Recombinant Macaca fascicularis UDP-glucuronosyltransferase 2B9 (UGT2B9)

What is UGT2B9 and what is its role in Macaca fascicularis?

UGT2B9 is a UDP-glucuronosyltransferase enzyme expressed in cynomolgus monkeys (Macaca fascicularis) that plays a crucial role in phase II detoxification. This enzyme catalyzes the transfer of glucuronic acid from UDP-glucuronic acid to various endogenous and exogenous substrates, including steroids and xenobiotics, making them more water-soluble and facilitating their elimination from the body . As a member of the UGT2B subfamily, UGT2B9 contributes to the metabolism of various compounds and represents an important component of the monkey's detoxification system.

How does UGT2B9 in cynomolgus monkeys compare structurally with human UGT enzymes?

The UGT2B9 cDNA from cynomolgus monkey is 2,648 bp in length and contains an open reading frame of 1,587 bp encoding a protein of 529 amino acid residues . In vitro transcription/translation of the cDNA produces a protein of approximately 52 kD . While the cynomolgus monkey UGT2B9 shares structural similarities with human UGT2B enzymes, it shows a unique substrate specificity profile. Northern blot analysis using human UGT2B cDNA probes has demonstrated the expression of homologous UGT2B transcripts in various monkey tissues, indicating structural conservation between human and cynomolgus monkey UGT2B enzymes . Western blot analyses using polyclonal antibodies raised against human UGT2B17 protein have also confirmed the expression of homologous UGT2B proteins in monkey tissues .

In which tissues is UGT2B9 expressed in Macaca fascicularis?

UGT2B9 shows a wide tissue distribution in cynomolgus monkeys. Northern blot analysis has demonstrated the expression of UGT2B transcripts in several tissues including:

• Liver

• Kidney

• Adrenal gland

• Breast

• Testis

• Prostate

This diverse tissue expression pattern suggests UGT2B9 plays important roles in multiple organs and physiological processes. The expression in steroid target tissues (prostate, testis, breast, adrenal) particularly indicates its involvement in steroid metabolism .

How does the expression of UGT2B9 vary during infection or disease states?

While the search results don't specifically address UGT2B9 expression during infection, they do provide insights into transcriptomic changes in Macaca fascicularis during Plasmodium knowlesi infection. During infection, cynomolgus macaques show distinct transcriptomic responses that differ from those of Macaca mulatta, potentially contributing to their ability to control parasitemia and survive the infection .

Though not directly linked to UGT2B9, these studies demonstrate that expression of various genes in cynomolgus monkeys can be significantly altered during infection states. Further research would be needed to specifically characterize how UGT2B9 expression changes during infection or disease.

What is the substrate specificity profile of UGT2B9?

UGT2B9 demonstrates a remarkably broad substrate specificity profile compared to other characterized UGT enzymes. It has been tested with over 60 compounds and shows activity toward:

• C18 steroids (estrogens)

• C19 steroids (androgens)

• C21 steroids (progestins and corticosteroids)

• Bile acids

• Xenobiotics including eugenol, 1-naphthol, and p-nitrophenol

Among UGT enzymes characterized to date, UGT2B9 is specific for more different classes of steroids than any other UGT enzyme, making it uniquely versatile in steroid metabolism .

What are the kinetic properties of UGT2B9 with different substrates?

Kinetic analysis has revealed that UGT2B9 glucuronidates steroids with high affinity and efficiency. The Km values for various substrates are:

These low Km values indicate high affinity for these steroid substrates, suggesting that UGT2B9 plays an important role in steroid metabolism in cynomolgus monkeys . The exceptionally high affinity for dihydrotestosterone and androsterone (Km = 0.2 μM) suggests these may be physiologically relevant substrates for this enzyme.

What methods are used to express and purify recombinant UGT2B9?

Recombinant UGT2B9 can be produced using several approaches:

• cDNA library construction and cloning: cDNA libraries can be constructed from monkey liver and prostate mRNA, and the UGT2B9 cDNA can be isolated from these libraries .

• In vitro transcription/translation: The cDNA clone can be used for in vitro transcription/translation to produce the UGT2B9 protein .

• Stable cell line establishment: The UGT2B9 cDNA can be transfected into cell lines such as HK293 cells to establish stable cell lines expressing the UGT2B9 protein .

• Commercial recombinant production: Recombinant UGT2B9 is also commercially available, which can be stored at -20°C, or at -80°C for extended storage, with working aliquots kept at 4°C for up to one week .

How can UGT2B9 activity be measured in experimental settings?

UGT2B9 activity can be assessed using various experimental approaches:

• Substrate glucuronidation assays: The activity of UGT2B9 can be tested with various compounds, including steroids, bile acids, and xenobiotics . Measurement of glucuronide formation allows for quantification of enzymatic activity.

