A Dissertation Entitled Investigation of Single Nucleotide

Home , Cholesterol sulfate, POLI, SULT1A1, SULT1B1, SULT1C4, SULT2B1

A Dissertation entitled

Investigation of Single Nucleotide Genetic Polymorphisms of the Human SULT2B1 Gene:

Functional Characterization of SULT2B1b Allozymes

Fatemah Alherz

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Experimental Therapeutics

______Dr. Ming-Cheh Liu, Committee Chair

______Dr. Ezdihar A. Hassoun, Committee Member

______Dr. Caren L. Steinmiller, Committee Member

______Dr. Zahoor Shah, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo May 2018

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Investigation of Single Nucleotide Genetic Polymorphisms of the Human SULT2B1 Gene: Functional Characterization of SULT2B1b Allozymes

Fatemah Alherz

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental Therapeutics

The University of Toledo

May 2018

Human cytosolic sulfotransferase enzymes (SULTs) are a family of Phase II metabolizing enzymes, which are responsible for catalyzing the transfer of the sulfonate moiety form 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to a hydroxyl or amino group of an acceptor substrate. SULT2B1b, also called the cholesterol sulfotransferase, catalyzes the transfer of the sulfonate moiety to a number of steroids/sterols, including cholesterol, pregnenolone, dehydroepiandrosterone (DHEA), and oxysterols. Genetic polymorphisms of SULT2B1 have been reported to be associated with several cancer types such as breast, liver, colorectal, gastric, prostate, and endometrial cancer, and esophageal squamous cell carcinoma. Variations in the sulfating activity of SULT2B1b may affect the sulfation of steroids/sterols and influence steroid-related pathophysiological events. This study was designed to evaluate the effect of the genetic polymorphisms of SULT2B1 on the sulfating activity of the coded SULT2B1b allozymes. A systemically search of three online single nucleotide polymorphism (SNP) databases for SULT2B1 missense coding SNPs (cSNPs) was first conducted. cDNAs

coded by the ten selected SULT2B1 non-synonymous coding single nucleotide polymorphisms (cSNPs) were generated by site-directed mutagenesis, and the coded

SULT2B1b allozymes were expressed and purified.

The sulfating activities of the recombinant SULT2B1b allozymes were analyzed toward several endogenous substrates including cholesterol, pregnenolone, dehydroepiandrosterone (DHEA), 7-ketocholesterol (7KC), 5α,6α-epoxycholesterol

(5,6α-EC), 5β,6β- epoxycholesterol (5,6β-EC), 25-hydroxycholesterol (25HC), and 24- hydroxycholesterol (24HC). Three of the examined allozymes (SULT2B1b-Gly72Val,

SULT2B1b-Arg147His, and SULT2B1b-Gly276Val) showed no detectable activity toward any of the tested substrates, while the other seven allozymes (SULT2B1b-

Pro69Ala, SULT2B1b-Thr73Met, SULT2B1b-Asp191Asn, SULT2B1b-Arg230His,

SULT2B1b-Ser244Thr, SULT2B1b-Arg274Gln, and SULT2B1b-Pro345Leu) displayed differential sulfating activity toward different substrates tested, in comparison to the wild- type SULT2B1b.

Kinetic studies of SULT2B1b allozymes with cholesterol, pregnenolone, and DHEA revealed further differences in their catalytic activity, substrate-binding affinity, and catalytic efficiency. Furthermore, the pH-dependence of the sulfation of DHEA indicated that most of the tested SULT2B1b allozymes showed an optimum pH (at pH 9.0) similar to that of the wild-type, except SULT2B1b-Pro69Ala, and SULT2B1b-Arg274Gln that exhibited activity pH optimum at pH 8.0. -Taken together, the results obtained showed clearly the impact of genetic polymorphisms on the sulfating activity of SULT2B1b

allozymes, which may underscore the differential metabolism of the steroid and sterols in individuals with different SULT2B1b genotypes.

Acknowledgements

I would like to express my gratitude for my wonderful adviser Dr. Ming-Cheh

Liu, who was always available for help, support, and excellent guidance. I attribute this research and my degree to his encouragement. His thoughtful mentorship has given me the ability to think critically and conduct a scientific research.

I also would like to thank my committee members: Dr. Ezdihar Hassoun, Dr.

Zahoor Shah, and Dr. Caren Steinmiller for their insightful comments, time, help, and encouragement. I also would like to thank my lab mates especially, Dr. Katsuhisa Kurogi,

Amal El Daibani, Maryam Abunaja, Ahsan Bairam, and Mohammed Rasool, thank you for helping, listening, and supporting me through this entire process.

Finally, it is nearly impossible to describe my greatest gratitude and love to my family especially my husband and my mother for their sacrifices, unconditional support, patience, and happiness they have been generously providing.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations ...... xv

List of Symbols ...... xviii

1 Introduction………………………………………………………………………..1

1.1 Biotransformation of low-molecular weight endogenous compounds and

xenobiotics………………………………………………………………………...1

1.1.1 Phase I reaction……………………………………………………...2

1.1.2 Phase II reaction ...... 3

1.2 Sulfation and Sulfotransferases………………………………………………4

1.2.1 Sulfotransferases family…………………………………………….5

1.3 SULT2B1b: a cholesterol sulfotransferase ………………………………….10

1.3.1 SULT2B1b structure……………………………………………….11

1.3.2 SULT2B1b tissue distribution ………………………………….....13

1.3.3 SULT2B1b substrates preference ……………………………...….14

1.4 Overview of Steroid Biosynthesis………………………………………...…15 vi

1.4.1 Role of SULT2B1b in hydroxysteroids and cholesterol sulfation…16

1.5 Genetic polymorphisms in SULT enzymes………………………………….18

1.5.1 Human SULT2B1 genetic polymorphisms and physiological

implications……………………………………………………………...20

1.6 rational and objectives…………………………………………………….…23

2 Materials and Methods……………………...……………………………………26

2.1 Materials……………………………………………………………………..26

2.2 Identification and analysis of human SULT2B1 SNPs………………………27

2.3 Generation of SULT2B1b allozymes cDNAs………………………………..34

2.4 The recombinant SULT2B1b allozymes expression, and purification………38

2.5 Sulfation Assay………………………………………………………………40

2.6 kinetic studies………………………………………………………………...41

2.7 pH-dependence Studies………………………………………………………41

2.8 Statistical analysis……………………………………………………………42

3 On the Role of Genetic Polymorphisms in the Sulfation of Cholesterol by Human

Cytosolic Sulfotransferase SULT2B1b……………………………………………….…43

3.1 Abstract………………………………………………………………………44

3.2. Introduction………………………………………………………………….45

3.4. Materials and Methods………………………………………………………47

3.4.1. Materials…………………………………………………………..47

3.4.2. Identification and analysis of human SULT2B1 SNPs…………....48

vii

3.4.3. Generation, expression, and purification of SULT2B1b

allozymes………………………………………………………………...48

3.4.4. Sulfotransferase assay……………………………………………..49

3.4.5. Statistical analysis………………………………………………...50

3.5. Results and Discussion……………………………………………………...51

3.5.1. Identification and categorization of SNPs of human SULT2B1

gene………………………………………………………………………51

3.5.2. Effects of SULT2B1 genetic polymorphism on the cholesterol-

sulfating activity of SULT2B1b allozymes……………………………..52

3.5.3. Characterization of the cholesterol-sulfating activity of human

SULT2B1b allozymes…………………………………………………..52

3.6. Conclusion……………………………………………………………….....60

4 Effect of SULT2B1 Genetic Polymorphisms on the Sulfation of

Dehydroepiandrosterone and Pregnenolone by SULT2B1b Allozymes……………….61

4.1. Abstract…………………………………………………………………....62

4.2. Introduction………………………………………………………………..63

4.3. Materials and Methods…………………………………………………….65

4.3.1. Materials…………………………………………………………65

4.3.2. Identification and analysis of human SULT2B1 SNPs………….66

4.3.3. Generation, expression, and purification of selected SULT2B1b

allozymes…………………………………………………………….…..66

viii

4.3.4. Sulfotransferase assay……………………………………………..68

4.3.5. pH-dependence……………………………………………………68

4.3.6. Statistical analysis…………………………………………………69

4.4. Results ………………………………………………………………………69

4.4.1. Identification of cSNPs of human SULT2B1 gene………………..69

4.4.2. Generation of cDNAs encoding SULT2B1b allozymes and bacterial

expression and purification of SULT2B1b allozymes…………………...70

4.4.3. Characterization of the DHEA-sulfating activity of human

SULT2B1b allozymes……………………………………………………70

4.4.4. Characterization of the pregnenolone-sulfating activity of human

SULT2B1b allozymes……………………………………………………75

4.4.5. The pH-dependence of DHEA-sulfating activity of human

SULT2B1b allozymes……………………………………………………79

4.5. Discussion…………………………………………………………………...83

4.6. Conclusion…………………………………………………………………..87

5 Role of SUL2B1 Genetic Polymorphisms in the Sulfation of Oxysterols by

Human Cytosolic Sulfotransferase SULT2B1b…………………………………………89

5.1. Abstract……………………………………………………………………...89

5.2. Introduction………………………………………………………………….90

5.3. Materials and Methods………………………………………………………93

5.3.1. Materials…………………………………………………………..94

5.3.2. Identification and analysis of human SULT2B1 SNPs……………94

5.3.3. Generation of selected SULT2B1b allozyme cDNAs ………..…..95

5.3.4. Expression and purification of SULT2B1b allozymes……………95

5.3.5. Oxysterol sulfation assay …………………………………………96

5.3.6. Statistical analysis………………………………………….……..97

5.4. Results ………………………………………………………………….…..97

5.4.1. Identification of cSNPs of the human SULT2B1 gene……………97

5.4.2. Generation of SULT2B1b allozyme cDNAs, and expression and

purification of recombinant SULT2B1b allozymes……………………..98

5.4.3. Characterization of the oxysterols-sulfating activity of human

SULT2B1b allozymes……………………………………………………98

5.5. Discussion………………………………………………………………….106

5.6. Conclusion…………………………………………………………………112

6 Xenoestrgens Sulfation by Human Cytosolic Sulfotransferase SULT2B1b…..114

6.1. Abstract…………………………………………………………………....114

6.2. Introduction……………………………………………………………….115

6.3. Materials and Methods……………………………………………………116

6.3.1. Materials………………………………………………………...117

6.3.2. Expression, and purification of human cytosolic SULT2B1b…..117

6.3.3. Enzymatic assay with the xenobiotics substrates………………..118

6.4. Results and discussion………………………………………………….…119

6.4.1. SULT2B1b recombinant enzyme expression, and purification…119

6.4.2. Sulfating Activities of the Human SULT2B1b …………………120

6.5. Conclusion…………………………………………………………………122

7 Summary.……… ...... 126

References ...... 128

List of Tables

1.1 SULTs enzyme substrate example and tissue distribution ...... 9

1.2 SULT2B1 SNPs that have been reported in scientific literature ...... 22

2.1 Numbers of SULT2B1 SNPs and their location in the gene...... 28

2.2 SULT2B1b missense cSNPs ...... 30

2.3 Primer sets used in the site-directed mutagenesis of the cDNA encoding human

SULT2B1b Allozymes……………………………………...... 36

3.1 Specific activities of the human SULT2B1b allozymes with cholesterol as

substrate ...... 54

3.2 Kinetic constants of the human SULT2B1b allozymes in catalyzing the sulfation

of cholesterol...... 57

4.1 Kinetic constants of the human SULT2B1b allozymes in catalyzing the sulfation

of DHEA ...... 75

4.2 Kinetic constants of the human SULT2B1b allozymes in catalyzing the sulfation

of pregnenolone……………….…………………………………………………79

6.1 Specific activities of human cytosolic SULT2B1b toward xenoestrogens ...... 121

xii

List of Figures

1-1 Chemical structure of 3’-phosphoadenosine 5’-phosphosulfate (PAPS) ...... 4

1-2 Two-step reaction in the formation of PAPS, in human these two steps carried out

by bifunctional PAPS synthetase………………………………………………….5

1-3 Nomenclature system for cytosolic SULT superfamily ...... 6

1-4 Amino acid alignment of human SULT2B1a and SULT2B1b enzymes ...... 11

1-5 The nucleotide and deduced amino acid of SULT2B1b ...... 13

1-6 Steroid hormone biosynthesis pathway...... 16

2-1 SULT2B1b structure and the location of the selected cSNPs as well as the location of the important residues in the enzyme function...... 29

2-2 Agarose gel electrophoresis of SULT2B1b-wt and allozymes colony PCR (in

DH5α E. coli) ...... 37

2-3 Agarose gel electrophoresis of SULT2B1b-wt and allozymes colony PCR (in

BL21 E. coli)...... 39

2-4 SDS gel electrophoretic pattern of the purified human SULT2B1b allozymes .....40

3-1 Kinetic analysis of the sulfation of cholesterol by wild-type SULT2B1b ...... 56

4-1 Specific activity of the human SULT2B1b allozymes toward DHEA ...... 72

4-2 Kinetic analysis of the sulfation of DHEA by human wild-type SULT2B1b ...... 74

4-3 Specific activity of the human SULT2B1b allozymes toward pregnenolone ...... 76

4-4 Kinetic analysis of the sulfation of pregnenolone by human wild-type SULT2B1b .

...... 78

xiii

4-5 pH-Dependence of the Sulfating Activity of Human SULT2B1b-wt and allozymes ...... 82

5-1 Oxysterols formation and metabolism ...... 93

5-2 Specific activities of the sulfation of 7KC by human SULT2B1b allozymes .....100

5-3 Specific activities of the sulfation of 24HC by human SULT2B1b allozymes ...102

5-4 Specific activities of the sulfation of 25HC by human SULT2B1b allozymes ...103

5-5 Specific activities of the sulfation of 5,6βEC by human SULT2B1b allozymes.104

5-6 Specific activities of the sulfation of 5,6αEC by human SULT2B1b allozymes 105

5-7 Location of the examined SNPs-based amino acid in the PAPS binding region.112

6-1 Analysis of the xenoestrogens sulfated product generated by SULT2B1b ...... 124

6-2 Chemical structure of the exogenous substrates ...... 125

xiv

List of Abbreviations

ADP...... Adenosine-5’-Diphosphate Ala/A ...... Alanine APS ...... Adenosine-5’-Phosphosulfate Arg /R ...... Arginine Asn/N ...... Asparagine Asp/D ...... Aspartic acid ATP ...... Adenosine-5’-Triphosphate

cDNA ...... Complementary Deoxyribonucleic Acid CHES ...... Sodium acetate, 2- (Cyclohexylamino) Ethanesulfonic Acid CYP ...... Cytochrome P-450 Cys/C ...... Cysteine

DHEA ...... Dehydroepiandrosterone DHEA-S ...... Dehydroepiandrosterone Sulfate DMSO ...... Dimethyl Sulfoxide DNA ...... Deoxyribonucleic Acid DTT ...... Dithiothreitol

E. coli ...... Escherichia Coli 5,6α-EC ……………..5α,6α-epoxycholesterol 5,6β-EC……………...5β,6β- epoxycholesterol

HEPES ...... N-2-Hydroxylpiperazine-N2-Ethanesulfonic 24HC…………………24-hydroxycholesterol 25HC…………………25-hydroxycholesterol His/H ...... Histidine

Ile/I ...... Isoleucine IPTG...... Isopropyl-β-D-thiogalactopyranoside

Gln/Q ...... Glutamine Glu/E ...... Glutamic acid Gly/G ...... Glycine GST...... Glutathione S-transferase

xv kDa...... Kilodalton 7KC………………….7-ketocholesterol

LB ...... Luria Broth LXR...... Liver X receptor Leu/L ...... Leucine Lys/K ...... Lysine

MAF...... Minor allele frequency MES ...... 2- Morpholinoethanesulfonic Acid Met/M ...... Methionine MOPS ...... β-naphthol, 3-(N-Morpholino) Propanesulfonic Acid

NCBI ...... National Center for Biotechnology Information

OD600 nm ...... Optical Density at 600 nm wavelength

PAPS ...... 3‘-phosphoadenosine-5‘-phosphosulfate PCR ...... Polymerase Chain Reaction Phe/F ...... Phenylalanine Pro/P ...... Proline

Ser/S ...... Serine S.D ...... Standard Deviation SDS ...... Sodium Dodecyl Sulfate SDS–PAGE ...... Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis SULTs ...... Human Cytosolic Sulfotransferases Enzymes SULT1A1 ...... Human Cytosolic Sulfotransferases Family 1A Member 1 SULT1A2 ...... Human Cytosolic Sulfotransferases Family 1A Member 2 SULT1A3 ...... Human Cytosolic Sulfotransferases Family 1A Member 3 SULT1B1 ...... Human Cytosolic Sulfotransferases Family 1B Member 1 SULT1C1 ...... Human Cytosolic Sulfotransferases Family 1C Member 1 SULT1C2 ...... Human Cytosolic Sulfotransferases Family 1C Member 2 SULT1C4 ...... Human Cytosolic Sulfotransferases Family 1C Member 4 SULT1E1 ...... Human Cytosolic Sulfotransferases Family 1E Member 1 SULT2A1 ...... Human Cytosolic Sulfotransferases Family 2A Member 1 SULT2B1a ...... Human Cytosolic Sulfotransferases Family 2B Member 1a SULT2B1b ...... Human Cytosolic Sulfotransferases Family 2B Member 1b SULT4A1 ...... Human Cytosolic Sulfotransferases Family 4A Member 1 SULT6B1 ...... Human Cytosolic Sulfotransferases Family 6B Member 1

TAPS ...... 3-[N-Tris-(hydroxymethyl) Methylamino[-propanesulfonic acid] Thr/T ...... Threonine TLC ...... Cellulose Thin-Layer Chromatography Tris-HCl ...... Trisaminomethane Hydrocholoride Trp/W ...... Tryptophan xvi

Tyr/Y ...... Tyrosine

Val/V ...... Valine

xvii

List of Symbols

L ...... Liter ml ...... Milliliter μl ...... Microliter g...... Gram mg ...... Milligram μg ...... Mirogram mM ...... Millimolar μM ...... Micromolar SO3-...... Sulfonate Group SO42-...... Inorganic Sulfate nmol ...... Nanomole mmol ...... Millimole Sec...... Second min ...... Minute oC...... Celsius Ci ...... Curie Vmax ...... Maximal Velocity Km ...... Michaelis Constant Kcat ...... Turnover Number Kcat/ Km ...... Specificity Constant “catalytic efficiency” α ...... Angle of incidence β ...... Angle of distortion

xviii

Chapter 1

1. Introduction

1.1 Biotransformation of low-molecular weight endogenous compounds and xenobiotics

The human body is constantly exposed to different types of xenobiotics, like drugs, pesticides, animal and plant toxins. The physical properties of xenobiotics, especially the lipophilicity, help them to enter the body easily through the lung, skin, and gastrointestinal tract (GIT). For our body to be protected from any harmful substances, metabolizing enzymes have to facilitate the removal of any unwanted chemicals through a process called biotransformation. Xenobiotic biotransformation is an essential mechanism for maintaining homeostasis and protecting the body from harmful chemicals.

The human body produces several metabolizing enzymes that have broad specificity, which help to maintain homeostasis by working on both endogenous compounds (such as hormones, vitamins, and fatty acids) and exogenous chemicals like drugs and toxins [1].

Metabolizing enzymes work by rendering xenobiotics to more hydrophilic forms, which facilitate their removal from the body. Even though the highest level of metabolizing enzymes is found in the gastrointestinal tract (GIT), most of the body tissues express them in differential levels. The reactions of xenobiotic biotransformation are classified

1 into two phases: phase I and phase II [1]. Phase I reaction is responsible for exposing or introducing functional groups (like SH, -NH2, -OH, and –COOH) through reduction, oxidation, and/or hydrolysis. The product of phase I reaction is usually slightly hydrophilic, which sometimes makes it necessary for the chemical to undergo a phase II reaction. Phase II reactions may or may not be preceded by phase I reactions. Phase II reactions include several conjugation reactions (such as glucuronidation, sulfation, acetylation, glutathione conjugation, methylation, and amino acid conjugation), which mostly result with more hydrophilic products that can be easily removed from the body

[1].

1.1.1 Phase I reaction

As mentioned earlier, phase I reactions help to remove xenobiotics through three major reactions including hydrolysis, reductions, and/or oxidation. Hydrolysis occurs in the body with the help of several enzymes like carboxylesterases, cholinesterases, and paraoxonases (lactonase). Hydrolyzing enzymes help to remove amide or ester groups from xenobiotics [1]. Reduction reactions facilitates the removal of the functional group from xenobiotics like aldehyde, ketone, and sulfoxide by the help of reducing agents (the reduced form of glutathione and NAD(P)) [1]. The oxidation reaction is the major and the most important phase I reaction which is catalyzed mainly by cytochrome P450 monooxygenases (CYP) enzymes [1]. CYP is a super family of heme-containing proteins that isare localized mainly in the endoplasmic reticulum and the mitochondria.

CYPs are grouped into about 18 families and 44 subfamilies. Out of all known CYP enzymes, only 12 enzymes from CYP1, CYP2, and CYP3 families are known to be

2 responsible for metabolizing drugs and xenobiotics [2, 3]. The remaining CYP enzymes are involved in endogenous steroid-synthesis and several endogenous substrate homeostasis [2]. The major CYPs subfamilies that are involved in metabolizing drugs and xenobiotics are CYP2C, CYP2D, and CYP3A [4]. The CYP3A4 enzyme is the most important CYP metabolizing enzyme because it is the most abundant CYP expressed in the liver and the intestine [2]. Further, CYP3A4 is responsible for metabolizing about

50% of clinically approved drugs [4].

1.1.2 Phase II reaction

Phase II reactions improve the hydrophilicity and thus facilitates elimination of the xenobiotics especially through glucuronidation, sulfation, glutathione conjugation, and/or conjugation with an amino acid, such as l glutamine, taurine, glycine [1, 4]. The main classes of phase II metabolizing enzymes are: UDP-glucuronosyltransferases

(UGTs); cytosolic sulfotransferases (SULTs); N-acetyltransferases (NATs), glutathione

S-transferases (GSTs); and methyltransferases (e.g. thiopurine S-methyl transferase

(TPMT); and catechol O-methyl transferase (COMT)) [5]. Most phase II reactions result in highly hydrophilic and biologically inactive metabolites that can be easily excreted from the body, yet they sometimes result in a metabolitewith metabolites that are pharmacologically active or even more toxic [1, 4]. For example, glutathione conjugation of hydroquinone and bromobenzene promotes their nephrotoxicity [6]. Most of the phase II metabolizing enzymes are localized in the cytosolic part of the cell, except

UDP-glucuronosyltransferases (UGTs) which is located in the endoplasmic reticulum [1,

4].

1.2 Sulfation and Sulfotransferases

Sulfation reactions involve the biosynthesis, biotransformation, and detoxification of several endogenous compounds and xenobiotics [7]. Sulfation reactions are catalyzed by sulfotransferases (SULTs) with the help of 3‘-phosphoadenosine-5‘-phosphosulfate

(PAPS) as a sulfate donor (cf. figure 1-1) [8]. In prokaryotes and lower eukaryotes PAPS is synthesized mainly by a two-step reaction which involves the formation of adenosine5’-phosphosulfate (APS) in the first step followed by the formation of the

PAPS molecule by the help of ATP-sulfurylase and APS-kinase enzymes in each step respectively (cf. figure 1-2) [8-10]. However, in the human body bifunctional PAPS synthetase carries out both steps of PAPS synthesis reaction [10, 11].

Figure 1-1 chemical structure of 3’-phosphoadenosine 5’-phosphosulfate (PAPS)

Figure 1-2 two-step reaction in the formation of PAPS, in human these two steps carried out by bifunctional PAPS synthetase.

SULT enzymes are responsible for transferring SO3-1 from PAPS molecule to an acceptor compound that has either the amino or hydroxyl group forming sulfamate or sulfate metabolite respectively [12]. Sulfonate conjugation mostly renders xenobiotics to biologically inactive metabolites and improves their hydrophilicity, which facilitates their removal from the body through biliary and urinary excretion [8, 13]. Sometimes, however, sulfation of some xenobiotics results in more active or toxic metabolite [8]. For example, SULT enzymes render minoxidil to its active form minoxidil sulfate [8].

Moreover, SULTs have been reported to be involved in activation of several procarcinogens like, 2-acetylaminofluorene, 2-hydroxymethylpyrene, N-hydroxy-2- aminofluorene, and 1-hydroxysafrole, which result in genotoxic metabolites [14].

1.2.1 Sulfotransferases family

5 Sulfotransferases are classified into two classes: membrane-bound (Golgi- associated) SULTs and soluble (cytosolic SULTs). Membrane-associated SULTs are involved in the post-translation modification of macromolecules such as proteins, peptides, and carbohydrates [15]. On the other hand, cytosolic SULTs are responsible for sulfating small molecules like hormones, drugs, and xenobiotics. Cytosolic SULTs superfamily consists of 13 enzymes, which classify into four gene families: SULT1,

SULT2, SULT4, and SULT6 [4]. SULTs enzymes have been named according to their identified substrates until a new nomenclature’s guideline has been developed [16, 17].

Members of each family, indicated by a number after (SULT), are at least 45% identical, while sub-family, indicated by a letter after the family number, are at least 60% identical

[17]. Members of at least 97% identical amino acid sequence are assigned to the same isoform names, indicated by a number after the subfamily letter (cf. figure 1-3) [17].

Figure 1-3 Nomenclature system for cytosolic SULT superfamily.

6 The SULT1 family is responsible for metabolizing small phenolic compounds, catecholamines, thyroid hormones, eicosanoids, and estrogens [7, 18]. The SULT1 family consists of eight enzymes including: SULT1A1, SULT1A2, SULT1A3,

SULT1B1, SULT1C1, SULT1C2, SULT1C4, and SULT1E1 [4]. The SULT1A subfamily is mainly responsible for small substrates that contain phenolic or catecholic group [10] and consist of three enzymes SULT1A1, SULT1A2, and SULT1A3, which are located on chromosome 16p11.2-12.1 [19]. SULT1A1 is considered the main xenobiotic metabolizing SULT enzyme because of its ability to sulfate a broad range of endogenous and exogenous substrates and it is widely expressed in several human tissues

(highly expressed in the liver) [20]. SULT1A2 is 96% identical to SULT1A1 and share similar substrates range yet with lower affinities [7]. SULT1A3 is known as a catechol sulfotransferase and able to sulfonate catecholamines and monoamine neurotransmitters such as dopamine, 3-methyldopamine, adrenaline, and noradrenaline [21-23]. The

SULT1B1 gene is located on chromosome 4q11-13 [24]. The SULT1B1 enzyme exhibits high affinity toward thyroid hormones such as 3,3'-diiodothyronine, triiodothyronine, reverse triiodothyronine and thyroxine and can also catalyze the sulfation of small phenolic compounds like 1-naphthol and p-nitrophenol [25, 26]. The

SULT1C subfamily gene is located on chromosome 2q12.2 and consists of three enzymes: SULT1C1, SULT1C2, and SULT1C4. The SULT1C subfamily enzymes are responsible for catalyzing the sulfation of hydroxylamines like N-hydroxy-2- acetylaminofluorene, N-hydroxy-4-aminobiphenyl, and b-naphthylamine [24, 27].

