CORTICOSTEROID-BINDING GLOBULIN (CBG): DEFICIENCIES AND THE ROLE OF CBG IN DISEASE PROCESSES by LESLEY ANN HILL B.Sc. University of Western Ontario, 2011 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Reproductive and Developmental Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2017 © Lesley Ann Hill, 2017 Abstract Corticosteroid-binding globulin (CBG, SERPINA6) is a serine protease inhibitor family member produced by hepatocytes. Plasma CBG transports biologically active glucocorticoids, determines their bioavailability to target tissues and acts as an acute-phase negative protein with a role in the delivery of glucocorticoids to sites of inflammation. A few CBG-deficient individuals have been identified, yet the clinical significance of this remain unclear. In this thesis, I investigated 1) the biochemical consequences of naturally occurring single nucleotide polymorphisms in the SERPINA6 gene, 2) the role of human CBG during infections and acute inflammation and 3) CBG as a biomarker of inflammation in rats. A comprehensive analysis of functionally relevant naturally occurring SERPINA6 SNP revealed 11 CBG variants with abnormal production and/or function, diminished responses to proteolytic cleavage of the CBG reactive center loop (RCL) or altered recognition by monoclonal antibodies. In a genome-wide association study, plasma cortisol levels were most closely associated with SERPINA6 SNPs and plasma CBG-cortisol binding capacity. These studies indicate that human CBG variants need to be considered in clinical evaluations of patients with abnormal cortisol levels. In addition, I obtained evidence that discrepancies in CBG values obtained by the 9G12 ELISA compared to CBG binding capacity and 12G2 ELISA are likely due to differential N-glycosylation rather than proteolysis, as recently reported. In relation to human inflammation, the bacterial protease Pseudomonas aeruginosa elastase was shown to cleave the RCL and α-2-macroglobulin specifically inhibited this. ICU patients with a variety of illnesses had significantly reduced plasma CBG levels, with the lowest levels in individuals with severe inflammation. Similar results were observed in a rodent model of inflammation, where significant reductions in plasma CBG levels were associated with CBG proteolysis and the down-regulation of hepatic Serpina6 ii expression. In addition, lower baseline plasma CBG levels in Harlan Sprague Dawley rats were linked with an increased susceptibility to inflammation. Together, the human and rodent studies highlight the importance of CBG in inflammatory reactions and suggest that CBG is a useful biomarker of inflammation onset and severity. iii Lay summary Corticosteroid-binding globulin (CBG) is a plasma protein that is known to bind and transport the steroid hormone cortisol within human blood. Cortisol controls the growth and development of our organs, helps us deal with stress, fights off infections, and assists in recovery after illness. In various disease states, CBG delivers and releases anti-inflammatory cortisol to sites of inflammation. A few individuals with DNA variations that cause deficits in CBG production and/or function have been identified, but the clinical significance is unclear. The objective of this research was to: (1) identify new CBG variants, and (2) investigate the role of CBG during inflammation. These studies have demonstrated how CBG variants influence the biological actions of cortisol and lay the foundation for further clinical investigations. Additionally, the results demonstrate that changes in CBG levels play a vital role in our bodies’ response to inflammation. iv Preface A version of chapter 3.2 has been published. Hill LA, Vassiliadi DA, Simard M, Pavlaki A, Perogamvros I, Hadjidakis D, Hammond GL. (2012). Two different corticosteroid-binding globulin variants that lack cortisol-binding activity in a Greek woman. Journal of Clinical Endocrinology and Metabolism. 97(11): 4260-7. I was responsible for experimental design, data acquisition and manuscript preparation. Dr. Vassiliadi was responsible for the acquisition of clinical endocrinology data (section 3.2.1.1). Dr. Simard produced the recombinant CBG variant proteins. All co-authors edited the manuscript. A version of chapter 3.3 has been published in an article of which I am a co-first author. Simard M*, Hill LA*, Lewis JG, Hammond GL. (2014). Naturally Occurring Mutations of Human Corticosteroid-binding Globulin. Journal of Clinical Endocrinology and Metabolism. 