Sialylated Keratan Sulfate Chains Are Ligands for Siglec-8 in Human Airways

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

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

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

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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.

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 2: The Physiologically-relevant Human Airway Siglec-8 Ligand…………20 2.1 Materials and Methods………………………………………….………….22 2.1.1 Siglec-8 Lectin Histochemical Overlay……………………………22 2.1.2 Anti-Aggrecan Immunofluorescence ……………………………...23 2.1.3 Extraction and Isolation of Siglec-8 Nasal Lavage Ligands……….24 2.2 Anti-Aggrecan Immunofluorescence……………………………………...26

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2.2.1 Aggrecan Found in Cartilage but not Mucosal Cells………………26 2.2.2 Aggrecanase I (ADAMTS-4) Sensitivity of Siglec-8 Ligands…….27 2.2.3 Nasal Lavage Extracted Siglec-8 Ligands…………………………29 2.3 Conclusions and Remarks………………………………………………….32 Chapter 3: Keratan Sulfate Chains in Human Airways………..….………………...34 3.1 Materials and Methods…………………………….…………….…………35 3.1.1 Pronase Digestion of Human Airway Tissue and Nasal Lavage ….35 3.1.2 Anion Exchange Chromatography……………………….………...36 3.1.3 Methanol Precipitation………………………………….………….36 3.1.4 Removal of Non-KS Glycosaminoglycans………………………...37 3.1.5 Neocuproine Reducing Sugar Assay………………………………38 3.1.6 Fluorometric Sialic Acid Quantification…………………………...40 3.1.7 KS Carbohydrate Compositional Analysis………………………...41 3.2 Analysis of Purified Keratan Sulfate Chains……………………………..42 3.2.1 Neocuproine Reducing Sugar Assay Data…………………………42 3.2.2 Fluorometric Sialic Acid Quantification …………………………..45 3.2.3 Carbohydrate Analysis on DIONEX………………………………46 3.3 Conclusions and Remarks………………………………………………….51 Chapter 4: Keratan Sulfate Chain Neoglycolipid Semi-synthesis….………………..53 4.1 Materials and Methods……………………………………………………..53 4.1.1 Conjugation of Azido Linker to Keratan Sulfate…………………..53 4.1.2 Synthesis of Dibenzocyclooctyne (DBCO)-Lipid…………………55 4.1.3 Anion Exchange Separation of Siglec-8-binding KS……………...55 4.1.4 Attachment of Azido-KS to DBCO-Lipid…………………………56 4.1.5 PVDF Dot-blot Analysis…………………………………………...56 4.1.6 Neoglycolipid Array Binding Analysis……………………………56 4.2 Keratan Sulfate Neoglycolipid Synthesis…………………….…….……...58 4.2.1 Keratan Sulfate Chains Linker Reaction…………………………...58 4.2.2 DBCO and DSPE Lipid Reaction………………………………….59 4.2.3 Keratan Sulfate Neoglycolipid Reaction…………………………..61

viii

4.2.4 Analysis of Keratan Sulfate Neoglycolipids……………………….61 4.3 Conclusions and Remarks………………………………………………….63 Chapter 5: Biotinylation of Keratan Sulfate Chains…………………………………65 5.1 Materials and Methods……………………………………………………..65 5.1.1 Biotinylation Reaction……………………………………………..65 5.1.2 Siglec-8-COMP Affinity Chromatography………………………...66 5.1.3 KS-Biotin Siglec-8-Fc Binding ELISA……………………………67 5.1.4 KS-Biotin Eosinophil Apoptosis Functional Assay………………..68 5.1.5 Size Exclusion Chromatography Analysis…..……………………..69 5.2 KS-Biotinylation and Isolation of Siglec-8 Binding KS……………….….70 5.2.1 KS-Biotin Reaction………………………………………………...70 5.2.2 Siglec-8-COMP Affinity Chromatography………………………...71 5.2.3 Affinity Column Fraction Analysis………………………………..72 5.2.4 Eosinophil Apoptosis Functional Assay…………………………...76 5.2.5 Size Exclusion Chromatography of Siglec-8-Binding KS-Biotin…77 5.3 Conclusions and Remarks………………………………………………….82 Chapter 6: Revisiting Siglec-8 and Siglec-F Ligands………………………………...84 6.1 Materials and Methods……………………………………………………..85 6.1.1 Siglec-F Overlay of Siglec-8 Ligands……………………………...85 6.1.2 Sensitive of Siglec-F Binding to Keratanase Enzymes…………….86 6.2 Siglec Cross-reactivity Analysis…………………………………………....86 6.2.1 Siglec-8 Tracheal Ligands Cross-reactivity with Siglec-F………...86 6.2.2 Siglec-F Ligands Sensitivity to Keratanase Enzymes……………..88 6.3 Conclusions and Remarks………………………………………………….89 Chapter 7: Conclusions and Discussion……………………………………………….91 References……………………………………………………………………………97-99 Curriculum Vitae…………………………………………………………………100-106

LIST OF FIGURES

Figure 1.1. Domain structures of the known Siglecs in humans and mice……………….5

Figure 1.2. Symbol nomenclature for glycan structures………………………………...10

Figure 1.3. Siglec-8-Fc overlay of a CFG glycan microarray…………………………..11

Figure 1.4. Binding of Siglec-Fc chimeras to custom glycolipid array…….…………...11

Figure 1.5. Siglec overlay of human and mouse trachea cross sections………………...13

Figure 1.6. Three distinct size classes of Siglec-8 ligands from human trachea………. 15

Figure 1.7. Aggrecan proteoglycan structure……………………………………………16

Figure 1.8. General schematic structures of KS and CS chains…………………………16

Figure 1.9. Keratanase and sialidase sensitivity of Siglec-8 ligands……………………17

Figure 1.10 Chondroitinase and keratanase treatments…………………………………18

Figure 1.11 Human airway histological section enzyme sensitivity….………...... 18

Figure 1.12 Tracheal Siglec-8 ligand induces eosinophil apoptosis…………………….20

Figure 2.1 Relative molar abundance of terminal KS sub-structures…………………...21

Figure 2.2 Aggrecan is not present in mucosal glands………………………………….27

Figure 2.3 Siglec-8 binding on histological sections insensitive to aggrecanase I……...28

Figure 2.4 Nasal lavage Siglec-8 ligand proteomic mass spectrometry………………...30

Figure 2.5 Nasal lavage Siglec-8 ligand is keratanase sensitive………………………...31

Figure 2.6 Affinity purification of nasal lavage Siglec-8 ligand………………………..31

Figure 2.7 DMBT1 predicted O-glycosylation sites…………………………………….34

Figure 3.1 Neocuproine reducing sugar assay depiction………………………………..40

Figure 3.2 Galactose and N-acetylglucosamine standard curves………………………..44

Figure 3.3 Human tracheobronchial KS reducing sugar assay………………………….44

Figure 3.4 Human nasal lavage KS reducing sugar assay………………………………45

Figure 3.5 Sialic acid quantification assay of human tracheobronchial KS…………….46

Figure 3.6 Standard neutral sugar analysis on DIONEX………………………………..49

Figure 3.7 Standard acidic sugar analysis on DIONEX…………………………………49

Figure 3.8 Neutral sugar analysis of hydrolyzed KS on DIONEX…………………...... 50

Figure 3.9 Acidic sugar analysis of hydrolyzed KS on DIONEX………………………50

Figure 4.1 Azido-KS reaction completion visualized with fluorescamine……………...58

Figure 4.2 Azido-KS reaction completion visualized with 1-ethynyl pyrene…………...59

Figure 4.3 Flow-chart of KS-Neoglycolipid formation reactions and conditions………60

Figure 4.4 KS-Neoglycolipid formation reaction visualization…………………………61

Figure 4.5 KS Neoglycolipid Dot-blot Analysis………………………………………...62

Figure 4.6 KS Neoglycolipid ELISA Binding Analysis………………………………...63

Figure 5.1 Biotinylation of purified keratan sulfate chains……………………………..71

Figure 5.2 Siglec-8-binding KS represent a minor species of tracheobronchial KS.…...74

Figure 5.3 Siglec-8-binding Assay of affinity-purified tracheobronchial KS…………..74

Figure 5.4 Siglec-8-binding KS represent a minor species of nasal lavage KS………....75

Figure 5.5 Siglec-8-binding assay of affinity-purified nasal lavage KS………………...76

Figure 5.6 Eosinophil apoptosis assay on a streptavidin-coated plate…………………..77

Figure 5.7 Enoxaparin sodium molecular weight calibrant size exclusion profile……...80

Figure 5.8 Size exclusion separated human airway KS and binding analysis…………..81

Figure 5.9 Keratanase I treated KS size exclusion profile and binding analysis………..82

Figure 6.1 Cross-reactivity of Siglec-F with Siglec-8 tracheal ligands…………………87

Figure 6.2 Siglec-8 and Siglec-F keratanase sensitivity on histological sections……….89

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LIST OF TABLES

Table 1.1 Comparison of Siglec-8-Fc and Siglec-F-Fc binding specificities…...………12

Table 3.1 Summary of KS sample carbohydrate analysis on DIONEX………………...51

xiii

Chapter 1: Introduction

1.1 Glycans as Immuno-regulatory Molecules

Glycans (carbohydrates, saccharides, or sugars) are complex biological molecules

that support various cellular functions necessary for survival and also encode cellular

ID’s for recognition by other cells in their immediate environment. This requirement for

glycan scavenging and biosynthesis is exemplified by the fact that no cell, eukaryotic or

prokaryotic, has ever been found devoid of a sugar coat or glycocalyx, which shields the

cell, provides structural support, and allows for cell-cell communication among many

other functions. Though seemingly complex, the human glycome consists primarily of

only 9 glycan building blocks that are combined by glycosyltransferase enzymes (writers)

to construct specific and highly regulated glycan patterns which compose a language read

functionally by glycan-binding proteins (readers). Deciphering this glycan language has

been most instructive towards understanding the human immune response, where glycans

play key roles in immune cell activation, trafficking, and regulation as well as

identification of “self” vs “non-self”.

The study of glycan structure and function, glycobiology, has provided a window into

the evolutionary processes occurring at the interface of pathogens and human hosts, a

constant wavering of biological success attributed by the selective modification of

glycans and glycan-binding proteins. The scientific contributions from this field are evident not only in understanding basic biological phenomena but also in the translational application of successful anti-infective and anti-inflammatory glycan-mimetic therapeutics.

This thesis provides direction for pharmacological intervention aimed towards targeting a human immuno-regulatory receptor Siglec-8 for resolving allergic airway inflammation by identifying the endogenous airway ligands for this immuno-regulatory receptor and specificity for Siglec-8 recognition. A functional paralog of Siglec-8 in mice, Siglec-F, similarly found on the surface of eosinophils is also analyzed during this thesis but proves to be of limited value towards modeling alleviation of human allergic diseases by targeting these eosinophilic Siglecs.