Furthermore, SULT1E1 is known as estrogen sulfotransferase and is located on

7 chromosome 4q13.1 [24, 28]. SULT1E1 shows high affinity toward estrogen hormones such as estrogens, estrone (E1), and 17-β-estradiol (E2) [9, 29].

The SULT2 family is located on chromosome 19q13.3 and consists of three enzymes: SULT2A1, SULT2B1a, SULT2B1b [30-32]. SULT2 enzymes are primarily responsible for sulfating steroids and hydroxysteroids such as DHEA, pregnenolone, oxysterols, and cholesterol [8]. SULT2 enzymes were named after their preferred substrate, for example, SULT2A1 is known as a dehydroepiandrosterone sulfotransferase, while SULT2B1a and SULT2B1b are known as a pregnenolone sulfotransferase and a cholesterol sulfotransferase, respectively [12, 33]. SULT2B1b and SULT2A1 have 48% identical amino acid sequences [34]. SULT2B1b and SULT2A1 show distinctive tissue distribution and substrate preferences. For instance, SULT2A1 is expressed mainly in the liver, adrenal glands, and intestines [35, 36], while SULT2B1b is highly expressed in different tissues like lung, prostate, breast, and endometrium (will be discussed in detail below). Moreover, SULT2A1 has a wide range of substrate specificity, it can sulfate 3α- and 3β- hydroxyl group in sterols as well as bile acids [37], whereas SULT2B1b selectively sulfates 3β- hydroxyl group sterols [8].

SULT4A1 and SULT6B1 enzymes are located on chromosome 22q13.31 and

2p22.3, respectively [19, 38]. To date, limited information is available about their substrates or tissue distribution. More information about all members of SULT enzymes such as tissue distribution and substrate examples are presented in table 1-1.

Table 1-1: SULTs enzyme substrate example and tissue distribution.

8 Enzyme Tissue distribution Substrate Reference

SULT1A1 Gastrointestinal tract, p-Nitrophenol (PNP), [10, 20] liver, kidney, lung, brain. acetaminophen, minoxidil. SULT1A2 Liver, stomach. PNP, minoxidil, b- [7, 39] naphthol. SULT1A3 Brain, platelet, salbutamol, [10, 13, 20] gastrointestinal tract, dobutamine, kidney. norepinephrine. SULT1B1 Colon, liver, small 1-Naphthol, PNP, [20] intestine, iodothyronines. leukocytes. SULT1C1 Kidney, stomach, PNP, N-hydroxy-2- [20, 39] thyroid gland, liver, fetal acetylaminofluorene liver. . SULT1C2 Fetal liver, spleen. PNP, N-hydroxy-2- [20, 39] acetylaminofluorene. SULT1C4 In fetal: liver, lung, Acetaminophen, 2- [39, 40] kidney, heart. naphthol, p- In the adult: kidney, Nitrophenol. ovary, spinal cord, stomach. SULT1E1 Liver, endometrium, Estrogens (E2), estrone [20] jejunum, mammary (E1), 17- epithelial cells. ethinylestradiol. SULT2A1 Liver, adrenal gland, dehydroepiandrosterone [20, 39] jejunum, brain, bone (DHEA), lithocholic marrow. Acid, pregnenolone. SULT2B1a prostate, placenta, Pregnenolone. [34] respiratory system, skin. SULT2B1b Human lung, prostate, cholesterol, oxysterols, [34, 41-43] breast, brain, placenta, DHEA, pregnenolone, and skin, human breast estrone, and and prostate cancer cell androstenediol. lines. SULT4A1 Brain and No substrate identified. [38] gastrointestinal tract, bladder, cervix, testis, trachea, prostate. SULT6B1 Testis, kideney. No substrate identified. [19]

1.3 SULT2B1b: a cholesterol sulfotransferase

9 The SULT2B1 gene was first cloned and identified by Her et. al., in the placenta, prostate, trachea and to lower extent in the small intestine and lung [44]. The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b [12, 45]. SULT2B1aand

SULT2B1b enzymes differ in their N-termini, unique 8 and 23 amino acid terminals, respectively as a result of alternative transcription initiation and splicing (c.f. figure1-4)

[12, 45]. The difference in their N-terminal region influence their substrate preference,

SULT2B1b shows a strong activity toward cholesterol, while SULT2B1a preferentially sulfates pregnenolone and shows weak activity toward cholesterol [46]. SULT2B1a and

SULT2B1b have unique proline-serine-rich carboxyl terminals of about 52 unique amino acids extending longer than other SULT enzymes [42, 47]. This extension is responsible for the enzyme thermostability, kinetic activity, and nuclear translocation [42].

SULT2B1a ------maspppfhsqklpgeyfrykgvpfpvglyslesislaentqdvrd SULT2B1b mdgpaepqipglwdtyeddiseisqklpgeyfrykgvpfpvglyslesislaentqdvrd . *************************************

SULT2B1a ddifiitypksgttwmieiiclilkegdpswirsvpiwerapwcetivgafslpdqyspr SULT2B1b ddifiitypksgttwmieiiclilkegdpswirsvpiwerapwcetivgafslpdqyspr ************************************************************

SULT2B1a lmsshlpiqiftkaffsskakviymgrnprdvvvslyhyskiagqlkdpgtpdqflrdfl SULT2B1b lmsshlpiqiftkaffsskakviymgrnprdvvvslyhyskiagqlkdpgtpdqflrdfl ************************************************************

SULT2B1a kgevqfgswfdhikgwlrmkgkdnflfityeelqqdlqgsvericgflgrplgkealgsv SULT2B1b kgevqfgswfdhikgwlrmkgkdnflfityeelqqdlqgsvericgflgrplgkealgsv ************************************************************

SULT2B1a vahstfsamkantmsnytllppslldhrrgaflrkgvcgdwknhftvaqseafdrayrkq SULT2B1b vahstfsamkantmsnytllppslldhrrgaflrkgvcgdwknhftvaqseafdrayrkq ************************************************************

SULT2B1a mrgmptfpwdedpeedgspdpepspepepkpslepntslereprpnsspspspgqasetp SULT2B1b mrgmptfpwdedpeedgspdpepspepepkpslepntslereprpnsspspspgqasetp ************************************************************

SULT2B1a hprps SULT2B1b hprps *****

Figure 1-4 amino acid alignment of human SULT2B1a and SULT2B1b enzymes using Clustal Omega multiple sequence alignment tool. * Indicate identical amino acid residues.

1.3.1 SULT2B1b structure

The majority of the human cytosolic sulfotransferase enzymes consist of 284 to

296 amino acids except for SULT2B1 isoforms SULT2B1a and SULT2B1b, which are

comprised of 350 and 365 amino acids, respectively. This is because SULT2B1 isoforms

have a unique proline-serine-rich carboxyl terminal of about 52 unique amino acids

extension longer than other SULT enzymes [42, 47]., and that extension is responsible for

the enzyme thermostability, kinetic activity, and nuclear translocation [42]. Moreover, it

has been suggested that the unique proline-serine-rich carboxyl terminal may be

subjected to post-translational modification, e.g. phosphorylation [42]. Another

11 characteristic of the SULTs is that they are mostly present in solution as dimers [7], the

KXXXTVXXE (known as KTVE motif) conserved amino acid sequence is responsible for SULTs dimerization in solution [48].

The SULT2B1b crystal structure in presence of PAP and pregnenolone or PAP and DHEA has been resolved by Lee et al. [47]. SULT2B1b is comprised of α and β motifs with a central 5-standard parallel β-sheet [47]. The amino acid residues from 19-

23 in the N-terminal region are necessary for the cholesterol sulfating activity [46]. In fact, a mutational study revealed that the isoleucine at positions 21 and 23 are crucial for cholesterol-sulfating activity [46]. SULT2B1b binds to the PAPS/PAP molecule within three important regions, the 5’-phosphosulphate binding (PSB) loop, 3’-phosphate binding region, and the PAP adenine group (c.f. Figure 1-5) [46]. The conserved amino acid residues 67TYPKSGT73 compose the PSB loop [46]. The amino acid residues (Lys70,

Ser71, Gly72, and Thr73) of the PSB loop and Thr74 are involved in the 5’-phosphate binding of PAP/PAPS, while the amino acid residues (Arg274, Lys275, Gly276, Arg147, and

Ser155) are involved in the 3’-phosphate binding of PAP/PAPS [46]. The interaction with the adenine group of the PAP involves the amino acid residues Ser244, Tyr 210, Trp75, and

Phe246 [46]. Histidine, at position 125, is responsible for the catalytic activity of

SULT2B1b [46]. Even though the carboxy-terminal of SULT2B1b was not solved in the crystal structure study, a mutational study has suggested that changes (e.g. Ser348Asp or

Ser348Gly) in the carboxy terminal may influence the enzyme sulfating activity, thermostability, and the enzyme localization in the nuclei [49].

atggacgggcccgccgagccccagatcccgggcttgtgggacacctatgaagatgacatc M D G P A E P Q I P G L W D T Y E D D I 20 tcggaaatcagccagaagttgccaggtgaatacttccggtacaagggcgtccccttcccc S E I S Q K L P G E Y F R Y K G V P F P 40 gtcggcctgtactcgctcgagagcatcagcttggcggagaacacccaagatgtgcgggac V G L Y S L E S I S L A E N T Q D V R D 60 gacgacatctttatcatcacctaccccaagtcaggcacgacctggatgatcgagatcatc D D I F I I T Y P K S G T T W M I E I I 80 tgcttaatcctgaaggaaggggatccatcctggatccgctccgtgcccatctgggagcgg C L I L K E G D P S W I R S V P I W E R 100 gcaccctggtgtgagaccattgtgggtgccttcagcctcccggaccagtacagcccccgc A P W C E T I V G A F S L P D Q Y S P R 120 ctcatgagctcccatcttcccatccagatcttcaccaaggccttcttcagctccaaggcc L M S S H L P I Q I F T K A F F S S K A 140 aaggtgatctacatgggccgcaacccccgggacgttgtggtctccctctatcattactcc K V I Y M G R N P R D V V V S L Y H Y S 160 aagatcgccgggcagttaaaggacccgggcacacccgaccagttcctgagggacttcctc K I A G Q L K D P G T P D Q F L R D F L 180 aaaggcgaagtgcagtttggctcctggttcgaccacattaagggctggcttcggatgaag K G E V Q F G S W F D H I K G W L R M K 200 ggcaaagacaacttcctatttatcacctacgaggagctgcagcaggacttacagggctcc G K D N F L F I T Y E E L Q Q D L Q G S 220 gtggagcgcatctgtgggttcctgggccgtccgctgggcaaggaggcactgggctccgtc V E R I C G F L G R P L G K E A L G S V 240 gtggcacactcaaccttcagcgccatgaaggccaacaccatgtccaactacacgctgctg V A H S T F S A M K A N T M S N Y T L L 260 cctcccagcctgctggaccaccgtcgcggggccttcctccggaaaggggtctgcggcgac P P S L L D H R R G A F L R K G V C G D 280 tggaagaaccacttcacggtggcccagagcgaagccttcgatcgtgcctaccgcaagcag W K N H F T V A Q S E A F D R A Y R K Q 300 atgcgggggatgccgaccttcccctgggatgaagacccggaggaggatggcagcccagat M R G M P T F P W D E D P E E D G S P D 320 cctgagcccagccctgagcctgagcccaagcccagccttgagcccaacaccagcctggag P E P S P E P E P K P S L E P N T S L E 340 cgtgagcccagacccaactccagccccagccccagccccggccaggcctctgagaccccg R E P R P N S S P S P S P G Q A S E T P 360 cacccacgaccctcataa H P R P S - 365

Figure 1-5 The nucleotide and deduced amino acid of SULT2B1b. Highlighted in red is the PSB region, orange is the amino acid residues that are involved in PAP/PAPS interaction, green indicates the highly conserve dimerization motif, and the blue area is the amino acid residues that are involved in substrate interaction.

1.3.2 SULT2B1b tissue distribution

13 SULT2B1a and SULT2B1b mRNAs have been detected in several tissues, yet only

SULT2B1b protein has been detected in human tissues and cell lines [34]. SULT2B1b mRNA has been detected in various human tissues like prostate, placenta, breast, endometrium, uterus, ovary, small intestine, colon, lung, platelet, brain, and skin [12, 34,

41, 44, 50, 51]. SULT2B1b protein expression has been characterized in several human tissues such as skin, platelet, lung, endometrium, prostate, and placenta [50-54].

SULT2B1b protein and mRNA also have been detected in several cancerous human tissues and cell lines like prostate adenocarcinoma, LNCaP prostate adenocarcinoma cells, T47D and MCF-7 breast cancer cell lines [34, 55]. Furthermore, SULT2B1b has been reported to be further localized in the nuclei of placental tissues, which may influence its activity in this organ [53].

1.3.3 SULT2B1b substrates preference

SULT2B1 isoforms display outstanding differences in substrate preferences. As pointed out previously, SULT2B1b displays strong sulfating activity toward cholesterol, whereas SULT2B1a shows higher sulfating activity toward pregnenolone [33]. In addition to cholesterol, SULT2B1b exhibits a specific preference for 3β-hydroxysteroids such as oxysterols, dehydroepiandrosterone (DHEA), pregnenolone, estrone, and androstenediol [42, 43]. Additionally, SULT2B1b has been reported to exhibit lower sulfating activity toward several drugs and xenobiotics, such as 3-OH-tibolone,

14 raloxifene, bisphenol A, 4-n-octylphenol, 4-n-nonylphenol, diethylstilbestrol, 17-α- ethynylestradiol, and p-nitrophenol (PNP) [30, 43, 56].

1.4 Overview of Steroid Biosynthesis.

Cholesterol is the main biosynthetic precursor of steroid hormones. In the adrenal gland, cholesterol is transformed into pregnenolone by the action of cytochrome P450scc, which is the rate-limiting step in the steroidogenesis pathway (cf. figure 1-6) [57, 58].

Once pregnenolone is synthesized, it can serve as a precursor for glucocorticoids, mineralocorticoids, and dehydroepiandrosterone (DHEA) [59]. In sex-steroid responsive tissues, the locally synthesized or the adrenally secreted DHEA serves as a precursor for estrogen and androgen hormones through a process called intracrinology, which describes the intracellular formation, action, and inactivation of sex steroids [59-62].

SULT2B1b Cholesterol Cholesterol-S

P450scc SULT2A1/ Preg-S SULT2B1b P450c17 17-OH P450c17 Pregnenolone DHEA-S Pregnenolone DHEA SULT2B1b/ 3β-HSD SULT2B1a 3β-HSD 3β-HSD Androstenedione

Progesterone 17-OH Estrone 17β-HSD Progesterone 21-OHase 21-OHase Testosterone Deoxycorticosterone 5α-reductase 11-Deoxycortisol Estradiol 11β-OHase Dihydrotestosterone C o r t i c o o 11s tβ e- rOHase o n e

Corticoosterone Cortisol 18-OHase

Aldosterone Figure 1-6 Steroid hormone biosynthesis pathway. The red boxes show the involvement

of SULT2B1b. OHase, hydroxylase; HSD, hydroxysteroid dehydrogenase.

1.4.1 Role of SULT2B1b in hydroxysteroids and cholesterol sulfation.

Since SULT2B1b is expressed in many steroid hormone-responsive tissues, (such

as prostate, breast, placenta, and endometrium), SULT2B1b may be involved in steroid

hormone regulation and homeostasis. SULT2B1b sulfate several steroids substrates such

as DHEA, pregnenolone, and sterols like cholesterol and oxysterols [34]. For instance,

SULT2B1b has been proposed to be involved in the regulation of DHEA, which is an

important precursor for androgen and estrogen synthesis [12]. In circulation, DHEA and

DHEA-S are known to be the most abundant steroid hormones in humans [63]. The pool

of DHEA is circulated as the inactive sulfate metabolite (DHEA-S) by the action of

SULT2A1, SULT2B1a, and SULT2B1b [12, 64, 65]. In the peripheral tissues, sulfatase

16 enzymes (STS) can de-sulfate steroid hormones to retain their bioactive forms and interact with their corresponding receptors [66]. Moreover, SULT2B1b is also able to sulfate pregnenolone, which is also an important precursor for all steroid hormones including DHEA [12]. DHEA and pregnenolone and their sulfated ester (DHEA-S and pregnenolone-sulfate), are neurosteroids, that are synthesized and released in the nervous system independently and act as neuromodulators [67-69].

In addition to DHEA and pregnenolone, SULT2B1b is considered the major

SULT in sulfating cholesterol (considering its higher affinity for cholesterol compared with the other SULTs) [70]. Cholesterol sulfation produces cholesterol sulfate, which is biologically active by itself [33]. For instance, cholesterol sulfate is involved in promoting keratinocyte differentiation, increasing cell membrane stability, regulating the activity of serine proteases involved in blood clotting, and regulating cholesterol synthesis [70]. Considering the multiple functions of cholesterol-sulfate and its distribution in many tissues, SULT2B1b expression and activity may be a key regulator of cholesterol metabolism and physiological function.

In the human body, cholesterol oxidation produces oxysterols that are involved in a myriad range of biological activities ranging from cytotoxicity to nuclear receptors regulation [71]. SULT2B1b has been reported to be involved in the sulfation of several oxysterols including, 7-ketocholesterol (7KC), 5α,6α-epoxycholesterol (5α,6α-EC),

5β,6β- epoxycholesterol (5 β,6 β -EC), 25-hydroxycholesterol (25HC), and 24- hydroxycholesterol (24HC) [15, 72, 73]. SULT2B1b sulfation of oxysterols may affect their biological activity and their elimination [15, 72]. For example, 7-ketocholesterol has been shown to be involved in atheromatous lesions and retinal macular degeneration

17 [15]. SULT2B1b was reported to be able to render 7-ketocholesterol to a less active sulfated metabolite, thereby preventing its cytotoxic effect. It has been indicated that induction of SULT2B1b expression in 293T cells attenuated the cytotoxic effect of 7- ketocholesterol by producing 7-ketocholesterol sulfate conjugate [15, 74]. Moreover,

SULT2B1b was suggested to play a role in intracellular lipid homeostasis by catalyzing

25HC to 25HC-sulfate, which acts as an inhibitor in the LXR/SREP-1 signaling pathway and reduces the cellular lipid level [74].

1.5 Genetic polymorphisms in SULT enzymes

SULT enzymes sulfating activities toward endogenous and exogenous compounds vary considerably in different individuals [75]. In fact, previous studies have revealed that some SULT genetic polymorphisms are implicated in many diseases and treatment responses [11, 76]. Genetic polymorphisms of SULT1A1 and SULT1A3 were commonly investigated because they are expressed in the platelets [13]. Out of all known

SULT enzymes, SULT1A1 was extensively studied because of its role in xenobiotics metabolism, procarcinogens bioactivation, and widespread expression in the body [7].

Thus, several epidemiological studies were conducted to investigate the role of

SULT1A1 genetic polymorphisms in relation to cancer susceptibility. Indeed, SULT1A1 genetic polymorphism has been implicated in different cancer types such as lung, colorectal, prostate, and breast cancer, especially among interethnic groups [77-83]. For instance, two SNPs in the 3'-flanking region (14A/G and 85C/T) have been reported to be associated with endometrial cancer [83]. Moreover, SULT1A1 non-synonymous coding

18 single nucleotide polymorphisms (cSNPs), which affects the coded protein product and thereby influences the enzyme sulfating activity toward different drugs, procarcinogens, and environmental toxins, [32, 84, 85]. An in vitro study of SULT1A1 reported at least

50-fold variation in SULT1A1 sulfation activity toward 4-nitrophenol in platelets from

905 individuals [86]. SULT1A1*2 (Arg213His) and SULT1A1*3 (Met223Val) are commonly reported SULT1A1 alleles with a minor allele frequency of 32% and 16% in

Caucasian-Americans and African-Americans, respectively [86-88]. SULT1A1*3 was reported to cause a significant decrease in Km values of 4-nitrophenol and PAPS [32], while SNPs in SULT1A1*2 alters the activation rate of different pro-mutagens such as N-

Hydroxy-2-acetylaminofluorene and 1-Hydroxymethylpyrene [13].

Besides SULT1A1, non-synonymous cSNPs have been also identified in the other members of the SULT1 family. For example, SULT1A2*2 (Ile7Thr, Asn235Thr) is a commonly reports SULT1A2 allele with a frequency of 0.287 [32]. Variation in

SULT1A2*2 was reported to cause a dramatic change in the enzyme affinity toward 4- nitrophenol and PAPS [32]. Interestingly, this allele is genetically linked to the

SUL1A1*2 variant [75]. Moreover, genetic variation in SULT1A3 (Lys234Asn) has been reported to cause reduction in the enzyme sulfating activity toward dopamine and reduce the immunoreactive protein to 28% and 54% of the wildtype, respectively [89].

Common missense cSNPs (Asp22Tyr and Ala32Val) of SULT1E1 have been reported to cause a decrease in the enzyme affinity toward 17-β estradiol and PAPS, as well as a decrease in the immunoreactive protein level [28]. In the SULT1C subfamily, SULT1C2

(Asp5Glu) variant was implicated in higher post-treatment relapse rates in acute myeloblastic leukemia [27].

19 In the SULT2 family a commonly reported SULT2A1 genetic variant

(Glu186Val) caused a decrease in DHEA-sulfating activity, reduction in the enzyme thermostability, and decrease in the level of immunoreactive protein when expressed in

COS-1 cells [13]. Genetic polymorphisms of SULT2B1 will be discussed in great details below. No data are available about SULT4A1 and SULT6B1 genetic polymorphisms to date.

1.5.1 Human SULT2B1 genetic polymorphisms and physiological implications

SULT2B1 polymorphisms have been suggested to be related to different types of cancer. For instance, SULT2B1 SNPs (rs4149455 intronic SNP, and rs1052131 synonymous SNP) have been associated with reduced risks of esophageal squamous cell carcinoma [90]. SULT2B1 SNPs in the intronic region (rs12460535, rs2665582 and rs10426628) have been reported to be associated with prostate cancer progression and overall survival [91]. SULT2B1 (rs2665582 and rs10426628) alleles were found to be associated with reduced risk of prostate cancer progression, while (rs12460535) allele is associated with prostate cancer progression [91]. In fact, the SULT2B1 (rs10426628) allele was proposed to reduce the risk of the prostate cancer progression by reducing the circulating steroid hormones and lowering reactive oxygen species accumulation by forming less reactive metabolites [91]. Furthermore, SULT2B1b non-synonymous coding cSNP (Arg274Gln and Pro149Leu) and in-frame deletion (rs16989366) were reported to be involved in autosomal recessive congenital ichthyoses [92]. These variants

20 cause a disturbance in cholesterol metabolism, which results in a decrease or even absence of cholesterol sulfate in the skin [92].

Generally, aberrant SULT2B1b expression levels have been reported in different cancer types including endometrial, breast, prostate, colorectal, and gastric, as well as hepatocellular carcinomas [55, 64, 93-95]. For example, SULT2B1b was reported to be down-regulated in prostate cancer, while it is up-regulated in colorectal, breast, liver, and endometrial cancers [94]. The down-regulation of SULT2B1b in prostate cancer was believed to be the reason behind prostate cancer progression due to the lake of the protective effect of SULT2B1b that is involved in the decrease of steroid hormone precursors like DHEA [55, 64]. Conversely, a recent study has shown that SULT2B1b is over-expressed in prostate cancer and positively correlated with prostate cancer progression and cell survival, whereas SULT2B1b knockdown in the LNCap cell line caused a reduction in cell viability and induced cell death [96]. Likewise, SULT2B1b overexpression has been shown to be involved in promoting hepatocellular carcinoma in vitro and in vivo [95]. SULT2B1b overexpression in Hepal-6 cells leads to up-regulation of the expression of pro-apoptotic factor (Fas) and downregulation of the expression of anti-apoptotic factor, BCL-2, that decreases cell growth. In contrast, knockdown of

SULT2B1b was fouind to result in cell cycle arrest and apoptosis [95]. Furthermore,

SULT2B1b up-regulation in colorectal cancer has been reported to be associated with promoting colorectal cancer progression, metastasis, invasion, while knockdown of

SULT2B1b prevents these events [94]. SULT2B1b is also overexpressed in gastric

21 cancer and was found to be positively correlated with angiogenesis and poor clinical feature [93].

Table 1-2: SULT2B1 SNPs that have been reported in scientific literature

No. SNP ID Location Effect

1 rs4149455 Non-coding region Associated with reduced risks of (intron variant) 51528 esophageal squamous cell carcinoma C>T [90]. 2 rs1052131 Coding region (synonymous variant) 52085 T>C 3 rs12460535 Non-coding region (intron variant) 48322 Positively associated with the risk of A>G prostate cancer progression [91]. 4 rs2665582 Non-coding region (intron variant) 37239 Associated with a reduced risk of A>G prostate cancer progression [91]. 5 rs10426628 Non-coding region (5’- UTR) 42002 A>G 6 rs3786749 Non-coding region Associated with colorectal cancer (intron variant) 44850 progression [97]. C>T 7 rs279451 Non-coding region Associated with larger prostate (upstream variant) G>T volume [98]. 8 rs1114167424 SULT2B1b Coding region (missense mutation) (Pro149Leu) Implicated in autosomal-recessive 9 rs762765702 SULT2B1b Coding congenital ichthyosis [92] region (missense mutation) (Arg274Gln) 10 rs16989366 In-frame deletion 11 rs16982149 SULT2B1b Coding region (missense mutation) (Leu51Ser) Cause changes in sulfating activity 12 rs16982158 SULT2B1b Coding toward DHEA and immunoreactive region (missense protein [45] mutation) (Asp191Asn) 13 rs16982169 SULT2B1b Coding region (missense mutation) (Arg230His)

22 14 rs17842463 SULT2B1b Coding region (missense mutation) (Pro345Leu)

1.6 Rational and Objectives

Human cytosolic sulfotransferase SULT2B1b, also called the cholesterol sulfotransferase, plays a crucial role in the hemostasis of cholesterol and related endogenous compounds, as well as in the human body. SULT2B1b catalyzes the transfer of the sulfate moiety to a number of steroids/sterols, including pregnenolone, DHEA, cholesterol, and oxysterols [42, 43]. It has been demonstrated that SULT2B1b is expressed mostly in steroid-responsive tissues such as placenta, prostate, breast, and endometrium [34, 41]. Genetic polymorphisms of SULT2B1b have been correlated with the progression and proliferation of different types of cancer including prostate cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, and colorectal cancer

[90, 91, 94, 95, 99].

The goal of this study was to identify and categorize human SULT2B1b polymorphisms and to investigate their effects on the sulfating activity of the resulting

SULT2B1b allozymes. Since SULT2B1b plays a crucial role in steroids homeostasis and sterols metabolism, we hypothesize that SULT2B1b SNPs will lead to SULT2B1b allozymes with differential sulfating activity toward endogenous steroids and xenobiotics, thereby influencing steroids homeostasis and metabolism of xenobiotics, including drugs. To verify this hypothesis, the specific aims are:

23 Aim 1. To identify and categorize different SNPs of the human SULT2B1b gene and gather epidemiological data that are related to these SNPs. To accomplish that, all reported human SULT2B1b SNPs have been identified by searching databases including

U.S. National Center for Biotechnology Information (NCBI), Ensembl Variation database, and The Universal Protein Resource (UniProt). Afterward, identified SULT2B1

SNPs were analyzed and categorized into SULT2B1b coding SNPs (synonymous, nonsense, and non-synonymous (missense)) and non-coding SNPs (introns, 5’- untranslated region (5’-UTR), and 3’-untranslated region (3’-UTR)). Moreover, the information derived from epidemiological studies that are related to the pathophysiological effect of SULT2B1 SNPs and SULT2B1b cSNPs were collected and analyzed.

