UDP-Glucuronosyltransferases (UGTs): Overview

Anna Radominska-Pandya

Department of Biochemistry and Molecular Biology

University of Arkansas for Medical Sciences

Little Rock, Arkansas, US

October 2010; Gdansk University of Technology

General Concept of Detoxification

Endobiotics

XenobioticsMetabolism

Inactive Excretion

Products

Urine Bile

Lipid Soluble Water Soluble

Phase I

Oxidation

(P450s)

OH

Phase II

Conjugation

(UGTs)

H

O

HO

O OHH

COOH

O

Phase 0: Absorption of Drugs and Endobiotics

Biotransformation of Drugs and Endobiotics via Oxidation and Conjugation Pathways

Phase I: OxidationCYP450s

Phase III: Excretion to Bile and Urine

Phase II: ConjugationUGTs

SULTs GSTs

Abbreviations: CYP450 (Cytochrome P450); UGT (UDP-Glucuronosyltransferase); SULT (Sulfotransferase); GST (Glutathione Sulfatransferase)

HO

7-OH-warfarin

glucuronide

Warfarin

Glucuronic

Acid

O

Biotransformation via Glucuronidation

UDP-GlcUA

Co-SubstrateUGT

Cytoplasm

Lumen

ER

_COO

-

+H3N_

Various Glucuronides

Bioactivated Glucuronides

Retinoic Acid-gluc

6-O-Morphine-gluc

3-O-Lithocholic Acid-gluc

D-ring glucuronides of estradiol,

testosterone, DHEA

Electophilic Glucuronides

Acyl glucuronides (NSAIDs)

N-O-glucuronides (Hydroxamic Acids)

Hydrophilic β-D-glucuronides

Therapeutic Drugs

Carcinogens

Environmental Toxins

Dietary Constituents

Lipophilic Substrates

Bilirubin Steroids

Bile Acids

Retinoic Acids

Fatty Acids

Prostaglandins

Substrate Specificity of UGTs

• Substrate specificity is broad and overlapping (promiscuous)

• Wide range of substrates:– Endogenous substrates

– Xenobiotic substrates• Drugs

• Dietary plant constituents

• Carcinogens

• Types of glucuronides:– O-glucuronides

• Including acyl glucuronides

– N-glucuronides

– S-glucuronides

– C-glucuronides

Tukey R.H. 2000.

Phenols

Aliphatic alcohols

Anthraquinones/flavones

Carboxylic acids

Billirubin

Bile acids

Amines

Coumarins

Opioids

Steroids

Sapogenins

Estrogens

Androgens

Progestin

Bile acids

Primary

Secondary

Tertiary

Heterocyclic

Simple

Complex

Glucuronidation Reactions Catalyzed by UGTs

O-Glucuronidation

S-Glucuronidation

N-Glucuronidation

C-Glucuronidation

UDP-Glucuronosyltransferases (UGTs)

• Large family of membrane bound glycosylatedproteins located in the ER as well as inner and outer nuclear membranes

• Conjugate a wide range of endobiotics and xenobiotics with glucuronic acid

– Glucuronic acid moiety can attach via a hydroxyl, carboxyl, amino, thiol or carbonyl group on the substrates

– Generate more polar, water soluble metabolites which can be excreted in urine and bile

Biological Significance of Glucuronidation

• Detoxification

– increases hydrophobic properties• soluble in blood/urine

– structure different from parent compound• no favorable interaction with pharmacological target

• Detoxification leads to:

– Excretion of catabolic products

– Elimination of nucleophilic metabolites of carcinogens

– Inactivation of biologically active components• AZT

Biological Significance of Glucuronidation

• Bioactivation of the parent compound– Increases toxicity

– Increases pharmacological properties

• Metabolic activation leads to:– Cholestatic glucuronides

• Lithocholic acid glucuronide

• Estradiol glucuronide

– Chemically reactive glucuronides:• Acyl-glucuronides

• NSAID glucuronides– Ketoprofen

– Zomepirax

– Metabolically-active glucuronides• Morphine-6-O-glucuronide

• Irinotecan glucuronide

• Retinoid glucuronides

Biological Significance of Glucuronidation

• Control of homeostasis of the body and cells– Control of steady state concentrations of ligands for nuclear

receptors and signaling molecules

• Downregulation of UGTs results in pathological conditions such as:– Cancer

– Alzheimer’s Disease

– Hyperbiliruminemia – Crigler-Najjar syndrome

– Obesity

– Other

• Alteration in Glucuronidation can be caused by:– Genetic defects

– Polymorphisms

– Tissue-specific regulation

Ligands for nuclear receptors

Regulation of gene expression

Up/down regulation of Protein

expression

UGTs

ENDOBIOTICS

XENOBIOTICS

Toxins and

Pollutants

DetoxificationIncreased

potency

Elimination

More undesired

effects

Olfactory Substances

Detoxification

Drugs

Detoxification

Elimination

Increased

potencyActivation

Increased toxicity and/or

immunological reactions

Elimination

Bilirubin, Steroids, etc

Detoxification

Elimination

Phylogenetic Tree of Mammalian UGTs

• Divergence of 49 mammalian UGT proteins – Known mammalian UGTs have been separated into two families,

UGT1 and UGT2.• UGT1 family is localized on chromosome 2q37 and is divided into 2

subfamilies, UGT1A and UGT1B. • UGT2 is localized on chromosome 4q13 and is divided into 3

subfamilies, UGT2A, UGT2B, and UGT2C (not shown).

