A Dissertation entitled Functional Genomic Studies On The Genetic Polymorphisms Of The Human Cytosolic Sulfotransferase 1A3 (SULT1A3) by Ahsan Falah Hasan Bairam 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 Hassoun, Committee Member ________________________________________ Dr. Zahoor Shah, Committee Member ________________________________________ Dr. Caren Steinmiller, Committee Member ________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2018 Copyright 2018, Ahsan Falah Hasan Bairam 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 Functional Genomic Studies On The Genetic Polymorphisms Of The Human Cytosolic Sulfotransferase 1A3 (SULT1A3) by Ahsan Falah Hasan Bairam Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental Therapeutics (Pharmacology/Toxicology) The University of Toledo May 2018 Abstract Previous studies have demonstrated the involvement of sulfoconjugation in the metabolism of catecholamines and serotonin (5-HT), as well as a wide range of xenobiotics including drugs. The study presented in this dissertation aimed to clarify the effects of coding single nucleotide polymorphisms (cSNPs) of the human SULT1A3 and SULT1A4 genes on the enzymatic characteristics of the sulfation of catecholamines, 5- HT, and selected drugs by SULT1A3 allozymes. Following a comprehensive search of different SULT1A3 and SULT1A4 genotypes, thirteen non-synonymous (missense) cSNPs of SULT1A3/SULT1A4 were identified. cDNAs encoding the corresponding SULT1A3 allozymes, packaged in pGEX-2T vector were generated by site-directed mutagenesis. Recombinant SULT1A3 allozymes were bacterially expressed and affinity-purified. iii Purified SULT1A3 allozymes were found to exhibit differential sulfating activities toward dopamine (DA), epinephrine (EP), norepinephrine (NE), 5-HT, acetaminophen (APAP), morphine, tapentadol, O-desmethyl tramadol (O-DMT), phenylephrine, and salbutamol, in comparison to the wild-type enzyme. Kinetic analyses further demonstrated differences in substrate affinity (as reflected by Km) and catalytic ativity (as reflected by Vmax) of different SULT1A3 allozymes. Collectively, the findings made provided useful information relevant to the differential metabolism of above-mentioned endogenous and xenobiotic compounds. Such information may eventually shed light on the correlation of particular SULT1A3/SULT1A4 genotypes to neuropathological disorders associated with abnormal levels of the monoamines that act as substrates for SULT1A3. Furthermore, these results obtained may in the future aid in designing personalized regimens of relevant drugs in order to optimize their efficacy and mitigate their adverse effects for individuals with distinct SULT1A3/SULT1A4 genotypes. iv Acknowledgements I would like to express my appreciation and sincere thanks to Dr. Ming-Cheh Liu for being a wonderful advisor, who has always been available for help, excellent guidance, and support. I attribute the level of my doctorate degree to his encouragement, and without him this thesis would not have been completed or written. I would also like to thank my committee members: Dr. Ezdihar Hassoun, Dr. Zahoor Shah, and Dr. Caren Steinmiller for their time, encouragement and insightful comments. I owe my deepest gratitude to my wife and my brother, who greatly supported me and my aspirations and dreams. To all my lab mates, especially, Dr. Katsuhisa Kurogi, thank you for listening and supporting me through this entire process. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables…. ............................................................................................................... xii List of Figures .................................................................................................................. xiv List of Abbreviations ...................................................................................................... xvii List of Symbols ................................................................................................................ xix 1. Introduction........................................................ ..............................................................1 1.1 Overview of Metabolism ..................................................................................1 1.2 Phases of Metabolism .......................................................................................2 1.2.1. Phase I Reactions ...................................................................2 1.2.2. Phase II Reactions..................................................................3 1.2.3. Phase III transporters.............................................................4 1.3. Sulfoconjugation...............................................................................................4 1.4. Sulfotransferases...............................................................................................6 1.4.1. SULT1....................................................................................9 1.4.2. SULT2..................................................................................11 1.4.3. SULT4..................................................................................12 1.4.4. SULT6.................................................................................13 vi 1.5. Genetic variations...........................................................................................13 1.6. SULT1A3........................................................................................................17 1.6.1. Role of human SULT1A3 in the sulfation of endogenous Substrates......................................................................................18 1.6.2. Role of SULT1A3 in the sulfation of xenobiotics...............19 1.6.3. Role of SULT1A3 in the sulfation of food compounds.......20 1.6.4. Pathophysiological association and etiological role of SULT1A3.......................................................................................20 2. Materials and Methods ...................................................................................................24 2.1. Materials.........................................................................................................24 2.2. Methods...........................................................................................................25 2.2.1. Database search....................................................................25 2.2.2. Generation of SULT1A3 cDNAs coding for SULT1A3 allozymes.......................................................................................26 2.2.3. Expression and purification of recombinant SULT1A3 allozymes.......................................................................................34 2.2.4. Analysis of differential sulfating activities of SULT1A3 allozymes toward endogenous and exogenous compounds...........36 2.2.5. Analysis of differential sulfating activities of SULT1A3 allozymes toward xenobiotics........................................................37 2.2.6. Statistical analysis................................................................37 3. Sulfation of Catecholamines and Serotonin by SULT1A3 Allozymes.........................38 3.1. Introduction.....................................................................................................40 vii 3.2. Materials and Methods....................................................................................42 3.2.1. Materials..............................................................................42 3.2.2. Methods................................................................................43 3.2.2.1. Database search.....................................................43 3.2.2.2. Generation, expression, and purification of SULT1A3 allozymes.........................................................43 3.2.2.3. Enzymatic assay....................................................44 3.2.2.4. Statistical analysis.................................................44 3.3. Results.............................................................................................................45 3.3.1. Analysis of human SULT1A3 and SULT1A4 single nucleotide polymorphisms.............................................................45 3.3.2. Expression and purification of recombinant human SULT1A3 allozymes.....................................................................48 3.3.3. Enzymatic characterization of the SULT1A3 allozymes....48 3.3.4. Kinetic Analyses..................................................................55 3.4. Discussion.......................................................................................................61 4.