Aim 2. To synthesize cDNAs that code for different human SULT2B1b allozymes, and express and purify the recombinant SULT2B1b allozymes. By employing site- directed mutagenesis techniques, cDNAs that encode SULT2B1b allozymes were generated. Briefly, the wild-type SULT2B1b cDNA packaged in pGEX-4T-2 prokaryotic expression vector was used as a template in conjunction with specific mutagenic primers.

The vectors harboring cDNAs encoding individual SULT2B1b allozymes were transformed into E. coli cells for recombinant protein expression. The expressed recombinant SULT2B1b allozyme was purified from the homogenates and analyzed for purity by SDS-polyacrylamide gel electrophoresis.

24 Aim 3. To delineate the functional consequences of human SULT2B1b allozymes by characterizing their sulfating activities toward representative endogenous and exogenous substrates. A sulfating activity assay was used to characterize the purified

SULT2B1b wild-type and allozymes with regard to their substrate specificity, pH- dependence, and kinetic characteristics.

25 Chapter 2

Materials and Methods

2.1 Materials

Pregnenolone, DHEA, cholesterol, hydroxypropyl-β-cyclodextrin, adenosine 5′- triphosphate (ATP), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-

(cyclohexylamino)-1 propanesulfonic acid (CAPS), 2-(N-morpholino) ethanesulfonic acid (MES), 2-(cyclohexylamino) ethanesulfonic acid (CHES), 3-[N-Tris-

(hydroxymethyl) methyl amino]-propane sulfonic acid (TAPS), dimethyl sulfoxide

(DMSO), Trizma base, and dithiothreitol (DTT) were products of Sigma-Aldrich (St.

Louis, MO, USA). 7-ketocholesterol, 5α,6α-epoxycholesterol, 5β,6β- epoxycholesterol,

25-hydroxycholesterol, and 24-hydroxycholesterol were products of Steraloids (Newport,

RI, USA). PrimeSTAR® Max DNA polymerase was a product of Takara Bio (Mountain

View, CA, USA). Oligonucleotide primers were synthesized by Eurofins Genomics

(Louisville, KY, USA). Dpn I and the DNA ladder were products of New England

Biolabs (Ipswich, MA, USA). QIAprep® Spin Miniprep Kit was a product of QIAGEN

(Germantown, MD, USA). Glutathione Sepharose 4B GST-tagged protein purification resin was a product of GE Healthcar Bio-Sciences (Pittsburgh, PA, USA). Cellulose

TLC plates, silica gel TLC plates, and Ultrafree-MC 5000 NMWL filter units were from

26 EMD Millipore (Billerica, MA, USA). Ecolume scintillation cocktail was purchased from MP Biomedicals, LLC. (Irvine, CA, USA). Protein molecular weight markers were from Bioland Scientific LLC. (Paramount, CA, USA). Carrier-free sodium [35S] sulfate was from American Radiolabeled Chemicals (St. Louis, MO, USA). Recombinant human bifunctional ATP sulfurylase/adenosine 5′-phosphosulfate kinase was prepared as previously described [1]. PAP[35S] was synthesized using recombinant human bifunctional PAPS synthase based on a previously established procedure [1]. All other chemicals were of the highest grade commercially available.

2.2 Identification and analysis of human SULT2B1 SNPs.

A systemic search of the keywords (homo sapiens or human SULT2B1) was conducted in three single nucleotide polymorphism databases including the U.S. National

Center for Biotechnology Information (NCBI), Ensembl Variation database, and the

Universal Protein Resource (UniProt). About 6,393 SULT2B1 SNPs were identified and categorized into SULT2B1b coding SNPs (synonymous, nonsense, frameshift, and non- synonymous (missense)) and non-coding SNPs (introns, 5’-untranslated region (5’-UTR), and 3’-untranslated region (3’-UTR)) (cf.Table 2-1). The missense SULT2B1b cSNPs were collected and are presented in Table 2-2. Moreover, the information collected from epidemiological studies in regard to the pathophysiological effect of SULT2B1 SNPs and the reported changes in SULT2B1b sulfating activity were collected and presented as previously mentioned in Chapter 1 (Table 1-2). SULT2B1b cSNPs were further analyzed for the location (in the substrate binding site, PAPS-binding sites, and

27 dimerization motif) of the amino acid changes, as well as the chemical nature of the changed amino acid (acidic to/or from basic, charged to/from uncharged, and turn- inducing to/from non-turn inducing residues) (1,18,34). Based on these characteristics, ten missense SULT2B1b cSNPs were selected for further studies as described below.

Figure 2-1 shows the location of the selected cSNPs in the overall structure of the

SULT2B1b enzyme.

Table 2-1 Numbers of SULT2B1 SNPs and their location in the gene.

SNPs location SNPs number

Non-coding SNPs 6,117 Intron region 6,088 3’-UTR region 6 5’-UTR region 23 SULT2B1b coding SNPs 276 synonymous 99 nonsense 4 frame shift 5 non-synonymous 168

f the important residues in in the f residues important

as well as the location o location the as as well

tool[2].

protter

The figure was generated by by was Thefigure generated

1 SULT2B1b structure and the location of structure SULT2B1b 1 cSNPs selected location the the and

Figure 2 Figure function. enzyme the

Table 2-2 SULT2B1b missense cSNPs. No. SNP code no. Position of nucleotide Nucleotide change Amino Amino acid change acid change position 1 rs904805770 83 T ⇒ G 1 M ⇒ R 2 rs970881383 85 G ⇒ A 2 D ⇒ N 3 rs1040692276 89 G ⇒ A 3 G ⇒ E 4 rs757865968 88 G ⇒ C 3 G ⇒ R 5 rs982337211 92 C ⇒ G 4 P ⇒ R 6 rs751154801 94 G ⇒ A 5 A ⇒ T 7 rs769598394 100 C ⇒ T 7 P ⇒ S 8 rs777714431 104 A ⇒ G 8 Q ⇒ R 9 rs527440291 110 C ⇒ T 10 P ⇒ L 10 rs989661502 109 C ⇒ T 10 P ⇒ S 11 rs560149467 112 G ⇒ A 11 G ⇒ S 12 rs560149467 112 G ⇒ T 11 G ⇒ C 13 rs769209838 125 C ⇒ T 15 T ⇒ I 14 rs776828696 128 A ⇒ G 16 Y ⇒ C 15 rs932006884 143 C ⇒ T 21 S ⇒ L 16 rs117476816 179 G ⇒ A 33 R ⇒ Q 17 rs113212465 190 G ⇒ A 37 V ⇒ I 18 rs544073330 202 G ⇒ A 41 V ⇒ I 19 rs145371861 205 G ⇒ A 42 G ⇒ S 20 rs377054892 220 G ⇒ A 47 E ⇒ K 21 rs377054892 220 G ⇒ C 47 E ⇒ Q 22 rs749847968 221 A ⇒ G 47 E ⇒ G 23 rs16982149 233 T ⇒ C 51 L ⇒ S 24 rs759048996 247 C⇒ G 56 Q ⇒ E 25 rs766992586 250 G ⇒ C 57 D ⇒ H 26 rs763833757 257 G ⇒ A 59 R ⇒ Q 27 rs753662500 260 A ⇒ C 60 D ⇒ A 28 rs146090633 262 G ⇒ A 61 D ⇒ N 29 rs750329743 265 G ⇒ A 62 D ⇒ N 30 rs750329743 265 G ⇒ C 62 D ⇒ H 31 rs1051070889 270 C ⇒ G 63 I ⇒ M 32 rs781330139 278 T ⇒ A 66 I ⇒ N 33 rs777924668 286 C ⇒ G 69 P ⇒ A

30 No. SNP code no. Position of nucleotide Nucleotide change Amino Amino acid change acid change position 34 rs746398875 296 G ⇒ T 72 G ⇒ V 35 rs527454384 299 C ⇒ T 73 T ⇒ M 36 rs768213131 312 C ⇒ G 77 I ⇒ M 37 rs140526640 313 G⇒ A 78 E ⇒ K 38 rs769753162 319 A ⇒ T 80 I ⇒ F 39 rs773038899 331 C ⇒ A 84 L ⇒ M 40 rs200817158 337 G ⇒ A 86 E ⇒ K 41 rs751390677 353 G ⇒ T 91 W ⇒ L 42 rs149116557 358 C⇒ T 93 R ⇒ C 43 rs372325021 359 G ⇒ A 93 R ⇒ H 44 rs372325021 359 G ⇒ T 93 R ⇒ L 45 rs200176089 364 G ⇒ A 95 V ⇒ M 46 rs765637140 367 C ⇒ T 96 P ⇒ S 47 rs138292714 380 G ⇒ A 100 R ⇒ Q 48 rs932416497 385 C ⇒ T 102 P ⇒ S 49 rs201511288 409 G ⇒ C 110 A ⇒ P 50 rs780615173 410 C ⇒ T 110 A ⇒V 51 rs747459444 415 A ⇒ G 112 S ⇒ G 52 rs777927515 422 C ⇒ T 114 P ⇒ L 53 rs780820646 427 C⇒ A 116 Q ⇒ K 54 rs558429244 431 A ⇒ C 117 Y ⇒ S 55 rs769594886 437 C ⇒ T 119 P ⇒ L 56 rs377120502 440 G ⇒ A 120 R ⇒ H 57 rs1027668633 439 C ⇒ T 120 R ⇒ C 58 rs749117260 445 A ⇒ G 122 M ⇒ V 59 rs986181196 468 G ⇒ T 129 Q ⇒ H 60 rs774401100 485 T ⇒ C 135 F ⇒ S 61 rs764196414 500 C ⇒ T 140 A ⇒ V 62 rs768145409 505 G ⇒ T 142 V ⇒ L 63 rs141909233 510 C ⇒ G 143 I ⇒ M 64 rs755712133 515 T ⇒ C 145 M ⇒ T 65 rs777140014 521 G ⇒ A 147 R ⇒ H 66 rs1114167424 527 C ⇒ T 149 P ⇒ L 67 rs550795943 530 G ⇒ A 150 R ⇒ Q 68 rs550795943 530 G ⇒ T 150 R ⇒ L 69 rs745691806 536 T ⇒ C 152 V ⇒ A

31 No. SNP code no. Position of nucleotide Nucleotide change Amino Amino acid change acid change position 70 rs776274742 551 A ⇒ G 157 Y ⇒ C 71 rs763041534 568 G ⇒ A 163 A ⇒ T 72 rs138616965 571 G ⇒ A 164 G ⇒ R 73 rs775399633 583 G ⇒ A 168 D ⇒ N 74 rs767984007 585 C ⇒ G 168 D ⇒ E 75 rs753326658 587 C⇒ A 169 P ⇒ Q 76 rs377692774 598 G ⇒ A 173 D ⇒ N 77 rs756748647 599 A ⇒ G 173 D ⇒ G 78 rs750020063 603 G ⇒ C 174 Q ⇒ H 79 rs1020958119 602 A ⇒ C 174 Q ⇒ P 80 rs779793800 617 T ⇒ A 179 F ⇒ Y 81 rs781146722 628 G ⇒ A 183 E ⇒ K 82 rs764776470 636 G ⇒ C 185 Q ⇒ H 83 rs16982158 652 G ⇒ A 191 D ⇒ N 84 rs16982158 652 G ⇒ T 191 D ⇒ Y 85 rs752620448 658 A ⇒ C 193 I ⇒ L 86 rs576779662 665 G⇒ A 195 G ⇒ D 87 rs544329093 673 C ⇒ T 198 R ⇒ W 88 rs376235752 674 G ⇒ A 198 R ⇒ Q 89 rs16982159 681 G ⇒ C 200 K ⇒ N 90 rs758482574 683 G ⇒ A 201 G ⇒ D 91 rs1051357870 686 A ⇒ G 202 K ⇒ R 92 rs780151698 692 A ⇒ G 204 N ⇒ S 93 rs139421205 697 C ⇒ A 206 L ⇒ I 94 rs777022651 703 A ⇒ G 208 I ⇒ V 95 rs891334773 707 C ⇒ A 209 T ⇒ N 96 rs370301606 727 G ⇒ A 216 D ⇒ N 97 rs778313193 734 A ⇒ G 218 Q ⇒ R 98 rs770390968 736 G ⇒ A 219 G ⇒ S 99 rs771791526 742 G ⇒ A 221 V ⇒ M 100 rs774942612 746 A ⇒ G 222 E ⇒ G 101 rs763970039 748 C ⇒ G 223 R ⇒ G 102 rs148634243 749 G ⇒ A 223 R ⇒ H 103 rs148634243 749 G ⇒ T 223 R ⇒ L 104 rs763970039 748 C ⇒ G 223 R ⇒ G 105 rs766419184 754 T ⇒ C 225 C ⇒ R

32 No. SNP code no. Position of nucleotide Nucleotide change Amino Amino acid change acid change position 106 rs751691295 769 C ⇒ T 230 R ⇒ C 107 rs16982169 770 G ⇒ A 230 R ⇒ H 108 rs781489535 773 C ⇒ T 231 P ⇒ L 109 rs542643724 784 G⇒ A 235 E ⇒ K 110 rs757800349 794 G ⇒ A 238 G ⇒ D 111 rs2302947 799 G⇒ A 240 V ⇒ I 112 rs528536213 802 G⇒ A 241 V ⇒ M 113 rs761474582 808 C ⇒ T 243 H ⇒ Y 114 rs765224593 811 T ⇒ A 244 S ⇒ T 115 rs546703156 815 C ⇒ T 245 T ⇒ I 116 rs199842613 823 G ⇒ A 248 A ⇒ T 117 rs764247607 833 C ⇒ T 251 A ⇒ V 118 rs757500882 844 T⇒ G 255 S ⇒ A 119 rs779491595 848 A ⇒ G 256 N ⇒ S 120 rs143866846 854 C ⇒ T 258 T ⇒ M 121 rs746709836 879 C ⇒ A 266 D ⇒ E 122 rs202103681 883 C ⇒ T 268 R ⇒C 123 rs146884008 884 G ⇒ A 268 R ⇒ H 124 rs199827788 892 G ⇒ C 271 A ⇒ P 125 rs201499986 893 C ⇒ T 271 A ⇒ V 126 rs762765702 902 G ⇒ A 274 R ⇒ Q 127 rs774212320 908 G ⇒ T 276 G ⇒ V 128 rs868532022 910 G ⇒ T 277 V ⇒ F 129 rs760760185 911 T ⇒ G 277 V ⇒ G 130 rs761911725 916 G ⇒ A 279 G ⇒ S 131 rs972714102 919 G ⇒ A 280 D ⇒ N 132 rs750776892 938 C ⇒ T 286 T ⇒ M 133 rs752098559 948 G ⇒ C 289 Q ⇒ H 134 rs201648343 961 G ⇒ A 294 D ⇒ N 135 rs920026531 965 C ⇒ T 295 R ⇒ C 136 rs777438571 965 G ⇒ A 295 R ⇒ H 137 rs749037593 973 C ⇒ T 298 R ⇒ C 138 rs770659899 974 G ⇒ T 298 R ⇒ L 139 rs778883635 980 A ⇒ G 300 Q ⇒ R 140 rs61748775 985 C ⇒ G 302 R ⇒ G 141 rs776553066 986 G ⇒ A 302 R ⇒ Q

33 No. SNP code no. Position of nucleotide Nucleotide change Amino Amino acid change acid change position 142 rs375751466 988 G ⇒ T 303 G ⇒ W 143 rs142168444 993 G ⇒ A 304 M ⇒ I 144 rs769942417 995 C ⇒ T 305 P ⇒ L 145 rs766844809 1021 G ⇒ A 311 E ⇒ K 146 rs752149713 1017 C ⇒ G 312 D ⇒ E 147 rs143292540 1019 C ⇒ A 313 P ⇒ Q 148 rs1052131 1029 C ⇒ A 316 D ⇒ E 149 rs763671518 1036 C ⇒ T 319 P ⇒ S 150 rs570769125 1042 C ⇒ A 321 P ⇒ T 151 rs757113850 1047 G ⇒ T 322 E ⇒ D 152 rs1045662705 1052 G ⇒ T 324 S ⇒ I 153 rs201143505 1055 C⇒ G 325 P ⇒ R 154 rs745856982 1059 G ⇒ T 326 E ⇒ D 155 rs758356699 1075 A ⇒ G 332 S ⇒ G 156 rs780001419 1091 C⇒ T 337 T ⇒ I 157 rs748181434 1099 G ⇒ A 340 E ⇒ K 158 rs141268538 1103 G ⇒ A 341 R ⇒ H 159 rs372338514 1105 G ⇒ A 342 E ⇒ K 160 rs866304725 1107 G ⇒ T 342 E ⇒ D 161 rs17842463 1115 C ⇒ T 345 P ⇒ L 162 rs771470847 1120 T ⇒ C 347 S ⇒ P 163 rs535907004 1121 C ⇒ T 347 S ⇒ F 164 rs759937237 1138 C ⇒ T 353 P ⇒ S 165 rs554318148 1141 G ⇒ A 354 G ⇒ S 166 rs760164635 1159 C ⇒ G 360 P ⇒ A 167 rs763659919 1160 C ⇒ G 360 P ⇒ R 168 rs566268776 1171 C ⇒ T 364 P ⇒ S

2.3 Generation of SULT2B1b allozymes cDNAs

The wild-type SULT2B1b (SULT2B1b-wt) cDNA ligated into a prokaryotic expression vector pGEX-4T-2 was used as a template to generate the mutated cDNAs

34 using site-directed mutagenesis techniques. PCR was performed with 0.25 μM of each sense and antisense mutagenic primers (cf. Table 2-3), 50 ng of the templet (SUL2B1b- wt/ pGEX-4T-2), and PrimeSTAR® Max DNA polymerase. The PCR amplification conditions were: pre-denaturation at 95°C for 2 min followed by 12 cycles of 30 s at

95°C, 1 min at 55°C, and 6 min at 72°C. To degrade the wild-type template, at the end of

PCR, the reaction mixtures were treated with 5 units of Dpn I and incubated at 37°C for an hour. The “mutated” SULT2B1b plasmid present in Dpn I-treated mixtures was transformed into competent DH5α E. coli cells to amplify the mutated plasmid. Briefly, 1

µL of the PCR mixture was added to the competent cells and placed in ice for 10 minutes. After that, the heat shock was carried out at 42°C for 30 seconds, then the mixture was placed in ice for 5 minutes. Subsequently, a 200 µl of the SOC medium was added to the mixture and incubated for 10 minutes at 37°C with continuous shaking. The mixture was then spread onto agar plate containing ampicillin and incubated at 37°C overnight.

In order to verify if the bacteria contain the right insert of the expected size; a colony PCR was conducted. The PCR was conducted using pGEX sense and antisense primers, pGEX 5' (GGGCTGGCAAGCCACGTTTGGTG) and pGEX 3'

(CCGGGAGCTGCATGTGTCAGAGG). The PCR conditions involved, pre- denaturation at 95°C for 2 min followed by 35 cycles of 30 s at 95°C, 1 min at 55°C, and

6 min at 68°C. To visualize the PCR product, ethidium bromide staining was added to the mixture, and the mixture was loaded onto a 1% agarose gel, and then separated by electrophoresis. The size of the PCR product (amplicon) was similar to the expected size of 1247 base pairs (bp) (cf. Figure 2-2). To amplify the plasmid of individual SUL2B1b

35 allozymes, the colony that carried each allozymes was grown in 10 ml LB medium overnight. The plasmid was purified following the protocol of the QIAprep® Spin

Miniprep Kit. The mutations in individual SULT2B1b allozyme plasmids were verified via nucleotide sequencing by Eurofins Genomics (Louisville, KY, USA).

Table 2-3 Primer sets used in the site-directed mutagenesis of the cDNA encoding human SULT2B1b Allozymes. SULT2B1b allozyme and corresponding MAFa Mutagenic primer set amino acid substitution SULT2B1b-Pro69Ala 0.000008 5’-ATCTTTATCATCACCTACGCCAAGTCAGGCACGACC-3’ 5’-GGTCGTGCCTGACTTGGCGTAGGTGATGATAAAGAT-3’

SULT2B1b-Gly72Val 0.000009 5’-ATCACCTACCCCAAGTCAGTCACGACCTGGATGATC-3’ 5’-GATCATCCAGGTCGTGACTGACTTGGGGTAGGTGAT-3’

SULT2B1b-Thr73Met 0.00002 5’-ACCTACCCCAAGTCAGGCATGACCTGGATGATCGAG-3’ 5’-CTCGATCATCCAGGTCATGCCTGACTTGGGGTAGGT-3’

SULT2B1b-Arg147His 0.000008 5’-AAGGTGATCTACATGGGCCACAACCCCCGGGACGTT-3’ 5’-AACGTCCCGGGGGTTGTGGCCCATGTAGATCACCTT-3’

SULT2B1b-Asp191Asn 0.008 AA 5’-CAGTTTGGCTCCTGGTTCAACCACATTAAGGGCTGG-3’ 5’-CCAGCCCTTAATGTGGTTGAACCAGGAGCCAAACTG-3’

SULT2B1b-Arg230His 0.008 AA 5’-ATCTGTGGGTTCCTGGGCCATCCGCTGGGCAAGGAG-3’ 5’-CTCCTTGCCCAGCGGATGGCCCAGGAACCCACAGAT-3’

SULT2B1b-Ser244Thr 0.0001 5’-GGCTCCGTCGTGGCACACACAACCTTCAGCGCCATG-3’ 5’-CATGGCGCTGAAGGTTGTGTGTGCCACGACGGAGCC-3’

SULT2B1b-Arg274Gln 0.00002 5’-CGTCGCGGGGCCTTCCTCCAGAAAGGGGTCTGCGGC-3’ 5-GCCGCAGACCCCTTTCTGGAGGAAGGCCCCGCGACG-3’

SULT2B1b-Gly276Val 5’-GGGGCCTTCCTCCGGAAAGTGGTCTGCGGCGACTGG-3’ 5’-CCAGTCGCCGCAGACCACTTTCCGGAGGAAGGCCCC-3’

SULT2B1b-Pro345Leu 0.025 CA 5’-CTGGAGCGTGAGCCCAGACTCAACTCCAGCCCCAGC-3’ 5’-GCTGGGGCTGGAGTTGAGTCTGGGCTCACGCTCCAG-3’ a Minor allele frequency. AA, African American population. CA, Caucasian American population.

A) Gly72Val Pro69Ala wt

- 3000 bp - 1500 bp

- 1000bp

Arg274Gln Ser244Thr Arg230His Asp191Asn Arg147His Thr73Met

- 3000 bp - 1500 bp

- 1000bp

C) Gly276Val Pro345Leu

- 3000 bp - 1500 bp

- 1000bp

Figure 2-2 Agarose gel electrophoresis of SULT2B1b-wt and allozymes colony PCR (in

DH5α E. coli).

2.4 The recombinant SULT2B1b allozymes expression, and purification.

To express SULT2B1b allozymes, the bacterial expression vector (pGEX-

4T-2) harboring individual SULT2B1b allozymes cDNAs were transformed into competent BL21 E. coli cells using the same conditions described above. The bacterial colonies were analyzed by colony PCR and visualized in 1% agarose gel as described above (cf. Figure 2-3). After that, the transformed cells (with the correct insert) were grown in 10 mL LB medium containing 100 μg/ml ampicillin and incubated at 25°C in a shaker overnight. The overnight culture was inoculated in 1 L LB medium with ampicillin at a final concentration of 100 μg/ml until OD600 nm ∼0.5 was reached.

Protein expression was induced with 0.1 mM Isopropyl-β-D-thiogalactopyranoside

(IPTG) and incubated overnight at 25°C with continuous shaking. The bacterial cells, were collected by centrifugation at 3,000× g for 20 minutes at 4°C , and then resuspended in 20 ml ice-cold lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mm NaCl, and

1 mM EDTA). The bacteria were homogenized using an Aminco French press. To collect the supernatant, the crude homogenates were centrifuged for 20 min at 10,000 × g at 4°C. The collected supernatants were individually fractionated using 2 ml of a 50% slurry of glutathione-sepharose resin. The resin was then washed several times with lysis buffer to remove unbound proteins. To cleave the enzyme without the fusion protein, the resin was treated with 2 ml of a thrombin digestion buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2) and 3.5 unit/ml bovine thrombin, and mixture was then incubated for 20 minutes at 25°C with constant agitation. Using centrifugation, the supernatant containing the recombinant SULT2B1b allozymes was collected and

38 analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). As shown in

Figure 2-4, the molecular weights of the purified SULT2B1b allozymes were all similar to the calculated molecular weight of the SULT2B1b-wt (41,307). To determine the concentration of the prepared allozymes, the Bradford method was used with bovine

serum albumin as a regular standard [3].

A) Thr73Met Gly72Val Pro69Ala wt

- 3000 bp

- 1500 bp

- 1000bp

Arg274Gln Ser244Thr Arg230His Asp191Asn Arg147His B)

- 3000 bp

- 1500 bp - 1000bp

Pro345Leu Gly276Val C)

- 3000 bp - 1500 bp - 1000bp

Figure 2-3 Agarose gel electrophoresis of SULT2B1b-wt and allozymes colony PCR (in

BL21 E. coli).

1 2 3 4 5 6 7 8 9 10 11 12

75 kDa

45 kDa

25 kDa

Figure 2-4. SDS gel electrophoretic pattern of the purified human SULT2B1b

allozymes. SDS-PAGE was performed on a 12% gel, followed by Coomassie

blue staining. Lane 1 indicates the migrating positions of protein molecular

weight markers co-electrophoresed. Samples analyzed in lanes 2 through 12

correspond to SULT2B1b-wt, SULT2B1bPro69Ala, SULT2B1b-Gly72Val,

SULT2B1b-Thr73Met, SULT2B1b-Arg147His, SULT2B1b-Asp191Asn,

SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, SULT2B1b-Arg274Gln,

SULT2B1b-Gly276Val, and SULT2B1b-Pro345Leu.

2.5 Sulfation Assay

To quantify the sulfating activity of the recombinant SULT2B1b allozymes toward the selected substrates, the 20 µL assay mixture consists of 14 µL radiolabeled

PAPS (PAP[35S]) as the sulfate donor, SULT2B1b-wt or allozyme, 1 mM DTT, 50 mM

HEPES buffer (pH 7.4), and a substrate. A control with the used vehicle was assayed in parallel. The reaction was started by incubating the mixture for 10 minutes at 37°C for

40 10 minutes and was terminated by placing the reaction’s tube in a heating block for 3 minutes at 100°C. Centrifugation at 13,000×g for 3 minutes was used to clear the precipitates in the reaction mixture. To analyze the radiolabeled sulfated product, a specific amount of the reaction (as indicated later under each substrate assay) was spotted in a TLC plate. The spotted TLC was developed by using a specific solvent system and was air dried. The sulfated product was visualized using autoradiography, cut from the

TLC plate, eluted with 0.5 ml water in a vial, and mixed thoroughly with 2 ml of

Ecolume scintillation liquid. A liquid scintillation counter was used to quantify the [35S] radioactivity. The obtained radioactivity of the sulfated product, in counts per minute (CPM), was used to calculate the specific activity, in the units of nmol of sulfated product per minute per mg enzyme.

