Sialylated Keratan Sulfate Chains are Ligands for Siglec-8 in Human Airways by Ryan Porell A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland September 2018 © 2018 Ryan Porell All Rights Reserved ABSTRACT Airway inflammatory diseases are characterized by infiltration of immune cells, which are tightly regulated to limit inflammatory damage. Most members of the Siglec family of sialoglycan binding proteins are expressed on the surfaces of immune cells and are immune inhibitory when they bind their sialoglycan ligands. When Siglec-8 on activated eosinophils and mast cells binds to its sialoglycan ligands, apoptosis or inhibition of mediator release is induced. We identified human airway Siglec-8 ligands as sialylated and 6’-sulfated keratan sulfate (KS) chains carried on large proteoglycans. Siglec-8- binding proteoglycans from human airways increase eosinophil apoptosis in vitro. Given the structural complexity of intact proteoglycans, target KS chains were isolated from airway tissue and lavage. Biological samples were extensively proteolyzed, the remaining sulfated glycan chains captured and resolved by anion exchange chromatography, methanol-precipitated then chondroitin and heparan sulfates enzymatically hydrolyzed. The resulting preparation consisted of KS chains attached to a single amino acid or a short peptide. Purified KS chains were hydrolyzed with either hydrochloric acid or trifluoroacetic acid to release acidic and neutral sugars, respectively, followed by DIONEX carbohydrate analysis. To isolate Siglec-8-binding KS chains, purified KS chains from biological samples were biotinylated at the amino acid, resolved by affinity and/or size- exclusion chromatography, the resulting fractions immobilized on streptavidin microwell plates, and probed for binding of Siglec-8-Fc. Siglec-8 affinity chromatography of the tagged KS chains was performed on a nickel column derivatized with a pentameric construct of Siglec-8. Most of the KS chains flowed through the column, whereas Siglec- 8 binding was retained through 150 mM NaCl and eluted with 1.5 M NaCl. Based on size ii separation, Siglec-8-binding KS chains are high molecular weight (>40 kD). Similarly, purified KS chains were cleaved with keratanase I, hydrazine-biotin tagged on their reducing ends, size-separated, and resulting fractions immobilized on streptavidin microwell plates. Keratanase I-cleaved KS chains showed a Siglec-8-binding peak at ~2 kD, indicating a terminal oligosaccharide epitope resistant to keratanase I that retained robust Siglec-8 binding. These data reveal a minor fraction of terminally sialylated KS chains as the Siglec-8 ligands in human airway tissues and secretions. Advisor: Dr. Ronald Schnaar Reader: Dr. Yuan Chuan Lee iii ACKNOWLEDGEMENTS This milestone in my scientific career and in my personal development was an endeavor that was made possible only with the assistance, support, and encouragement of my many colleagues, friends, and family members. I would like to start by thanking those collaborators who assisted me with training and sample analysis as well as being wonderful friends and mentors during this journey. I am fortunate to have met and worked with Drs. Corwin Nycholat, Daniela Janevska Carroll, Jeremy O’Sullivan, Simone Kurz, Kazuhiro Aoki, Bruce Bochner, Jean Kim, Jim Paulson, Michael Tiemeyer, Robert Linhardt, Shukti Chakravarti, Kevin Yarema, Natasha Zachara, Yuan Chuan Lee and Anthony Leung. Each of you have been of tremendous help throughout my doctoral career and have been there when I needed mentorship, resources and technologies. Many of you have also been researchers that have analyzed samples important to the research presented in this thesis and for that I am immensely grateful. Also, a special acknowledgement to Dr. Jim Stivers and Dr. Steve Rokita for acknowledging my interest in chemical biology and directing me towards the chemistry-biology interface program. This has been the most rewarding experience for me and I am so thankful that I was accepted into the CBI program. I was lucky to be able to work with an amazing group of colleagues in the Schnaar lab. I would like to thank Anabel Gonzalez-Gil Alvarenga for all of her guidance, protocols, buffers, and light-hearted attitude which was a blessing during stressful times. It was wonderful working side-by-side with you on this project and we have been able to accomplish a lot of astounding work in our field through this combined effort. I would like to thank Steve M. Fernandes for his friendship, mentorship, support, iv and combined effort on the Siglec-9 project. Thank you for ordering each of the reagents for the lab and keeping everything running smoothly for us while still pumping out some bench