1.2 Eosinophilic Airway Inflammation

1.2.1 Eosinophilic Airway Inflammation Prevalence and Pathogenesis

Chronic respiratory diseases are among the leading causes of mortality and morbidity worldwide with asthma and chronic obstructive pulmonary disease (COPD) as the most common. These respiratory diseases are characterized by infiltration of immune cells which are central to their pathophysiology and can contribute to the manifestation of alveolar wall destruction (emphysema), stiffening of bronchioles, and mucus hypersecretion ultimately resulting in airflow obstruction and respiratory failure in severe cases. Based on a Global Burden of Disease (GBD) meta-analysis conducted in 2015, the prevalence of COPD was estimated to be 174.5 million individuals worldwide, an increase of 44.2% from 1990, and asthma was estimated to have twice the number of cases with a prevalence of 358.2 million individuals, an increase of 12.6% since 1990. The death rate attributed to COPD was estimated to be 3.2 million individuals (11.6% increase from 1990) which was eight times the death rate from asthma.1

Although COPD and asthma have similar clinical features, there are marked differences

in their pathophysiology with differential recruitment of subsets of immune cells to the

airways and subsequently a diverse set of expressed and secreted inflammatory mediators

such as chemokines, cytokines, growth factors, and receptors. Asthma typically involves the recruitment and activation of eosinophils and mucosal mast cells, which are both

terminally differentiated myeloid-derived immune cells.2 Increased eosinophil migration

into the airways is a key component of allergic asthma and typically the increase in number

of eosinophils correlates with the disease severity. The functional role of eosinophils in

asthma is not as clear as for mast cells which release several bronchoconstrictors such as

histamine and some lipid-derived mediators like leukotrienes which are stored in pre- formed granules in the cytoplasm of activated mast cells.3

COPD has traditionally been characterized by infiltration and activation of neutrophils

and is typically referred to as a neutrophilic disease. Neutrophils are also myeloid-derived

and terminally differentiated granulocytes that release enzymes such as elastase and several

matrix metalloproteinases (MMPs) that degrade connective tissue of alveolar walls leading

to emphysema when improperly regulated. A recently proposed but still controversial

subset of COPD may be eosinophilic in disease progression and even neutrophilic- predominant but severe cases of COPD exacerbations are showing increased numbers of eosinophils in induced sputum and bronchial biopsies analyzed by Saetta et al.,4 which may indicate a role for eosinophils in COPD severity. Resolution of airway inflammation with corticosteroids, which are known for their effect on eosinophils and less apparent for neutrophils, has been effective in treating and preventing COPD exacerbations.

Currently, inhaled corticosteroids represent the centerpiece of anti-inflammatory therapy for asthma and COPD. Along with these, include leukotriene receptor antagonists, theophylline, an HDAC2 activator which presumably functions by regulating inflammatory genes, and a humanized monoclonal anti-IgE antibody labeled omalizumab.5

These current therapies have shown variable effectiveness in treating eosinophilic airway

inflammation and thus far there is no therapy which selectively targets activated eosinophils in the airways.

1.3 Siglec-8 and the Siglec Family of Lectins

1.3.1 Siglec Family of Lectins

Siglecs (sialic acid binding type-1 transmembrane lectin receptors) are the only well- characterized group from the I-type lectin immunoglobulin superfamily (IgSF). Siglecs are specifically expressed on subsets of similar cells, typically the hematopoietic immune cells encompassing both the myeloid and lymphocyte lineages. Members of the IgSF contain at least one immunoglobulin (Ig)-like fold in the form of a C1/C2 constant domain and/or an amino-terminal V-set Ig domain responsible for sialoglycan ligand recognition

(Figure 1.1). As their name implies, binding to sialic acid is a requirement for classification into this group and each of the 14 human Siglecs contain an essential arginine residue in the V-set domain which forms a salt-bridge with the carboxylate functional group of sialic acid. The cytoplasmic C-terminal domains are variable and determine overall function, however, 9 of the human Siglecs (Siglecs 2, 3, and 5-11) carry immunoinhibitory domains

(ITIM or ITIM-like) which have been directly shown or are hypothesized to regulate ongoing inflammatory responses.6

Mice carry a set of conserved Siglecs (Siglecs 1, 2, 4, and 15) along with a set of CD-

33 related Siglecs designated as Siglec-3, -E, -F, -G, and –H. Direct homologs of human and mouse Siglecs have not been identified presumably due to rapid evolution of sialoglycans and subsequently sialoglycan-binding proteins on the forefront of host- pathogen interactions.

Figure 1.1. Domain structures of the known Siglecs in humans and mice. There are two subgroups of Siglecs: One group contains sialoadhesin (Siglec-1), CD22 (Siglec-2), MAG (Siglec-4), and Siglec-15, and the other group contains CD33-related Siglecs. The plus sign indicates the presence of a charged residue in the transmembrane domain, which has been shown to interact with the immunoreceptor tyrosine-based activatory motif (ITAM)-containing adaptor proteins DAP12 and DAP10. (ITIM) Immunoreceptor tyrosine- based inhibitory motif (Essentials of Glycobiology 3rd edition).

1.3.2 Siglec-8 Receptor

Bochner et al. have identified an inhibitory lectin, Siglec-8, selectively expressed

on the surface of allergic inflammatory cells (eosinophils, basophils and mast cells)

which may provide a unique target for modulating the survival of these immune cells.

Eosinophilic expression of Siglec-8 was first discovered through a cDNA library screen

created from a subject with hypereosinophilic syndrome using high-throughput

sequencing of expressed sequence tags.7 Although initially labeled as SAF-2 due to its

homology to sialoadhesin family members, it was later properly characterized by Floyd et

al as the eighth human Siglec.8

Siglec-8 contains two Ig-like constant domains which extend extracellularly from the

surface of the immune cell plasma membrane. A V-set, Ig-like domain, at the most distal portion of the receptor is required for sialic acid binding and conveys specificity for sialoglycan recognition. Past the single transmembrane domain towards the cell lumen,

Siglec-8 contains a cytoplasmic ITIM motif (immunoreceptor tyrosine-based inhibitory motif) which recruits certain SH2-domain-containing effectors, most notably the protein tyrosine phosphatases SHP-1 and SHP-2. These effectors negate activating signals and can function to down-regulate an immune response when Siglec-8 is cross-linked on the cell surface.

Upon Siglec-8 receptor crosslinking by either a synthetic multi-valent ligand or an

antibody specific for the lectin, eosinophils undergo apoptosis and mast cell mediator

release is inhibited.9 The function of Siglec-8 ligation on the surface of basophils has not

yet been investigated. Furthermore, activated eosinophils, primed with either IL-5, GM-

CSF, or IL-33, survive longer in vitro and demonstrate enhanced cell death with cross-

linking of Siglec-8 when compared to nascent eosinophils. Late-phase activated

eosinophils purified from human blood have also shown enhanced cell apoptosis when

Siglec-8 is ligated compared to early phase eosinophils. Ligation of Siglec-8 on mast

cells does not induce apoptosis but does limit the immune response through inhibition of

prostaglandin D2 and histamine release as well as inhibition of FcεRI-mediated Ca2+ flux.10 These qualities highlight Siglec-8 as an allergic immune cell specific receptor to target for therapeutic intervention of eosinophilic airway inflammatory diseases and shows enhanced effectiveness towards activated eosinophils.

1.4 Human and Mouse Eosinophilic Siglecs

1.4.1 Murine Siglec-8 Functional Paralog: Siglec-F

Although mice do not express Siglec-8, they do express a functional paralog

designated Siglec-F which shares surface expression on eosinophils but can also be found

on myeloid precursor cells and tissue macrophages. By sequence homology, Siglec-F is

more closely related to Siglec-5, however, overlap of eosinophil expression and binding

selectivity makes it a potential target for modeling alleviation of eosinophil-based airway

inflammatory diseases.

1.4.2 Siglec-F Proof-Of-Concept

Zimmermann et al. (2008) tested the ability of an anti-Siglec-F monoclonal mouse

IgG1 isotype antibody to reduce eosinophil numbers in vivo. Administration of anti-

Siglec-F intravenously into mice engineered to over-express IL-5, a cytokine which

activates eosinophils, induced a rapid decline of peripheral blood eosinophils which was

sustained for at least 48 hours and was not observed with an isotype-matched control nor

an eosinophil-binding control (anti-CCR3). With eosinophils predominantly residing in

tissues, Zimmermann et al. then tested the levels of eosinophils in the jejunum which

serves as a reservoir for eosinophils in the gastrointestinal tract. They observed a 34.5 ±

9% decrease in the number of eosinophils 48-72 h post-administration of anti-Siglec-F.11

Importantly, there was no decrease in the number of peritoneal and jejunal mast cells

which reflects the inability of Siglec-8 to affect mast cell viability. These data were also

reproduced in wild-type mice which rejects the possibility that high levels of circulating

IL-5 were required for the Siglec-F-mediated effect. Siglec-F engagement did not reduce

the total level of white blood cells which indicates its specificity for regulating eosinophil

survival.

Also, induction of allergic lung inflammation in previously ovalbumin-sensitized

mice by intranasal ovalbumin challenges demonstrated an up-regulation of Siglec-F on

blood eosinophils as well as a quantitative increase of Siglec-F ligands expressed in

mouse airways.12 Zhang et al. also created Siglec-F-null mice which showed exacerbated infiltration of eosinophils into the airways. Kiwamoto et al. (2014) used mice lacking a sialyltransferase gene product (ST3Gal-III), shown previously to be required for Siglec-F

lung ligand synthesis, and observed increased allergic eosinophilic airway inflammation

upon loss of Siglec-F ligand production.13 Together these data show enhanced

eosinophilic allergic airway inflammation and infiltration of eosinophils into the airways

with loss of Siglec-F expression on eosinophils or loss of Siglec-F sialoglycan ligand

biosynthesis in mouse airways. The in vitro and in vivo functional role of Siglec-F to

modulate eosinophil survival and limit airway inflammation provides a proof-of-concept

for clinical targeting of Siglec-8 to selectively treat human eosinophilic diseases.

1.4.3 Published Data on Siglec-8 and Siglec-F Ligand Specificity

As an initial screen for Siglec-8 and Siglec-F ligand specificity, Fc-containing

chimeric constructs of these Siglecs were utilized as probes on a glycan microarray with

610 covalently linked synthetic glycan structures of which 208 were either α2-3 or α2-6

terminally sialylated. Glycan microarrays were developed by Blixt et al and analyzed on

the Consortium for Functional Glycomics Version 5.1 microarray

(http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh8.shtml).1

4 Siglec-8 binding to the glycan array was highly specific and only recognized two

glycan structures on the array (Figure 1.3). Siglec-8 binding requires an α2-3 linked

terminal sialic acid to a galactose with a C6-sulfate and a β1-4 linkage to an N- acetylglucosamine (6’Su-3-sialyl-LacNAc) with or without an α1-3 fucose (6’Su-3- sialyl-Lex). Siglec-F was shown to bind to these two structures as well as sialylated tri-

and tetra-antennary N-linked glycans without sulfation on either the galactose or N-

acetylglucosamine (Table 1.1).

These findings were published by our lab in Yu et al. (2017), and the binding

specificity was further validated with a microplate glycolipid array using synthetic

glycolipids containing a 6 or 6’-sulfated and 3-sialylated N-acetyllactosamine termini.15

Again, Siglec-8 bound the 3-sialylated and 6’-sulfated LacNAc on the synthetic neoglycolipid. Siglec-8 did not bind to any of the ganglio-series glycosphingolipid structures on the array, however Siglec-F surprisingly showed robust binding to major natural gangliosides GD1a and GT1b among others on the array (Figure 1.4).