2.6 Kinetic Studies.

For the kinetic studies, varying concentrations of the substrate were used with individual SULT2B1b allozymes with 50 mM HEPES at pH 7.4 (similar to the physiological pH), according to the procedure described above. Moreover, to determine the PAPS km values of each SULT2B1b allozymes, varying concentrations of PAPS were used at pH 7.4.

2.7 pH-Dependence Studies.

To examine the pH profile, dehydroepiandrosterone (DHEA) was examined as substrates with individual SUL2B1b-wt or the allozymes. To analyze the pH-dependence of the DHEA-sulfation by each enzyme, different buffers were used (50 mM sodium

41 acetate at 4.5,5, or 5.5; MES at 6 or 6.5; HEPES at 7, 7.5, or 8; TAPS at 8.5 or 9; CHES at 9.5 or10; CAPS at 10.5, 10.5, 11, or 11.5). The experimental procedure for pH- dependence studies were the same as described above, except for the used buffers.

2.8 Statistical Analysis.

Based on Michaelis-Menten kinetics, GraphPad Prism® v 6.0 software was used to generate non-linear regression curves and to calculate the kinetic constants. For inter- group comparisons, one-way ANOVA was used, followed by Dunnett’s test to determine statistical differences between the wild-type SULT2B1b and individual allozymes. P- values less than 0.05 were considered statistically significant.

Chapter 3

On the Role of Genetic Polymorphisms in the Sulfation of Cholesterol by Human Cytosolic Sulfotransferase SULT2B1b

Running Title: Sulfation of Cholesterol by Human SULT2B1b Allozymes

Fatemah A. Alherz1, Maryam S. Abunnaja1, Amal A. El Daibani1, Ahsan F. Bairam1,2, Mohammed I. Rasool1,3, Katsuhisa Kurogi1,4, Yoichi Sakakibara4, Masahito Suiko4, Ming- Cheh Liu1,*

1Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, University of Toledo Health Science Campus, Toledo, OH 43614 USA 2Department of Pharmacology, College of Pharmacy, University of Kufa, Najaf, Iraq 3Department of Pharmacology, College of Pharmacy, University of Karbala, Karbala, Iraq 4Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki 889-2192 Japan

*Corresponding Author: Ming-Cheh Liu, Ph.D. Professor Department of Pharmacology College of Pharmacy and Pharmaceutical Sciences University of Toledo Health Science Campus 3000 Arlington Avenue Toledo, OH 43614 USA Tel: (419) 383-1918 Fax: (419) 383-1909 E-mail: [email protected]

43 3.1 Abstract: Sulfated cholesterol, like its unsulfated counterpart, is known to be biologically active and serves a myriad of biochemical/physiological functions. Of the thirteen human cytosolic sulfotransferases (SULTs), SULT2B1b has been reported as the main enzyme responsible for the sulfation of cholesterol. As such, SULT2B1b may play the role as a key regulator of cholesterol metabolism. Variations in the sulfating activity of SULT2B1b may affect the sulfation of cholesterol and, consequently, the related physiological events. The current study was designed to evaluate the impact of the genetic polymorphisms on the sulfation of cholesterol by SULT2B1b. Ten recombinant

SULT2B1b allozymes were generated, expressed, and purified. Purified SULT2B1b allozymes were shown to display differential cholesterol-sulfating activities, compared with the wild-type enzyme. Kinetic studies revealed further their distinct substrate affinity and catalytic efficiency toward cholesterol. These findings showed clearly the impact of genetic polymorphisms on the cholesterol-sulfating activity of SULT2B1b allozymes, which may underscore the differential metabolism of cholesterol in individuals with different SULT2B1b genotypes.

44 3.2. Introduction

Cholesterol is known to be involved in a variety of critical physiological functions such as the biosynthesis of steroid hormones and the maintenance of cell membrane integrity (1,2). Studies have demonstrated the presence of sulfoconjugated form of cholesterol, cholesterol sulfate, in human tissues and body fluids, with plasma concentrations ranging 253 - 690 µM (3-6). Importantly, cholesterol sulfate has been shown to be biologically active, being involved in the increase of cell membrane stability, the regulation of the activity of serine proteases involved in blood clotting, the support for platelet adhesion, and the regulation of cholesterol synthesis (3). Studies have shown that cholesterol sulfate is able to inhibit cholesterol synthesis by inhibiting 3-hydroxy-3- methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol synthesis pathway (7, 8). Moreover, cholesterol sulfate appears to play a role in keratinocyte differentiation. Studies have shown that the ratio of cholesterol sulfate to cholesterol is much higher in the skin (1:10 to 1:5) than in the blood (1:500)

(3). Like cholesterol, cholesterol sulfate can also be used as a biosynthetic precursor for steroid hormones without prior removal of sulfate moiety, particularly in fetal adrenal mitochondria (10, 11). Moreover, cholesterol sulfate has been reported to play a regulatory role in steroidogenesis by regulating the rate of conversion of cholesterol to pregnenolone (10). It is noted that cholesterol sulfate deficiency has been reported to be associated with pathological conditions such as autosomal recessive congenital ichthyosis and atherosclerosis (12, 13).

45 Sulfation, a major conjugation reaction in humans and other mammals, is involved in the homeostasis of key endogenous compounds and the inactivation and removal of xenobiotics (14). Sulfation occurs under the action of the cytosolic sulfotransferase (SULT) enzymes that catalyze the transfer of a sulfonate group from the sulfonate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to the hydroxyl or amino group of the substrate compound (15, 16). Of the known SULT enzymes, members of the SULT2 family have been shown to be responsible for the sulfation of steroids and sterols (17-19). In humans, the SULT2 family consists of three members,

SULT2Al, SULT2Bla, and SULT2Blb (20, 21). Interestingly, SULT2Bla and

SULT2B1b have been shown to be coded by the same gene (designated SULT2B1) and are generated as a result of alternative transcription initiation and alternative splicing

(22). Consequently, SULT2Bla and SULT2B1b differ only in their N-terminal regions, with 8- and 23-amino acid extensions, respectively (23). SULT2A1 is commonly known as the dehydroepiandrosterone (DHEA) sulfotransferase; whereas SULT2Bla and

SULT2B1b are known as pregnenolone and cholesterol sulfotransferase, respectively (20

21). SULT2B1b mRNA was first reported to be present in human placenta, prostate, trachea, lung, and small intestine tissues, and has since been shown to be also expressed in the skin, ovary, uterus, brain, liver, colon and platelet (22, 24-26). The genetic polymorphisms of the SULT2B1 gene have been reported (27-33). An important question is whether the genetic polymorphisms of the gene encoding SULT2Blb may influence its cholesterol-sulfating activity and thus the multitude of physiological functions related to unsulfated and sulfated cholesterol in different individuals.

46 In this study, we performed a systematic search of SULT2B1 SNPs deposited in several SNP databases. Ten SULT2B1b allozymes coded by missense SNPs, selected based on predicted importance of the amino acid variations, were generated, expressed and purified. The sulfating activity of the purified SULT2B1b allozymes toward cholesterol was examined. Kinetic studies were performed to analyze their differential substrate affinity and catalytic efficiency with cholesterol as a substrate.

3.4. Materials and Methods

3.4.1. Materials.

Cholesterol, hydroxypropyl-β-cyclodextrin, adenosine 5′-triphosphate (ATP), N-

2-hydroxylpiperazine-N′-2-ethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO),

Trizma base, and dithiothreitol (DTT) were products of Sigma-Aldrich (St. Louis, MO,

USA). Silica gel thin-layer chromatography (TLC) plates and Ultrafree-MC 5000

NMWL filter units were from EMD Millipore (Billerica, MA, USA). Carrier-free sodium [35S]sulfate was from American Radiolabeled Chemicals (St. Louis, MO, USA).

Ecolume scintillation cocktail was purchased from MP Biomedicals, LLC. (Irvine, CA,

USA). Recombinant human bifunctional ATP sulfurylase/adenosine 5′-phosphosulfate kinase was prepared as previously described (34). PrimeSTAR® Max DNA polymerase was a product of Takara Bio (Mountain View, CA, USA). Protein molecular weight markers were from Bioland Scientific LLC. (Paramount, CA, USA). Oligonucleotide primers were synthesized by Eurofins Genomics (Louisville, KY, USA). PAP[35S] was

47 synthesized using recombinant human bifunctional PAPS synthase as described previously (34). All other chemicals were of the highest grade commercially available.

3.4.2. Identification and analysis of human SULT2B1 SNPs.

Since SULT2B1b is coded by the SULT2B1 gene, three online databases, located at the websites of U.S. National Center for Biotechnology Information (NCBI), the

Ensembl Variation database, and the Universal Protein Resource (UniProt), were systematically searched using the keyword “human SULT2B1”. The human SULT2B1

SNPs identified were analyzed and categorized based on the locations of the nucleotide variations in the region specifically encoding SULT2B1b.

3.4.3. Generation, expression, and purification of SULT2B1b allozymes.

PrimeSTAR® Max DNA polymerase was used to generate cDNAs encoding

SULT2B1b allozymes via PCR. Briefly, the wild-type SULT2B1b cDNA packaged in pGEX-4T-2 prokaryotic expression vector was used as a template in conjunction with specific mutagenic primers (see Table 2-3 for the mutagenic primer sets). The PCR amplification conditions were 12 cycles of 30 s at 95°C, 1 min at 55°C, and 6 min at

72°C. At the end of PCR, the reaction mixtures were treated with Dpn I to degrade the wild-type SULT2B1b cDNA/pGEX-4T-2. The “mutated” SULT2B1b cDNA/pGEX-4T-

2 plasmids present in Dpn I-treated reaction mixtures were individually introduced to competent DH5 E. coli cells for the amplification and purification of the plasmids.

48 Specific “mutations” in individual SULT2B1b cDNA/pGEX-4T-2 plasmids prepared were verified by nucleotide sequencing. pGEX-4T-2 vector harboring individual

“mutated” SULT2B1b cDNAs were introduced to competent BL21 E. coli cells for the expression of recombinant SULT2B1b allozymes. Transformed cells were grown in 1 liter of LB medium containing 100 μg/ml ampicillin to A600 nm = ∼0.5, and then induced with 0.1 mM IPTG overnight at 25°C. The IPTG-treated cells, collected by centrifugation and resuspended in 20 ml aliquots of an ice-cold lysis buffer (10 mM Tris-

HCl, pH 8.0, 150 mm NaCl, and 1 mM EDTA). The resuspended cells were homogenized using an Aminco French press. The crude homogenates were subjected to centrifugation at 10,000 × g for 20 min at 4°C, and the supernatants collected were individually fractionated using 1 ml aliquots of glutathione-Sepharose. For each recombinant SULT2B1b allozyme clone, the bound protein fusion was treated with 2 ml of a thrombin digestion buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2.5 mM

CaCl2) containing 3.5 unit/ml bovine thrombin. The preparation was incubated for 20 minutes at room temperature with constant agitation. Afterwards, the preparation was subjected to centrifugation. The recombinant SULT2B1b allozyme present in the supernatant was collected and analyzed by SDS-polyacrylamide gel electrophoresis

(SDS-PAGE).

3.4.4. Sulfotransferase assay.

The sulfating activity of the recombinant SULT2B1b allozymes was assayed using PAP[35S] as the sulfate donor. The standard assay mixture, with a final volume of

20 L, consists of 0.5 µg of a SULT2B1b allozyme, 50 mM HEPES buffer (pH 7.4), 1

49 mM DTT, 14 M PAP[35S], and cholesterol dissolved in hydroxypropyl-β-cyclodextrin.

A control with hydroxypropyl-β-cyclodextrin alone was assayed in parallel. The reaction mixture was incubated at 37°C for 10 minutes and then terminated by heating at 100°C for 3 minutes. The analysis of the [35S]sulfated cholesterol was carried out by spotting 2

μL of the final reaction mixture on a silica gel TLC plate. The spotted TLC plate was subjected to TLC analysis using a solvent system containing acetic acid: n-butanol in a ratio of 2:1 (by volume). The [35S]sulfated cholesterol spot identified by autoradiography was cut from the TLC plate and eluted with 0.5 ml water. Afterwards, the

[35S]radioactivity was counted using a liquid scintillation counter as previously described

(27). For the kinetic studies on the sulfation of cholesterol by individual SULT2B1b allozymes, varying concentrations of cholesterol ranging 0.5 to 50 μM were used with 50 mM HEPES (pH 7.4), according to the procedure described above. Moreover, to determine the PAPS km values of each SULT2B1b varying concentration of PAPS ranging 0.1 to 50 μM were used at pH 7.4 following the assay procedure described above.

3.4.5. Statistical analysis.

To calculate the kinetic constants, data were processed based on Michaelis-

Menten kinetics using non-linear regression curve generated by GraphPad Prism® v 6.0 software. One-way ANOVA was used for inter-group comparison followed by Dunnett’s test to calculate statistical differences between SULT2B1b-wt (SULT2B1b wild-type) and individual allozymes. P-values < 0.05 were considered statistically significant.

3.5. Results and Discussion

As a first step toward clarifying the mechanisms underlying the phenotypic consequences of SULT2B1 genetic polymorphisms, the current study was carried out to examine the differential cholesterol-sulfating activity of SULT2B1b allozymes resulting from missense coding SNPs of the SULT2B1 gene.

3.5.1. Identification and categorization of SNPs of human SULT2B1 gene.

A systematic search of three databases, including the NCBI SNP database, the

Ensembl Variation database, and the UniProt database, yielded a total of 6,393 SULT2B1

SNPs, which were grouped into coding (synonymous, non-synonymous (missense), and nonsense) SNPs and non-coding (introns, 5’-untranslated region (5’UTR), and 3’- untranslated region (3’UTR)) SNPs. Of the 3,370 SULT2B1 SNPs, 168 were found to be

SULT2B1b missense coding SNPs (cSNPs) that resulted in amino acid changes in the protein products. These SULT2B1b cSNPs were further scrutinized for the location (in the substrate binding site, PAPS-binding sites, and dimerization motif) of the amino acid changes, as well as the chemical nature of the amino acid changes (acidic to/or from basic, charged to/from uncharged, and turn-inducing to/from non-turn inducing residues)

(23, 36, 37). Based on these criteria, ten missense SULT2B1b cSNPs were selected for further studies as described below. Table 2-3 shows the amino acid changes and the

51 locations, as well as the sense and antisense mutagenic primers designed for use in the

PCR-amplification of the corresponding SULT2B1b cDNAs.

3.5.2. Effects of SULT2B1 genetic polymorphism on the cholesterol-sulfating activity of SULT2B1b allozymes.

cDNAs encoding different SULT2B1b allozymes packaged in pGEX-4T-2 prokaryotic expression vector, generated as described in the Materials and Methods, were individually introduced to BL21 E. coli cells for expressing the recombinant enzymes.

The recombinant SULT2B1b allozymes were fractionated from the E. coli cell homogenates using glutathione-Sepharose, followed by thrombin digestion to release the untagged recombinant SULT2B1b allozymes. Recombinant SULT2B1b allozymes thus prepared were analyzed by SDS-polyacrylamide gel electrophoresis and found to be highly homogeneous (cf. Fig. 2-4). The apparent molecular weights of the ten

SULT2B1b allozymes were similar to the predicted molecular weight (41,307) of the wild-type SULT2B1b (cf. lane 2 in Fig. 2-4).

3.5.3. Characterization of the cholesterol-sulfating activity of human SULT2B1b allozymes.

The sulfating activity of purified SULT2B1b allozymes was analyzed using cholesterol as a substrate. As shown in Table 3-1, three of the ten SULT2B1b allozymes,

SULT2B1b-Gly72Val, SULT2B1b-Arg147His, and SULT2B1b-Gly276Val, showed no

52 detectable activity, while the other seven SULT2B1b allozymes exhibited differential sulfating activity toward cholesterol. It was noted that while four (SULT2B1b-

Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, and SULT2B1b-

Pro345Leu) of these seven allozymes displayed a considerable (2-fold or greater) decrease in cholesterol-sulfating activity, the other three (SULT2B1b-Pro69Ala,

SULT2B1b-Thr73Met, and SULT2B1b-Arg274Gln) exhibited a much greater (more than

20-fold) decrease in cholesterol-sulfating activity compared with the wild-type enzyme

(SULT2B1b-wt). Among them, SULT2B1b-Arg274Gln showed the lowest cholesterol- sulfating activity (0.04 nmol/min/mg) which is only 0.50% of the SULT2B1b-wt.

Among the SULT2B1b allozymes tested, SULT2B1b-Arg274Gln had previously implicated in autosomal recessive congenital ichthyosis caused by elevated cholesterol level and absence of cholesterol sulfate in the skin (12). It should be pointed out also that the sulfating activity of SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, and

SULT2B1b-Pro345Leu are considerably lower than the sulfating activity of the same allozymes previously characterized using DHEA as a substrate (33). This discrepancy could be due to the use of different substrates (DHEA vs. cholesterol) and/or the use of different enzyme preparation (enzymes expressed in COS-1 cells vs. purified recombinant enzymes) (33).

53 Table 3-1 Specific activities of the human SULT2B1b allozymes with cholesterol as substrate. Enzyme Specific activity Relative activity (nmol/min/mg) (% of wild type) SULT2B1b-wt 8.03 ± 0.2 100 %

SULT2B1b-Pro69Ala 0.36 ± 0.01*** 4.48 %

SULT2B1b-Gly72Val N.D. N.D.

SULT2B1b-Thr73Met 0.28 ± 0.03*** 3.49 %

SULT2B1b-Arg147His N.D. N.D.

SULT2B1b-Asp191Asn 4.38 ± 0.04*** 54.55 %

SULT2B1b-Arg230His 3.94 ± 0.36*** 49.07 %

SULT2B1b-Ser244Thr 2.32 ± 0.18*** 28.89 %

SULT2B1b-Arg274Gln 0.04 ± 0.01*** 0.50 %

SULT2B1b-Gly276Val N.D. N.D.

SULT2B1b-Pro345Leu 2.99 ± 0.06*** 37.24 %

Concentration of cholesterol used in the enzymatic assay was 50 µM. Results shown represent mean ± s.d. derived from 3 independent analyses. N.D. refers to no detected activity. * Statistical significance from SULT2B1b-wt (***P-value < 0.0001) using one- way ANOVA followed by Dunnett’s post hoc analysis.

To investigate further the effects of genetic polymorphisms on the cholesterol- sulfating activity of SULT2B1b allozymes, kinetic experiments were performed using varying concentrations (ranging from 0.5 - 50 μM) of cholesterol as a substrate and

HEPES buffer at pH 7.4. As shown in Fig. 3-1, the sulfation of cholesterol appeared to

54 follow the Michaelis-Menten kinetics. Table 3-2 shows the kinetic constants, Km, Vmax,

Kcat, and Kcat/Km, determined for the wild-type and SULT2B1b allozymes. Compared with SULT2B1b-wt, the Km values were found to be at least 3 times higher for most of the variants (SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, SULT2B1b-Arg230His,

SULT2B1b-Ser244Thr, and SULT2B1b-Pro345Leu), indicating that these amino acid changes may have led to a decrease in cholesterol binding affinity. All SULT2B1b allozymes examined displayed lower Vmax compared with that of the wild-type enzyme.

Three allozymes (SULT2B1b-Asp191Asn, SULT2B1b-Ser244Thr, and SULT2B1b-

Pro345Leu) exhibited a more than 30% decrease in Vmax, while the other two

(SULT2B1b-Pro69Ala and SULT2B1b-Thr73Met) displayed a more than 90% reduction in Vmax compared with the wild-type. Consequently, the catalytic efficiency as reflected by calculated kcat/km was lower for all SULT2B1b allozymes analyzed. Four of them

(SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, and

SULT2B1b-Pro345Leu) showed a greater than 45% decrease than the wild-type

(SULT2B1b-wt), while two (SULT2B1b-Pro69Ala and SULT2B1b-Thr73Met) showed a much more dramatic decrease (greater than 95%) compared with the wild-type. To determine the affinity of each SULT2B1b allozymes toward the cofactor (PAPS), the km values of the recombinant SULT2B1b allozymes toward PAPS were determined and listed in Table 3-2. Only three allozymes show a significant difference in the PAPS km values compared to the wild-type. SULT2B1b-Pro69Ala and SULT2B1b-Thr73Met showed a 1.8 and 2.5-fold increase in km, respectively, while SULT2B1b-Asp191Asn displayed 2-fold reduction in the km compared to SULT2B1b-wt.

55 To investigate further the effects of genetic polymorphisms on the PAPS binding affinity of SULT2B1b allozymes, the km values were determined using varying concentrations (ranging from 0.1 - 50 μM). As shown in Table III, only three allozymes show a significant difference in the PAPS km values compared to the wild-type. Of them two (SULT2B1b-Pro69Ala and SULT2B1b-Thr73Met) showed a 1.8 and 2.5-fold increase in PAPS km, respectively, while the other allozyme (SULT2B1b-Asp191Asn) displayed a 2-fold reduction in PAPS km compared to SULT2B1b-wt.

Figure 3-1 Kinetic analysis of the sulfation of cholesterol by wild-type SULT2B1b.

The figure shows the saturation curve analysis of the sulfation of cholesterol. The fitting curve was generated based on Michaelis-Menten kinetics. Data shown represent calculated mean ± standard deviation derived from three experiments.

56 Table 3-2 Kinetic constants of the human SULT2B1b allozymes in catalyzing the sulfationTable 3- 2of a Kineticcholesterol. constants of the human SULT2B1b allozymes in catalyzing the sulfation of cholesterol.

a a a a Enzyme Km Vmax Kcat Kcat/Km PAPS Km

(µM) (nmol/min/mg) (sec-1) ×10-3 (sec-1 M-1) (µM)

SULT2B1b-wt 21.9 ± 3.6 10.2 ± 0.8 7.0 ± 0.5 324.2 ± 27.2 3.5 ± 0.7

SULT2B1b-Pro69Ala 69.6 ± 7.7 *** 0.8 ± 0.1 *** 0.6 ± 0.0 *** 8.0 ± 0.7 *** 6.5 ± 0.9 **

SULT2B1b-Thr73Met 79.7 ± 8.5 *** 0.7 ± 0.0 *** 0.5 ± 0.0 *** 5.8 ± 1.0 *** 9.4 ± 1.6 ***

SULT2B1b-Asp191Asn 23.6 ± 2.2 6.0 ± 0.2 *** 4.2 ± 0.1 *** 175.2 ± 12.8 *** 1.5 ± 0.2 *

SULT2B1b-Arg230His 69.6 ± 6.1 *** 8.7 ± 0.5 * 6.0 ± 0.3 * 86.4 ± 12.0 *** 3.5 ± 0.6

SULT2B1b-Ser244Thr 84.9 ± 21.1 *** 5.9 ± 1.0 *** 4.1 ± 0.7 *** 51.4 ± 21.3 *** 1.8 ± 0.2

SULT2B1b-Pro345Leu 66.7 ± 10.6 *** 6.8 ± 0.7 *** 4.6 ± 0.5 *** 69.3 ± 4.3 *** 3.9 ± 0.3

a ResultsKinetic shown constants represent of mean the human± s.d. derived SULT2B1b from 3 independent allozymes determinants. with cholesterol * Statistical. Results significance shown from SULT2B1b-wt (*p-value<0.05), (**p-value<0.001), (***p-value<0.0001) using one-way ANOVA followed by Dunnett’s post hoc analysis. SULT2B1b-Arg274Gln the kinetic constants could not be representdetermined mean accurately ± s.d. due derived to the weak from activity 3 independent of this allozyme determinants. toward cholesterol. * Stati stical significance

from SULT2B1b-wt (*p-value<0.05), (**p-value<0.001), (***p-value<0.0001) using one-

way ANOVA followed by Dunnett’s post hoc analysis. SULT2B1b-Arg274Gln the

kinetic constants could not be determined accurately due to the weak activity of this

allozyme toward cholesterol.

The crystal structure of human SULT2B1b has been solved (23). In view of the reported crystal structure, the above-mentioned results are not surprising since most of the SULT2B1b allozymes examined have amino acid changes either within or close to the PAPS binding pocket (Figure 3-2) (23). Moreover, most of the amino acid changes in these allozymes were dramatic from the biochemistry point of view, with potential for

57 affecting substrate affinity or catalytic activity (38). For example, the change from proline (a turn-inducing amino acid residue) to alanine (a non-turn-inducing residue) in

SULT2B1b-Pro69Ala could be the reason for the significant increase in the Km for PAPS, indicating a lowered affinity toward the PAPS molecules. As a result, the catalytic

-1 -1 efficiency (with a kcat of 7.96 sec M ) of SULT2B1b-Pro69Ala became dramatically lower than that (324.20 sec-1 M-1) of the wild-type enzyme (38). In the case of

SULT2B1b-Thr73Met, the replacement of threonine (a polar amino acid residue) with methionine (a nonpolar residue) might have affected the hydrogen-bonding between the hydroxyl group of threonine with the oxygen atom O4P of 5’-phosphate in the PAPS, which resulted in a lower affinity for PAPS as judged by an increase in the Km value (9.4

µM), compared with that (3.5 µM) of the wild-type enzyme (23, 38). Moreover, the replacement of arginine by glutamine in SULT2B1b-Arg274Gln, which showed a dramatically lower specific activity compared with the wild-type enzyme; (Table 3-1) might have weakened the polar interaction between the positively charged nitrogen atom of arginine with O3P phosphate oxygen of the PAPS molecule (23). On the other hand, the amino acids substitutions that occur in SULT2B1b-Gly72Val, SULT2B1b-

Arg147His, and SULT2B1b-Gly276Val were found to completely abolish the cholesterol sulfating activities. The substitution of glycine (which presumably provides conformation flexibility) to valine (which may cause a restriction in the conformation) in

SULT2B1b-Gly72Val and SULT2B1b-Gly276Val might have disrupted the hydrogen- bonding with the O4P and O2P phosphate oxygens, respectively, of the PAPS (23, 38).

In the case of SULT2B1b-Arg147His, the replacement of the arginine residue, an amino acid which is necessary to form the hydrogen bond with the oxygen atom O3P of the

58 3’phosphate of the PAPS, with histidine might be the reason for the loss of the sulfating activity (23). In general, the activity data obtained for the SULT2B1b allozymes analyzed indicated clearly the importance of the amino acid residues in the proper functioning of the SULT2B1b enzyme.

Figure 3-2 The location of the SNP-based amino acid exchanges in the PAPS binding region. The figure shows a close view of the PAPS binding region, PAPS molecules, and the location of the SNP-based amino acid residues that were characterized in this study.

The dashed lines represent the hydrogen-bonding between the amino acid side chains and the PAPS molecule. The figure is generated using PyMOL software and the reported

SULT2B1b crystal structure by Lee et al., 2003 with the PDB ID: 1q22.

