compounds via glucuronidation pathways (e.g., displaying the highest intrinsic clearance of GPC among several species at 2121 μL/min/mg), the specific contribution of UGT2B31 to this pattern needs further characterization .

• Pharmacokinetic implications: The high activity of canine glucuronidation enzymes suggests that dogs may eliminate certain drugs more rapidly than humans, with UGT2B31 likely contributing significantly to this difference. This has important implications for dosing when using dogs in preclinical studies .

Understanding UGT2B31's role is essential for interpreting preclinical pharmacokinetic data from canine models, especially for drugs known to undergo glucuronidation in humans.

How does UGT2B31 activity compare between different dog breeds or within canine populations?

While the search results don't provide direct information about UGT2B31 polymorphisms across dog breeds, we can extrapolate from research on human UGT polymorphism patterns:

Although specific UGT2B31 polymorphism data isn't provided in the search results, research on human UGT polymorphisms indicates substantial variation (e.g., UGT2B15*2 with allele frequencies of 66.2% G and 33.8% T in one study population) , suggesting similar variation might exist in canine populations.

How does canine UGT2B31 compare to UGT enzymes in other species in terms of substrate specificity and kinetics?

Comparative analysis of UGT enzymes across species reveals interesting patterns in substrate specificity and kinetics:

• Phylogenetic relationships: Phylogenetic analysis has determined that canine UGT2B31 is most closely related to rat UGT2B1, despite sharing high sequence identity with several human UGT enzymes .

• Substrate specificity comparison:

  • UGT2B31 displays substrate specificity similar to human UGT2B7 and rat UGT2B1

  • All three enzymes catalyze glucuronidation of phenols, opioids, and carboxylic acid-containing drugs

  • UGT2B31 specifically forms morphine-3-glucuronide, showing regioselectivity in its catalytic action

• Kinetic parameters across species: While specific comparative kinetic data for UGT2B31 across species is limited, studies of glucuronidation of compounds like GPC show significant species differences:

  Species	Km (μM)	CLint (μL/min/mg)
  Dog	9.56	2121
  Pig	9.58	147
  Mouse	10.04	690
  Bovine	12.24	259
  Human	15.36	27
  Rabbit	16.19	304
  Rat	94.07	40
  Monkey	107.10	77

  This data shows that dogs generally have both high affinity (low Km) and high intrinsic clearance for glucuronidation reactions compared to other species .

Understanding these interspecies differences is critical when using animal models to predict human drug metabolism or when developing veterinary medications.

What is the evolutionary significance of UGT2B31 in canines compared to UGT enzymes in other mammals?

The evolutionary positioning of UGT2B31 provides several insights into its biological significance:

• Sequence conservation: The high degree of sequence identity between canine UGT2B31 and human UGT enzymes (75-76% with UGT2B15, UGT2B4, and UGT2B7) suggests evolutionary conservation of critical functional domains .

• Phylogenetic relationship: Despite sequence similarities with human enzymes, phylogenetic analysis shows UGT2B31 is most closely related to rat UGT2B1, indicating complex evolutionary patterns in the UGT family that may reflect adaptation to different ecological niches or dietary patterns .

• Functional convergence: The similar substrate specificity between canine UGT2B31 and human UGT2B7 represents an example of functional convergence in xenobiotic detoxification mechanisms across mammalian species .

• Species-specific adaptations: The particularly high glucuronidation activity in dogs (highest CLint for GPC among eight species studied) suggests possible evolutionary adaptation to specific dietary or environmental challenges faced by canids throughout their evolution .

These evolutionary insights help contextualize the role of UGT2B31 within the broader adaptive landscape of mammalian detoxification systems and highlight its potential value in comparative studies of xenobiotic metabolism.

Can canine UGT2B31 serve as a model for studying human UGT-mediated drug metabolism?

Canine UGT2B31 offers both advantages and limitations as a model for human UGT-mediated drug metabolism:

Advantages:

• Sequence homology: UGT2B31 shares 75% sequence identity with human UGT2B7, suggesting structural similarities that may translate to comparable catalytic mechanisms .

• Functional similarity: UGT2B31 displays substrate specificity similar to human UGT2B7, catalyzing the glucuronidation of phenols, opioids, and carboxylic acid-containing drugs .

• Kinetic parallels: For specific substrates like morphine, UGT2B31 exhibits similar kinetic parameters to human UGT2B7 (Km ≈ 1300 μM for both enzymes), indicating comparable binding affinities .

Limitations:

• Metabolic rate differences: Dogs generally exhibit much higher glucuronidation clearance rates than humans. For example, intrinsic clearance of GPC in dogs (2121 μL/min/mg) is nearly 80 times higher than in humans (27 μL/min/mg) .