100(1):E129-139. Together, Dr. Simard and I were responsible for the experimental design, data acquisition and manuscript preparation under the guidance of Dr. Hammond. I purified CBG variants using FPLC and biochemically characterized their steroid binding properties and measured CBG protein levels by ELISAs (sections 3.3.1.1 and 3.3.1.3). Dr. Simard produced the proteins in CHO cells, characterized their production and secretion (section 3.3.1.2) and completed the proteolytic digests (section 3.3.1.4). All co-authors edited the manuscript. A portion of the work presented in chapter 3.4 is used with permission from a published article of which I am an author. Bolton JL, Hayward C, Direk N, Lewis JG, Hammond GL, Hill LA, et al. (2014). Genome wide association identifies common variants at the SERPINA6/SERPINA1 locus influencing plasma cortisol and corticosteroid binding globulin. PLoS Genetics. 10(7): v e1004474. For the manuscript, I completed CBG-cortisol binding capacity measurements described in section 3.4.1.2. Genetic investigations (section 3.4.1.1), plasma cortisol measurements (total and free) and CBG ELISA values (section 3.4.1.2) were completed by co- authors. Data presented in section 3.4.1.3 is part of an ongoing research study that I am conducting under Dr. Hammond’s guidance. A portion of the work presented in chapter 4.2 is from a published article of which I am an author. Simard M, Hill LA, Underhill CM, Keller BO, Villanueva I, Hancock RE, Hammond GL. (2014). Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin. Endocrinology. 155(8): 2900-2908. I completed the initial bacterial culture screen (section 4.2.1.1), time-course incubations (section 4.2.1.2), PAE characterization and Protease IV/TLCK inhibitor experiments (section 4.2.1.4). Dr. Simard completed the pH and protease inhibitor assays (section 4.2.1.2), as well as the PAE deficient strain experiments (section 4.2.1.5). Caroline Underhill was responsible for all FPLC purifications and Dr. Keller obtained and analyzed all mass spectrometry data (section 4.2.1.3). Pseudomonas aeruginosa media was kindly provided by Dr. Hancock. The manuscript was largely prepared by Dr. Simard. All co-authors edited the manuscript. Work presented in chapter 4.3 is part of an ongoing research study. Dr. Vassiliadi collected the ICU plasma samples and I conducted all experiments under Dr. Hammond’s guidance. A version of chapter 5.2 is used with permission from a published article of which I am an author. Bodnar TS, Hill LA, Taves MD, Yu W, Soma KK, Hammond GL, Weinberg J. (2015). vi Colony specific differences in endocrine and immune responses to an inflammatory challenge in female Sprague Dawley rats. Endocrinology. 156(12):4604-17. I was responsible for the acquisition and manuscript preparation of all CBG related data. Dr. Bodnar conducted the animal experiments (section 5.2.1.1), measured plasma corticosterone levels (section 5.2.1.2) and led the preparation of the manuscript. A version of chapter 5.3 has been published. Hill LA, Bodnar TS, Weinberg J, Hammond GL. (2016). Corticosteroid-Binding Globulin is a biomarker of inflammation onset and severity in female rats. Journal of Endocrinology. 230(2):215-225. Dr. Hammond and I were responsible for the experimental design. Animal experiments were completed by Dr. Bodnar and myself. Dr. Bodnar measured plasma cytokine levels (section 5.3.1.5) and I analyzed the data. I completed all of the remaining experiments and prepared the manuscript. All co-authors edited the manuscript. Unless otherwise stated, all tables and figures consist of work completed exclusively by myself. vii Table of Contents Abstract ........................................................................................................................................... ii Lay summary ................................................................................................................................. iv Preface ..............................................................................................................................................v Table of Contents ......................................................................................................................... viii List of Tables ............................................................................................................................... xvi List of Figures ............................................................................................................................. xvii List of Abbreviations .....................................................................................................................xx Acknowledgements .................................................................................................................... xxiii Chapter 1: Introduction ..................................................................................................................