3.6. Conclusion. This study represented the first attempt to gather information concerning the effects of SULT2B1b genetic polymorphisms on the cholesterol-sulfating activity of SULT2B1b allozymes. Activity data obtained indicated that seven of the ten

SULT2B1b allozymes examined displayed lower and differential sulfating activity toward cholesterol in comparison to the wild-type enzyme, with the other three showing no detectable activity. While the information concerning the allelic frequencies of the tested SULT2B1b genotypes in the population is incomplete and remains to be clarified, this study provided convincing evidence that the coded SULT2B1b allozymes exhibited significant and sometimes dramatic differences in their enzymatic activities. These results imply that individuals with different SULT2B1b genotype may have differential capacity in sulfating cholesterol. Pending additional studies, such information may have significance in predicting risk for diseases, as well as aiding in the formulation of personalized regimens for drugs that may be metabolized by SULT2B1b for individuals with distinct SULT2B1b genotypes.

Funding: This work was supported in part by a grant from National Institutes of Health

(Grant # R03HD071146).

60 Chapter 4

Effect of SULT2B1 Genetic Polymorphisms on the Sulfation of Dehydroepiandrosterone and Pregnenolone by SULT2B1b Allozymes

Running Title: Sulfation of DHEA and pregnenolone by Human SULT2B1b allozymes

Fatemah A. Alherz1, Amal A. El Daibani1, Ahsan F. Bairam1,2, Maryam S. Abunnaja1, Mohammed I. Rasool1,3, Katsuhisa Kurogi1,4, Yoichi Sakakibara4, Masahito Suiko4, Ming- Cheh Liu1,*

61 4.1. Abstract:

Background: Pregnenolone and dehydroepiandrosterone (DHEA) serve as biosynthetic precursors for steroid hormones in the body. SULT2B1b has been reported to be critically involved in the sulfation of pregnenolone and DHEA, particularly in sex steroid-responsive tissues. The current study was designed to investigate the impact of the genetic polymorphisms of SULT2B1 on the sulfation of DHEA and pregnenolone by

SULT2B1b allozymes.

Methods: Online single nucleotide polymorphism (SNP) databases were systematically searched for SULT2B1 missense coding SNPs (cSNPs). cDNAs coded by selected

SULT2B1 cSNPs were generated by site-directed mutagenesis, and the corresponding

SULT2B1b allozymes were expressed and purified using established protocols.

Enzymatic characteristics of SULT2B1b allozymes toward DHEA and pregnenolone were analyzed using an established assay procedure.

Results: Purified SULT2B1b allozymes exhibited differential sulfating activities toward

DHEA and pregnenolone in comparison to the wild-type enzyme. Kinetic studies showed further significant changes in their substrate-binding affinity and catalytic activity toward DHEA and pregnenolone.

Conclusions: Taken together, these results indicated clearly a profound effect of

SULT2B1 cSNPs on the sulfating activities of SULT2B1b allozymes toward DHEA and pregnenolone.

Keywords: Sulfation; cytosolic sulfotransferase; SULT; SULT2B1b; single nucleotide polymorphisms, dehydroepiandrosterone, DHEA; pregnenolone.

62 4.2. Introduction

Dehydroepiandrosterone (DHEA), the main biosynthetic precursor of sex steroids

[1], is synthesized mainly in the adrenal glands and, to a lower extent, in ovaries and testes [2]. In the adrenal glands, cholesterol is first transformed to pregnenolone under the action of cytochrome P450scc [1]. The conversion of cholesterol to pregnenolone is the rate limiting step in the steroid hormone biosynthesis pathway [3]. Pregnenolone then serves as a precursor for glucocorticoids, mineralocorticoids, and DHEA [3]. In peripheral tissues, the adrenal secreted DHEA acts as a precursor for estrogen and androgen hormones through a process called intracrinology [1,3], which involves the intracellular formation, inactivation, and action of sex steroids [2]. Sulfation of DHEA forming DHEA-S limits the amount of DHEA that is available for the biosynthesis of androgen hormones [4]. In the body, the sulfation of DHEA and pregnenolone has been shown to be mediated by the cytosolic sulfotransferase (SULT) enzymes, particularly

SULT2A1, SULT2B1a, and SULT2B1b [5]. It is noted that in addition to serving as steroid hormones precursors, DHEA, pregnenolone, and their sulfated metabolites synthesized independently in the nervous system are considered neurosteroids that act as neuromodulators [6].

The cytosolic sulfotransferases (SULTs) are a group of phase II conjugation enzymes that are involved in the homeostasis and detoxification of numerous exogenous and xenobiotic compounds [7]. The SULTs catalyze the transfer of a sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyl or amino group of

63 acceptor compounds, leading to their increased hydrophilicity and facilitated urinary and biliary excretion from the body [8, 9]. In humans, there are 13 distinct SULTs that are classified into four gene families, designated SULT1, SULT2, SULT4, and SULT6 [10,

11]. SULT2 family is previously known as the hydroxysteroid sulfotransferase family and consists of three isoforms: SULT2A1 (previously called a DHEA sulfotransferase),

SULT2B1a (a pregnenolone sulfotransferase), and SULT2B1b (a cholesterol sulfotransferase) [5]. SULT2B1a and SULT2B1b isoforms are coded by the same gene, designated SULT2B1, and are generated as a result of alternative initiation and splicing, leading to the formation of the two isoforms with distinct N-terminal regions [12].

Although all SULT2 family isoforms display overlapping substrate specificity toward different hydroxysteroids, such as DHEA and pregnenolone, they exhibit tissue-specific distribution [13-16]. For example, SULT2A1 is expressed mainly in the liver, adrenal glands, and intestine [15,16], whereas SULT2B1b is highly expressed in the placenta, prostate, breast, endometrium, ovary, uterus, small intestine, colon, lung, platelet, brain, and skin [13, 14]. SULT2B1b thus is more likely to be the main enzyme responsible for the sulfation of DHEA and pregnenolone in steroid-responsive tissues as well as in the brain. SULT2B1 genetic polymorphisms have been reported [17-24]. It is an interesting question whether SULT2B1 missense coding SNPs (cSNPs) may influence the sulfating activity of the resulting SULT2B1b allozymes and thus affect steroid-related physiology and pathology in different individuals.

In this study, a systematic search of SULT2B1 SNPs deposited in several SNP databases was performed. Ten SULT2B1 missense cSNPs were selected based on the

64 potential importance of the predicted amino acid variations. The corresponding

SULT2B1b allozymes were generated, expressed and purified. The sulfating activity of the purified SULT2B1b allozymes toward DHEA and pregnenolone was examined.

Kinetic parameters of SULT2B1b allozymes in mediating the sulfation of DHEA and pregnenolone were determined to delineate their differential substrate affinity and catalytic activity toward DHEA and pregnenolone in comparison to the wild-type enzyme.

4.3. Materials and Methods

4.3.1. Materials.

Pregnenolone, DHEA, adenosine 5′-triphosphate (ATP), N-2-hydroxylpiperazine-

N′-2-ethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), Trizma base, 3-

(cyclohexylamino)-1 propanesulfonic acid (CAPS), 2-(N-morpholino) ethanesulfonic acid (MES), 2-(cyclohexylamino) ethanesulfonic acid (CHES), 3-[N-Tris-

(hydroxymethyl) methyl amino]-propane sulfonic acid (TAPS), and dithiothreitol (DTT) were products of Sigma-Aldrich (St. Louis, MO, USA). PrimeSTAR® Max DNA polymerase was a product of Takara Bio (Mountain View, CA, USA). Oligonucleotide primers were synthesized by Eurofins Genomics (Louisville, KY, USA). Cellulose TLC plates and Ultrafree-MC 5000 NMWL filter units were from EMD Millipore (Billerica,

MA, USA). Ecolume scintillation cocktail was purchased from MP Biomedicals, LLC.

(Irvine, CA, USA). Protein molecular weight markers were from Bioland Scientific

65 LLC. (Paramount, CA, USA). Carrier-free sodium [35S]sulfate was from American

Radiolabeled Chemicals (St. Louis, MO, USA). Recombinant human bifunctional ATP sulfurylase/adenosine 5′-phosphosulfate kinase was prepared as previously described

[25]. PAP[35S] was synthesized using recombinant human bifunctional PAPS synthase based on a previously established procedure [25]. All other chemicals were of the highest grade commercially available.

4.3.2. Identification and analysis of human SULT2B1 SNPs.

Three SNP databases located, respectively, at the websites of the U.S. National

Center for Biotechnology Information (NCBI), the Ensembl Variation database, and the

Universal Protein Resource (UniProt), were comprehensively searched for human

SULT2B1 genotypes. The identified SULT2B1 SNPs were categorized and analyzed based on the positions of the nucleotide alterations in the region that encode SULT2B1b.

4.3.3. Generation, expression, and purification of selected SULT2B1b allozymes.

To generate SULT2B1b allozymes cDNAs via PCR, PrimeSTAR® Max DNA polymerase was used. The wild-type SULT2B1b cDNA ligated into prokaryotic expression vector pGEX-4T-2 was used as a template, in conjunction with specific sense and antisense mutagenic primers (Table 2-3). The PCR amplification conditions were: pre-denaturation at 95°C for 2 min followed by 12 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 6 min. Upon PCR completion,

66 the reaction mixture was treated with 5 units of Dpn I to degrade the wild-type template.

To amplify and purify the SULT2B1b allozyme plasmid, the mutated SULT2B1b cDNA/pGEX-4T-2 present in Dpn I-treated reaction mixture was transformed into competent DH5 E. coli cells and purified using a miniprep kit. Nucleotide sequencing was performed to verify the “mutation” in the “mutated” SULT2B1b cDNA/pGEX-4T-2 plasmid.

To express the recombinant SULT2B1b allozyme, the mutated SULT2B1b cDNA/pGEX-4T-2 was transformed into competent BL21 E. coli cells. Transformed E. coli cells were grown in 1 liter of Luria broth (LB) medium with 100 μg/ml ampicillin.

Upon reaching 0.5 OD600nm, the cells were induced with 0.1 mM IPTG overnight at 25°C.

Afterwards, the cells were collected by centrifugation and resuspended in 20 ml ice-cold lysis buffer (containing 10 mM Tris-HCl, pH 8.0, 150 mm NaCl, and 1 mM EDTA). The resuspended cells were homogenized using an Aminco French press. The crude homogenate was centrifuged at 10,000 × g for 20 min at 4°C. The supernatant collected was subjected to fractionation using 2 ml of a 50% slurry of glutathione Sepharose. The glutathione S-transferase (GST)-SULT2B1b fusion protein bound to glutathione

Sepharose was treated with 2 ml of a thrombin digestion buffer (50 mM Tris-HCl, pH

8.0, 150 mM NaCl, and 2.5 mM CaCl2) containing 3.5 unit/ml bovine thrombin. The mixture was incubated for 20 minutes at 25°C with constant agitation. The recombinant

SULT2B1b allozyme released into solution was recovered by centrifugation. The

SULT2B1b allozyme thus purified was analyzed for purity by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

4.3.4. Sulfotransferase assay.

To quantify the sulfating activity of the recombinant SULT2B1b allozymes,

PAP[35S] was used as the sulfate donor. The standard assay mixture, with a final volume of 20 L, contained 50 mM HEPES buffer (pH 7.4), 1 mM DTT, 14 M PAP[35S], 0.5

µg of wild-type or SULT2B1b allozyme, and DHEA or pregnenolone (dissolved in

DMSO) as a substrate. A control with DMSO alone was installed in parallel. The reaction was performed for 10 minutes at 37°C and terminated by incubating the reaction mixture at 100°C for 3 minutes. To analyze the production of [35S]sulfated DHEA or pregnenolone, 1 μL of the final reaction mixture was spotted on a cellulose TLC plate, followed by TLC using a solvent system containing n-butanol: isopropanol: formic acid: water in a ratio of 3:1:1:1 (by volume). The [35S]sulfated DHEA or pregnenolone spot was located by autoradiography, cut out from the TLC plate, and eluted with 0.5 ml H2O.

The [35S]radioactivity of the eluate was quantified using a liquid scintillation counter as described previously [26]. To determine the kinetic parameters of individual SULT2B1b allozymes, varying concentrations of DHEA or pregnenolone were used, based on the same procedure described above.

4.3.5. pH-dependence

To examine the pH-dependence of the sulfation of DHEA by SULT2B1b or SULT2B1b allozymes, 50 mM of different buffers (sodium acetate at 4.5,5, or 5.5; MES at 6 or 6.5;

HEPES at 7, 7.5, or 8; TAPS at 8.5 or 9; CHES at 9.5 or10; CAPS at 10.5, 10.5, 11, or

11.5), were used instead of 50 mM HEPES (pH 7.4). Substrate concentration used was 10

68 µM except with SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, and SULT2B1b-

Arg274Gln 100 µM due to weak enzyme activity of these allozymes with DHEA. The experimental procedure for the pH-dependence studies was the same as described above in Sulfotransferase assay, except for the buffer used.

4.3.6. Statistical analysis.

GraphPad Prism® v 6.0 software was used for calculating the kinetic constants,

Km, Vmax, Kcat, and Kcat/ Km based on Michaelis-Menten kinetics using non-linear regression. To determine statistical differences between the wild-type SULT2B1b and individual SULT2B1b allozymes, one-way ANOVA was used for inter-group comparisons, followed by Dunnett’s test, with p-value < 0.05 considered being statistically significant.

4.4. Results

4.4.1. Identification of cSNPs of human SULT2B1 gene.

A systematic search for SULT2B1 SNPs in three databases, the NCBI SNP database, the UniProt database, and the Ensembl Variation database, was performed. Of all gathered SULT2B1 SNPs, 168 were found to be non-synonymous cSNPs, which may result in amino acid changes in the coded SULT2B1b protein products. These

SULT2B1b cSNPs were analyzed based on the location of the amino acid alteration in the substrate-binding site [31], dimerization motif [32], and PAPS-binding sites [31], as well

69 as the physicochemical properties of the changed amino acid (charged to/from uncharged, proton donor to/or from proton acceptor, and turn-inducing to/from non-turn inducing residues). Based on potential impact of the amino acid changes, ten non-synonymous cSNPs were selected for further studies. Table 2-3 shows the sense and antisense mutagenic primers sets designed for PCR-amplification, the amino acid changes and locations, as well as the documented allelic frequency of respective SULT2B1 genotypes.

4.4.2. Generation of cDNAs encoding SULT2B1b allozymes and bacterial expression and purification of SULT2B1b allozymes.

To express the recombinant SULT2B1b allozymes, SULT2B1b allozyme cDNAs ligated to pGEX-4T-2 prokaryotic expression vectors were individually transformed into

BL21 E. coli cells. Upon induction of recombinant protein expression in transformed cells by IPTG, the cells were homogenized. Glutathione Sepharose was used to fractionate the recombinant SULT2B1b allozymes from the E. coli cell homogenates.

Afterwards, bovine thrombin was used to release the recombinant SULT2B1b allozymes from the Glutathione Sepharose-bound GST fusion proteins. Recombinant SULT2B1b allozymes thus prepared were analyzed by SDS-PAGE. As shown previously in Figure

2-4, the apparent molecular weights of purified SULT2B1b allozymes were all similar to that of the wild-type SULT2B1b, which has a predicted molecular weight of 41,307.

4.4.3. Characterization of the DHEA-sulfating activity of human SULT2B1b allozymes.

70 The sulfating activity of the recombinant SULT2B1b allozymes toward DHEA was examined. Three of the ten tested SULT2B1b allozymes (SULT2B1b-Gly72Val,

SULT2B1b-Arg147His, and SULT2B1b-Gly276Val) showed no detectable activity, whereas the other seven SULT2B1b allozymes displayed differential sulfating activity toward DHEA (Figure 4-1). Among these seven SULT2B1b allozymes, SULT2B1b-

Arg274Gln showed the greatest decrease in DHEA-sulfating activity, with a 27-fold reduction compared with the SULT2B1b-wt. Of the other six allozymes, four

(SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, and

SULT2B1b-Pro345Leu) displayed a considerable decrease (1.7-fold or greater) in

DHEA-sulfating activity, while the other two (SULT2B1b-Pro69Ala and SULT2B1b-

Thr73Met) exhibited a much greater (more than 8-fold) decrease in DHEA-sulfating activity compared with the wild-type enzyme.

Figure 4-1 Specific activity of the human SULT2B1b allozymes toward DHEA.

Concentration of DHEA used in the enzymatic assays was 50 μM. Data shown

represent mean ± standard deviation derived from three independent

determinations. One-way ANOVA was performed followed by Dunnett’s post

hoc analysis. *** Statistical significant p<0.001 from SULT2B1b-wt.

To investigate further the effects of genetic polymorphisms on the DHEA- sulfating activity of SULT2B1b allozymes, kinetic experiments were performed using varying concentrations (ranging from 7 - 150 μM) of DHEA as a substrate. The sulfation of DHEA appeared to follow the Michaelis-Menten kinetics (cf. Figure 4-2). The determined kinetic constants, Km, Vmax, Kcat, and Kcat/ Km, for the wild-type and

SULT2B1b allozymes are compiled in Table 4-1. Of the seven SULT2B1b allozymes

72 examined, two (SULT2B1b-Thr73Met and SULT2B1b-Arg274Gln) showed dramatic increases in Km value (at least 5 times) compared with SULT2B1b-wt, indicating that the amino acid changes resulted in decreased DHEA binding affinity. All tested SULT2B1b allozymes exhibited lower catalytic activity (Vmax) compared with the wild-type enzyme.

Among them, SULT2B1b-Asp191Asn displayed the smallest decrease (a 15% reduction) in the catalytic activity, while SULT2B1b-Pro345Leu and SULT2B1b-Arg230His showed more than 35% decrease in Vmax compared with the wild-type enzyme. In contrast, the other four allozymes exhibited much greater reduction. SULT2B1b-

Ser244Thr and SULT2B1b-Thr73Met showed a more than 52% decrease in Vmax, whereas SULT2B1b-Pro69Ala and SULT2B1b- Arg274Gln exhibited a more than 87% decrease in Vmax compared with SULT2B1b-wt. Consequently, the catalytic efficiency as reflected by kcat/km was significantly lower for all seven SULT2B1b allozymes. Four of them, SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, and

SULT2B1b-Pro345Leu, showed more than 28% decrease compared with the wild-type

(SULT2B1b-wt), while the other three (SULT2B1b-

Pro69Ala, SULT2B1b-Thr73Met, and SULT2B1b-Arg274Gln) showed more dramatic reduction (up to 92%) compared with the wild-type enzyme.

Figure 4-2 Kinetic analysis of the sulfation of DHEA by human wild-type SULT2B1b.

The figure shows the saturation curve analysis of the sulfation of DHEA. The fitting curve was generated based on Michaelis-Menten kinetics. Data shown represent calculated mean ± standard deviation derived from three experiments.

Enzyme Km Vmax Kcat Kcat/Km (μM) (nmol/min/mg) (sec-1) ×10-3 (sec-1 M-1) SULT2B1b-wt 35.9 ± 3.1 8.3 ± 0.2 5.7 ± 0.2 160.0 ± 9.6

SULT2B1b-Pro69Ala 62.4 ± 6.4 1.1 ± 0.0*** 0.7 ± 0.0*** 11.6 ± 0.9***

SULT2B1b-Thr73Met 221.5 ± 24.2*** 2.8 ± 0.1*** 1.9 ± 0.1*** 8.8 ± 0.6***

SULT2B1b-Asp191Asn 61.7 ± 7.6 7.0 ± 0.3*** 4.8 ± 0.2*** 78.9 ± 6.2***

SULT2B1b-Arg230His 35.4 ± 0.2 5.4 ± 0.2*** 3.7 ± 0.2*** 105.0 ± 5.7***

SULT2B1b-Ser244Thr 24.3 ± 3.8 4.0 ± 0.2*** 2.7 ± 0.1*** 113.7 ± 12.7***

SULT2B1b-Arg274Gln 190.2 ± 25.2*** 0.8 ± 0.4*** 0.6 ± 0.0*** 2.9 ± 0.2***

SULT2B1b-Pro345Leu 38.0 ± 3.2 4.6 ± 0.3*** 3.1 ± 0.2*** 83.5 ± 2.5***

Table 4-1. Kinetic constants of the human SULT2B1b allozymes in catalyzing the sulfation of DHEA Data shown represent mean ± SD derived from 3 independent experiments. *Statistical significance from SULT2B1b-wt (***p-value<0.0001) using one-way ANOVA followed by Dunnett’s post hoc analysis.

4.4.4. Characterization of the pregnenolone-sulfating activity of human SULT2B1b allozymes.

In addition to DHEA sulfation, previous studies have shown that SULT2B1b can also sulfate pregnenolone [5]. In an initial experiment, the sulfating activity of

SULT2B1b allozymes were examined using 10 µM of pregnenolone as substrate. Of the ten SULT2B1b allozymes analyzed, three (SULT2B1b-Gly72Val, SULT2B1b-

Arg147His, and SULT2B1b-Gly276Val) showed no detectable sulfating activity. The other seven SULT2B1b allozymes exhibited differential and significantly lower sulfating activity toward pregnenolone. As shown in Figure 4-3, four (SULT2B1b-Asp191Asn,

75 SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, and SULT2B1b-Pro345Leu) displayed a more than 1.2-fold reduction in pregnenolone-sulfating activity, while two (SULT2B1b-

Pro69Ala and SULT2B1b-Thr73Met) showed a much greater reduction (more than 5- fold) in pregnenolone-sulfating activity compared with SULT2B1b-wt. Above all,

SULT2B1b-Arg274Gln displayed the lowest (greater than 35-fold lower) pregnenolone- sulfating activity compared with the wild-type enzyme.

Figure 4-3 Specific activity of the human SULT2B1b allozymes toward

pregnenolone. Concentration of pregnenolone used in the enzymatic assays was

10 μM. Data shown represent mean ± standard deviation derived from three

independent determinations. One-way ANOVA was performed followed by

Dunnett’s post hoc analysis. *** Statistical significant p<0.001 from SULT2B1b-

wt.

76 To analyze further the effect of genetic polymorphisms on the pregnenolone- sulfating activity of SULT2B1b allozymes, kinetic experiments were performed using varying concentrations (ranging from 0.5 to 20 µM) of pregnenolone as substrates. As shown in Figure 4-4, pregnenolone sulfation appeared to follow the Michaelis-Menten kinetics. Kinetic constants determined for the wild-type and SULT2B1b allozymes are compiled in Table 4-2. Of the seven SULT2B1b allozymes analyzed, four (SULT2B1b-

Pro69Ala, SULT2B1b-Thr73Met, SULT2B1b-Ser244Thr, and SULT2B1b-Arg274Gln) showed significant differences in Km compared with the wild-type enzyme. SULT2B1b-

Pro69Ala and SULT2B1b-Ser244Thr both showed a 1.7-fold reduction in Km value compared to the wild-type SULT2B1b. In contrast, SULT2B1b-Thr73Met and

SULT2B1b-Arg274Gln displayed 1.6 and 2.8-fold increase in Km value, respectively.

All seven SULT2B1b allozymes examined showed a significant reduction in Vmax compared with that of SULT2B1b-wt. SULT2B1b-Pro69Ala and SULT2B1b-

Arg274Gln exhibited the greatest reduction in the catalytic activity toward pregnenolone

(more than 92% decrease) compared with SULT2B1b-wt. Of the other five SULT2B1b allozymes, three (SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, and SULT2B1b-

Pro345Leu) displayed a greater than 17% decrease in Vmax, while two (SULT2B1b-

Thr73Met and SULT2B1b-Ser244Thr) showed a more than 55% reduction in Vmax compared with the wild-type. Based on these results, five (SULT2B1b-Pro69Ala,

SULT2B1b-Thr73Met, SULT2B1b-Ser244Thr, SULT2B1b-Arg274Gln, and

SULT2B1b-Pro345Leu) showed a significant reduction in the catalytic efficiency

(kcat/km). Two of them (SULT2B1b-Ser244Thr and SULT2B1b-Pro345Leu) showed a more than 17% decrease in the catalytic efficiency compared with the wild-type

77 (SULT2B1b-wt), while three (SULT2B1b-Pro69Ala, SULT2B1b- Arg274Gln, and

SULT2B1b-Thr73Met) showed a much greater reduction (more than 84%) in catalytic efficiency as compared with the wild-type.

Figure 4-4 Kinetic analysis of the sulfation of pregnenolone by human wild-type

SULT2B1b. The figure shows the saturation curve analysis of the sulfation of

pregnenolone. The fitting curve was generated based on Michaelis-Menten

kinetics. Data shown represent calculated mean ± standard deviation derived

from three experiments.

Table 4-2. Kinetic constants of the human SULT2B1b allozymes in catalyzing the

Enzyme Km Vmax Kcat Kcat/Km (μM) (nmol/min/mg) (sec-1) ×10-3 (sec-1 M-1) SULT2B1b-wt 7.86 ± 1.09 11.53 ± 0.69 7.94 ± 0.48 1017 ± 81

SULT2B1b-Pro69Ala 4.58 ± 0.55* 0.85 ± 0.02*** 0.61 ± 0.02*** 135 ± 12***

SULT2B1b-Thr73Met 12.76 ± 1.72** 2.89 ± 0.15*** 1.99 ± 0.10*** 157 ± 13***

SULT2B1b-Asp191Asn 5.87 ± 1.11 8.14 ± 0.5*** 5.60 ± 0.34*** 970 ± 127

SULT2B1b-Arg230His 7.04 ± 0.77 9.55 ± 0.54*** 6.57 ± 0.37*** 937 ± 50

SULT2B1b-Ser244Thr 4.53 ± 1.21* 5.09 ± 0.66*** 3.50 ± 0.45*** 793 ± 116**

SULT2B1b-Arg274Gln 21.74 ± 2.40*** 0.83 ± 0.05*** 0.57 ± 0.03*** 26 ± 1***

SULT2B1b-Pro345Leu 7.84 ± 0.74 9.58 ± 0.59*** 6.60 ± 0.41*** 843 ± 28* sulfation of pregnenolone

Results shown represent mean ± SD derived from 3 independent experiments. * Statistical significance from SULT2B1b-wt (*p-value<0.05, **p-value<0.001, ***p-value<0.0001) using one-way ANOVA followed by Dunnett’s post hoc analysis.

4.4.5. The pH-dependence of DHEA-sulfating activity of human SULT2B1b allozymes.

In the pH-dependence study, the wild-type SULT2B1b as well as five allozymes

(SULT2B1b-Thr73Met, SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-

Ser244Thr, and SULT2B1b-Pro345Leu) exhibited optimum pH at 9.0, while SULT2B1b-

Pro69Ala and SULT2B1b-Arg274Gln display an optimum pH at 8.0 with DHEA as a substrate.

79 A)

80 D)

(F)

81 G)

Figure 4-5 pH-Dependence of the Sulfating Activity of Human SULT2B1b-wt (A), SULT2B1b-Pro69Ala (B), SULT2B1b-Thr73Met (C), SULT2B1b-Asp191Asn (D), SULT2B1b-Arg230His (E), SULT2B1b-Ser244Thr (F), SULT2B1b-Arg274Gln (G), and SULT2B1b-Pro345Leu (H) with DHEA as Substrate. The enzymatic assays were carried out under standard assay conditions as described in Materials and Methods, using different buffer systems as indicated. Data shown represent calculated mean ±S.D. derived from three independent determinants.

82 4.5. Discussion

In humans, DHEA and its sulfate ester, DHEA-S, are known to be the most abundant steroids in circulation [5]. DHEA is synthesized and secreted mainly from the adrenal glands and, to a lower extent, in brain, gonads, and skin [27]. DHEA has been proposed to be effective in reducing cardiovascular risk, alleviating insulin resistance, stimulating endothelial proliferation, and improving memory and cognitive function [2,

28]. More importantly, DHEA is a major precursor for the biosynthesis of sex steroid hormones [1]. In peripheral tissues, the adrenally secreted DHEA is taken up and converted to estrogens and androgens via a process known as intracrinology [1].