Using solution NMR spectroscopy of synthetic sialylated and sulfated LacNAc carbohydrates, Propster et al. (2016), explored the molecular basis of Siglec-8’s binding

site specificity.16 Along with the expected salt bridge formed between the conserved and essential R109 of Siglec-8 and the carboxylate on sialic acid, the C6-sulfate on the galactose formed a salt bridge with R56 and hydrogen bonding with Q59. These two side chains appeared to clamp onto the sulfated-galactose from opposite sides and simultaneously contribute to recognition of this glycan structure. Furthermore, disrupting the sulfated-galactose salt bridge by mutating R56 to an alanine, showed an eightfold drop in affinity using NMR titration without disrupting the overall structure of Siglec-8

as determined by comparative 2D 1H, 15N-HSQC with wild-type.

Figure 1.2. Symbol nomenclature for glycan structures. Adapted from Essentials of Glycobiology 3rd edition.

Siglec-8 Glycan Microarray

20000

15000

10000

5000 x 6’Su-sialyl-LacNAc 6’Su-sialyl-Le Binding (fluorescence) Binding 0

Glycans (>600)

Figure 1.3. Siglec-8-Fc overlay of a CFG glycan microarray. Emory University CFG array consisting of 610 glycan structures, www.functionalglycomics.org. Siglec-8 specifically recognizes two distinct glycan structures, 6’Su-3-sialyl-LacNAc and 6’Su-3-sialyl-Lex.

Figure 1.4. Binding of human Fc chimeras of Siglec-8, -F, -9 and -E to a custom glycolipid microplate array. Glycolipids were co-adsorbed with carrier lipids (phosphatidylcholine and cholesterol) as a monolayer on polystyrene 96-well microwells (Lopez and Schnaar 2006). Glycans included phosphatidylethanolamine- based synthetic neoglycolipids (6’-Su-SLacNAc, 6-Su-SLacNAc), synthetic ceramide-based glycosphingolipids (GD1α, GQ1bα, GM1b and di-Su-GM1b) and naturally sourced ceramide-based gangliosides (GM3, GD3, GM1, GD1a, GD1b, GT1b and GQ1b). Control wells were adsorbed with carrier lipids only. Binding of each siglec is normalized to its maximum binding glycan. Values are reported as mean ± SEM for triplicate wells. Average maximum and background binding (relative colorimetric values, background in parentheses) for each of the siglecs was: Siglec-8, 59 (0.7); Siglec-F, 254 (2); Siglec-9, 256 (3) and Siglec-E, 305 (4).

Table 1.1. Comparison of Siglec-8-Fc and Siglec-F-Fc binding specificities. Adapted from Kiwamoto et al. (2015).

1.4.4 Published Data on Siglec-8 and Siglec-F Ligand Expression

To further explore the differences in Siglec-8 and Siglec-F ligand specificity and cross-species ligand expression, human and mouse tracheal cross-sections were overlaid

with each Fc-chimera (Figure 1.5). Siglec-8 showed robust binding to human submucosal glands and cartilage, whereas no Siglec-8 binding was observed on mouse tracheal sections. However, Siglec-F showed robust binding to mouse airway epithelium and submucosal glands and showed broader specificity for human tracheal sections with connective tissue and cartilage also showing intense staining. Each Siglec-Fc overlay was validated for sialidase-sensitive binding with all staining reversed by sialidase treatment

(data not shown). These data indicate more promiscuous binding specificity for Siglec-F

and a potentially evolutionarily restricted binding pattern for Siglec-8. Of evolutionary

interest, though not of direct relevance to this thesis, an apoptosis-inducing neutrophilic

12 surface receptor, Siglec-9, shows a binding pattern more similar to Siglec-F than Siglec-8 showed.15 Mouse airways do not make Siglec-8 ligands which precludes future Siglec-8 ligand analysis from mouse airway tissue and necessitates a focus on human airway tissue ligands. This finding also prevents use of mouse models for Siglec-8 mediated alleviation of airway inflammation and rather requires a Siglec-8 transgenic knock-in mouse for in vivo studies.

Figure 1.5. Siglec overlay of human and mouse trachea cross sections. Cross sections of human and mouse trachea were stained with Siglec-8-Fc or Siglec-F-Fc precomplexed with AP-conjugated anti-human- Fc. Lectin binding was detected using Vector Red stain and sections counterstained using Hematoxylin QS. Images captured using different siglec-Fc chimeras were linearly adjusted to maximize the dynamic staining range within the section. Arrowheads: airway epithelium; arrows: submucosal glands; asterisks: cartilage. Scale bars, 250 μm top row (human), 50 μm bottom row (mouse). This figure is adapted from Yu et al. (2017).

1.4.5 Characterization of Siglec-8 Human Airway Ligands

Using post-mortem human airway tissue as our source of Siglec-8 ligands, we began by extracting the tracheal tissue using 6 M guanidinium hydrochloride and resolving the crude extract by denaturing sodium dodecyl sulfate gel electrophoresis on composite 2% agarose/ 1.5% acrylamide denaturing gels due to the high molecular weight of these ligands and their inability to migrate in SDS-PAGE gels.15 Our most recent publication, Gonzalez Gil A. and Porell R.N. et al. (2018), characterizes three distinct size classes of Siglec-8 ligands (ranging 250 kDa – 1 MDa) from human airway tissue that are each sialylated keratan sulfate chains found on the proteoglycan aggrecan

(Figure 1.6).17

Aggrecan, identified through proteomic mass spectrometry analysis and validated by sensitivity of tracheal ligands to aggrecanase and co-migration of resolved ligands with anti-aggrecan monoclonal antibody, carries two types of glycosaminoglycan chains in the form of sulfated poly-N-acetyllactosamine (keratan sulfate, KS) and repeats of heterogeneously sulfated N-acetylgalactosamine and glucuronic acid disaccharides

(chondroitin sulfate, CS) (Figures 1.7 & 1.8). Ligand digestion with keratanase I, an endo-β-galactosidase, or keratanase II, an endo-β-N-acetylglucosaminidase, abrogated all

Siglec-8-Fc binding to electrophoretically resolved tracheal ligands, whereas chondroitinase ABC digestion shifted the largest molecular weight ligand (S8-1M) and slightly increased Siglec-8-Fc binding (Figures 1.9 and 1.10). Interestingly, binding of

Siglec-8-Fc to commercial bovine articular cartilage aggrecan was absent, however, chondroitinase ABC digestion revealed a very minor Siglec-8-Fc-binding component that

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Figure 1.9. Keratanase and sialidase pretreatments diminish Siglec-8 binding to purified Siglec-8 ligands. Siglec-8 ligands were extracted from human trachea, purified by sequential size-exclusion and Siglec-8 affinity chromatography and subjected to keratanase or sialidase treatments. Samples were resolved on 1.5% acrylamide, 2% agarose composite gels, blotted to PVDF membranes and probed with precomplexed Siglec-8-Fc or anti-aggrecan antibody (7D4) as indicated. Lanes: (1) incubation without enzyme; (2) sialidase (67 mU/mL, 90 min); (3) keratanase II (6 mU/mL, 16 h). HiMark molecular weight standards are shown at the left (Stds). Figure from Gonzalez Gil A. and Porell R.N. et al. (2018).

Figure 1.10. Chondroitinase ABC (ChABC) and keratanase treatments of a human tracheal Siglec-8 ligand. Reveals a chondroitin sulfate (CS) proteoglycan with Siglec-8-binding keratan sulfate (KS) chains. Siglec-8 ligands were extracted from human trachea and S8-1M purified by sequential size-exclusion and Siglec-8 affinity chromatography. Equal aliquots containing the isolated ligand (S8-1M) or commercial bovine articular cartilage aggrecan (bovine aggrecan) were treated with buffer alone (no enzyme), ChABC, keratanase I or both enzymes for 20 h at 37°C. Samples were denatured and resolved on 1.5% acrylamide, 2% agarose composite gels, blotted to PVDF membranes and probed with precomplexed Siglec-8-Fc overlay or anti-aggrecan antibody. Lanes (all enzyme treatments 20 h at 37°C): (1) no incubation; (2) ChABC (500 mU/mL); (3) keratanase I (21 mU/mL); (4) ChABC (500 mU/mL) plus keratanase I (21 mU/mL); (5) incubation without enzymes. Migration positions of HiMark molecular weight markers are shown. Figure from Gonzalez Gil A. and Porell R.N. et al. (2018).

Figure 1.11. Chondroitinase ABC (ChABC) treatment of human trachea tissue sections enhances Siglec-8-Fc binding. Cross sections of human trachea were incubated with Siglec-8-Fc precomplexed with AP-conjugated anti-human-Fc. Lectin binding was detected using Vector Red stain and sections counterstained using hematoxylin QS. (A–C) Human tracheal cartilage from sections treated: (A) for 44 h with buffer alone; (B) for 18 h with 150 mU/mL ChABC followed by 25 h with buffer alone, or (C) for 18 h with 150 mU/mL ChABC followed by a 1-h wash and further incubation for 25 h with 10 mU/mL keratanase II. (D–F) Human trachea submucosal glands from the same experiment: (D), buffer incubation; (E) ChABC followed by buffer incubation; (F) ChABC followed by keratanase II. Figure from Gonzalez Gil A. and Porell R.N. et al. (2018).

To confirm these human trachea extracted, purified, and characterized Siglec-8

ligands as functional ligands, the S8-250K (smallest ligand) was subjected to selective

mild periodate oxidation of the terminal sialic acid glycerol group followed by reduction

with sodium borohydride. An equal portion of S8-250K was left intact and only treated

with sodium borohydride without prior oxidation. The oxidized and intact ligands were

each dialyzed against RPMI medium prior to incubation with freshly isolated and IL-5

activated primary human eosinophils. After 24 hours of ligand incubation with the

eosinophils, apoptosis was quantified by flow cytometry and showed a modest but

significantly increased percentage of eosinophil apoptosis after incubation with the intact

ligand (Figure 1.12). The oxidized ligand which no longer binds Siglec-8 did not

significantly increase apoptosis above control eosinophils in media alone. Presumably,

though not yet analyzed, the sialylated keratan sulfate chains on the intact ligands

increased apoptosis of primary eosinophils through surface Siglec-8 cross-linking. This

thesis aims to further compare Siglec-8 and Siglec-F ligand specificity, to explore the

potential for aggrecanS8 to be the physiologically relevant ligand in human airways, but to predominantly focus on the isolation and characterization of these Siglec-8-binding keratan sulfate chains to better understand Siglec-8 recognition of these endogenous sialylated keratan sulfate ligands.

