Recombinant Macaca fascicularis UDP-glucuronosyltransferase 2B18 (UGT2B18)

How does UGT2B18 differ structurally from related UGT2B family members like UGT2B9?

When comparing UGT2B18 (UniProt ID: O97951) with the related UGT2B9 (UniProt ID: O02663) from the same species, several key structural differences become evident despite their similar length and expression regions (22-529 for both proteins):

These differences, particularly in the N-terminal domains responsible for substrate recognition, likely contribute to the distinct substrate specificities between these enzymes. Researchers investigating substrate selectivity should focus experimental designs on these divergent regions .

What expression systems are most effective for producing functional recombinant UGT2B18?

Based on available data, the following expression systems have proven effective for UGT2B18 production:

• E. coli Expression System: Similar to protocols used for UGT2B9, recombinant UGT2B18 can be expressed with an N-terminal His-tag in E. coli, allowing for efficient purification via nickel affinity chromatography. When using this system, expression should be optimized at lower temperatures (16-18°C) to enhance protein solubility .

• Insect Cell Systems: Though not explicitly mentioned in the search results for UGT2B18, baculovirus-infected insect cells often provide better post-translational modifications for UGT family proteins compared to bacterial systems.

For optimal functionality, the expression construct should include amino acids 22-529, representing the mature protein without the signal peptide. Researchers should consider that membrane-associated proteins like UGTs may require detergent solubilization to maintain enzymatic activity after purification .

What are the recommended storage conditions to maintain long-term stability of purified UGT2B18?

For optimal stability of purified recombinant UGT2B18, the following storage protocols are recommended:

• Short-term storage (up to one week): Store working aliquots at 4°C to maintain activity while avoiding repeated freeze-thaw cycles .

• Long-term storage: Store at -20°C or preferably -80°C in small aliquots to prevent repeated freezing and thawing, which significantly reduces enzymatic activity .

• Storage buffer composition: Use Tris-based buffer with 50% glycerol optimized for protein stability. For lyophilized preparations, a buffer containing 6% trehalose at pH 8.0 has been shown to maintain stability .

• Reconstitution protocol: When using lyophilized protein, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Add glycerol to a final concentration of 50% for optimal stability during freeze-thaw cycles .

Researchers should verify protein stability after storage by assessing enzyme activity using standard glucuronidation assays with known substrates.

How can researchers design kinetic assays to characterize UGT2B18 substrate specificity?

To properly characterize UGT2B18 substrate specificity and kinetic parameters, researchers should implement the following methodological approach:

• Substrate screening assay:

  • Test a panel of potential substrates including phenols, alcohols, carboxylic acids, amines, and steroids

  • Use HPLC-UV or LC-MS/MS for detection of glucuronide formation

  • Include both endogenous compounds and xenobiotics in the screening panel

• Kinetic parameter determination:

  • Measure initial reaction velocities at various substrate concentrations (typically 5-8 concentrations ranging from 0.1× to 10× the estimated Km)

  • Maintain constant UDP-glucuronic acid concentration at saturating levels (usually 5 mM)

  • Plot data using Michaelis-Menten, Lineweaver-Burk, or Eadie-Hofstee transformations to determine Km and Vmax

• Comparative analysis with human UGTs:

  • Include parallel assays with human UGT2B enzymes to identify species differences

  • Focus particularly on pharmaceutically relevant substrates to assess the macaque model's relevance for drug metabolism studies

The protein sequence features of UGT2B18 suggest it may have unique substrate preferences compared to other UGT2B family members, making comprehensive kinetic characterization essential for researchers using this enzyme in xenobiotic metabolism studies .

What methods are effective for studying potential inhibitory effects on UGT2B18 activity?