SULT2B1b has been shown to be capable of sulfating specifically 3β-hydroxysteroids including cholesterol, DHEA, and pregnenolone, and is highly expressed in steroid hormone-responsive tissues such as prostate, breast, placenta, and endometrium [13].

Genetic polymorphisms of SULT2B1 have been correlated with the progression and proliferation of several different types of cancer including prostate cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, and colorectal cancer

[17-23]. SULT2B1b mRNA has been detected in a number of cancerous human tissues and cell lines including prostate adenocarcinoma cells, LNCaP prostate adenocarcinoma cells, as well as T47D and MCF-7 breast cancer cell lines [13, 29]. While the exact role of SULT2B1b in different type of cancers has not been fully elucidated, studies have suggested that its involvement in the sulfation of hydroxysteroids may limit their availability for the sex steroids biosynthesis and their capacity to bind to corresponding androgen receptors [29, 30]. Interestingly, the down-regulation of SULT2B1b in prostate cancer has been proposed to be the reason behind prostate cancer progression due to the

83 lake of the protective effect of SULT2B1b in decreasing steroid hormone precursors like

DHEA [29, 30].

The current study was designed to investigate functional relevance of SULT2B1 cSNPs on the sulfating activity of the resulting SULT2B1b allozymes toward two major steroids precursors, DHEA and pregnenolone. The ten selected SULT2B1b cSNPs were generated via site-directed mutagenesis and the corresponding SULT2B1b allozymes were expressed and purified. As shown in the Results section, three allozymes

(SULT2B1b-Gly72Val, SULT2B1b-Arg147His, and SULT2B1b-Gly276Val) showed no detectable activity with both DHEA and pregnenolone. The other seven allozymes

(SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, SULT2B1b-Asp191Asn, SULT2B1b-

Arg230His, SULT2B1b-Ser244Thr, SULT2B1b- Arg274Gln, and SULT2B1b-

Pro345Leu) exhibited significant decreases in their sulfating activity toward DHEA and pregnenolone. It should be pointed out that the sulfating activity of SULT2B1b-

Asp191Asn, SULT2B1b-Arg230His, and SULT2B1b-Pro345Leu in this study were noticeably lower than the sulfating activity of the same allozymes previously reported using DHEA as a substrate [24]. It is possible that this discrepancy could have been due to the use of different enzyme preparation (purified recombinant enzymes vs. enzymes expressed in COS-1 cells [24]). Subsequent kinetic analysis showed that amino acid variations in the seven allozymes caused significant decrease in the catalytic activity and efficiency toward DHEA, whereas with pregnenolone, only five allozymes (SULT2B1b-

Pro69Ala, SULT2B1b-Thr73Met, SULT2B1b-Ser244Thr, SULT2B1b- Arg274Gln, and

SULT2B1b-Pro345Leu) showed significant decrease in the Vmax and Kcat/Km. These

84 results indicate clearly that SULT2B1b cSNPs indeed have great effect on the enzyme function.

The crystal structure of human SULT2B1b determined previously has revealed a number of structural elements that are critical to the functioning of the enzyme [31]. In relation to the interaction with the co-substrate, PAPS, the elements include a 3’- phosphate-binding region, a 5’-phosphosulphate-binding (PSB) loop, and a PAP adenine- binding region [31]. The PSB loop is composed of the conserved amino acid sequence

67TYPKSGT73, of which the amino acid residues Lys70, Ser71, Gly72, and Thr73, as well as Thr74, are involved in binding the 5’-phosphate of PAPS. The amino acid residues Arg274, Lys275, Gly276, Arg147, and Ser155 are involved in binding the 3’- phosphate of PAPS. Moreover, Ser244, Tyr 210, Trp75, and Phe246 are involved in the interaction with the adenine group of the PAPS molecule [31]. It is noted that seven

(SULT2B1b-Pro69Ala, SULT2B1b-Gly72Val, SULT2B1b-Thr73Met, SULT2B1b-

Arg147His, SULT2B1b-Ser244Thr, SULT2B1b-Arg274Gln, and SULT2B1b-

Gly276Val) of the ten SULT2B1b allozymes examined in this study contain amino acid variations in the PAP/PAPS-binding pocket [31]. Of these seven SULT2B1b allozymes, the amino acid changes in three (SULT2B1b-Gly72Val, SULT2B1b-Arg147His, and

SULT2B1b-Gly276Val) were found to exert the most drastic effects, leading to the complete loss of sulfating activity toward DHEA or pregnenolone. For SULT2B1b-

Gly72Val and SULT2B1b-Gly276Val, Gly72 and Gly276 in the wild-type enzyme have been proposed to form hydrogen-bonding with the O4P and O2P phosphate oxygens of the co-substrate, PAPS, respectively. Replacement of these glycine residues, which has

85 more conformational flexibility, with valine residues, which carry a bulkier side chain, may place more conformational restriction and interfere with the interaction, and thus the binding, with PAPS. In the case of SULT2B1b-Arg147His, the substitution of the arginine residue with histidine may also affect the hydrogen-bonding to the oxygen atom of the 3’phosphate of the PAPS and thus the loss of the sulfating activity [31, 33]. In contrast to the three SULT2B1b allozymes mentioned above, the amino acid changes in

SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, SULT2B1b-Ser244Thr, and SULT2B1b-

Arg274Gln caused only differential decreases in the sulfating activity. Among them,

SULT2B1b-Ser244Thr resulted with the smallest reductions (28% and 22%, respectively) in the catalytic efficiency toward DHEA and pregnenolone, compared with the wild-type enzyme. These relatively small decreases in sulfating activity could have been due to the replacement of the serine residue with a highly similar amino acid residue, threonine, which also carries a hydroxyl group in the side chain [33]. On the other hand, the amino acid changes in SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, and SULT2B1b-

Arg274Gln caused more dramatic differences in the sulfating activity. For SULT2B1b-

Pro69Ala, the proline residue in the wild-type enzyme, though having no direct interaction with PAPS, may contribute to the PAPS binding by imposing a kink in the polypeptide that helps place the lysine70 properly for interaction with the O6P oxygen atom of the 5’-phosphate of the PAPS [31, 33]. The replacement of the proline with alanine in SULT2B1b-Pro69Ala may weaken the hydrogen bond formation with the

PAPS and cause the dramatic decrease (with a more than 86% reduction compared to the wild-type) in the catalytic efficiency toward both DHEA and pregnenolone. In the case of SULT2B1b-Thr73Met, the decrease in the sulfating activity was due to the

86 replacement of the threonine residue that carries a polar side chain with methionine that carries a hydrophobic side chain, which might have disrupted the hydrogen bonding with the O4P oxygen atom of the 5’-phosphate of the PAPS [31,33]. For SULT2B1b-

Arg274Gln, the substitution of arginine by glutamine may have weakened the interaction between O3P oxygen of the PAPS molecule with the positively charged nitrogen atom of arginine, causing a dramatic decrease in the sulfating activity toward DHEA or pregnenolone. Interestingly, SULT2B1b-Arg274Gln has recently been linked to autosomal-recessive congenital ichthyosis, a genetic skin disorder [34].

4.6. Conclusion. This study represents the first effort to gather information concerning the effect of the genetic polymorphisms on the sulfating activity of human SULT2B1b toward DHEA and pregnenolone. Results obtained showed that three of the ten

SULT2B1b allozymes examined displayed no detectable activities with either of the two substrates, while the other seven allozymes exhibited lower and differential sulfating activities toward DHEA and pregnenolone, compared with the wild-type SULT2B1b.

Although the allelic frequencies of some of the SULT2B1b variants examined remain to be determined, the information derived from this study demonstrated clearly the impact of genetic polymorphisms on the enzymatic properties of the coded SULT2B1b protein products. Pending additional epidemiological studies, such information may eventually become useful in helping predict the risk for certain SULT2B1b-associated disorders such as colorectal cancer and autosomal-recessive congenital ichthyosis, as previously proposed [21, 34].

87 Acknowledgments:

This work was supported in part by a grant from National Institutes of Health (Grant #

R03HD071146).

Chapter 5

Role of SUL2B1 Genetic Polymorphisms in the Sulfation of Oxysterols by Human Cytosolic Sulfotransferase SULT2B1b

5.1. Abstract:

Oxysterols, generated during the metabolism of cholesterol to form bile acids, serve a myriad of functions in the body by regulating the liver X receptors (LXRs). Oxysterols were also proposed to participate in numerous pathological process including inflammation, necrosis, apoptosis, and atherosclerosis. Several oxysterols have been reported to undergo sulfation under the action of the cytosolic sulfotransferase (SULT)

SULT2B1b. As a first step toward clarifying the effects of genetic polymorphisms on the sulfating activity of SULT2B1b, ten SULT2B1b allozymes were generated, expressed, and purified. Their sulfating activities toward oxysterols, including 7-ketocholesterol

(7KC), 5α,6α-epoxycholesterol (5,6α-EC), 5β,6β- epoxycholesterol (5,6β-EC), 25- hydroxycholesterol (25HC), and 24-hydroxycholesterol (24HC), were analyzed. Three of the ten allozymes (SULT2B1b-Gly72Val, SULT2B1b-Arg147His, and SULT2B1b-

Gly276Val) showed no detectable activity toward any of the tested oxysterols, while the other seven (SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, SULT2B1b-Asp191Asn,

89 SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, SULT2B1b-Arg274Gln and

SULT2B1b-Pro345Leu) displayed differential sulfating activities toward the tested oxysterols in comparison to the wild-type SULT2B1b. These results showed a profound impact of genetic polymorphisms on the sulfating activity of SULT2B1b toward oxysterols, which may affect the metabolism of oxysterol in individuals with different

SULT2B1 genotypes.

5.2. Introduction

Oxysterols are oxidized products of cholesterol that are generated as end products or as intermediates of cholesterol metabolism leading to the formation of bile acids [1].

As shown in Figure 1, the classic pathway for bile acid synthesis starts with the transformation of cholesterol into 7α-hydroxycholesterol by the action of CYP7A1 [2].

While in the alternative pathway, cholesterol is metabolized into 25-hydroxycholesterol

(25HC) or 24-hydroxycholesterol (24HC) by the action of cholesterol 25-hydroxylase and CYP46A1 enzymes, respectively [3, 4]. Oxysterols that results from the classic and alternative pathway then transformed into cholic acid and Chenodeoxycholic acid, respectively [5]. On the other hand, a free radical attack of cholesterol in the cell membrane, lipoprotein, or food sources produces 7-ketocholesterol (7KC), 5β,6β- epoxycholesterol (5,6β-EC), and 5α,6α-epoxycholesterol (5,6α-EC) [4]. The transformation of cholesterol into oxysterols facilitates its removal and controls its level inside the cells [6]. 24HC, for example, is produced mainly in the brain, and has been

90 proposed to be responsible for regulating cholesterol level in the brain [7]. Besides the biotransformation of oxysterols into bile acids, studies have demonstrated that oxysterols can be eliminated from the body by forming more polar metabolites through sulfation and glucuronidation [8-10]. Oxysterols sulfation is mediated by several members of the cytosolic sulfotransferases (SULTs) with SULT2B1b showing the highest activity toward most oxysterols forming 3- sulfate derivatives [10-14].

Oxysterols have been proposed to be implicated in numerous biological activities ranging from cytotoxicity to nuclear receptors regulation [4]. For instance, oxysterols such as 25HC, 24HC, 5,6α-EC have been reported to be capable of activating the liver X receptors (LXRs), which regulate the expression of genes involved in cholesterol synthesis and transport [15, 16]. Furthermore, oxysterols have been also suggested to be implicated in several pathological conditions such as, multiple sclerosis (24HC), atherosclerosis (7KC), age-related macular degradation (7KC), Alzheimer disease

(24HC), and inflammatory bowel diseases (7KC, 5,6α-EC, and 5,6β-EC) [2, 5, 7].

The human hydroxysteroid sulfotransferase SULT2B1b is a member of the cytosolic sulfotransferases superfamily, which comprises a large group of enzymes that catalyze the transfer of a sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyl or amino group of the acceptor substrates, forming sulfamates or sulfate esters [17]. The human SULT2B1 gene encodes two SULT2B1isoforms with distinct N- terminal amino acid sequences due to alternative exon I and differential splicing [18].

The two resulting isoforms, SULT2B1a and SULT2B1b, were shown to exhibit substrate

91 preference toward pregnenolone and cholesterol, respectively [18]. Out of all SULTs enzymes, SULT2B1b has been reported to exhibit a high affinity toward sterols including, cholesterol, 7KC, 5,6β-EC, 5,6α-EC, 25HC, and 24HC, forming 3- monosulfate products [10-12]. Since SULT2B1b mediated the sulfation of several oxysterols, it may also play an important role in regulating the cellular lipid level as well.

Interestingly, single nucleotide genetic polymorphisms (SNPs) of the SULT2B1 gene have been reported [19-24]. Therefore, it is possible that the activity of SULT2B1b allozymes, coded by the non-synonymous coding SNPs (cSNPs) of SULT2B1 may display differential sulfating activities toward oxysterols, thereby affecting their metabolism in individuals with different SULT2B1 genotypes.

In this report, three databases were comprehensively searched for SULT2B1 cSNPs. Ten SULT2B1 cSNPs that affect the coding region for SULT2B1b were selected based on the intramolecular locations and/or the physicochemical characteristics of the amino acid changes. cDNAs corresponding to the selected SULT2B1 genotypes were generated by site-directed mutagenesis, and the SULT2B1b allozymes were expressed in

E-coli, and purified. The sulfating activities of purified SULT2B1b allozymes were assessed toward several oxysterols (7KC, 25HC, 24HC, 5,6α-EC, and 5,6β-EC) in comparison to the wild-type SULT2B1b.

Figure 5-1. Oxysterols formation and metabolism. The diagram show cholesterol oxidation by cytochrome P450 (CYP), cholesterol 25-hydroxylase (CH25H), and reactive oxygen species (ROS), bile acids formation, and the involvement of SULT2B1b in oxysterol sulfation. 7KC, 7-ketocholesterol; 7KCS, 7-ketocholesterol sufate; 5,6α-EC

,5α,6α-epoxycholesterol; 5,6α-ECS ,5α,6α-epoxycholesterol sulfate; 5,6β-EC, 5β,6β- epoxycholesterol; 5,6β-ECS, 5β,6β- epoxycholesterol sulfate; 25HC, 25- hydroxycholesterol; 25HCS, 25-hydroxycholesterol sulfate; 24HC, 24- hydroxycholesterol; 24HCS, 24-hydroxycholesterol sulfate; 27HC, 27- hydroxycholesterol; 27HCS, 27-hydroxycholesterol sulfate; 7α-HC, 7α- hydroxycholesterol.

5.3. Materials and Methods

5.3.1. Materials.

7-ketocholesterol, 5α,6α-epoxycholesterol, 5β,6β- epoxycholesterol, 25- hydroxycholesterol, and 24-hydroxycholesterol were from Steraloids (Newport, RI,

USA). PrimeSTAR Max DNA polymerase was a product of Takara Bio (Mountain

View, CA, USA). Dpn I was obtained from New England Biolabs (Ipswich, MA, USA).

Oligonucleotide primers were synthesized by Eurofins Genomics (Louisville, KY, USA).

Adenosine 5′-triphosphate (ATP), N-2-hydroxylpiperazine-N′-2-ethanesulfonic acid

(HEPES), dimethyl sulfoxide (DMSO), Trizma base, and dithiothreitol (DTT) were products of Sigma-Aldrich (St. Louis, MO, USA). Silica gel thin-layer chromatography

(TLC) plates and Ultrafree-MC 5000 NMWL filter units were from EMD Millipore

(Billerica, MA, USA). Ecolume scintillation cocktail was purchased from MP

Biomedicals, LLC. (Irvine, CA, USA). Carrier-free sodium [35S]sulfate was from

American Radiolabeled Chemicals (St. Louis, MO, USA). Protein molecular weight markers were from Bioland Scientific LLC. (Paramount, CA, USA). Recombinant human bifunctional ATP sulfurylase/adenosine 5′-phosphosulfate kinase was prepared as previously described [25]. PAP[35S] was synthesized using recombinant human bifunctional PAPS synthase based on a previously established procedure [25]. All other chemicals were of the highest grade commercially available.

5.3.2. Identification and analysis of human SULT2B1 SNPs.

94 The online SNPs databases, the Ensembl Variation database, the U.S. National

Center for Biotechnology Information (NCBI)SNP database, and the Universal Protein

Resource (UniProt), were extensively searched for homo sapiens (human) SULT2B1 genetic variations. The SULT2B1 SNPs identified were classified and analyzed based on the positions of the altered nucleotides in the SULT2B1 gene.

5.3.3. Generation of selected SULT2B1b allozyme cDNAs.

Site-directed mutagenesis was used to generate SULT2B1b allozyme cDNAs.

The wild-type SULT2B1b cDNA packaged in pGEX-4T-2 was used as a template in conjunction with SULT2B1b allozyme-specific sense and antisense primers (Table 2-3).

The PCR was performed using PrimeSTAR Max DNA polymerase and amplification conditions were as follows: pre-denaturation at 95°C for 2 min, followed by 12 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 6 min. Upon completion of the PCR reaction, restriction endonuclease Dpn I was added to the reaction mixture to degrade the wild-type SULT2B1b- pGEX-4T-2 plasmid.

Thereafter, the treated reaction mixture was transformed into competent DH5α E. coli cells for plasmid amplification and purification. To confirm the nucleotide changes in the

“mutated” SULT2B1b allozyme cDNAs, nucleotide sequencing was performed.

5.3.4. Expression and purification of SULT2B1b allozymes.

SULT2B1b allozyme cDNAs ligated to pGEX-4T-2 prokaryotic expression vectors were individually transformed into competent E. coli BL21 cells. The transformed cells were grown in 1 liter of Luria broth medium (LB) containing 100

95 g/ml ampicillin. Expression of recombinant SULT2B1b allozymes was induced by adding Isopropyl-β-D-thiogalactopyranoside (IPTG) when the cell density reached 0.5

OD600nm, and the cells were incubated overnight at 25°C with continuous shaking.

Subsequently, the cells were collected by centrifugation, resuspended in ice-cold lysis buffer (containing 150 mm NaCl, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA), and homogenized using an Aminco French press. The crude homogenate was centrifuged at

10,000 × g for 20 min at 4°C, and the supernatant collected was mixed with 2 ml of

Glutathione Sepharose and incubated for 30 min with continuous agitation. Afterwards, the SULT2B1b-GST fusion protein bound on the glutathione sepharose resin was treated with 2 ml of a thrombin digestion buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and

2.5 mM CaCl2). To cut out the SULT2B1b allozyme, 0.5 unit/ml bovine thrombin was added to the mixture and incubated at 25°C for 20 minutes with constant agitation.

Recombinant SULT2B1b allozyme released into the solution was collected by centrifugation and analyzed for purity by SDS-polyacrylamide gel electrophoresis (SDS-

PAGE).

5.3.5. Oxysterol sulfation assay.

To determine the sulfating activity of the recombinant SULT2B1b allozymes toward oxysterols, radiolabeled PAPS (PAP[35S]) was used as the sulfate donor. The 20

μl assay mixture contained equal amount 1 µg of the purified wild-type or SULT2B1b allozyme, 50 mM HEPES buffer (pH 7.4), 14 μM PAP[35S], 1 mM DTT, specific oxysterol (dissolved in hydroxypropyl-β-cyclodextrin) as substrate. A control with hydroxypropyl-β-cyclodextrin alone was assayed in parallel. The reaction was carried

96 out at 37°C for 10 min and stopped by placing the tube containing the reaction mixture on a heating block at 100°C for 3 minutes. Upon centrifugation to remove any precipitates, 4 μl of the reaction mixture was applied onto a silica gel TLC plate, which was then subjected to ascending TLC using a solvent system consisted of acetic acid: n- butanol in a ratio of 2:1 (by volume). Afterwards, the radioactive spot corresponding to

[35S]sulfated oxysterol was located by autoradiography, cut out from the plate, eluted

35 with H2O, and counted for [ S]radioactivity using a liquid scintillation counter as described previously [26].

5.3.6. Statistical analysis.

One-way ANOVA was used for inter-group comparison, followed by Dunnett’s test to analyze the difference in sulfating activity between wild-type SULT2B1b and

SULT2B1b allozymes. The statistical analyses were performed with GraphPad Prism 6.0 software, with p-values < 0.05 considered statistically significant.

5.4. Results

5.4.1. Identification of cSNPs of the human SULT2B1 gene.

A systematic search for SULT2B1 SNPs in the Ensembl Variation database, the

NCBI SNP database, and the UniProt database, has yielded a total of 276 SULT2B1b cSNPs, of which 168 were non-synonymous missense cSNPs that result in amino acid

97 changes in the coded SULT2B1b proteins. By examining the reported SULT2B1b crystal structure [27] and the physicochemical properties of the altered amino acid residue, ten SULT2B1 cSNPs were selected for further analysis. Sense and antisense oligonucleotides primers designed for the PCR-amplification of individual SULT2B1 cSNPs, as well as the reported allelic frequency of SULT2B1 genotypes are shown in

Table 2-3.

5.4.2. Generation of SULT2B1b allozyme cDNAs, and expression and purification of recombinant SULT2B1b allozymes.

Upon PCR amplification, pGEX-4T-2 plasmids harboring different SULT2B1b allozyme cDNAs were individually transformed into BL21 E. coli cells. Following IPTG induction for recombinant protein expression, each crude homogenate that contained a specific SULT2B1b allozyme was treated with glutathione sepharose to fractionate the recombinant SULT2B1b allozymes. The bound fusion protein was treated with bovine thrombin to release the SULT2B1b allozyme, free of the GST tag. SULT2B1b allozymes thus prepared were analyzed by SDS-PAGE. The apparent molecular weights of the recombinant SULT2B1b allozymes appeared similar to the calculated molecular weight

(41,307) of the wild-type SULT2B1b (cf. Figure 2-4).

5.4.3. Characterization of the oxysterols-sulfating activity of human SULT2B1b allozymes.

98 The sulfating activities of the purified SULT2B1b allozymes along with the wild- type SULT2B1b were analyzed using 7KC, 24HC, 25HC, 5,6α-EC, and 5,6β-EC at three different concentrations (5 µM, 25 µM, 100 µM). The activity data are shown in Figures

5-1 to 5-6. Three (SULT2B1b-Gly72Val, SULT2B1b-Arg147His, and SULT2B1b-

Gly276Val) of the ten tested SULT2B1b allozymes showed no detectable activity with any of the oxysterols tested. The other seven SULT2B1b allozymes exhibited differential sulfating activity toward the tested oxysterols.

With 7KC as a substrate, four allozymes (SULT2B1b-Asp191Asn, SULT2B1b-

Arg230His, SULT2B1b-Ser244Thr, and SULT2B1b-Pro345Leu) exhibited a considerable lower (by 50% or more) specific activity, compared to SULT2B1b-wt, at all three substrate concentrations (cf. Figure 5-2). On the other hand, SULT2B1b-

Pro69Ala, SULT2B1b-Thr73Met, and SULT2B1b-Arg274Gln showed no detectable activity with the lowest substrate concentration (5µM) tested, while at 25 µM and 100

µM of 7KC as substrates, SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, and

SULT2B1b-Arg274Gln displayed very low levels of activities (greater than 97% decrease), compared with the wild-type enzyme.

Figure 5-2. Specific activities of the sulfation of 7KC by human SULT2B1b allozymes.

Concentrations of morphine used in the enzymatic assays were 5 μM (black), 25 μM

(gray) and 100 μM (white). Specific activity refers to nmol of 7KC sulfate /min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. wt refers to wild-type SULT2B1b. Statistical significance from

SULT2B1b-wt (***P-value < 0.0001) using one-way ANOVA followed by Dunnett’s post hoc analysis.

With 24HC and 25HC as substrates, all SULT2B1b allozymes exhibited similar specific patterns of activity (cf. Figure 4 and 5). At all three concentrations of 24HC and

100 25HC, six SULT2B1b allozymes (SULT2B1b-Pro69Ala and SULT2B1b-Thr73Met,

SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, and

SULT2B1b-Pro345Leu) showed lower specific activity than with the wild-type

SULT2B1b at varying extent. On the other hand, SULT2B1b-Arg274Gln showed no detectable activity at the low and mid concentrations (5 µM and 25 µM) of 24HC and

25HC and displayed a greater than 97% reduction of sulfating activity compared with the wild-type with the highest concentration (100 µM) of these two substrates.

101

*** ***

*** *** *** *** ****** ****** *** ***

Figure 5-3. Specific activities of the sulfation of 24HC by human SULT2B1b allozymes.

Concentrations of morphine used in the enzymatic assays were 5 μM (black), 25 μM

(gray) and 100 μM (white). Specific activity refers to nmol of 24HC sulfate /min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. wt refers to wild-type SULT2B1b. Statistical significance from

SULT2B1b-wt (***P-value < 0.0001) using one-way ANOVA followed by Dunnett’s post hoc analysis.

102 *** ***

*** ***

*** *** *** *** *** *** ****** ****** *** *** *** *** ***

Figure 5-4. Specific activities of the sulfation of 25HC by human SULT2B1b allozymes.

Concentrations of morphine used in the enzymatic assays were 5 μM (black), 25 μM

(gray) and 100 μM (white). Specific activity refers to nmol of 25HC sulfate /min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. wt refers to wild-type SULT2B1b. Statistical significance from

SULT2B1b-wt (***P-value < 0.0001) using one-way ANOVA followed by Dunnett’s post hoc analysis.

With 5,6β-EC as a substrate, At low and high substrate concentrations (5 and 100 μM, respectively), lower 5,6β-EC-sulfating activities were found for the analyzed SULT2B1b allozymes compared to the wild-type SULT2B1b (cf. Figure 6). Among them, three

(SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met, and SULT2B1b-Arg274Gln) exhibited

103 the lowest specific activity. Interestingly, an inhibitory effect was observed with

SULT2B1b-Pro345Leu at 100 μM of 5,6β-EC. At mid substrate concentration (25 μM),

SULT2B1b-Pro345Leu showed a slightly higher specific activity than the wild-type enzyme, while the specific activity of SULT2B1b-Arg230His were comparable to that of the wild-type, the specific activities of all other allozymes were lower than the wild-type enzyme, with SULT2B1b-Arg274Gln exhibiting the lowest specific activity (90% less than the wild-type SULT2B1b).

*** *** ** ***

*** *** *** ***

*** ***

Figure 5-5. Specific activities of the sulfation of 5,6βEC by human SULT2B1b allozymes. Concentrations of morphine used in the enzymatic assays were 5 μM (black),

25 μM (gray) and 100 μM (white). Specific activity refers to nmol of 5,6βEC sulfate

/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. wt refers to wild-type SULT2B1b. Statistical significance

104 from SULT2B1b-wt (***P-value < 0.0001) using one-way ANOVA followed by

Dunnett’s post hoc analysis.

With 5,6α-EC as a substrate, At all three substrate concentrations (5, 25 and 100

μM), SULT2B1b-Arg274Gln displayed a specific activity 98% less than SULT2B1b-wt

(cf. Figure 7). The other six SULT2B1b allozymes all displayed lower specific activities than the wild-type at varying degrees.