work for the lab and towards our projects. All of your organizational and managerial efforts allowed me to stay focused on my thesis and made it a lot easier to have the tools I needed to keep going. Thank you to August Li for being a great friend, a critical thinker, and a hardworking colleague who is always there to help out in any way possible. You have been an excellent addition to the lab and I appreciate your honesty and encouragement. Thank you Steve Arbitman for all of your support, friendship, and intellectualism. It was great having the opportunity to discuss ideas with you, whether scientific, political, abstract, or whatever may have come up. Also, thank you for helping to keep the lab running smoothly, for taking care of all of the cell cultures, for ordering reagents, and for providing some divergence from the normal day to day lab work. I am truly going to miss having a peer that shares as many interests and similar views on life. I would like to thank my wonderful wife Cassandra for all of her love, understanding, and support throughout my graduate school career. I know it has been very stressful and challenging, especially with getting married and buying a house along with multiple moves throughout my graduate career, but I truly appreciate your continued encouragement. You have been my rock during this whole process and have always showed me how proud you are of me and kept me pushing forward even when things got rough. I am so lucky to have you in my life and it is amazing how far we have come since we first met. I love you with all of my heart. I would like to thank my parents and all of the family members who cheered me on and helped motivate me to keep pushing on. Mom and dad, you have been beyond v supportive and I really appreciate you taking the time to listen to me talk about my research even when half of it probably sounded like a different language. It brings me great joy and honor to see how proud you are of me and I am lucky to have had you there to help me grow throughout my life and to show me how to be hardworking, respectful, responsible, and ultimately self-aware which has allowed me to progress so far. Thank you for all you have done for me throughout my entire life; it hasn’t gone unappreciated. Finally, I would like to acknowledge and thank Dr. Ronald Schnaar for being an amazing mentor and for advising me towards completion of this dissertation. I could not have asked for a more complementary mentor and I am grateful to have been trained by someone with such a wealth of knowledge, experience, and patience. Even before I applied to Hopkins I knew I wanted to work in your lab and that was my goal since I got accepted. It’s such an interesting coincidence that I first rotated with your graduate mentor Y.C. Lee whom prepared me for your lab and became another resource for completing my thesis work. I am so appreciative that you took the chance to let me rotate in your lab and ultimately join your lab even after you told me multiple times that you didn’t have the funding at the moment. I cannot say enough great things about you. You taught me how to think critically about science and data, how to write publications and posters, how to handle all kinds of situations inside and outside of the work environment, how to become the best presenter I could be, and taught me many valuable lessons to accelerate my development as a scientist. If I had to pick one trait that I wish I could mirror it would be your ability to select your verbiage to most accurately convey your thoughts. I look forward to collaborating with you in the future and I appreciate all that you have done for me during these past three and a half years. vi TABLE OF CONTENTS Abstract………………………………………………………………………………ii-iii Acknowledgements…………………………………………………………………...iv-vi Table of Contents……………………………………………………………………vii-ix List of Figures………………………………………………………………………...x-xii List of Tables…………………………………………………………………………...xiii Chapter 1: Introduction…………………………………………………………………1 1.1 Glycans as Immunoregulatory Molecules………………………………….1 1.2 Eosinophilic Airway Inflammation……….………………………………...2 1.2.1 Eosinophilic Airway Inflammation Prevalence and Pathogenesis..2 1.3 Siglec-8 and the Siglec Family of Lectins………….………………………..4 1.3.1 Siglec Family of Lectins…….………………………………………4 1.3.2 Siglec-8 Receptor……………………………………………………6 1.4 Human and Mouse Eosinophil Siglecs……………………………………...7 1.4.1 Murine Siglec-8 Functional Paralog: Siglec-F……………………...7 1.4.2 Siglec-F Proof-of-Concept…………………………………………..7 1.4.3 Published Data on Siglec-8 and Siglec-F Ligand Specificity……….9 1.4.4 Published Data on Siglec-8 and Siglec-F Ligand Expression……..12 1.4.5 Characterization of Siglec-8 Human Airway Ligands……………..14 Chapter