Figure 1.12. Purified human tracheal Siglec-8 ligand induces human eosinophil apoptosis. Siglec-8 ligands were extracted from human trachea and purified by size-exclusion chromatography followed by Siglec-8 affinity chromatography. A portion of purified S8-250K was treated with cold periodate to selectively oxidize the glycerol sidearm of its sialic acid followed by sodium borohydride reduction (negative control). An equal portion was incubated without periodate and treated with sodium borohydride (intact ligand). Oxidized and intact S8-250K were dialyzed against RPMI medium and added at equal concentrations to freshly isolated primary human eosinophils. After 24 h in culture, eosinophil apoptosis was quantified by flow cytometry. (A) Oxidized and intact S8-250 K were electrophoretically resolved on a composite agarose–acrylamide gel, blotted to PVDF membranes. Replicate blots were probed with Siglec- 8-Fc to reveal Siglec-8 ligands or anti-aggrecan antibody (7D4). Lanes: (1) untreated S8-250K; (2) periodate oxidized and reduced S8-250K; (3) reduced S8-250K (intact ligand). (B) After isolation and overnight interleukin 5 (IL-5) priming, human eosinophils were incubated with equal portions of oxidized and intact S8-250K and apoptosis was assessed 18–24 h later. Results are expressed relative to untreated eosinophils, which had average apoptosis of 42 ± 5% (SEM). Data are displayed as mean and SEM of six replicates performed on three separate primary human eosinophil preparations. Figure from Gonzalez Gil A. and Porell R.N. et al. (2018). Chapter 2: The Physiologically-relevant Human Airway Siglec-8 Ligand

Discovering sialylated keratan sulfate chains carried on a subpopulation of aggrecan molecules, of three different size classes, to be the Siglec-8 ligands from human airway trachea extracts was initially a surprise, though the glycan microarray data was suggesting a terminal structure consistent with keratan sulfate chains. Sialylation of human articular cartilage aggrecan KS has been noted in the literature, though the methods used did not allow for a quantification of the relative proportion of sialic acids on the capping regions 18. The same group also published KS chain characterization from

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aggrecan on human airway tissue. Since aggrecanase I (ADAMTS-4) is able to cleave

human trachea extracted Siglec-8 ligands, trachea tissue sections were pre-treated with aggrecanase prior to Siglec-8-Fc overlay to observe any loss in Siglec-8-binding which could indicate aggrecanase-sensitive ligands in mucosal glands. Finally, human nasal lavage samples were acquired from a collaborator at Johns Hopkins Bayview Medical

Center, Dr. Jean Kim, to identify Siglec-8 ligands in human airway secreted samples and compare their properties with human trachea extracted ligands for Siglec-8.

2.1 Materials and Methods

2.1.1 Siglec-8 Lectin Histochemical Overlay

Post-mortem human trachea tissues were fixed in neutral 4% paraformaldehyde in

phosphate-buffered saline (PBS) at 4°C for 16 h, embedded in paraffin, sectioned to 5 µm

and captured on glass slides. Following deparaffinization, the slides were heated briefly in

10 mM sodium citrate (pH 6.0) for antigen retrieval. Subsequent steps were performed at

ambient temperature unless otherwise noted. Slides were incubated in PBS supplemented

with 0.1% Tween 20 (PBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St.

Louis, MO) for 30 min, endogenous enzyme Blocking Reagent (Dako North America,

Carpinteria, CA) for 10 min, and then in Fc Receptor Blocker (Innovex Biosciences,

Richmond, CA) for 30 min. Siglec-8-Fc (15 μg/ml) was pre-incubated in PBST

supplemented with 1% BSA with alkaline phosphatase (AP)-conjugated goat anti-human

IgG and IgM heavy and light chains (2 μg/ml, Jackson Immunoresearch, West Grove, PA) for 30 minutes at 4°C. Incubation of pre-complex on the slides was for 16 h at 4°C. Slides were washed with PBST, equilibrated with 100 mM Tris-HCl (pH 8.3) supplemented with

0.1% Tween 20 for 10 min, bound lectin conjugate detected with Vector Red alkaline

phosphate substrate (Vector Laboratories, Burlingame, CA), counterstained with

Hematoxylin QS (Vector Laboratories), slides dehydrated, mounted in Krystalon (EMD

Millipore) and imaged using a Nikon Eclipse 90i microscope (Figure 2.2).

2.1.2 Anti-Aggrecan Immunofluorescence

Paraffin-embedded human airway tissues were deparaffinized and heated for antigen retrieval as above. Before blocking the slides, the tissues were incubated with

250 mU/mL Chondroitinase ABC enzyme in 50 mM Tris-HCl, 50 mM sodium acetate

(pH 8.0) at 37°C for 24 hours. Following enzymatic treatment, tissue slides were washed

for 20 minutes in PBS supplemented with 0.1% Tween 20 (PBST). Slides were

incubated in PBST supplemented with 1% bovine serum albumin (BSA, Sigma-Aldrich,

St. Louis, MO) for 30 min and then in Fc Receptor Blocker (Innovex Biosciences,

Richmond, CA) for 30 min. Slides were overlaid with one of three anti-aggrecan

antibodies which targeted different domains of the aggrecan core protein (G3-domain

specific, Pierce, 0.01 µg/µL; Biolegend Clone M4004A03, 0.01 µg/uL; BioRad, 7D4, mouse monoclonal IgG1, 0.01 µg/uL) for 16 h at 4°C. Slides were washed with PBST

for 10 minutes followed by incubation with 0.005 µg/µL alexa-488-conjugated goat anti-

rabbit IgG (H&L) at ambient temperature for 2 h. Slides were then washed with PBST

for 5 minutes, PBST supplemented with 2 mM DAPI for 5 minutes, and then with PBS

(calcium and magnesium free) for 5 minutes. Tissues were mounted in VectaShield,

covered, and imaged using a Nikon Eclipse 90i microscope (Figure 2.2).

For aggrecanase I (ADAMTS-4) enzymatic digestion, the tissues were incubated in isotonic phosphate buffer containing 0.5 mM calcium chloride for 24 h at 37°C directly

after antigen retrieval and were subsequently washed in PBST for 20 minutes prior to

blocking (Figure 2.3).

2.1.3 Extraction and Isolation of Siglec-8 Nasal Lavage Ligands

Nasal lavage samples from normal human volunteers were acquired by Dr. Jean

Kim at the Johns Hopkins Sinus Center. All samples were stored at -80°C. After

thawing the nasal lavage samples on ice, 10 mL of 6 M guanidinium hydrochloride, 20

mM sodium phosphate, 20 mM dithiothreitol (DTT), 5 mM EDTA, pH 6.5 (extraction

buffer) was added for each 1 mL of nasal lavage sample. After rotating end-over-end at

4°C for 24 h in the extraction buffer, the sample was concentrated and buffer-exchanged to 4 M guanidinium hydrochloride, 10 mM phosphate, pH 7.0 (column running buffer) using a 10 K molecular weight cut-off centrifugal filter (Millipore UFC901024, 10K-

Ultracel) spun for 30 minutes at 3,700 x g. Prior to loading onto a Sephacryl S-500 size exclusion column, the sample was centrifuge-filtered through a 0.22 µm sterile filter.

Using an injection loop of 5 mL, the entire extracted sample was loaded onto the S-500 column and run with a flow rate of 1 mL/min in column running buffer described above with 1.8 mL fractions collected in a 2 mL deep-well 96-well plate. Size-separated fractions were analyzed by blotting to nitrocellulose membranes, blocking with 5% nonfat blotto in Dulbecco’s PBS supplemented with 0.1% Tween-20 (PBST) for 1 h at ambient temperature, and overlaid with Siglec-8-Fc (0.5 μg/mL) pre-complexed with

HRP-conjugated goat anti-human IgG (0.2 μg/mL). Dot-blots were visualized by enhanced chemiluminescence. Fractions which showed robust Siglec-8-Fc binding were combined and dialyzed three times against 1.5 L of 1 M urea, 20 mM sodium phosphate,

pH 7.4 in 100 kDa cut-off dialysis cassettes (Spectra/Por G235059, Float-a-lyzer G2

cassettes).

To further isolate Siglec-8 ligands from nasal lavage samples, combined size- excluded and urea dialyzed samples were bound to a Siglec-8-Fc affinity column and eluted with high salt. Using protein A/G magnetic agarose beads (Pierce, Cat: 78609),

400 μL of magnetic bead slurry was transferred into each of two 1.5 mL low-bind eppendorf tubes (Eppendorf, Cat: 022431081) and beads were washed twice with 600 μL

of 150 mM sodium chloride, 10 mM sodium phosphate, pH 7.4 wash buffer. One set of

beads was incubated with 10 μg of recombinant human IgG1 (R&D, Cat: 110HG) in

wash buffer listed above and the other set incubated with 100 μg of Siglec-8-Fc in wash

buffer. After incubation on an end-over-end rotator for 24 h at 4°C, both sets of beads

were washed three times with Siglec-8-column elution buffer (1 M urea, 1 M sodium

chloride, 20 mM sodium phosphate, pH 7.4) to remove unbound or loosely bound

protein. Both sets of beads were then equilibrated three times with Siglec-8-column wash

buffer (1 M urea, 150 mM sodium chloride, 20 mM sodium phosphate, pH 7.4). To

preclear the sample 500 μL of combined nasal lavage sample was added to the beads

containing human IgG1, the slurry transferred to a 5 mL low-bind Eppendorf tube, and

residual beads were transferred over with another 4.5 mL of combined nasal lavage

sample to provide a final volume of 5 mL nasal lavage sample. These beads were mixed

end-over-end at 4°C for 3 h, after which the slurry was transferred back to a 1.5 mL low-

bind Eppendorf tube, placed on a magnetic stand, and the pre-cleared sample collected,

free of human IgG1 beads. This pre-cleared sample was then incubated for 16 h at 4°C

end-over-end with the Siglec-8-Fc containing beads, which had been stored on ice in the

Siglec-8-column wash buffer. After incubation, the solution was transferred to a 1.5 mL

low-bind Eppendorf tube, placed and a magnetic stand, and the solution removed and

stored as flow-through. Siglec-8-Fc beads were washed five times with 500 μL of Siglec-

8-column wash buffer followed by three elutions with 500 μL of Siglec-8-column elution

buffer. A fraction of each sample was mixed with denaturing loading buffer, samples

resolved on a 2% agarose/1.5% acrylamide composite gel, transferred to PVDF, blocked

in 5% nonfat milk protein in PBST, and overlaid with Siglec-8-Fc precomplexed with

HRP-conjugated goat anti-human IgG to track nasal lavage Siglec-8 ligands throughout

the affinity purification (Figure 2.6).

2.2 Anti-Aggrecan Immunofluorescence

2.2.1 Aggrecan Found in Cartilage but not Mucosal Cells

Human airway tissue overlaid with a C-terminal localized, G3-specific (globular domain) polyclonal antibody for aggrecan showed intense binding to the cartilaginous tissue with alexa-488 conjugated secondary (Figure 2.2, green). However, there is no observed anti-aggrecan binding to the mucosal region of the airway tissue, which is outlined beautifully with DAPI staining of nuclei, suggesting aggrecan may not be the physiologically relevant proteoglycan for eosinophil homeostasis in human airways.

Anti-aggrecan staining was replicated with two more anti-aggrecan antibodies which both

showed intense staining to cartilaginous tissue and an absence of mucosal gland staining

(data not shown).

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2.2.3 Nasal Lavage Extracted Siglec-8 Ligands

In collaboration with Anabel Gonzalez-Gil in our lab, we set out to extract and identify Siglec-8 ligands from human nasal lavage samples and compare them to our trachea tissue extracted ligands. Anabel initiated nasal lavage sample extraction with 6

M guanidinium hydrochloride, size exclusion separation of extracted material, affinity chromatography purification to isolate Siglec-8 ligands, and proteomic mass spectrometric analysis which revealed a protein named DMBT1 or deleted in malignant brain tumors 1 (Figure 2.4). Proteomic mass spectrometry did not detect aggrecan in the affinity-purified nasal lavage samples and anti-DMBT1 staining co-migrated with the

Siglec-8-binding material after resolution by composite gel electrophoresis and western blotting. The nasal lavage Siglec-8 ligand was a single band with an estimated molecular weight of ~900 kDa based on similar migration with an in-house IgM cross-linked high molecular weight ladder. We subjected this ligand to enzymatic treatments and found sensitivity to sialidase (data not shown) and keratanase I (Figure 2.5) which again supported sialylated KS chains as the Siglec-8 ligands in human airways.