Figure 5-6. Specific activities of the sulfation of 5,6αEC by human SULT2B1b allozymes. Concentrations of morphine used in the enzymatic assays were 5 μM (black),

25 μM (gray) and 100 μM (white). Specific activity refers to nmol of 5,6αEC sulfate

/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived

105 from three determinations. wt refers to wild-type SULT2B1b. Statistical significance from SULT2B1b-wt (***P-value < 0.0001) using one-way ANOVA followed by

Dunnett’s post hoc analysis.

5.5. Discussion

The major role of oxysterols in the body is to facilitate cholesterol excretion from the body as end products or intermediates by further transforming into bile acids [1].

Oxysterols are also proposed to be implicated in several other pathological processes in the body like inflammation, immunosuppression, necrosis, and apoptosis [2]. More importantly, they have been reported to be involved in cholesterol homeostasis through their action on the sterol regulatory element binding protein (SREBP), LXR receptors, or directly by affecting the activity of the enzymes that are involved in cholesterol biosynthesis pathway like 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase

(HMGCR) [4].

Oxysterols can also be eliminated as sulfated or glucuronide conjugates [9]. In fact, sulfated forms of several oxysterols such as 24HC, 25HC, and 7KC have been reported in several human tissues and specimens [8-10]. In vitro studies have shown that

SULT2B1b can mediate the sulfation of many oxysterols such as 7KC, 5,6α-EC, 5,6β-

EC, 25HC, and 24HC [10-12]. Oxysterols sulfation has been believed to be an elimination pathway until the discovery of a novel oxysterol sulfate (25HC-sulfate)

106 which was shown to play a regulatory role in regulating cholesterol and lipids metabolism [28, 29]. Since SULT2B1b mediated the sulfation of several oxysterols, it may also play an important role in regulating the cellular lipid level as well. In fact,

SULT2B1b was suggested to be involved in intracellular lipid homeostasis by mediating the conversion of 25HC to 25HC-sulfate, which in turn acts as an inhibitor in the

LXR/SREP-1 signaling pathway and reduces the cellular cholesterol and lipid levels [29].

SULT2B1b is also involved in 24-HC sulfation, a major metabolite and regulator of cholesterol synthesizes mainly in the brain, forming 24-HC sulfate that acts as an antagonist to LXRα [11]. Moreover, the sulfation of 7KC by SULT2B1b has shown to promote its elimination and attenuate its cytotoxic activity when overexpressed in 293T cells [10]. SULT2B1b involvement in catalyzing the sulfation of 5,6α-EC to 5,6α-EC- sulfate was shown to promote the cytotoxic effect of tamoxifen by modulating the activity of LXRβ in MCF7 cells [14]. In view of the SULT2B1b role in catalyzing the sulfation of oxysterols and its widespread expression in several tissues in the body, this study was designed to investigate the effect of ten SULT2B1 missense cSNPs on the sulfating activity of the resulting SULT2B1b allozymes toward oxysterols.

The ten identified SULT2B1b missense cSNPs were generated by site-directed mutagenesis and the coded SULT2B1b allozymes were expressed and affinity-purified.

As indicated in the Results section, three allozymes (SULT2B1b-Gly72Val, SULT2B1b-

Arg147His, and SULT2B1b-Gly276Val) exhibited no detectable activity with all the tested oxysterols. The other seven allozymes (SULT2B1b-Pro69Ala, SULT2B1b-

Thr73Met, SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr,

107 SULT2B1b- Arg274Gln, and SULT2B1b-Pro345Leu) displayed a significant reduction in their sulfating activity toward the examined oxysterols (except SULT2B1b-Arg230His and SULT2B1b-Pro345Leu with 25 μM 5,6β-EC). Interestingly, it was noticed that

SULT2B1b and SULT2B1b allozymes exhibited higher activity toward the low and mid concentration (5 and 25µM) of 5,6β-EC compared with the other tested oxysterols

(especially with its diastereomer 5,6α-EC), that could be explained by the strict structural requirements and stereo-selectivity of SULT2B1b towards toward 3β-hydroxysteroid as well as a planar arrangement of the fused ring (perhydrocyclopentanophenanthrene) [17,

30]. A previously proven that small changes in the substrate structure caused may influence SULT2B1b sulfating activity [17]. For instance, a change from a planer structure like cholesterol to 5α-reduced form like cholestenol or a non-planer molecule with several bends like coprostanol caused a 30% or 75-80% reduction, respectively, in

SULLT2B1b sulfating activity [17]. However, the change of the spatial orientation of the

3β-hydroxy group into 3α-hydroxyl stereoisomer of cholesterol (epicholesterol) makes it a poor substrate for SULT2B1b [17]. Moreover, 25µM of 5,6β-EC seems to be a saturation concentration especially for SULT2B1b-Arg230His and SULT2B1b-

Ser244Thr since no increase in these enzymes activity was observed with the higher concentration tested (100 µM). Over all, these results indicate clearly that SULT2B1b missense cSNPs have a significant impact on the enzyme function.

The crystal structure of SUL2B1b was reported by Lee et. al. [30]. The identified essential residues in SULT2B1b function and structure as follow: the residues that are involved in the catalysis (His125), dimerization motif (282KNHFTVAQSE291),

108 phosphosulfate binding loop (PSB) (67TYPKSGT73), the PAPS/PAP binding residues

(Thr74, Arg274, Lys275, Gly276, Arg147, and (Lys70, Ser71, and Thr73) from the PSB region ) , the residues that involve in the interaction with the adenine group of the PAPS molecule (Ser244, Tyr210, Trp75, and Phe246), the residues that surround the substrate binding pocket (Ile20, Tyr44, Leu43, Trp98, Trp103, Thr106, Val108, Tyr159, Gln165,

Tyr257,Leu260, Leu264, and Phe272), and the N-terminal residues (19DISEI23) which are responsible for the cholesterol-sulfation activity [27,31,32].

Seven of the examined allozymes (SULT2B1b-Pro69Ala, SULT2B1b-Gly72Val,

SULT2B1b-Thr73Met, SULT2B1b-Arg147His, SULT2B1b-Ser244Thr, SULT2B1b-

Arg274Gln, and SULT2B1b-Gly276Val) have changes in a residue that is involved in the

PAPS/PAP binding region (cf. Figure 5-7), which may influence their sulfating activity negatively [27]. Moreover, the altered amino acid residues in these allozymes have different chemical characteristics or structures. For example, a turn-inducing amino acid to non-turn inducing residue (SULT2B1b-Pro69Ala), aliphatic amino acid to heterocyclic amino acid (SULT2B1b-Arg147His), a small amino acid to a branched (bulker) residue

(SULT2B1b-Gly72Val, SULT2B1b-Gly276Val), from a basic amino acid to non-basic amino acid (SULT2B1b-Arg274Gln), and hydroxyl-containing side chain (polar) to sulfur-containing side chain residue (non-polar) (SULT2B1b-Thr73Met). All these allozymes exhibited a drastic decrease in the oxysterol-sulfating activity or even cause a complete loss of the enzymatic activity. The minor variation between these allozymes could be attributed to the chemical nature of the altered amino acid residues as well as their location in the overall structure of SULT2B1b. For example, SULT2B1b-Thr73Met

109 allozyme involves an alteration of the threonine to methionine at position 73 in the

PAPS/PAP binding region, which may lead to the reduction in the sulfating activity [27].

The hydroxyl group of the threonine at position 73 was proposed to form a hydrogen bond with the O4P oxygen atom of the 5’-phosphate of the PAPS [27]. Such a role might not be accomplished by an amino acid with a non-polar side chain like methionine.

Arginine residues in SULT2B1b-Arg147His and SULT2B1b-Arg274Gln are implicated in forming a hydrogen bond with oxygen atoms of the 3’phosphate the PAPS molecule

[27]. Arginine was reported to be able to form multiple hydrogen bond especially with negatively charged atoms on phosphates, due to the presence of a guanidinium group on its side chain [33]. In SULT2B1b-Arg147His and that SULT2B1b-Arg274Gln, the change of arginine to another amino acid in these allozymes could be the reason behind the total loss or a drastic decrease in the sulfating activity, respectively. Indeed, the variation in SULT2B1b-Arg274Gln was reported recently to be implicated in autosomal recessive ichthyosis, which resulted from the absence of cholesterol sulfate in the skin

[34]. The glycine at position 72 and 276 in SULT2B1b-Gly72Val and SULT2B1b-

Gly276Val, were proposed to form hydrogen-bond with the O4P and O2P phosphate oxygens of the PAPS/PAP, respectively [27]. Thus, the replacement of the glycine in these positions to a bulker residue (valine) may cause the complete loss of the sulfating activity toward all tested oxysterols. In case of SULT2B1b-Pro69Ala, the proline residue, which locates in the PSB region, changed into alaninee [27]. Proline is known to be responsible for introducing a sharp turn in the protein structure especially at the edge of β-sheet [33]. Thus, in SULT2B1b proline at position 69 could be responsible for the proper positioning of the highly conserved lysine70 for its polar interaction with the O6P

110 oxygen atom of the 5’-phosphate of the PAPS [27]. The change of proline into serine in

SULT2B1b-Pro69Ala probably cause a displacement of the lysine70, which could influence the sulfating activity toward oxysterols. In SULT2B1b-Ser244Thr, the serine at position 244, was proposed to form a hydrogen bond with the nitrogen atom (N6) of the adenine in the PAPS molecule [27]. The substitution of serine in this allozyme with a highly similar amino acid residue (threonine) could be the reason of the smaller reduction oxysterols-sulfating activity compared to the other allozymes that are located in the PAPS binding region. On the other hand, the other tested allozymes (SULT2B1b-Asp191Asn,

SULT2B1b-Arg230His, and SULT2B1b-Pro345Leu) have shown a smaller decrease in the sulfating activity, which could be because the amino acid alterations in these allozymes are not involved or closed to catalytic activity, PAPS binding, or substrate binding regions.

111

Figure 5-7. Location of the examined SNPs-based amino acid in the PAPS binding region. The figure shows the amino acid residues in the PAPS binding site A) Proline at position 69, B) Glycine at position 72, C) Threonine at position 73, D) Arginine at position 148, E) Arginine at position 274, F) Glycine at position 276. The yellow dashed lines represent the hydrogen bonds. The figure was generated using PyMOL software and the SULT2B1b crystal structure PDB ID:1q22.

5.6. Conclusion

This study aimed to gather information of the effect of SULT2B1 genetic polymorphisms on the sulfating activity of the coded SULT2B1b allozymes toward 7KC,

24HC, 25HC, 5,6β-EC, and 5,6α-EC. Of the examined SULT2B1b allozymes, three

112 (SULT2B1b-Gly72Val, SULT2B1b-Arg147His, and SULT2B1b-Gly276Val) showed no activity, while the other seven (SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met,

SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, SULT2B1b-

Arg274Gln, and SULT2B1b-Pro345Leu) displayed differential sulfating activities in comparison to the wild-type SULT2B1b. The obtained data prove the significant effect of SULT2B1 genetic polymorphisms on the sulfating activity of the coded SULT2B1b allozymes toward the five examined oxysterols, which may influence oxysterol metabolism in individuals with different SULT2B1b genotypes. Pending further epidemiological studies, such information may help to identify potential health problems that may be implicated with SULT2B1b-associated disorders.

113

Chapter 6

Xenoestrgens Sulfation by Human Cytosolic Sulfotransferase SULT2B1b

6.1. Abstract

Xenoestrogens have been a growing concern since they can mimic the estrogen effect and disrupt the endocrine hormones by binding the estrogenic receptors or interfere with the enzymes that regulate their levels in the body. They have been reported to be implicated in human reproductive disorders and malignancy. Human cytosolic sulfotransferases have been proposed to be involved in their detoxification. In this study,

SULT2B1b was expressed, affinity cleaved, purified, and its sulfating activity was characterized toward representative xenoestrogens. The activity data collected showed that SULT2B1b displayed activity toward six substrates (4-n-octylphenol, 4-n- nonylphenol, diethylstilbestrol, 17-α-ethynylestradiol, estrone, and p-nitrophenol) at the low concentration tested, while at the elevated concentration SULT2B1b exhibited activity toward four of them only (diethylstilbestrol, 17-α-ethynylestradiol, estrone, and p-nitrophenol).[1]

114

6.2. Introduction

Environmental xenoestrogens have been a growing concern since they can mimic the estrogen effect, which may cause extra burden to the endocrine system and arise several health hazards [2]. Xenoestrogens are a group of chemicals able to disrupt the endocrine hormone by directly bind to the estrogenic receptors or interfere with the enzymes that regulate the level of endogenous estrogens and exert their adverse effects, especially on steroid hormone responsive organs [3]. Xenoestrogens have been reported to be implicated in increasing the incidence of breast cancer, testicular cancer, as well as reduce the sperm quality and semen volume [4]. Among the widely used xenoestrogens are bisphenol A (used in food packaging and dental fillings), 4-n-octylphenol, 4-n- nonylphenol (used in detergent and plastic additives), 17-α-ethynylestradiol, diethylstilbestrol (synthetic estrogens used in oral contraceptives and in prostate cancer treatments, respectively), and raloxifene (an estrogen receptor modulator used in osteoporosis treatments) [2, 5, 6]. Xenoestrogens metabolism may alleviate their adverse effects by improving their removal from the body. In fact, xenoestrogen conjugation through glucuronidation and sulfation have been reported [3, 7, 8]. Human cytosolic sulfotransferase enzymes (SULTs) such as SULT1A1, SULT1A3, SULT1B1, SULT1C2,

SULT1E1, SULT2B1a, SULT2B1b, and SULT4A1, have been reported to be able to mediate the sulfation of several xenoestrogens [3, 9].

115 Sulfation is an essential phase II conjugation reaction, which is involved in regulating endogenous homeostasis and xenobiotic removal from the body [10].

Sulfation occurs under the action of the cytosolic sulfotransferase enzymes (SULTs), which are responsible for catalyzing the transfer of sulfate group from phosphoadenosine

5'-phosphosulfate to several endogenous compounds and xenobiotics [10, 11]. In humans, there are thirteen sulfotransferases enzymes that are categorized into four SULT gene families SULT1, SULT2, SULT4, and SULT6 [10, 11]. The human cytosolic

SULT2 family consists of three isoforms: SULT2A1 SULT2B1a, and SULT2B1b [12,

13]. SULT2B1a and SULT2B1b isoforms are coded by the SULT2B1 gene as a result of an alternative exon I initiation and splicing [13]. Although both SULT2B1a and

SULT2B1b mRNAs have been detected in several tissues, only SULT2B1b protein has been detected in human tissues and cell lines [14]. SULT2B1b protein has been detected in human lung, prostate, breast, brain, placenta, and skin as well as human breast and prostate cancer cell lines [14, 15]. Additionally, SULT2B1b has been reported to show sulfating activity toward several drugs and xenobiotics such as raloxifene, bisphenol A, 4- n-octylphenol, 4-n-nonylphenol, diethylstilbestrol, 17-α-ethynylestradiol, and p- nitrophenol [3, 16]. Since SULT2B1b is expressed in steroid-responsive tissues (where most xenoestrogens exert their activities), it may play an important role in xenoestrogen elimination. In this study, the SULT2B1b enzyme was expressed, affinity cleaved, purified, and its sulfating activity was examined toward representative xenoestrogens.

6.3. Materials and Methods

116

6.3.1. Materials

p-nitrophenol, bisphenol A, diethylstilbestrol, 4-n-octylphenol, 4-n-nonylphenol,

17-α-ethynylestradiol, estrone, N-2-hydroxylpiperazine-N′-2-ethanesulfonic acid

(HEPES), thrombin, sodium dodecyl sulfate (SDS), Trizma base, and dithiothreitol

(DTT) were obtained from Sigma-Aldrich Sigma-Aldrich (St. Louis, MO, USA). Silica gel thin-layer chromatography (TLC) plates, and Ultrafree-MC 5000 NMWL filter units were from EMD Millipore (Billerica, MA, USA). Carrier-free sodium [35S]sulfate was from American Radiolabeled Chemicals (St. Louis, MO, USA). Ecolume scintillation cocktail was purchased from MP Biomedicals, LLC. (Irvine, CA, USA). Recombinant human bifunctional ATP sulfurylase/adenosine 5′-phosphosulfate kinase was prepared as previously described [17]. Protein molecular weight markers were from Bioland

Scientific LLC. (Paramount, CA, USA). PAP[35S] was synthesized using recombinant human bifunctional PAPS synthase as described previously [17]. All other chemicals were of the highest grade commercially available.

6.3.2. Expression, and purification of human cytosolic SULT2B1b

SULT2B1b ligated to prokaryotic vector pGEX-4T-2 was transformed into a competent bacterial cells BL21. The transformed bacterial cells were inoculated into one- liter LB medium containing 100 μg/ml ampicillin until an optical density of OD600 nm =

∼0.5, then the enzyme expression was induced by adding Isopropyl β-D-1-

117 thiogalactopyranoside (IPTG). The mixture was incubated overnight at 25°C with continuous shaking. The bacterial cells were collected by centrifugation and resuspended in ice-cold lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mm NaCl, and 1 mM EDTA).

Aminco French press was used to homogenize the resuspended cells. By using centrifugation at 10,000 × g for 20 min at 4°C, the supernatant was collected and fractionated using 2ml of 50% aliquots of glutathione-Sepharose 4B. After washing with lysis buffer to remove the unbound proteins, 2 ml of a thrombin digestion buffer (50 mM

Tris-HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2) containing 3.5 unit/ml bovine thrombin, was added to cleave the enzyme from the fusion protein. The mixture was incubated for 20 minutes at 25°C with constant agitation. Upon centrifugation, the recombinant SULT2B1b present in the supernatant was collected and analyzed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE).

6.3.3. Enzymatic assay with the xenobiotics substrates

To quantify the amount of the sulfated product a radiolabeled PAPS (PAP[35S]) was used in the assay mixture. The enzymatic assay mixture of a final volume of 20 µL contains 50 mM HEPES buffer (pH 7.4), 1 mM DTT, 14 μM PAP[35S], substrate dissolved in DMSO, a control with DMSO alone was assayed as well. The reaction was started by adding 1µg of SULT2B1b and incubated for ten minutes at 37οC. The reaction was terminated by heating the reaction mixture to 100οC for three minutes. Centrifugation at 13,000 rpm for 3 minutes was used to clear the precipitate that formed in the reaction.

After that, 4 µL of the reaction mixture was spotted in a silica gel TLC plate and developed in a solvent system containing glacial acetic acid: n-butanol in a ratio of 2:1

118 (by volume). The visualized sulfated product by autoradiography was cut from the TLC plate and eluted with H2O and mixed with 2 ml of Ecolume scintillation liquid. The radioactivity of the sulfated product was counted by a liquid scintillation counter. The readings obtained in count per minute (CPM) were used to calculate the specific activity in units of pmol of the sulfated product formed per minute per mg of SULT2B1b.

6.4. Results and discussion

As mentioned earlier, xenoestrogens act as endocrine disruptors by disturbing normal hormone actions, which arise potential health problems [2]. Xenoestrogens have been proposed to be implicated in the development of hormone-dependent cancers such as breast and testicular cancers [2, 4]. Sulfation through SULTs enzymes, which are a major phase II detoxifying pathway may help to mitigate their effect by enhancing their removal from the body. SULT2B1b was reported to exhibit sulfating activity toward several drugs and xenobiotics such as raloxifene, bisphenol A, 4-n-octylphenol, 4-n- nonylphenol, diethylstilbestrol stilbestrol, 17-α-ethynylestradiol, and p-nitrophenol [3,

16]. In this study, the activity of recombinant SULT2B1b was characterized toward raloxifene, bisphenol A, 4-n-octylphenol, 4-n-nonylphenol, diethylstilbestrol, 17-α- ethynylestradiol, and estrone as well as p-nitrophenol.

6.4.1. SULT2B1b recombinant enzyme expression, and purification.

The plasmid harboring SULT2B1b as was overexpressed in BL21 E. coli cells as a glutathion S-transferase fusion protein to facilitate the protein expression and

119 purification. Glutathione Sepharose resin was used to fractionate the recombinant

SULT2B1b without the fusion protein. SULT2B1b cleavage from the fusion protein was facilitated by treating the mixture with bovine thrombin. After that, the collected enzyme was analyzed by SDS-PAGE. As shown in lane 2 Figure2-4, The apparent molecular weights of the recombinant SULT2B1b was similar to the calculated molecular weight of

(41,307).

6.4.2. Sulfating Activities of the Human SULT2B1b

To characterize the activity of SULT2B1b toward the selected xenoestrogen as well as p-nitrophenol, two concentrations (50 µM and 500 µM) of the substrates were used. As shown in Table 6.1, at the low concentration (50 µM), SULT2B1b showed no sulfating activity toward two substrates (raloxifene and bisphenol A) and showed differential sulfating activity toward the other six substrates. Among them, SULT2B1b showed the highest activity toward diethylstilbestrol with a specific activity of 28.2 ± 0.3 pmol/min/mg. While at the higher concentration (500 µM), SULT2B1b showed sulfating activity toward four substrates only (diethylstilbestrol, 17α-ethynylestradiol, p- nitrophenol, and estrone) with the highest activity toward p-nitrophenol (105.0 ± 3.1 pmol/min/mg). Interestingly, at 500 µΜ estrone seems to exert an inhibitory effect since

SULT2B1b activity dropped from 18.1 pmol/min/mg at 50 µΜ to 15.6 pmol/min/mg at

500 µΜ. Moreover, SULT2B1b showed no activity with 4-n-octylphenol and 4-n- nonylphenol at the higher concentration tested (500 µΜ). It should be pointed out that in contrast to previous studies, which reported SULT2B1b sulfating activity toward

120 raloxifene and bisphenol A [3, 16], in this study SULT2B1b did not display detectable activity toward these xenoestrogens at either concentration tested as shown in Figure 6-1.

Table 6.1 Specific activities of human cytosolic SULT2B1b toward xenoestrogens.

Specific Activity (pmol/min/mg) Substrate 50 μM 500 μM

28.2 ± 0.3 39.7 ± 3.5 Diethylstilbestrol 19.8 ± 10.0 40.4 ± 2.8 17α-Ethynylestradiol 19.0 ± 9.6 105.0 ± 3.1 p-Nitrophenol 18.1 ± 9.1 15.6 ± 1.2 Estrone N.D. N.D. Raloxifene 11.2 ± 5.9 N.D. 4-n-Nonylphenol 10.8 ± 0.5 N.D. 4-n-Octylphenol N.D. N.D. Bisphenol A

The weak activity of SULT2B1b toward the examined substrates was not surprising considering the chemical structures of the tested substrates (cf. Figure 6-2) and the fact that SULT2B1b exhibits strict structural requirements toward its substrate. In fact, SULT2B1b was reported to exhibit stereo-selectivity toward 3β-hydroxysteroid as well as a planar arrangement of the attached ring (perhydrocyclopentanophenanthrene)

121 [18, 19]. As a previous study has reported that any small change in the substrate structure

may influence SULT2B1b sulfating activity [19]. For example, the change of the planer

structure of the cholesterol to its 5α-dihydro derivative (cholestenol) or to a non-planer

molecule with several bends like coprostanol caused a 30% or 75-80% reduction,

respectively, in SULLT2B1b sulfating activity compared [19]. Furthermore, since

SULT2B1b showed a reduction in estrone-sulfating activity and a complete loss of the

activity with 4-n-octylphenol and 4-n-nonylphenol at the higher concentration tested,

these substrates could be good candidates as competitive inhibitor for SULT2B1b. This

could then be expected to interfere with the metabolism of the endogenous substrates of

SULT2B1b such as pregnenolone or DHEA, which may disturb the endocrine

homeostatic mechanisms [20, 21]. In fact, most of the examined substrates have been

reported to exert an inhibitory effect with other sulfotransferases enzymes [20, 21]. For

example, estrone and 17-α-ethynylestradiol caused 90-100% inhibition of the SULT1E1

activity, while 4-n-nonylphenol showed about 15-50% inhibition of the SULT1E1

sulfating activity toward 1-hydroxypyrene [20]. p-nitrophenol was also reported to cause

SULT1A1 inhibition when elevated concentration used [21].

6.5. Conclusion. The current study aimed to gather information concerning SULT2B1b

activity toward xenoestrogens. The activity data obtained indicated that SULT2B1b

showed no activity toward two of the tested substrate (raloxifene and bisphenol A) and

displayed differential sulfating activity toward the other six substrates (4-n-octylphenol,

4-n-nonylphenol, estrone, diethylstilbestrol, 17-α-ethynylestradiol, and p-nitrophenol)

especially at the lower tested concentration. Although SULT2B1b activity toward the

122 examined substrates was weak, they could serve as SULT2B1b inhibitors, which may disrupt its sulfating activity with endogenous steroids.

1 2 3 4

Sulfated product

Origin 5 6 7 8

123

Sulfated product

1 2 3 4 5

Origin

5 6

7 8

Figure 6-1. Analysis of the xenoestrogens sulfated product generated by SULT2B1b. The figure shows the autoradiograph taken from the TLC plate used for TLC analysis of the

SULT2B1b sulfating activity. A) show the generation of the sulfated xenoestrogens using

50 µM of 1, raloxifene; 2, bisphenol A; 3, estrone; 4, 4-n-octylphenol;5, 17-α- ethynylestradiol; 6, diethylstilbestrol; 7, p-nitrophenol; 8, 4-n-nonylphenol; 9, DMSO. B) show the generation of the sulfated xenoestrogens using 500 µM of 1, p-nitrophenol; 2,

124 17-α-ethynylestradiol; 3, diethylstilbestrol; 4, estrone; 5, 4-n-octylphenol; 6, 4-n- nonylphenol; 7, raloxifene; 8, bisphenol A.

Figure 6-2. Chemical structure of the exogenous substrates.

125 Chapter 7

Summary

This study was aimed to gather information of the effect of SULT2B1 genetic polymorphisms on the sulfating activity of the coded SULT2B1b allozymes toward steroids and sterols. With all tested substrates (cholesterol, pregnenolone, DHEA, and oxysterols), three allozymes (SULT2B1b-Gly72Val, SULT2B1b-Arg147His, and

SULT2B1b-Gly276Val) that are located in the PAPS binding region displayed no detectable activity, while the other seven (SULT2B1b-Pro69Ala, SULT2B1b-Thr73Met,

SULT2B1b-Asp191Asn, SULT2B1b-Arg230His, SULT2B1b-Ser244Thr, SULT2B1b-

Arg274Gln, and SULT2B1b-Pro345Leu) exhibited differential sulfating activities in comparison to the wild-type SULT2B1b. Among them, the data collected for

SULT2B1b-Arg274Gln with the cholesterol provided a biochemical basis for the role of this allozyme in the autosomal recessive ichthyosis. Although information concerning the allelic frequencies of the tested SULT2B1b genotypes in the population is incomplete and remains to be clarified, this study provided convincing evidence that the coded

SULT2B1b allozymes exhibited significant and sometimes dramatic differences in their

126 enzymatic activities. The obtained data imply that individuals with different SULT2B1b genotype may have differential capacity in sulfating activity.