To effectively investigate inhibition of UGT2B18 activity, researchers should implement the following methodological approaches:

• Inhibition screening protocol:

  • Select a well-characterized substrate with reliable detection methods

  • Use recombinant UGT2B18 protein at concentrations that produce linear reaction rates

  • Test potential inhibitors at multiple concentrations (typically 1-100 μM)

  • Include positive control inhibitors such as known UGT inhibitors (e.g., diclofenac)

• Inhibition mechanism determination:

  • Perform substrate concentration-dependent studies in the presence of different fixed inhibitor concentrations

  • Generate Dixon and Lineweaver-Burk plots to distinguish between competitive, non-competitive, and uncompetitive inhibition

  • Calculate Ki values to quantify inhibition potency

• Data analysis and interpretation:

  • Use appropriate enzyme kinetic software for data fitting

  • Consider potential species differences when extrapolating to human UGT enzymes

  • Assess potential for drug-drug interactions based on inhibition constants

The specific amino acid sequence of UGT2B18 includes key catalytic residues that might interact differently with inhibitors compared to human UGT enzymes, necessitating careful interpretation of results when using this model for drug interaction studies .

How does UGT2B18 compare functionally to UGT2B9 from the same species, and how should researchers account for these differences?

UGT2B18 and UGT2B9 from Macaca fascicularis share significant structural similarities but exhibit key differences that affect their functional properties:

When designing experiments involving these enzymes, researchers should:

• Perform direct comparative assays using identical experimental conditions to accurately assess substrate preferences

• Consider sequence differences in the substrate-binding domains when interpreting binding affinities and catalytic efficiencies

• Include both enzymes as controls in inhibition studies to identify inhibitor selectivity

• Account for potential differences in post-translational modifications when using different expression systems

The sequence-based differences between these closely related enzymes provide an excellent opportunity to investigate structure-function relationships in UDP-glucuronosyltransferases through site-directed mutagenesis and chimeric protein construction .

What approaches can researchers use to extrapolate findings from macaque UGT2B18 studies to human UGT metabolism?

To effectively translate research findings from Macaca fascicularis UGT2B18 studies to human UGT metabolism, researchers should employ the following methodological approaches:

• Orthologous protein identification and comparison:

  • Perform detailed phylogenetic analysis to identify the closest human ortholog(s)

  • Use bioinformatic tools to generate sequence alignments and calculate sequence identity/similarity percentages

  • Create 3D structural models to compare catalytic sites and substrate-binding domains

• Comparative functional analysis:

  • Conduct parallel substrate metabolism studies using both macaque UGT2B18 and its human ortholog(s)

  • Generate comparative kinetic parameters (Km, Vmax, catalytic efficiency) for key substrates

  • Develop scaling factors for extrapolation based on empirical data

• Species difference characterization:

  • Identify substrates with significant species differences in glucuronidation

  • Perform structure-activity relationship analyses to determine molecular features responsible for species differences

  • Use site-directed mutagenesis to confirm key amino acid residues responsible for species differences

The detailed sequence information available for UGT2B18 (O97951) enables researchers to identify regions of conservation and divergence compared to human UGTs, facilitating more accurate cross-species extrapolation of metabolism data .

What are common technical challenges when working with recombinant UGT2B18 and how can researchers address them?

Researchers working with recombinant UGT2B18 commonly encounter several technical challenges that can impact experimental outcomes. The following methodological solutions are recommended:

• Protein solubility issues:

  • Challenge: UGT2B18, like other membrane-associated UGTs, may exhibit poor solubility

  • Solution: Express with solubility-enhancing tags (e.g., SUMO, MBP) or optimize buffer conditions with appropriate detergents (0.1-0.5% Triton X-100 or CHAPS) to maintain native conformation without disrupting activity

• Low enzyme activity:

  • Challenge: Loss of activity during purification or storage

  • Solution: Include 50% glycerol in storage buffer as indicated in the product specifications, maintain pH between 7.5-8.0, and add reducing agents like DTT (1-5 mM) to prevent oxidation of critical cysteine residues

• Inconsistent glucuronidation assay results:

  • Challenge: Variable activity across experiments

  • Solution: Standardize UDP-glucuronic acid concentration (typically 5 mM), control incubation temperature precisely (37°C is standard), and normalize results to protein concentration determined by validated methods

• Detection of glucuronide products:

  • Challenge: Low sensitivity in detecting glucuronide formation

  • Solution: Optimize LC-MS/MS methods with authentic standards when available, or use multiple reaction monitoring (MRM) to increase sensitivity for novel glucuronides

The amino acid sequence of UGT2B18 includes several cysteine residues that may form disulfide bonds critical for stability, supporting the recommendation to include reducing agents in experimental buffers .

How should researchers design experiments to investigate the role of UGT2B18 in comparative drug metabolism studies?