• 20 human UGTs have been identified

Guillemette C, DMR 2009

Guillemette C, DMR 2009

Ritter JK, 1992

UGT1A Gene Cluster and Putative Protein Structure in Humans

Radominska-Pandya A, 1999

Transmembrane

fragment

Substrate binding domain UDP-GlcUA binding domain

Variable N-terminal domain

amino acids 25 - 286

NH2 COO-

Retention

signal

Signal

peptideConserved C-terminal domain

amino acids 287 - 530

UDP-GlcUASubstratesUDP-

GlcUA

UDP-GT

UDP-

GlcUA

binding

domain

Substrate

binding

domain

ER

COO-

N+H3

cytosol

lumen

HO

Cytosol

Lumen

Gluc-O

Gluc-O

UDP-GlcUA

UGT

Transporter

??

UDP

Hypothetical Model of UGT Topology

Transmembrane

fragment

Tissue-Specific Expression of Human UGTsIsoform

Tissue

Protein Expression mRNA expression

UGT1A1 Biliary tissue, colon, intestine, liver, stomach,Kidney, trachea, adrenal gland, lung, prostate, testis, thymus,

thyroid

UGT1A3 Biliary tissue, colon, liver, stomach, brain

UGT1A4 Biliary tissue, colon, liver, intestine, lung,

UGT1A6 Biliary tissue, colon, liver, stomach, brain, kidney, larynx, lungAdrenal gland, placenta, prostate, salivary gland, small intestine,

testis, thymus, thyroid gland, trachea, uterus.

UGT1A7 Orolaryngeal tissue, esophagus, stomach

UGT1A8 Colon, esophagus, intestine, kidney, larynx

UGT1A9 Breast, colon, esophagus, liver, kidney, ovary, prostate, skin, testis

UGT1A10Orolaryngeal tissue, colon, biliary tissue, esophagus, intestine,

stomachBreast

UGT2B4Adipose tissue, adrenals, breast, ovary, liver, lung, placenta,

prostate, skin, testis, kidney

UGT2B7Breast, brain, colon, esophagus, intestine, kidney, liver, lung, and

pancreasTestis, uterus

UGT2B10 Liver Adrenal gland, colon, heart, skeletal muscle, testis, uterus

UGT2B11 Lung

UGT2B15Breast, testis, uterus, prostate, lung, ovary, esophagus, kidney, liver,

skin Colon, pancreas, small intestine, stomach, testis, trachea

UGT2B17 Liver, prostateAdrenal gland, bone marrow, brain, colon, lung, pancreas, peripheral

leukocytes, salivary gland, small intestine, spinal cord, spleen, stomach, testis, thymus, trachea

UGT2A1 Lung Trachea

UGT Polymorphisms and Genetic Deficiencies

Examples of UGT Polymorphisms

• UGT1A1– UGT1A1*28

• A common variant [A(TA)7TAA] in the TATA-box region of the UGT1A1 promoter

– UGT1A1*1 • Results in:

– Decrease level of UGT1A1 gene expression

– Increased breast cancer risk (due to estrogen metabolism), specifically in African American women

• UGT1A6– Metabolizes aspirin and other NSAIDs

– Two missense mutations leading to T181A and R184S amino acid substitution• UGT1A6*2

– Has a frequency of 30% in Caucasian pop.

– Positively modified protective effect of aspirin (decreased glucuronidation leads to higher levels of aspirin)

Examples of UGT Polymorphisms

• UGT1A7– Glucuronidates polycyclic aromatic hydrocarbons and dietary

heterocyclic aromatic amines– Three missense mutations in exon 1 result in four alleles:

• UGT1A7*1 (N129, R131, W208)• UGT1A7*2 (K129, K131, W208)• UGT1A7*3 (K129, K131, R208)

– Increased risk of orolaryngeal, liver, and colon cancer

• UGT1A7*4 (N129, R131, R208)– Increased risk of orolaryngeal, liver, and colon cancer

• UGT2B7 – Single nucleotide polymorphisms in coding and regulatory

region of UGT2B7 gene are thought to play a role in morphine glucuronidation• Cytosine to thymine polymorphism at 802 bp• UGT2B7*1 (Y268)

– 3 times more likely Asian pop than 2B7*2.

• UGT2B7*2 (H268)

Genetic Deficiencies of UGT1A1

• Crigler-Najjar disease:– Severe, chronic, non-hemolytic, unconjugated

hyperbilirubinemia– Defect in the gene encoding bilirubin UGT1A1– Caused by mutations to common exons 2-5 or by a mutation to

exon 1– Type I

• Complete loss of bilirubin-conjugating activity

– Type II• Partial loss of bilirubin-conjugating activity (typically <10% of normal) • Responds to phenobarbital treatment

• Gilbert’s syndrome:– Mild, unconjugated hyperbilirubinemia– Defect in the gene encoding bilirubin UGT1A1– Missense mutation in the coding region– Homozygous insertion into promoter

Conclusions

Practice Questions