• Kinetic analysis: By varying substrate concentrations and measuring reaction rates, kinetic parameters such as Km and Vmax can be determined, providing insights into the affinity and efficiency of UGT2B9 for different substrates .

• Inhibition studies: Similar to experiments with other UGTs like UGT1A9 and UGT2B7, inhibition studies can help characterize UGT2B9 activity. For example, diclofenac, a known substrate for UGT2B7, has been used to inhibit glucuronidation reactions .

• Western blot analysis: Using antibodies raised against UGT2B proteins, Western blot analysis can be used to detect and quantify UGT2B9 protein expression .

How does UGT2B9 in Macaca fascicularis differ from similar enzymes in humans and other primates?

UGT2B9 from cynomolgus monkeys shows both similarities and differences compared to human UGT enzymes:

• Substrate range: UGT2B9 appears to have a broader substrate specificity than most human UGT enzymes, being active on C18, C19, and C21 steroids, bile acids, and various xenobiotics . Unlike many human enzymes active primarily on C19 steroids, UGT2B9 can also glucuronidate estrogens .

• Evolutionary relationship: Simians, including cynomolgus monkeys, represent the most appropriate animal model to study the glucuronidation of steroids in extrahepatic steroid target tissues, suggesting evolutionary conservation of UGT functions between humans and these primates .

• Polymorphism patterns: While specific UGT2B9 polymorphisms aren't detailed in the search results, studies on UGT1A9 and UGT2B7 show different polymorphism patterns across human populations (West African, Papua New Guinean, and North American) . This suggests that UGT enzymes, including potentially UGT2B9, may show population-specific variations.

What role does UGT2B9 play in the differential response of Macaca fascicularis to infections compared to other macaque species?

While the search results don't directly link UGT2B9 to infection responses, they do highlight important differences in how Macaca fascicularis (Mf) and Macaca mulatta (Mm) respond to Plasmodium knowlesi infections. Mf co-evolved with P. knowlesi in Southeast Asia and shows signs of disease but survives with low-level infection that can become chronic, whereas Mm becomes severely ill with escalating parasitemia .

The molecular basis for this stark difference lies in their transcriptomic responses. Mf initiates its transcriptomic response earlier than Mm and makes favorable adjustments around the time of acute blood-stage infection that could contribute to parasitemia control . While not specifically addressed, differential expression or activity of detoxification enzymes like UGT2B9 could potentially contribute to these species-specific responses to infection.

How is UGT2B9 utilized in drug metabolism and pharmacokinetic studies?

UGT2B9, as a key enzyme in phase II metabolism, has important applications in drug metabolism and pharmacokinetic studies:

• Glucuronidation of therapeutics: UGT2B9 can metabolize various drugs and xenobiotics, making it relevant for understanding drug metabolism in primate models. For example, UGT enzymes are involved in the metabolism of antimalarial dihydroartemisinin and antiretroviral zidovudine .

• Model for human drug metabolism: Given that simians represent appropriate animal models for studying glucuronidation of steroids, cynomolgus monkey UGT2B9 can serve as a model for predicting human drug metabolism, especially for compounds metabolized by glucuronidation .

• Experimental medicine applications: The cynomolgus macaque is increasingly selected for experimental medicine due to the ease with which it is maintained and bred in captivity, and its closer phylogenetic relationship to humans compared to rodents . Understanding UGT2B9 function contributes to the refinement of these experimental medicine models.

How can differences in UGT2B9 function affect the interpretation of experimental results from Macaca fascicularis models?

Researchers should consider several factors related to UGT2B9 when interpreting experimental results from cynomolgus monkey models:

• Broader substrate specificity: UGT2B9 has a broader substrate specificity than many human UGT enzymes, potentially resulting in different metabolism patterns for certain compounds compared to humans . This could affect the extrapolation of pharmacokinetic or toxicological data from monkey models to humans.

• High plasma levels of glucuronidated steroids: The high plasma levels of glucuronidated C19 steroids found in cynomolgus monkeys may be related to UGT2B9's unique specificity for multiple steroid classes . This physiological difference should be considered when interpreting hormone-related studies.

• Genetic differentiation: Cynomolgus macaques show distinct genetic differentiation between continental and island populations, which could affect enzyme expression and function . Studies should account for the origin of the monkeys used.

• MHC polymorphism effects: While not directly related to UGT2B9, the search results indicate that MHC polymorphism in cynomolgus macaques affects numerous biological parameters and the ability to mount immune responses against infectious pathogens and vaccines . Such genetic variability may interact with metabolism and should be considered in experimental design.