When designing experiments to investigate UGT2B18's role in comparative drug metabolism studies, researchers should implement the following methodological framework:

• Species comparison panel preparation:

  • Express and purify recombinant UGT2B18 from Macaca fascicularis using standardized methods

  • Include the closest human ortholog(s) expressed under identical conditions

  • Optionally include UGT2B18 from other non-human primate species to establish evolutionary relationships

  • Normalize protein concentrations using validated quantification methods

• Substrate metabolism assessment:

  • Select pharmaceutically relevant substrates representing diverse chemical structures

  • Design concentration ranges that span physiologically relevant levels (typically 1-100 μM)

  • Include both phase I metabolites and parent compounds to assess sequential metabolism

  • Perform time-course studies to establish linearity of glucuronidation reactions

• Data analysis and interpretation:

  • Calculate and compare kinetic parameters (Km, Vmax, CLint) across species

  • Generate correlation plots to assess predictive value of macaque UGT2B18 for human metabolism

  • Develop scaling factors based on empirical data to improve in vitro to in vivo extrapolation

• Validation with tissue fractions:

  • Complement recombinant enzyme studies with experiments using liver microsomes from both species

  • Use selective inhibitors or antibodies to confirm the contribution of specific UGT isoforms

  • Compare results between recombinant systems and tissue preparations to assess physiological relevance

The sequence information from the search results provides essential structural details that can inform interpretation of species differences in substrate specificity and metabolism rates .

What methodologies should researchers employ to investigate the effect of UGT2B18 genetic polymorphisms on enzyme function?

To investigate the impact of UGT2B18 genetic polymorphisms on enzyme function, researchers should implement the following comprehensive methodological approach:

• Polymorphism identification and characterization:

  • Sequence the UGT2B18 gene from multiple Macaca fascicularis individuals to identify natural variants

  • Use genomic databases to identify known polymorphisms in the Macaca fascicularis population

  • Select polymorphisms resulting in amino acid substitutions for functional analysis

  • Create site-directed mutants of the recombinant UGT2B18 expression construct to reproduce identified polymorphisms

• Functional characterization protocol:

  • Express wild-type and variant UGT2B18 proteins under identical conditions

  • Verify expression levels and protein integrity through Western blotting

  • Compare enzyme kinetics (Km, Vmax, CLint) across a panel of relevant substrates

  • Assess protein stability under various temperature and pH conditions

• Structural impact assessment:

  • Generate homology models of wild-type and variant UGT2B18 using the amino acid sequence provided in the search results

  • Perform molecular dynamics simulations to predict structural changes

  • Identify potential alterations in substrate binding pocket or catalytic residues

  • Correlate structural predictions with experimental findings

The detailed amino acid sequence of UGT2B18 available in the search results provides the foundation for generating accurate structural models and identifying functionally important regions that might be affected by polymorphisms .

How can researchers utilize UGT2B18 to develop improved in vitro-to-in vivo extrapolation models for drug metabolism?

To develop more accurate in vitro-to-in vivo extrapolation (IVIVE) models for drug metabolism using UGT2B18, researchers should implement the following methodological approach:

• Comparative enzyme kinetics framework:

  • Determine enzyme kinetic parameters for UGT2B18 and human UGT orthologs using standardized methods

  • Develop substrate-specific scaling factors based on relative activities

  • Generate a database of comparative activities across diverse chemical structures

  • Establish mathematical relationships between macaque and human glucuronidation rates

• Integrated metabolism model development:

  • Incorporate UGT2B18 activity data into physiologically-based pharmacokinetic (PBPK) models

  • Account for species differences in UGT expression levels across tissues

  • Consider the impact of protein sequence differences on substrate specificity and catalytic efficiency

  • Validate models using in vivo pharmacokinetic data from both species

• Application to drug development pipeline:

  • Use validated models to predict human glucuronidation clearance from macaque data

  • Apply correction factors based on sequence differences in substrate recognition sites

  • Integrate with other metabolism pathways for comprehensive clearance prediction

  • Refine models iteratively based on experimental validation

The amino acid sequence information for UGT2B18 provided in the search results enables researchers to identify key structural differences from human UGTs that may affect substrate specificity and catalytic efficiency, critical factors in developing accurate IVIVE models .