To determine the functional ability of this nasal lavage extracted Siglec-8 ligand, another batch of size-separated and Siglec-8-Fc affinity-isolated Siglec-8 ligands was purified. Western blot analysis of the affinity-captured nasal lavage Siglec-8 ligands

(Figure 2.6), showed all of the Siglec-8 ligands bound to the Siglec-8-Fc-conjugated magnetic beads, a negligible amount was eluted in the low-salt washes, and the majority of the ligands were eluted in the first high-salt elution. Analysis of these samples with anti-DMBT1 showed that all of the DMBT1 in the sample was captured onto the beads, half of the DMBT1 was eluted during wash steps, and the first high-salt elution contained

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2.3 Conclusions and Remarks

The expression of Siglec-8 ligands on trachea tissue are confined to mucosal and

cartilaginous tissues, with a sub-type of mucosal cells called serous cells predominantly expressing Siglec-8 ligands in the mucosa. Although published data suggest aggrecan

purified from human airway tissue is a ligand for Siglec-8, aggrecan cannot be found in

the mucosal regions of airway tissues, even with multiple antibodies spanning regions

across the aggrecan core protein backbone. Aggrecanase I treatment does not reduce

Siglec-8 binding to mucosa though does slightly reduce Siglec-8 binding to cartilage and

eliminates anti-aggrecan binding to human airway histological sections. These data

suggest that another protein may be the physiologically relevant carrier of sialylated KS

chains which engages immune cells at the airway lumen and that the over-abundance of

aggrecan from the cartilage tissue may be limiting the detection of this potential

alternative carrier protein found in the mucosal glands.

Similar extraction, purification, and analysis of Siglec-8 ligands from human

nasal lavage samples performed in collaboration with Anabel Gonzalez-Gil in our lab

have revealed not aggrecan, but another protein carrying sialylated KS chains named

DMBT1 or deleted in malignant brain tumors 1. This mucin-like glycoprotein is

localized to skin and mucosal surfaces and is reported to function as an innate immune

system regulatory and defense molecule. The protein backbone consists of a repeating

pattern of scavenger receptor cysteine-rich (SRCR) domains separated by serine and

threonine rich and therefore potentially heavily O-glycosylated scavenger interspersed

domains (SID). Tissue-specific alterations in glycosylation and post-translational

modifications have been hypothesized to produce the different size classes observed for

this protein, though most purified DMBT1 molecules have been found to be of a high

molecular weight of several hundred daltons. Keratan sulfate chains carried on DMBT1

have not been published in the literature and neither has a DMBT1 with an estimated

molecular weight of ~900 kDa. In comparison to aggrecanS8 which was affinity-purified

using a penta-valent Siglec-8-COMP construct, nasal lavage DMBT1S8 could not be

similarly salt-eluted off of the pentameric construct and we had to instead use a bivalent

Siglec-8-Fc construct to salt-elute the Siglec-8 ligands. This finding suggests a tighter

binding interaction of Siglec-8 to DMBT1S8 than aggrecanS8 which we hypothesize to be based on differences of the glycosylation pattern along the core proteins which would allow for more efficient cross-linking of Siglec-8 and higher avidity of binding.

Aggrecan is known to contain a sparsely populated KS region in the interglobular domain between globular domains 1 and 2, along with a short KS-rich region adjacent to globular domain 2. This type of KS landscape may not be as efficient for Siglec-8 binding.

DMBT1 glycosylation has not been adequately assessed to compare with aggrecan and the diversity in DMBT1 alternatively spliced variants and tissue-specific glycosylation patterns makes it challenging to predict the DMBT1S8 domains carrying sialylated KS

chains.

Using NetOGlyc 4.0 Server database to predict O-glycosylated regions of human

DMBT1 (UniProtKB – Q9UGM3-1), we overlaid DMBT1 peptides detected by

proteomic mass spectrometry with regions of predicted O-glycosylation and found a

pattern of highly O-glycosylated stretches separated by non-O-glycosylated peptides

(Figure 2.8). If this pattern of O-glycosylation mimics the expression of sialylated KS

chains, then we can imagine a more efficiently spaced KS landscape which would allow

33 for the engagement of multiple Siglec-8 receptors along a single DMBT1S8 molecule.

Since we consistently observe sialylated KS chains as the ligands for Siglec-8 regardless of the protein carrier, I decided to focus on isolating the KS chains which bind Siglec-8 and explore this interaction without the complexity of a protein carrier.

Figure 2.7. Predicted O-glycosylation Sites on DMBT1 Overlaid with Peptide Regions Detected by Proteomic Mass Spectrometry. NetOGlyc 4.0 Server database used to predict GalNAc mucin-like O- glycosylation sites on DMBT1 with peptide sequence acquired from UniProtKB as isoform Q9UGM3-1. Peptide coverage from mass spec data shown in green. Predicted O-glycosylated stretches shown in yellow.

Chapter 3: Keratan Sulfate Chains in Human Airways

From our recent findings in isolating Siglec-8 ligands from human airways, we propose that sialylated keratan sulfate chains are the endogenous Siglec-8 ligands from human airways. To explore this hypothesis, we isolated the KS chains and analyzed their ability to retain Siglec-8 binding when stripped from a protein backbone. Dr. Robert

Linhardt has been a pioneer in the isolation and characterization of keratan sulfate chains from various animal sources. Utilizing Linhardt’s published protocol,20 human trachea

tissue or human nasal lavage was extensively proteolyzed to generate KS chains,

presumably still O-linked to a single serine or threonine. Through the subsequent steps,

glycosaminoglycan chains are purified from the other tissue components by anion

exchange chromatography followed by enzymatic treatment to recover only keratan

sulfate chains. Total yield of KS chains can be approximated by a reducing sugar assay

using a colorimetric assay to quantify the differential cleavage products of KS with

keratanase I and keratanase II enzymes. Sialic acid quantification can be analyzed

through the use of a commercial fluorescence assay. Carbohydrate composition is

assessed by acid hydrolysis and analysis on a DIONEX ICS 6000.

3.1 Materials and Methods

3.1.1 Pronase Digestion of Human Airway Tissue and Nasal Lavage

The keratan sulfate isolation protocol is adapted from Robert Linhardt’s lab as published in Weyers et al. (2013). Frozen trachea-bronchus tissues and nasal lavage samples were thawed on ice. Wet weight of trachea-bronchus combined tissue or volume of nasal lavage sample was recorded and used to determine the amount of pronase enzyme and reaction buffer. A fresh solution pronase Reaction Buffer (50 mM ammonium bicarbonate, 5 mM CaCl2, pH 7.5) was made for the pronase digestion. The

amount of pronase (Roche Diagnostics 10165921001, Streptomyces griseus) required was

calculated by taking 2% of the wet tissue weight or lavage sample volume. Reaction buffer was added to the tissue with the ratio of 10 mL buffer/ 1 g of tissue or 1 mL of lavage which provides a working concentration of 2 mg/mL pronase. After vortexing the solution in a 50 mL conical tube, the solution was placed in a 60°C incubator for at least

24 h for limit-digestion. After incubation, the digested material was centrifuged for 10

minutes at 5,000 x g in 2 mL Eppendorf tubes to pellet any cell debris. Supernatants

were transferred to a PolyPrep (BioRad, Cat: 731-1550) chromatography column and

allowed to elute through the filtered column by gravity to clear residual particulates

larger than 30 µm. The eluted solution is then passed through a 0.22 µm filter to remove

any remaining particulate matter and stored in a 15 mL conical tube at 4°C.

3.1.2 Anion Exchange Chromatography

Separation of keratan sulfate chains (and other sulfated glycosaminoglycans) from

the tissue/lavage pronase supernatant is achieved by a Vivapure Q Maxi H strong anion

exchange column (Sartorius, VS-1X20QH08). The column is equilibrated with 5 mL of

Column Wash Buffer (50 mM sodium acetate, 50 mM sodium chloride, pH 4.5), by

centrifuging at 500 x g for 5 minutes to pass the buffer through the column. The pronase

supernatant is then added to the column in 10 mL increments and again centrifuged.

With the highly anionic glycosaminoglycans (GAGs) bound to the column, the flow-

through is discarded and the column is washed three times (10 mL each) with Column

Wash Buffer. The anion exchange centrifugal column is then transferred to a fresh 50

mL conical tube and the GAG chains are eluted with 5 mL of a 16% sodium chloride

solution.

3.1.3 Methanol Precipitation

The anion exchange chromatography purified GAGs are then precipitated with

80% methanol by adding 20 mL of distilled methanol to the 5 mL of salt-eluted sample.

The sample is placed at 4°C to accelerate precipitation. The precipitate is visualized after

16 hours to be a fluffy, white material that is isolated by pelleting by centrifugation at

5,000 x g for 20 minutes at 4°C. The methanol solution is carefully poured out and the

pelleted glycosaminoglycans are solubilized in a small volume of water (0.5 – 1 mL),

transferred to a 2 mL Eppendorf tube, and dried by a SpeedVac Concentrator.

3.1.4 Removal of Non-KS Glycosaminoglycans

The dried glycosaminoglycan sample is then resolubilized in 100 μL of a 5X

reaction buffer consisting of 250 mM Tris HCl, 250 mM sodium acetate, 250 mM sodium

chloride, 10 mM calcium chloride, pH 7.5. To this reconstituted sample is added 10 μL

of a 10 mU/μL stock of Chondroitinase ABC enzyme (AmsBio 100330-1A, Proteus

vulgaris) and 10 μL of a 10 mU/μL stock of Heparinase I and III enzyme blend (Sigma

H3917, Flavobacterium heparinum). The solution is then brought up to 500 μL final

volume with water to give the proper concentration of reaction buffer and a final enzyme

activity of 200 mU/mL. The sample reacts in a 37°C incubator for a minimum of 24 h to

limit-digest chondroitin sulfate, dermatan sulfate, and heparan sulfate glycosaminoglycan

chains. After incubation, the sample is applied to a 3K molecular weight cut-off

centrifugal filter (Millipore, Amicon Ultra – 0.5 mL, UFC500324) to filter out the

disaccharides generated upon enzymatic cleavage and retain the larger molecular weight

KS chains. The centrifugal filter tubes are centrifuged at 14,000 x g for 15 minutes and

the flow-through is discarded. Initially, 450 μL of a 1 M sodium chloride solution is

added to the concentrated sample to exchange the Tris-sulfate interactions with sodium

sulfate adducts, which is necessary for further chemical modification. After one buffer

exchange with 1 M sodium chloride, 450 μL of water is added to the centrifugal filter and

buffer-exchanged at least 5X to dilute the sodium chloride concentration. After several

37 rounds of buffer-exchange, the solution is collected into a pre-weighed Eppendorf tube by a reverse spin at 1,000 x g for 2 minutes. The sample is then dried by SpeedVac and a dry weight is recorded.

3.1.5 Neocuproine Reducing Sugar Assay

Quantification and characterization of the KS chains is accomplished through a reducing sugar colorimetric assay after differential enzymatic cleavage with keratanase I or keratanase II, which produces galactose and N-acetylglucosamine reducing ends, respectively (Figure 3.1). The keratan sulfate samples to be quantified are first reconstituted in a known volume of water before adding to the reaction solution (10 mM sodium acetate, pH 6.0). Keratanase I and II enzymes are at stock activities of 0.167

U/mL which is added to the reaction solution to a final activity of 16.7 mU/mL. The volume is then brought up to a final volume of 25 μL with Dulbecco’s 1X PBS without calcium and magnesium. In total, each keratan sulfate sample to be analyzed will have a control tube without enzyme, a keratanase I digest, and a keratanase II digest. The reaction Eppendorf tubes are centrifuged briefly and incubated at 37°C for a minimum of

24 h for a limit digest. Each reaction tube is then added to water and 10 mM sodium acetate, pH 6.0 buffer in increasing concentrations to provide a range of digested sample.

