A Dissertation entitled Investigation of Single Nucleotide Genetic Polymorphisms of the Human SULT2B1 Gene: Functional Characterization of SULT2B1b Allozymes by 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 Copyright 2018, Fatemah Alherz 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 by 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. v 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