• Regioselectivity differences: UGT2B31 forms only morphine-3-glucuronide, while human enzymes may form different glucuronide isomers depending on the substrate .

• Species-specific pathways: Dogs may utilize different metabolic pathways for certain compounds compared to humans, potentially limiting translational relevance.

Methodological considerations for researchers:
When using canine UGT2B31 as a model, researchers should:

• Validate findings with human enzymes whenever possible

• Account for quantitative differences in metabolic rates when extrapolating to humans

• Consider using rat models for certain studies, as rats had the most similar intrinsic clearance rate to humans for some compounds

What are the most effective methods for studying the regulatory mechanisms controlling UGT2B31 expression?

To investigate the regulatory mechanisms controlling UGT2B31 expression, researchers should consider these advanced methodological approaches:

• Promoter analysis and transcriptional regulation:

  • Cloning the UGT2B31 promoter region into reporter constructs

  • Deletion and mutation analysis to identify key regulatory elements

  • Chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the promoter

  • Electrophoretic mobility shift assays (EMSA) to confirm specific protein-DNA interactions

• Epigenetic regulation:

  • Bisulfite sequencing to analyze DNA methylation patterns in the promoter region

  • ChIP assays for histone modifications associated with gene activation/repression

  • ATAC-seq to assess chromatin accessibility in different tissues/conditions

• Tissue-specific expression mechanisms:

  • Single-cell RNA sequencing to characterize cell-type specific expression

  • Tissue-specific knockout models to understand physiological consequences

  • Analysis of enhancer regions that may contribute to liver-specific expression

• Genetic variants affecting expression:

  • Identification of eQTLs (expression quantitative trait loci) that influence UGT2B31 expression

  • Functional analysis of regulatory SNPs like rs344053754, which regulates UGT2B31 expression in liver by potentially affecting enhancer activity

• Environmental and physiological regulation:

  • Effects of xenobiotics, hormones, or disease states on expression levels

  • Cross-talk with other metabolic pathways and nuclear receptors

These methodologies can be applied in combination to develop a comprehensive understanding of UGT2B31 regulation in different physiological and pathological contexts.

What advanced techniques can be used to study structure-function relationships in canine UGT2B31?

To elucidate structure-function relationships in canine UGT2B31, researchers can employ these advanced techniques:

• Structural biology approaches:

  • X-ray crystallography of purified UGT2B31 (challenging for membrane proteins)

  • Cryo-electron microscopy for structural determination

  • NMR spectroscopy for dynamic structural elements

  • Homology modeling based on crystallized UGT structures or related proteins

• Site-directed mutagenesis studies:

  • Systematic mutation of conserved residues to identify catalytic sites

  • Chimeric constructs with other UGT enzymes to identify substrate specificity determinants

  • Alanine scanning mutagenesis to map the substrate binding pocket

  • Introduction of mutations corresponding to known polymorphisms in human UGTs

• Advanced binding and kinetic analyses:

  • Isothermal titration calorimetry (ITC) to determine thermodynamic binding parameters

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon substrate binding

  • Stopped-flow kinetics to resolve rapid catalytic steps

• Computational approaches:

  • Molecular dynamics simulations to study protein flexibility and substrate interactions

  • Quantum mechanics/molecular mechanics (QM/MM) methods to model the catalytic mechanism

  • Virtual screening to identify novel substrates or inhibitors

  • Machine learning models to predict substrate specificity based on molecular descriptors

• Functional analysis with advanced readouts:

  • Metabolomics approaches to identify novel endogenous substrates

  • Integrated multi-omics to understand broader metabolic impacts

  • In vivo CRISPR-mediated gene editing to study physiological consequences of specific structural alterations

These techniques, especially when applied in combination, can provide comprehensive insights into how UGT2B31's structure dictates its catalytic function, substrate specificity, and regulatory properties.

How can researchers optimize recombinant UGT2B31 expression for large-scale enzymatic studies?

For researchers seeking to optimize recombinant UGT2B31 expression for large-scale enzymatic studies, the following methodological considerations are critical:

• Expression system selection:

  • Mammalian cell systems (e.g., V79 cells): Provide proper post-translational modifications and membrane integration but may have lower yields

  • Baculovirus-insect cell systems: Balance between yield and proper folding/modification

  • Yeast expression systems: Can offer higher yields while maintaining eukaryotic processing

  • Bacterial systems with membrane fractions: Highest yield but may require refolding protocols

• Construct optimization:

  • Codon optimization for the selected expression system

  • Addition of purification tags (His, FLAG, etc.) that don't interfere with activity

  • Inclusion of optimal Kozak sequence for mammalian expression

  • Consideration of fusion partners to enhance solubility or expression

• Culture and induction optimization:

  • Determination of optimal cell density for induction

  • Optimization of inducer concentration and timing

  • Temperature modulation during expression phase

  • Media supplementation with factors that enhance protein folding

• Extraction and purification strategies:

  • Gentle membrane solubilization using appropriate detergents

  • Affinity chromatography based on incorporated tags

  • Size exclusion and ion exchange chromatography for further purification

  • Consideration of lipid nanodisc incorporation for maintaining native-like membrane environment

• Activity preservation:

  • Optimal buffer composition for storage (pH, ionic strength)

  • Addition of stabilizing agents (glycerol, specific lipids)

  • Flash-freezing protocols to maintain activity during storage

  • Activity assays at multiple stages to monitor preservation of function

• Scale-up considerations:

  • Bioreactor parameters for large-scale culture

  • Automation of purification processes

  • Quality control metrics for batch consistency

  • Cost-effectiveness analysis of different approaches

By systematically optimizing these parameters, researchers can develop protocols that balance yield, purity, and enzymatic activity for large-scale studies of UGT2B31, enabling applications from drug metabolism screening to detailed mechanistic investigations.

How can UGT2B31 research contribute to improving preclinical drug development?

UGT2B31 research offers several valuable applications for enhancing preclinical drug development:

• Species difference understanding:

  • Characterizing UGT2B31 helps explain why dogs metabolize certain drugs differently than humans

  • This knowledge allows more accurate interpretation of canine toxicology studies

  • Understanding that dogs have significantly higher glucuronidation clearance (e.g., CLint for GPC: 2121 μL/min/mg in dogs vs. 27 μL/min/mg in humans) helps contextualize exposure data

• Predictive model development:

  • Comparative studies between UGT2B31 and human UGT2B7 can improve in vitro-to-in vivo extrapolation (IVIVE) models

  • Knowledge of substrate specificity overlaps between UGT2B31 and human UGTs helps identify potential metabolic pathways

  • Development of species-specific scaling factors for glucuronidation pathways

• Screening system implementation:

  • Recombinant UGT2B31 systems can be used to screen drug candidates for glucuronidation liability

  • Identifying compounds that are rapidly metabolized by UGT2B31 but not by human UGTs could help deprioritize problematic compounds early

  • Development of high-throughput assays using recombinant UGT2B31

• Animal model selection guidance:

  • Data showing that rats have glucuronidation rates more similar to humans than dogs helps guide species selection for preclinical studies

  • Understanding canine-specific metabolism helps researchers select the most appropriate animal models for specific drug classes

By advancing our understanding of species-specific drug metabolism through UGT2B31 research, preclinical data can be more accurately translated to human outcomes, potentially reducing late-stage drug development failures due to unexpected metabolic differences.

What are the cutting-edge research directions for UGT2B31 in comparative metabolism studies?

Several cutting-edge research directions are emerging for UGT2B31 in comparative metabolism studies:

• Multi-species UGT atlases:

  • Development of comprehensive databases comparing UGT expression, activity, and substrate specificity across species

  • Integration of -omics data to create predictive models of species differences

  • Correlation of genetic variations with functional differences across species

• Precision animal models:

  • Creation of humanized UGT models in dogs through CRISPR/Cas9 technology

  • Development of transgenic models expressing human UGTs in place of canine enzymes

  • Validation of these models for improved prediction of human drug metabolism

• Systems biology approaches:

  • Integration of UGT2B31 activity with broader metabolic networks

  • Modeling of interspecies differences in metabolic flux

  • Exploration of co-regulation patterns with other drug-metabolizing enzymes

  • Investigation of UGT2B31's role in tissue-specific regulation of endogenous compounds

• Physiologically-based pharmacokinetic (PBPK) modeling:

  • Incorporation of UGT2B31-specific parameters into multi-species PBPK models

  • Development of algorithms to translate dog data to human predictions

  • Validation of these models with clinical data

  • Integration with machine learning approaches for improved prediction

• Endogenous substrate identification:

  • Exploration of UGT2B31's role in metabolizing endogenous signaling molecules

  • Comparative metabolomics to identify species-specific endogenous substrates

  • Investigation of potential regulatory feedback loops involving UGT2B31 substrates

  • Correlation of UGT2B31 activity with physiological parameters such as litter weight at weaning

These research directions leverage advanced technologies to develop a deeper understanding of UGT2B31's role in both xenobiotic metabolism and normal physiology, with applications ranging from basic science to applied drug development.

How can gene editing technologies be applied to study UGT2B31 function in vivo?

Gene editing technologies offer powerful approaches to investigate UGT2B31 function in vivo, providing insights not possible with traditional methods:

These gene editing approaches can significantly advance our understanding of UGT2B31's physiological roles beyond what can be learned from in vitro studies alone, while providing valuable models for drug metabolism and comparative biology research.