A typical assay includes a background control tube with no sample, followed by tubes containing 2.5 μL, 5 μL, or 12.5 μL of either enzymatically digested KS sample or undigested control KS sample. To each sample for analysis, 100 μL of a copper sulfate solution (4 g sodium carbonate, 1.63 g glycine, 47 mg copper sulfate pentahydrate, dissolved in 100 mL water) and 100 μL of a neocuproine solution (0.151 g neocuproine hydrochloride in 100 mL water) are added. The mechanism of colorimetric assay

development is shown in Figure 3.1. Briefly, copper II is reduced to copper I by the

reducing end aldehyde of the generated disaccharide which is oxidized to a carboxylic

acid. Copper I complexes with neocuproine to give a yellow solution which is measured

by absorbance at 405 nm. All tubes to be analyzed are boiled for 12 minutes at 100°C

followed by a 1:1 dilution into 100 μL of water in the wells of a 96-well clear polystyrene

plate (Corning, Costar assay plate, 9017) in duplicates. To allow for quantification, two

standard curves are generated with varying concentrations of galactose or N-

acetylglucosamine from 5-50 nmoles of glycan (Figure 3.2). The standard curve samples

are processed in a similar way with the same buffers and volumes but with concentrations

of 5 μg/mL, 10 μg/mL, 25 μg/mL, and 50 μg/mL of reducing sugar. The 96-well plate is

read for absorbance at 405 nm on a FilterMax F3 multi-mode microplate reader

(Molecular Devices, 0084427).

For data analysis, the slope of the control samples without enzymatic digestion

(background) are subtracted from the slopes of the keratanase I or II digested samples

which is then divided by the slopes of the standard curves (galactose or N-

acetylglucosamine) to give a value of reducing sugar (nmol) per sample volume (μL). A

ratio can be calculated between the keratanase II digested sample and the keratanase I

digested sample to provide a measure of sulfation with di-sulfated disaccharides to mono- sulfated disaccharides. Multiplying by the volume of the original sample as well as accounting for the initial sample dilution for the reactions results in the total amount of

KS disaccharide (nmol) in the sample (Figure 3.3 & 3.4).

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40 mix contains assay buffer (44 μL), sialic acid converting enzyme (2 μL), sialic acid development mix (2 μL), and a sialic acid probe (2 μL); all of which are proprietary formulas. The master mix is added to each well (50 μL) and the plate is incubated at room temperature for 30 minutes, protected from light. After incubation, the plate is analyzed for fluorescence on a microplate reader (Tecan Safire2, I112942) run with

Magellan 7.2 software, with an excitation wavelength of 535 nm and an emission wavelength of 587 nm.

3.1.7 KS Carbohydrate Compositional Analysis

To determine the carbohydrate composition of the purified KS samples, KS chains were hydrolyzed with strong acid and subjected to analysis on a DIONEX ICS

6000. For acidic carbohydrate analysis, KS samples were diluted in degassed and filtered water and combined 1:1 with 200 mM hydrochloric acid, 500 mM sodium chloride and subsequently heated for 3 h at 80°C. These samples were either further diluted with degassed and filtered water or injected directly into the 20 μL injection loop attached to a

DIONEX ICS 6000. These samples were resolved on a PA-1 column with a multi- gradient wash protocol consisting of the following buffers [A) 0.75 mM NaOH; B) 200 mM NaOH; C) 400 mM NaOAc] and the following proportions of buffer over time [0-20 minutes = 21.9% A, 40.6% B, 37.5% C; 20-21 minutes = 100% B; 21-31 minutes =

100% B; 31-32 minutes = 21.9% A, 40.6% B, 37.5% C; 32-40 minutes = 21.9% A,

40.6% B, 37.5% C]. The data collections rate was set at 2 Hz and the waveform used is a

DIONEX pre-set function labelled as *Gold, carbo, quad.

For neutral carbohydrate analysis, KS samples were diluted in degassed and filtered water and combined 1:1 with 4 M trifluoroacetic acid (TFA) in a glass 13 x 100

41 mm screw-cap tube and subsequently heated for 3 h at 100°C. After heating, acid hydrolyzed samples were placed on a SpeedVac to remove TFA, with a sodium hydroxide pellet in an adjacent tube to neutralize gaseous TFA, and re-solubilized in degassed and filtered water to inject onto the DIONEX. These samples were also resolved on a PA-1 column with a multi-gradient wash protocol consisting of the following buffers [A) 0.75 mM NaOH; B) 200 mM NaOH; C) 400 mM NaOAc]. The following multi-gradient elution profile was used [0-25 minutes = 92.5% A, 7.5% B; 25-

26 minutes = 100% B; 26-35 minutes = 100% B; 35-36 minutes = 92.5% A, 7.5% B; 36-

45 minutes = 92.5% A, 7.5% B]. The data collections rate was set at 2 Hz and the waveform used is a DIONEX pre-set function labelled as *Gold, carbo, quad.

Elution peak profiles from DIONEX-resolved KS samples were processed and analyzed in comparison with carbohydrate standards run using the same conditions consisting of 20 pmoles each of N-acetylneuraminic acid, N-glycolylneuraminic acid, and galacturonic acid for acidic sugars and 25 pmoles each of fucose, galactosamine, glucosamine, galactose, glucose, and mannose for neutral sugars.

3.2 Analysis of Purified Keratan Sulfate Chains

3.2.1 Neocuproine Reducing Sugar Assay Data

Galactose and N-acetylglucosamine have a slight difference in their reduction potentials (Figure 3.2), with equal concentrations of the glycans showing an increased absorbance of neocuproine for the galactose reducing end, implicating the aldehyde of galactose as a more effective reducing agent.

From a combined total of 9.8 g of human tracheobronchial tissue was purified

39.2 mg of lyophilized KS, a 0.4% (w/w) total amount of tissue-extracted KS.

Neocuproine reducing sugar assay quantified the human tracheobronchial KS

disaccharides as 27.3 μmoles, with a ratio of 0.4 di-sulfated to mono-sulfated

disaccharides based on keratanase enzyme differential sensitivity. However, nasal lavage

KS was more sensitive to keratanase II digestion and showed a ratio of 2.7 di-

sulfated/mono-sulfated disaccharides. These data implicate nasal lavage KS as carrying

either more di-sulfated disaccharide units or carrying more fucosylated regions which are

resistant to keratanase I treatment but may not be resistant to keratanase II.

Quantification of the nasal lavage KS provided a value of 1.7 μmoles total disaccharide

units from a purified stock of 6.7 mg of dried KS, which was isolated from 9.4 mL of

human nasal lavage (0.07% w/v). Total percent of sample-purified KS was higher for the

tracheobronchial tissue sample, which we hypothesize to be due to the high expression of

KS proteoglycans in the cartilaginous tissue that would not be expected to be found in such high expression in nasal lavage samples. The pattern of sulfation was notably different between tracheobronchial KS and nasal lavage KS. Tissue-derived KS was

more keratanase I sensitive and implicates less di-sulfated disaccharides on the KS chains. Nasal lavage KS was more keratanase II sensitive and implicates KS chains with a higher proportion of di-sulfated disaccharides.

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neutral sugars (Figure 3.6) and three acidic sugars (Figure 3.7) which together comprise

the majority of expressed carbohydrates found in mammalian tissues. A pattern of

elution times was obtained which allowed us to determine which carbohydrates were

eluted from the KS samples. Chromeleon 7.2.8 program software was utilized to set up

the protocols, collect the elution profiles, and analyze the data from each sample run.

Elution times were recorded, peaks selected, and peak areas were calculated using this

same software. After assigning each peak’s identity and calculating the peak area, a ratio

of carbohydrates could be obtained to hypothesize the composition of the KS chains.

Although some of the carbohydrates detected in the KS samples ran at slightly

different elution times than the standard 6-mix, an expected pattern of carbohydrate peak elution times was observed which allowed us to assign each peak. Both the tracheobronchial and nasal lavage KS samples showed distinct peaks for fucose, galactosamine, glucosamine, and galactose which compose the major neutral glycans of

O-linked KS chains. Each KS chain is O-linked by an N-acetylgalactosamine (GalNAc) to either a serine or threonine amino acid. For DIONEX sample run analysis, peak area of galactosamine is used as the denominator when calculating the composition of a KS chain since each KS chain is expected to contain only one GalNAc which is converted to galactosamine upon acid hydrolysis. KS chains are primarily disaccharide repeating units composed of galactose and N-acetylglucosamine (glucosamine after acid hydrolysis) and so the observed peak areas of these two sugars are expected to be equal. Both KS samples show two adjacent peaks which are roughly identical in peak area (Figure 3.8) and their elution pattern matches with glucosamine and galactose from the neutral sugar

6-mix standard. The elution times, peak areas, and ratio to galactosamine for the two KS

47 samples are summarized in Table 3.1. Tracheabronchial KS chains are calculated to be composed of 5-6 LacNAc disaccharide units, with only one fucose per KS chain. Nasal lavage KS chains are calculated to be composed of 1-2 LacNAc disaccharide units, with again a single fucose per KS chain. As for the acidic sugars, the expected elution time for sialic acid from the KS samples shows two peaks of 3.3 and 3.7 minutes, which are not clearly identifiable. To address this uncertainty, KS samples were sialidase treated for 1.5 h at 37°C with 25 mU/mL of V. cholera sialidase prior to DIONEX analysis and without acid hydrolysis and the resulting spectrum was identical to the acid hydrolyzed chromatograms, implicating each of these peaks as a species of sialic acid (data not shown). The peak eluting at 6.8 minutes also appears in the sialidase-treated DIONEX chromatograms which verifies its identity as a sialic acid but remains currently unknown as to its exact structure. Since both the acidic and the neutral sugar analyses were performed on equal amounts of sample, a ratio of sialic acid to galactosamine can be calculated and provides approximately one sialic acid per tracheobronchial KS chain and one sialic acid per every three nasal lavage KS chains based on the two peaks that elute near the sialic acid standard. This calculation does not take into account the peak at 6.8 minutes, which is released by sialidase, but is not yet structurally identified.

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Table 3.1. Summary of KS Sample Carbohydrate Analyses on DIONEX.

3.3 Conclusions and Remarks

The modified neocuproine reducing sugar assay with differential keratanase treatment provides a new analytical technique for quantifying keratan sulfate

disaccharides and deciphering patterns of sulfation along the keratan sulfate chains, albeit

currently in a heterogeneous KS population. Typical yields of low milligram amounts of

KS are purified from gram quantities of human airway tissue or nasal lavage with a selective amino functional group at the reducing end to allow for further derivatization.

The fluorometric sialic acid assay kit indicates high sialylation, with approximately one

sialic acid/KS disaccharide, which is more than we observe for the DIONEX analyses,

though not drastically different. DIONEX analysis of the two KS samples implicates a

tracheobronchial KS chain of 5-6 LacNAc disaccharides, a single fucose, and a single sialic acid, and a nasal lavage KS chain of 1-2 LacNAc disaccharides, maybe a single fucose, and a sialic acid on every three KS chains. Although there is another sialidase- sensitive acidic sugar peak eluting at 6.8 minutes, this structure cannot be determined yet because it appears to be some modified sialic acid that is resistant to 1 M hydrochloric acid, 80°C incubation for 3 h which is sufficient to hydrolyze sulfate groups and O- acetylated groups.