The activity of SULT2B1b-wt was also assessed toward xenoestrogens. The activity data collected revealed that SULT2B1b displayed no detectable activity toward two of the tested substrates (raloxifene and bisphenol A), but displayed differential and low sulfating activity towards the other six , including 4-n-octylphenol, 4-n-nonylphenol, estrone, diethylstilbestrol, 17-α-ethynylestradiol, and p-nitrophenol. Compared to the examined endogenous substrates, SULT2B1b showed significantly lower activity towards the tested xenoestrogens which made it difficult to examine the activity of SULT2B1b allozymes towards those substrates.

Pending additional epidemiological studies, such information may eventually become useful in helping predict the risk for certain SULT2B1b-associated disorders, such as colorectal cancer, stomach cancer, and autosomal-recessive congenital ichthyosis, as previously proposed.

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96. Vickman, R. E., Crist, S. A., Kerian, K., Eberlin, L., Cooks, R. G., Burcham, G. N.,

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1. Yanagisawa, K., Sakakibara, Y., Suiko, M., Takami, Y., Nakayama, T., Nakajima, H.,

Takayanagi, K., Natori, Y., Liu, M.C. (1998) cDNA cloning, expression, and characterization of the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate kinase enzyme. Biosci. Biotechnol. Biochem. 62, 1037-1040.

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141 3- Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

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142 8. Williams, M.L., Hughes-Fulford, M. and Elias, P.M. (1985) Inhibition of 3-hydroxy-3-

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Lathrop, M., Hausser, I., Zimmer, A.D. and Fischer, J. (2017) Mutations in SULT2B1

Cause Autosomal-Recessive Congenital Ichthyosis in Humans. Am. J. Hum. Genet.

100, 926-939.

13. Seneff, S., Davidson, R.M., Lauritzen, A., Samsel A., Wainwright, G. (2015) A novel

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18. Kohjitani, A., Fuda, H., Hanyu, O. and Strott, C.A. (2008) Regulation of SULT2B1a

(pregnenolone sulfotransferase) expression in rat C6 glioma cells: relevance of

AMPA receptor-mediated NO signaling. Neurosci. Lett. 430, 75-80.

19. Freimuth, R.R., Wiepert. M., Chute, C.G., Wieben, E.D. and Weinshilboum, R.M.

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21. Fuda, H., Lee, Y.C., Shimizu, C., Javitt, N.B. and Strott, C.A. (2002) Mutational

analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that

exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon

1A yields pregnenolone sulfotransferase. J. Biol. Chem. 277, 36161-6.

22. Her, C., Wood, T.C., Eichler, E.E., Mohrenweiser, H.W., Ramagli, L.S., Siciliano,

M.J. and Weinshilboum, R.M. (1998) Human hydroxysteroid sulfotransferase

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284-95.

144 23. Lee, K.A., Fuda, H., Lee, Y.C., Negishi, M., Strott, C.A. and Pedersen, L.C. (2003)

Crystal structure of human cholesterol sulfotransferase (SULT2B1b) in the presence

of pregnenolone and 3'-phosphoadenosine 5'-phosphate. Rationale for specificity

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Chem. 278, 44593-9.

24. Geese, W.J. and Raftogianis, R.B. (2001) Biochemical characterization and tissue

distribution of human SULT2B1. Biochem. Biophys. Res. Commun. 288, 280-9.

25. Yanai, H., Javitt, N.B., Higashi, Y., Fuda, H. and Strott, C.A. (2004) Expression of

cholesterol sulfotransferase (SULT2B1b) in human platelets. Circulation 109, 92-6.

26. Higashi, Y., Fuda, H., Yanai, H., Lee, Y., Fukushige, T., Kanzaki, T. and Strott, C.A.

(2004) Expression of cholesterol sulfotransferase (SULT2B1b) in human skin and

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13.

27. Hyland, P.L., Freedman, N.D., Hu, N., Tang, Z.Z., Wang, L., Wang, C., Ding, T.,

Fan, J.H., Qiao, Y.L., Golozar, A., Wheeler, W., Yu, K., Yuenger, J., Burdett, L.,

Chanock, S.J., Dawsey, S.M., Tucker, M.A., Goldstein, A.M., Abnet, C.C. and

Taylor, P.R. (2013) Genetic variants in sex hormone metabolic pathway genes and

risk of esophageal squamous cell carcinoma. Carcinogenesis 34, 1062-8.

28. Levesque, E., Laverdiere, I., Audet-Walsh, E., Caron, P., Rouleau, M., Fradet, Y.,

Lacombe, L. and Guillemette, C. (2014) Steroidogenic germline polymorphism

predictors of prostate cancer progression in the estradiol pathway. Clin. Cancer. Res.

20, 2971-83.

145 29. Mostaghel, E.A. (2013) Steroid hormone synthetic pathways in prostate cancer.

Transl. Androl. Urol. 2, 212-227.

30. Yang, X., Xu, Y., Guo, F., Ning, Y., Zhi, X., Yin, L. and Li, X. (2013)

Hydroxysteroid sulfotransferase SULT2B1b promotes hepatocellular carcinoma cells

proliferation in vitro and in vivo. PloS one 8, e60853.

31. Hu, L., Yang, G.Z., Zhang, Y., Feng, D., Zhai, Y.X., Gong, H., Qi, C.Y., Fu, H., Ye,

M.M., Cai, Q.P. and Gao, C.F. (2015) Overexpression of SULT2B1b is an

independent prognostic indicator and promotes cell growth and invasion in colorectal

carcinoma. Lab. Invest. 95, 1005-18.

32. Vickman, R.E., Crist, S.A., Kerian, K., Eberlin, L., Cooks, R.G., Burcham, G.N.,

Buhman, K.K., Hu, C.D., Mesecar, A.D., Cheng, L. and Ratliff, T.L. (2016)

Cholesterol sulfonation enzyme, SULT2B1b, modulates AR and cell growth

properties in prostate cancer. Mol. Cancer Res. 14, 776-86.

33. Ji, Y., Moon, I., Zlatkovic, J., Salavaggione, O.E., Thomae, B.A., Eckloff, B.W.,

Wieben, E.D., Schaid, D.J. and Weinshilboum, R.M. (2007) Human hydroxysteroid

sulfotransferase SULT2B1 pharmacogenomics: gene sequence variation and

functional genomics. J. Pharmaco. Exp. Ther. 322, 529-40.

34. Yanagisawa, K., Sakakibara, Y., Suiko, M., Takami, Y., Nakayama, T., Nakajima,

H., Takayanagi, K., Natori, Y. and Liu, M.C. (1998) cDNA cloning, expression, and

characterization of the human bifunctional ATP sulfurylase/adenosine 5'-

phosphosulfate kinase enzyme. Biosci. Biotechnol. Biochem. 62, 1037-40.

146 35. Hui ,Y. and Liu, M.C. (2015) Sulfation of ritodrine by the human cytosolic

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36. Falany, C.N. and Rohn-Glowacki, K.J. (2013) SULT2B1: unique properties and

characteristics of a hydroxysteroid sulfotransferase family. Drug. Metab. Rev. 45,

388-400.

37. Petrotchenko, E.V., Pedersen, L.C., Borchers, C.H., Tomer, K.B. and Negishi, M.

(2001) The dimerization motif of cytosolic sulfotransferases. FEBS Lett. 490, 39-43.

38. Betts, J.M. and Russell, B.R. (2003) Amino acid properties and consequences of

substitutions in Bioinformatics for Geneticists (Barnes, R.M. and Gray, C.I., eds.) pp.

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Chapter 4

1. Schiffer L, Arlt W, Storbeck KH, Intracrine androgen biosynthesis, metabolism and action revisited, Mol. Cell. Endocrinol. 2017; doi:10.1016/j.mce.2017.08.016

2. Labrie F, DHEA, important source of sex steroids in men and even more in women,

Prog. Brain Res. 2010;182: 97-148.

3. Neunzig J, Bernhardt R, Dehydroepiandrosterone sulfate (DHEAS) stimulates the first step in the biosynthesis of steroid hormones, PloS one. 2014;9: e89727.

147 4. Noordam C, Dhir V, McNelis JC, Schlereth F, Hanley NA, Krone N, Smeitink JA,

Smeets R, Sweep FC, Claahsen-van der Grinten HL, Arlt W, Inactivating PAPSS2 mutations in a patient with premature pubarche, N. Engl. J Med. 2009;360: 2310-2318.

5. Falany CN, Rohn-Glowacki KJ, SULT2B1: unique properties and characteristics of a hydroxysteroid sulfotransferase family, Drug Metab. Rev. 2013;45: 388-400.

6. Vallee M, Mayo W, Le Moal M, Role of pregnenolone, dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging, Brain Res. Rev. 2001;37:

301-312.

7. Falany CN, Enzymology of human cytosolic sulfotransferases, FASEB J. 1997;11:

206-216.

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Man. Florida: CRC Press Inc; 1993, pp. 101-115.

9. Weinshilboum R, Otterness D, Sulfotransferase enzymes. In: Kauffman FC editor.

Handbook of Experimental Pharmacology, vol. 112, Berlin, Heidelberg: Springer-Verlag;

1994, pp. 45-78.

10. Glatt H, Boeing H, Engelke CE, Ma L, Kuhlow A, Pabel U, Pomplun D, Teubner W,

Meinl W, Human cytosolic sulphotransferases: genetics, characteristics, toxicological aspects, Mutat. Res. 2001;482: 27-40.

148 11. Freimuth RR, Wiepert M, Chute CG, Wieben ED, Weinshilboum RM, Human cytosolic sulfotransferase database mining: identification of seven novel genes and pseudogenes, Pharmacogenomics J. 2004;4: 54-65.

12. Her C, Wood TC, Eichler EE, Mohrenweiser HW, Ramagli LS, Siciliano MJ,

Weinshilboum RM, Human hydroxysteroid sulfotransferase SULT2B1: two enzymes encoded by a single chromosome 19 gene, Genomics. 1998;53: 284-295.

13. Falany CN, He D, Dumas N, Frost AR, Falany JL, Human cytosolic sulfotransferase

2B1: isoform expression, tissue specificity and subcellular localization, J. Steroid

Biochem. Mol. Biol. 2006;102: 214-221.

14. Geese WJ, Raftogianis RB, Biochemical characterization and tissue distribution of human SULT2B1, Biochem. Biophys. Res. Commun. 2001;288: 280-289.

15. Otterness DM, Weinshilboum R, Human dehydroepiandrosterone sulfotransferase: molecular cloning of cDNA and genomic DNA, Chem. Biol. Interact. 1994;92: 145-159.

16. Thomae BA, Eckloff BW, Freimuth RR, Wieben ED, Weinshilboum RM, Human sulfotransferase SULT2A1 pharmacogenetics: genotype-to-phenotype studies,

Pharmacogenomics J. 2002;2: 48-56.

17. Hyland PL, Freedman ND, Hu N, Tang ZZ, Wang L, Wang C, Ding T, Fan JH, Qiao

YL, Golozar A, Wheeler W, Yu K, Yuenger J, Burdett L, Chanock SJ, Dawsey SM,

Tucker MA, Goldstein AM, Abnet CC, Taylor PR, Genetic variants in sex hormone metabolic pathway genes and risk of esophageal squamous cell carcinoma,

Carcinogenesis. 2013;34: 1062-1068.

149 18. Levesque E, Laverdiere I, Audet-Walsh E, Caron P, Rouleau M, Fradet Y, Lacombe

L, Guillemette C, Steroidogenic germline polymorphism predictors of prostate cancer progression in the estradiol pathway, Clin. Cancer Res. 2014;20: 2971-2983.

19. Mostaghel EA, Steroid hormone synthetic pathways in prostate cancer, Transl.

Androl. Urol. 2013;2: 212-227.

20. Yang X, Xu Y, Guo F, Ning Y, Zhi X, Yin L, Li X, Hydroxysteroid sulfotransferase

SULT2B1b promotes hepatocellular carcinoma cells proliferation in vitro and in vivo,

PloS one. 2013;8: e60853.

21. Hu L, Yang GZ, Zhang Y, Feng D, Zhai YX, Gong H, Qi CY, Fu H, Ye MM, Cai

PQ, Gao CF, Overexpression of SULT2B1b is an independent prognostic indicator and promotes cell growth and invasion in colorectal carcinoma, Lab Invest. 2015;95: 1005-

1018.

22. Vickman RE, Crist SA, Kerian K, Eberlin L, Cooks RG, Burcham GN, Buhman KK,

Hu CD, Mesecar AD, Cheng L, Ratliff TL, Cholesterol sulfonation enzyme, SULT2B1b, modulates AR and cell growth properties in prostate cancer, Mol. Cancer Res. 2016;14:

776-786.

23. Chen W, Zhou H, Ye L, Zhan B, Overexpression of SULT2B1b Promotes

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150 pharmacogenomics: gene sequence variation and functional genomics, J. Pharmacol.

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25. Yanagisawa K, Sakakibara Y, Suiko M, Takami Y, Nakayama T, Nakajima H,

Takayanagi K, Natori Y, Liu MC, cDNA cloning, expression, and characterization of the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate kinase enzyme, Biosci.

Biotechnol. Biochem. 1998;62: 1037-1040.

26. Hui Y, Liu MC, Sulfation of ritodrine by the human cytosolic sulfotransferases

(SULTs): effects of SULT1A3 genetic polymorphism, Eur. J. Pharmacol. 2015;761: 125-

129.

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33. Betts MJ, Russell RB. Amino acid properties and consequences of substitutions. In:

Barnes MR, Gray IC, editors. Bioinformatics for Geneticists, Chichester, England: John

Wiley & Sons, Ltd.; 2003. pp. 289-316.

34. Heinz L, Kim GJ, Marrakchi S, Christiansen J, Turki H, Rauschendorf MA, Lathrop

M, Hausser I, Zimmer AD, Fischer J, Mutations in SULT2B1 cause autosomal-recessive congenital ichthyosis in humans, Am. J. Hum. Genet. 2017;100: 926-939.

Chapter 5

1. Crosignani, A., Zuin, M., Allocca, M. & Del Puppo, M. (2011) Oxysterols in bile acid metabolism. Clin. Chim. Acta. 412, 2037-45.

2. Bjorkhem, I. & Diczfalusy, U. (2002) Oxysterols: friends, foes, or just fellow passengers? Arterioscler. Thromb Vascu Biol. 22, 734-42.

152 3. Schwarz, M., Russell, D. W., Dietschy, J. M. & Turley, S. D. (2001) Alternate pathways of bile acid synthesis in the cholesterol 7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding. J. Lipid Res. 42,

1594-603.

4. Luu, W., Sharpe, L. J., Capell-Hattam, I., Gelissen, I. C. & Brown, A. J. (2016)

Oxysterols: Old Tale, New Twists. Annu. Rev. Pharmacol. Toxicol. 56, 447-67.

5. Poli, G., Biasi, F. & Leonarduzzi, G. (2013) Oxysterols in the pathogenesis of major chronic diseases. Redox. Biol. 1, 125-30.

6. Bjorkhem, I. (2002) Do oxysterols control cholesterol homeostasis? J. Clin Invest.

110, 725-30.

7. Leoni, V., Masterman, T., Diczfalusy, U., De Luca, G., Hillert, J. & Bjorkhem, I.

(2002) Changes in human plasma levels of the brain specific oxysterol 24S- hydroxycholesterol during progression of multiple sclerosis. Neurosci. Lett. 331, 163-6.

8. Ren, S., Li, X., Rodriguez-Agudo, D., Gil, G., Hylemon, P. & Pandak, W. M. (2007)

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes. Biochem. Biophys. Res. Commun. 360, 802-8.

9. Bjorkhem, I., Andersson, U., Ellis, E., Alvelius, G., Ellegard, L., Diczfalusy, U.,

Sjovall, J. & Einarsson, C. (2001) From brain to bile. Evidence that conjugation and

153 omega-hydroxylation are important for elimination of 24S-hydroxycholesterol

(cerebrosterol) in humans. J Biol. Chem. 276, 37004-10.

10. Fuda, H., Javitt, N. B., Mitamura, K., Ikegawa, S. & Strott, C. A. (2007) Oxysterols are substrates for cholesterol sulfotransferase. J. Lipid Res. 48, 1343-52.

11. Cook, I. T., Duniec-Dmuchowski, Z., Kocarek, T. A., Runge-Morris, M. & Falany,

12. Ren, S. & Ning, Y. (2014) Sulfation of 25-hydroxycholesterol regulates lipid metabolism, inflammatory responses, and cell proliferation. Am. J. Physiol. Endocrinol.

Metab. 306, E123-30.

13. Javitt, N. B., Lee, Y. C., Shimizu, C., Fuda, H. & Strott, C. A. (2001) Cholesterol and hydroxycholesterol sulfotransferases: Identification, distinction from dehydroepiandrosterone sulfotransferase, and differential tissue expression.

Endocrinology. 142, 2978-2984.

14. Segala, G., de Medina, P., Iuliano, L., Zerbinati, C., Paillasse, M. R., Noguer, E.,

Dalenc, F., Payre, B., Jordan, V. C., Record, M., Silvente-Poirot, S. & Poirot, M. (2013)

5,6-Epoxy-cholesterols contribute to the anticancer pharmacology of tamoxifen in breast cancer cells. Biochem. Pharmacol. 86, 175-89.

154 15. Berrodin, T. J., Shen, Q., Quinet, E. M., Yudt, M. R., Freedman, L. P. & Nagpal, S.

(2010) Identification of 5alpha, 6alpha-epoxycholesterol as a novel modulator of liver X receptor activity, Mol. Pharmacol. 78, 1046-58.

16. Cha, J. Y. & Kim, Y. B. (2012) Sulfated oxysterol 25HC3S as a therapeutic target of non-alcoholic fatty liver disease. Metabolism. 61, 1055-7.

17. Strott, C. A. (2002) Sulfonation and molecular action, Endocr Rev. 23, 703-32.

18. Her, C., Wood, T. C., Eichler, E. E., Mohrenweiser, H. W., Ramagli, L. S., Siciliano,

M. J. & Weinshilboum, R. M. (1998) Human hydroxysteroid sulfotransferase SULT2B1: two enzymes encoded by a single chromosome 19 gene. Genomics. 53, 284-95.

19. Hyland, P. L., Freedman, N. D., Hu, N., Tang, Z. Z., Wang, L., Wang, C., Ding, T.,

Fan, J. H., Qiao, Y. L., Golozar, A., Wheeler, W., Yu, K., Yuenger, J., Burdett, L.,

Chanock, S. J., Dawsey, S. M., Tucker, M. A., Goldstein, A. M., Abnet, C. C. & Taylor,

P. R. (2013) Genetic variants in sex hormone metabolic pathway genes and risk of esophageal squamous cell carcinoma. Carcinogenesis. 34, 1062-8.

20. Levesque, E., Laverdiere, I., Audet-Walsh, E., Caron, P., Rouleau, M., Fradet, Y.,

Lacombe, L. & Guillemette, C. (2014) Steroidogenic germline polymorphism predictors of prostate cancer progression in the estradiol pathway. Clin. Cancer Res. 20, 2971-83.

155 21. Yang, X., Xu, Y., Guo, F., Ning, Y., Zhi, X., Yin, L. & Li, X. (2013) Hydroxysteroid sulfotransferase SULT2B1b promotes hepatocellular carcinoma cells proliferation in vitro and in vivo. PloS one. 8, e60853.

22. Hu, L., Yang, G. Z., Zhang, Y., Feng, D., Zhai, Y. X., Gong, H., Qi, C. Y., Fu, H.,

Ye, M. M., Cai, Q. P. & Gao, C. F. (2015) Overexpression of SULT2B1b is an independent prognostic indicator and promotes cell growth and invasion in colorectal carcinoma. Lab. Invest. 95, 1005-18.

23. Vickman, R. E., Crist, S. A., Kerian, K., Eberlin, L., Cooks, R. G., Burcham, G. N.,

Buhman, K. K., Hu, C. D., Mesecar, A. D., Cheng, L. & Ratliff, T. L. (2016) Cholesterol

Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate

Cancer. Mol. Cancer Res. 14, 776-86.

24. Ji, Y., Moon, I., Zlatkovic, J., Salavaggione, O. E., Thomae, B. A., Eckloff, B. W.,

Wieben, E. D., Schaid, D. J. & Weinshilboum, R. M. (2007) Human hydroxysteroid sulfotransferase SULT2B1 pharmacogenomics: gene sequence variation and functional genomics. J. Pharmaco. Exp. Ther. 322, 529-40.

25. Yanagisawa, K., Sakakibara, Y., Suiko, M., Takami, Y., Nakayama, T., Nakajima,

H., Takayanagi, K., Natori, Y. & Liu, M. C. (1998) cDNA cloning, expression, and characterization of the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate kinase enzyme. Biosci. Biotechnol. Biochem. 62, 1037-40.

156 26. Hui, Y. & Liu, M. C. (2015) Sulfation of ritodrine by the human cytosolic sulfotransferases (SULTs): Effects of SULT1A3 genetic polymorphism. Eur. J.

Pharmacol. 761, 125-9

27. Lee, K. A., Fuda, H., Lee, Y. C., Negishi, M., Strott, C. A. & Pedersen, L. C. (2003)

28. Li, X., Pandak, W. M., Erickson, S. K., Ma, Y., Yin, L., Hylemon, P. & Ren, S.

(2007) Biosynthesis of the regulatory oxysterol, 5-cholesten-3beta,25-diol 3-sulfate, in hepatocytes. J. Lipid Res. 48, 2587-96.

29. Bai, Q., Xu, L., Kakiyama, G., Runge-Morris, M. A., Hylemon, P. B., Yin, L.,

30. Meloche, C. A. & Falany, C. N. (2001) Expression and characterization of the human

3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b). J. Steroid

Biochem. Mol. Biol. 77, 261-9.

157 31. Petrotchenko, E. V., Pedersen, L. C., Borchers, C. H., Tomer, K. B. & Negishi, M.

(2001) The dimerization motif of cytosolic sulfotransferases. FEBS Lett. 490, 39-43.

32. Fuda, H., Lee, Y. C., Shimizu, C., Javitt, N. B. & Strott, C. A. (2002) Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon

1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase. J. Biol. Chem. 277, 36161-6.

33. Betts, J. M. & Russell, B. R. (2003) Amino Acid Properties and Consequences of

Substitutions in Bioinformatics for Geneticists (Barnes, R. M. & Gray, C. I., eds) pp. 289-

316, John Wiley & Sons, Ltd., Chichester, England.

34. Heinz, L., Kim, G. J., Marrakchi, S., Christiansen, J., Turki, H., Rauschendorf, M.

A., Lathrop, M., Hausser, I., Zimmer, A. D. & Fischer, J. (2017) Mutations in SULT2B1

Cause Autosomal-Recessive Congenital Ichthyosis in Humans. Am. J. Hum. Genet. 100,

926-939

Chapter 6

1. Kallionpaa, R. A., Jarvinen, E. & Finel, M. (2015) Glucuronidation of estrone and

16alpha-hydroxyestrone by human UGT enzymes: The key roles of UGT1A10 and

UGT2B7, J Steroid Biochem Mol Biol. 154, 104-11.

158 2. Fernandez, S. V. & Russo, J. (2010) Estrogen and xenoestrogens in breast cancer,

Toxicol Pathol. 38, 110-22.

3. Pai, T. G., Sugahara, T., Suiko, M., Sakakibara, Y., Xu, F. & Liu, M. C. (2002)

4. Toppari, J., Larsen, J. C., Christiansen, P., Giwercman, A., Grandjean, P., Guillette, L.

J., Jr., Jegou, B., Jensen, T. K., Jouannet, P., Keiding, N., Leffers, H., McLachlan, J. A.,

Meyer, O., Muller, J., Rajpert-De Meyts, E., Scheike, T., Sharpe, R., Sumpter, J. &

Skakkebaek, N. E. (1996) Male reproductive health and environmental xenoestrogens,

Environmental health perspectives. 104 Suppl 4, 741-803.

5. Bonefeld-Jorgensen, E. C., Long, M., Hofmeister, M. V. & Vinggaard, A. M. (2007)

Endocrine-disrupting potential of bisphenol A, bisphenol A dimethacrylate, 4-n- nonylphenol, and 4-n-octylphenol in vitro: new data and a brief review, Environmental health perspectives. 115 Suppl 1, 69-76.

6. Malkowicz, S. B. (2001) The role of diethylstilbestrol in the treatment of prostate cancer, Urology. 58, 108-13.

7. Ritter, J. K. (2000) Roles of glucuronidation and UDP-glucuronosyltransferases in xenobiotic bioactivation reactions, Chem Biol Interact. 129, 171-93.

159 8. Metzler, M. (1981) The metabolism of diethylstilbestrol, CRC Crit Rev Biochem. 10,

171-212.

9. Suiko, M., Sakakibara, Y. & Liu, M. C. (2000) Sulfation of environmental estrogen- like chemicals by human cytosolic sulfotransferases, Biochemical and biophysical research communications. 267, 80-4.

10. Falany, C. & Roth, J. (1993) Properties of human cytosolic sulfotransferases involved in drug metabolism in Human Drug Metabolism: From Molecular Biology to

Man (EH, J., ed) pp. 101-115, CRC Press, Boca Raton, FL.

11. weinshilboum, R. & Otterness, D. (1994) sulfotransferase enzymes in conjugation- deconjugation reactions in Drug Metabolism and Toxicity (Kaufmann, F., ed) pp. 45-78,

Springer-Verlag, Berlin.

12. Neunzig, J. & Bernhardt, R. (2014) Dehydroepiandrosterone Sulfate (DHEAS)

Stimulates the First Step in the Biosynthesis of Steroid Hormones, PloS one. 9.

13. Fuda, H., Lee, Y. C., Shimizu, C., Javitt, N. B. & Strott, C. A. (2002) Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon

1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase, The Journal of biological chemistry. 277, 36161-6.

160 14. Falany, C. N., He, D., Dumas, N., Frost, A. R. & Falany, J. L. (2006) Human cytosolic sulfotransferase 2B1: isoform expression, tissue specificity and subcellular localization, J Steroid Biochem Mol Biol. 102, 214-21.

15. Geese, W. J. & Raftogianis, R. B. (2001) Biochemical characterization and tissue distribution of human SULT2B1, Biochemical and biophysical research communications.

288, 280-9.

16. Falany, J. L., Pilloff, D. E., Leyh, T. S. & Falany, C. N. (2006) Sulfation of raloxifene and 4-hydroxytamoxifen by human cytosolic sulfotransferases, Drug metabolism and disposition: the biological fate of chemicals. 34, 361-8.

17. Yanagisawa, K., Sakakibara, Y., Suiko, M., Takami, Y., Nakayama, T., Nakajima,

18. Meloche, C. A. & Falany, C. N. (2001) Expression and characterization of the human

3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b), J Steroid Biochem

Mol Biol. 77, 261-9.

19. Strott, C. A. (2002) Sulfonation and molecular action, Endocr Rev. 23, 703-32.

161 20. Reinen, J., Vriese, E., Glatt, H. & Vermeulen, N. P. (2006) Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase, Analytical biochemistry. 357, 85-92.

21. Gamage, N. U., Duggleby, R. G., Barnett, A. C., Tresillian, M., Latham, C. F.,

Liyou, N. E., McManus, M. E. & Martin, J. L. (2003) Structure of a human carcinogen- converting enzyme, SULT1A1. Structural and kinetic implications of substrate inhibition,

The Journal of biological chemistry. 278, 7655-62.

162