These various analytical techniques allow for the quantification and initial carbohydrate compositional analysis of an isolated KS chain sample. Having an approximate quantification of the KS chains will provide an effective starting point for chemical modification of the KS chains with an estimate of molar ratios and appropriate stoichiometry of reagents. Also, this pool of KS chains can be further separated into

Siglec-8-binding and non-binding fractions and again subjected to these and other analytical methods to understand the structural subtleties of Siglec-8-binding sialylated

KS chains.

Chapter 4: Keratan Sulfate Chain Neoglycolipid Semi-synthesis

Quantification of the KS disaccharides allows for approximation of molar

equivalents of reagents to tag the KS chains, specifically targeting the amino group of the

single amino acid remaining at the non-reducing terminus of the chains. One of the most

popular conjugation reagents are the “click” reagents which attach through a 1, 3-dipolar

cycloaddition of an azido functional group and an alkyne, either catalyzed with copper or

through steric-hindrance of a cyclo-alkyne. Azide-tagged KS chains could be attached to several platforms for binding and functional studies. Our primary interest was in

generating KS-neoglycolipids by “clicking” the KS chains to lipids, which would be

useful for functional assays by incorporating the lipid tails into nano-liposomes. This

approach has been used extensively in the Paulson lab to display CD22 (Siglec-2)

synthetic glyco-lipid ligands for B-cell apoptosis studies.21 This conjugation technique

was applied to human airway tissue purified KS chains and due to issues with subsequent

purification and solubility was not applied to nasal lavage KS chains.

4.1 Materials and Methods

4.1.1 Conjugation of Azido Linker to Keratan Sulfate Chains

After extensive proteolysis and isolation from human airway tissue as described

in Chapter 3, intact KS chains, glycosidically linked primarily to a single serine or

threonine, were derivatized at the remaining primary amine using azidoacetic acid NHS

ester. KS chains were dried on a centra-vap and reconstituted in 100 mM sodium

phosphate buffer, pH 8.0. An equal volume of freshly prepared azidoacetic acid-NHS

ester (BroadPharm, BP-22467) dissolved in 30% THF/water was added to the KS chains

53 and the reaction proceeded for 16 h at ambient temperature with final concentrations of 1 mM total KS disaccharides (based on keratanase release of reducing sugars, see Chapter

3) and 50 mM azidoacetic acid-NHS ester. The reaction solution (1 µL) was dotted onto a fluorescent thin-layer chromatography (TLC) plate adjacent to 1 µL each of azidoacetic acid NHS-ester starting material, unreacted keratan sulfate starting material, and ethylamine as a positive control. The TLC plate was run for 40 minutes in 60:35:8 chloroform, methanol, 0.25 % aqueous potassium chloride. The plate was subsequently sprayed with 1 mg/mL fluorescamine in acetone followed by 1% triethylamine in dichloromethane to visualize primary amino functional groups remaining at the origin

(KS chains will not migrate). Released N-hydroxysuccinimide from the reaction is detectable by short-wave UV (Figure 4.1).

Remaining azido-derivatized KS chains were further reacted with 1-ethynyl pyrene to fluorescently tag the azido group on the attached linker through 1, 3-dipolar cycloaddition (Huisgen cycloaddition; “click” chemistry). Prior to conjugation, the linker reaction product was filtered of excess azido-linker reagent through a 3K MWCO centrifugal spin filter and buffer exchanged with water three times. Intact azido-KS product (equivalent to 1 mM of KS disaccharides) was added to a solution of 25 mM potassium phosphate buffer, pH 7.7, 10 mM 1-ethynyl pyrene in DMSO, 1 mM copper sulfate, and 1.25 M sodium ascorbate. The reaction proceeded for two hours at ambient temperature. The reaction was run on a non-fluorescent silica-based TLC plate in

60:35:8 chloroform, methanol, 0.25 % aqueous potassium chloride along with 1-ethynyl pyrene stock, and KS-linker unreacted (1 µL of each). Visualization by long and short

wave UV light for fluorescence at the baseline, since the KS chains are too polar to move

at all (Figure 4.2).

4.1.2 Synthesis of Dibenzocyclooctyne (DBCO)-Lipid

Dibenzocyclooctyne-amine (DBCO, 18 µmol; Sigma 761540) was added to 9

µmol of distearoyl-phosphatidylethanolamine-NHS ester (Coatsome, FE8080SU5) and

dissolved in 500 µL of chloroform and reacted for 16 h at ambient temperature. The

reaction was then extracted five times with 1.5 mL acetonitrile to remove the unreacted

DBCO and the released N-hydroxysuccinimide. The acetonitrile washes were spotted on

a non-fluorescent silica-coated TLC plate and visualized by long and short wave UV light

for absorbance of NHS and fluorescence of DBCO. The extracted reaction product was

then run on a non-fluorescent silica-coated TLC plate in 60:35:8 chloroform, methanol,

0.25 % aqueous potassium chloride along with stock solutions of the starting materials to

show reaction completion and purity.

4.1.3 Anion Exchange Separation of Siglec-8-binding KS

Untagged human airway KS chains were reloaded onto a Vivapure Q Maxi H

strong anion exchange column, the same type of column used during initial KS

purification. The column is centrifuged at 500 x g for 5 minutes to pass the buffer

through the column and washed three times (10 mL each) with the wash buffer described

in Chapter 3. The anion exchange column is then transferred to a fresh 50 mL conical

tube and the keratan sulfate chains are eluted with 3 mL of a step-wise increasing

gradient of sodium chloride solutions (200 mM, 600 mM, 1 M, 1.75 M, 2.7 M sodium

chloride). Step-wise salt eluted KS were dialyzed against water in 1 kD molecular

55 weight cut-off (MWCO) dialysis tubing with several exchanges to remove all of the salt.

Each eluted fraction was centra-vapped to a dry powder, reacted with the azidoacetic acid

NHS ester as above, and purified from excess reagents with 3 kDa MWCO centrifugal filtering with buffer exchange to water followed by drying to powder by centra-vap.

4.1.4 Attachment of Azido-KS to DBCO-Lipid

DBCO-Lipid (10 molar eq.) prepared above was dissolved in tetrahydrofuran and reacted with dried Azido-KS for 16 h at 37°C through a sterically hindered copper-free

“click” reaction. Reaction completion was assessed by running a non-fluorescent silica- coated TLC plate in 60:35:8 chloroform, methanol, 0.25 % aqueous potassium chloride along with stock solutions of the starting reagents. Each fraction salt-eluted from the anion exchange column was separately tagged with the azido-linker and conjugated to

DBCO-lipid to form KS neoglycoplipids (Figure 4.4).

4.1.5 PVDF Dot-Blot Analysis

After synthesizing the KS neoglycolipids, 5 µL of each salt-eluted fraction was dotted onto a methanol-activated PVDF membrane along with 2 µL of S8-1M tracheal extract ligand as a positive control. The membrane was dried in a chemical hood for 1 h, blocked in 5% non-fat milk (blotto) in 0.1% PBST for 1 h, and overlaid with pre- complexed Siglec-8-Fc with HRP-conjugated goat anti-human IgG for 16 h at 4°C. The membrane was then washed three times with 0.1% PBST (5 minutes each), incubated with enhanced chemiluminescence reagent, and visualized on the GeneSys imaging system (Figure 4.5).

4.1.6 Neoglycolipid Array Binding Analysis

The 1 M and 1.75 M salt-eluted KS fractions conjugated to lipid were diluted in

water to bring the final KS concentration to approximately 100 μM based on disaccharide

quantification. Aqueous dilutions of KS-lipids were diluted 1:1 with a solution of 1 µM

phosphatidylcholine (PC) and 4 µM cholesterol in ethanol, vortexed, and 50 μl added to

each of triplicate wells of a butanol- and ethanol-washed 96-well polystyrene microwell

plate22 to give 50, 125, 250, or 500 pmoles KS (based on disaccharide)/well. The solvent

was allowed to evaporate in a chemical fume hood for 1.5 hours, the plate briefly

immersed in water three times, shaking off between each wash, and blocked by adding

100 μl/well of 2 mg/mL BSA in Dulbecco’s PBS (cation free) for 1 hour at 37°C. In

triplicate wells on the same plate negative and positive control glycolipids were adsorbed

as follows. Ganglioside GT1b, 6-sulfo-3-sialylLacNAc neoglycolipid, or 6’-sulfo-3-

sialylLacNAc neoglycolipid were prepared at 1 μM in ethanol and added to the above

plate at50 µL/well (50 pmol/well). After drying under a stream of nitrogen 50 µL of the

above PC/cholesterol solution was added to each well prior to subjecting to air-drying,

washing, and blocking as above.

Siglec-8-Fc (2 ng/µL final) and anti-human IgG AP-conjugated secondary

antibody (1 ng/µL final) were pre-complexed on ice for 30 minutes in blocking buffer.

After blocking, the plate was submerged three times in Dulbecco’s PBS, incubated with pre-complexed Siglec-8-Fc for 1 h at ambient temperature, washed twice in PBS and once in water, followed by incubation with p-nitrophenyl phosphate at 2 mg/mL and visualization by kinetic absorbance readings at 405 nm every 5 minutes for 10 cycles.

Absorbance slopes were calculated and the average of background triplicates of

PC/cholesterol alone were subtracted (Figure 4.6).

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activated eosinophils for functional assays. The azido-linker attachment reaction shows a

high yield of azido-KS product based on the complete loss of fluorescein detection of the

starting material KS chains. The KS-lipid that was initially separated by step-wise salt elution off of an anion exchange column before chemical derivatization is now sufficiently hydrophobic to immobilize into polyvinylidene fluoride membranes (PVDF) for semi-quantitative Siglec-8-Fc binding assays in the form of a dot-blot experiment.

The 1 M salt elution did contain Siglec-8-binding KS chains and so we moved forward with this sample as well as the 1.75 M salt elution sample as a negative control for more quantitative binding analyses. Although Siglec-8-Fc was able to bind to KS-lipids adsorbed to PVDF membranes, it could not bind to KS-lipids in an ELISA format on polystyrene plates. We believe this may be due to the heterogeneity of the salt-eluted samples and we determined that a more stringent and selective method for isolating

Siglec-8-binding KS chains needs to be employed. Anion exchange chromatography will separate KS chains with different degrees of sulfation, but will not necessarily isolate the

Siglec-8-binding chains from other sub-populations.

The KS-lipids showed low solubility in entirely aqueous solutions, which make functional assays challenging because of the sensitivity of eosinophils in culture to the media that they are cultured in. Low aqueous solubility also limits the ability to separate these KS-lipids by affinity purification and other methods. Also, the unpredictability of the lipid tail with respect to the formation of micelles or non-specific attachment to affinity resins makes these samples more challenging. These limitations, along with the necessity for quantitative binding analyses, motivated us to begin investigating alternate ways to tag purified KS chains and isolate only the Siglec-8-binding KS chains.

Chapter 5: Biotinylation of Keratan Sulfate Chains

Due to the issues with KS-lipid solubility and binding experiments, we decided to

tag the remaining amino acid with a highly water soluble sulfo-NHS-biotin reagent which

provided a more effective means of following the KS chains through subsequent

purification and analyses. Biotin – streptavidin interactions are some of the tightest in

biology and the ability to bind biotinylated-KS chains to a streptavidin-coated 96-well plate would allow for high throughput analysis of KS-chains isolated by Siglec-8 affinity

separation and size exclusion chromatography.

5.1 Materials and Methods

5.1.1 Biotinylation Reaction

Purified KS chains from human tracheobronchial tissue or human nasal lavage

were incubated with sulfo-N-hydroxysuccinimide-biotin to covalently attach a biotin tag

to the KS chains. KS chains (2 µmoles based on disaccharide) were centra-vapped to a

powder in a 500 µL Eppendorf tubes, then were dissolved in 250 μl of 95 mM sulfo-

NHS-biotin in 25 mM HEPES buffer, pH 8.3(12 molar equivalents) and reacted 16 h at

ambient temperature. The reaction product was purified by several rounds of centrifugal filtering from distilled water using a 3K-molecular weight cut-off spin column to remove

biotinylation reagent byproducts and retain the biotinylated-KS chains. Each wash step

with distilled water on the centrifugal filter was saved to be analyzed for removal of the

excess unreacted biotin. Purified KS-biotin was eluted with a 2 min, 1,000 x g inverted

spin.

Equivalent volumes of reactants, crude product, washes, and purified product

were spotted onto two methanol-activated PVDF membranes, each with positive controls

for detection of biotin and Siglec-8 ligand. Each membrane was air dried in a fume hood and then was blocked for 1 h in 5% nonfat dry milk (blotto) in PBS containing 0.1%

Tween-20 (PBST), shaking at ambient temperature. One blot contained an equal volume of trachea-extracted Siglec-8 ligand as a positive control and was subsequently overlaid with Siglec-8-Fc pre-complexed with HRP-conjugated goat anti-human IgG. The second blot contained an equal volume of biotinylated-MAL-II lectin (1 mg/mL stock, Vector

Labs) as a positive control and was subsequently overlaid with HRP-streptavidin to detect biotinylation of the KS chains. After incubating the blots with overlay antibody or streptavidin for 16 h at 4°C, the blots were washed three times with PBST (5 minutes/wash) and analyzed with enhanced chemiluminescence reagent. Equivalent volumes of the above samples were similarly spotted onto a non-fluorescent TLC plate and visualized for excess biotin removal using an iodine vapor chamber (Figure 5.1).

5.1.2 Siglec-8-COMP Affinity Chromatography

To isolate the Siglec-8-binding KS chains, we engineered a chimeric pentavalent

Siglec-8-COMP construct and immobilized it on nickel Sepharose beads to generate an affinity column. Using a 2 mL bed volume of pre-loaded hexa-histidine tagged Siglec-8-

COMP chimera on nickel Sepharose beads to provide a multivalent affinity capture system, the purified KS-biotin chains were separated into Siglec-8-binding and non- binding fractions and eluted directly onto a 96-well streptavidin-coated microwell plate

(Pierce, High binding capacity, 15500).

The Siglec-8 affinity column was attached to a peristaltic pump to regulate the

flow of solutions through the column and the purification protocol was conducted at 4°C.

The column was initially equilibrated with 15 mL of 10 mM sodium chloride, 10 mM

sodium phosphate, pH 7.0 before adding 100 µL of the KS-biotin which was dried and

reconstituted in the above equilibration buffer prior to loading onto the column. After the

KS-biotin was pushed onto the column, the column was washed with 2 mL of

equilibration buffer with 1 mL collected into an Eppendorf tube and the subsequent

elutions collected onto the streptavidin-coated plate in 150 µL fraction volumes (~3

drops). The KS-biotin was then eluted from the column with 2 mL of 150 mM sodium

chloride, 10 mM sodium phosphate, pH 7.0 and finally with 4 mL of 1.5 M sodium

chloride, 10 mM sodium phosphate, pH 7.0. After each of the elutions were collected, 50

µL of each fraction was transferred to a second streptavidin-coated microwell plate and the positive and negative synthetic controls for Siglec-8-binding were added in duplicate to the two plates. As a positive control, 200 pmol of synthetic 6’sulfo-sialyl-LacNAc-

biotin was added and 200 pmol of synthetic 6-sulfo-sialyl-LacNAc-biotin was added as

the negative control. The streptavidin plates incubated at ambient temperature while

shaking for 2 h to allow efficient binding. After incubation, the plate was washed three

times with Dulbecco’s PBS.

5.1.3 KS-Biotin Siglec-8-Fc Binding ELISA

The streptavidin-coated plate used to collect the KS-biotin elution fractions was

analyzed for Siglec-8 binding. Siglec-8-Fc was pre-complexed with alkaline

phosphatase-conjugated goat anti-human IgG + IgM secondary antibody in 2 mg/mL

BSA/PBS for 30 minutes at 4°C. After pre-complexing, the plate was incubated with the

Siglec-8-Fc pre-complex mixture (50 µL/well; 100 ng Siglec-8-Fc/well) at ambient

temperature for 1.5 h. The plate was washed twice in 1X PBS and once in water. 20 mg

of the alkaline phosphatase colorimetric substrate, PNPP, was dissolved in 10 mL of 100

mM Tris, 100 mM sodium chloride, 5 mM magnesium chloride, pH 9.5 and 50 µL of this

solution was added to each well. The plate was analyzed on a microplate reader at an

absorbance of 405 nm with a kinetic cycle of 5 minutes and a total of 10 cycles. The data

was processed by taking the slope of absorbance readings per cycle number for each well

(Figure 5.3 & 5.5). Alternatively, the streptavidin-coated plates with bound KS-biotin

were overlaid with anti-KS mAb 5D4 pre-complexed with HRP-conjugated anti-mouse

IgG in 10 mg/mL BSA/PBS and visualized by absorbance at 370 nm with Ultra TMB substrate (Thermo Scientific, 34028)(Figure 5.2 & 5.4).

5.1.4 KS-Biotin Eosinophil Apoptosis Functional Assay

The second streptavidin plate containing bound KS-Biotin was shipped to our collaborators Bruce Bochner and Daniela Janevska Carroll at Northwestern University,

Chicago, Illinois for functional eosinophil apoptosis assays. Siglec-8 has been shown to induce eosinophil apoptosis upon cross-linking with our airway tissue derived Siglec-8 ligand, but it has not been shown for the individual KS chains. Upon arrival, the plate was washed five times with PBS heated to 37°C and three times with complete media with a final incubation of 100 µL of media/well at 37°C for 5 minutes to equilibrate the plate before adding the eosinophils. The eosinophils were plated at 200,000 eosinophils/well and incubated for 20 h at 37°C. Apoptosis was analyzed by FACS with

Annexin V and DAPI staining (Figure 5.6). After eosinophil apoptosis analysis, the plate

was shipped back to us and probed for Siglec-8 binding as above, to validate the presence

of bound Siglec-8-specific KS-biotin chains.

5.1.5 Size Exclusion Chromatography Analysis

Affinity-purified KS chains were separated by size exclusion chromatography to

approximate the molecular weight and the length of the Siglec-8-binding KS chains. The

purified KS chains were lyophilized and reconstituted in 10 mM sodium chloride, 10 mM

sodium phosphate, pH 7.0 buffer prior to injecting onto a Superdex 200 Increase 10/300

size exclusion column with a total column volume (CV) of 24 mL. The column was

equilibrated for 0.1 CV while manually injecting 150 µL KS-biotin sample into the 180

µL injection loop. The injection loop was then flushed onto the column with 500 µL of

the column running buffer and the separation proceeded for 1.5 CV after injection while

absorbance at 214 nm (amide bond absorbance for N-acetyl containing sugars) was

collected to provide a real-time chromatogram. Fractions of 500 µL were collected in a

96-well polypropylene 1 mL deep-well plate. The column was held at 4°C with a flow rate of 0.1 mL/min throughout the entire run. After completion of the size exclusion separation, 100 µL of each fraction was loaded onto a 96-well streptavidin plate for

Siglec-8 and anti-KS (5D4) binding ELISA analyses as described above (Figure 5.8).

Siglec-8-binding KS chains were also treated with keratanase I enzyme, biotinylated on the reducing end of each cleavage product, and separated by size exclusion chromatography as above. KS chains were incubated with keratanase I enzyme

(16.7 mU/mL) in 10 mM sodium acetate, pH 5.9 for 22 h at 37°C. After incubation with the KS chains, keratanase enzyme was inactivated by heating the sample to 90°C for 5

minutes. A solution of hydrazine-biotin reagent (Sigma, B7639) and sodium

cyanoborohydride (Acros Organics, 168550100) in 1.3 M acetic acid and 42% DMSO

(v/v) were added to the KS sample to a final concentration of 5 mM hydrazine-biotin and

500 mM sodium cyanoborohydride. KS chains reacted with hydrazine biotin for 24 h at

37C, after which the solution was brought up to a pH of 6.0 with 2 M sodium hydroxide

and the biotin-tagged sample was injected onto the sizing column as above and analyzed

for Siglec-8 binding on a streptavidin-coated microwell plate as above (Figure 5.9).

Briefly, the hydrazine-biotin solution was prepared by dissolving 26.5 mg of hydrazine-

biotin in 6.89 mL DMSO. After 20 minutes of periodically inverting the solution in a 15

mL conical tube (Thermo, 339651), 1.22 mL of glacial acetic acid was added. In a

separate 15 mL conical tube, 770 mg of sodium cyanoborohydride was dissolved in 9.8

mL water and inverted periodically for 20 minutes. After which, 8 mL of each solution

were combined and aliquots stored at -80°C. Final concentration of reagents in stock

solution is 6.25 mM hydrazine-biotin and 625 mM sodium cyanoborohydride.

5.2 KS-Biotinylation and Isolation of Siglec-8 Binding KS

5.2.1 KS-Biotin Reaction

Biotinylation of keratan sulfate chains and removal of excess biotin reagent was analyzed by PVDF membrane dot-blots overlaid with Siglec-8-Fc or with streptavidin-

HRP, as well as iodine vapor TLC analysis. Siglec-8-Fc binds selectively to the KS-

biotin crude and purified samples which indicates that biotinylated-KS adsorb to the

PVDF membrane more efficiently than untagged KS and that none of the KS was lost

during the wash steps (Figure 5.1). The streptavidin-HRP membrane overlay shows a

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column was collected 42 fractions of 150 μL/fraction in a serpentine elution pattern

directly into a 96-well streptavidin-coated plate. These fractions were transferred to a second streptavidin-coated plate and these plates have a theoretical capacity of 125 pmoles of biotinylated sample. The use of a streptavidin plate will wash away any KS that was not biotinylated which simplifies the data analysis and allows us to observe only the tagged KS chains. To compare the population of Siglec-8-binding and non-binding fractions, each fraction was dotted onto an activated PVDF membrane and overlaid with

AP-Siglec-8-Fc and Streptavidin-HRP, similar to the analysis of the biotinylation reaction samples listed above. With both probes conjugated to different enzymes, we could use the same dot-blot to observe both Siglec-8-binding and streptavidin-binding, which eliminates the variability between dotting the small sample volumes onto multiple membranes.

5.2.3 Affinity Column Fraction Analysis

Since each KS chain presumably has only a single biotin, dot-blot analysis with streptavidin overlay allows for relative quantification of KS chains in each fraction eluted off of the affinity column. Streptavidin overlay shows the majority of KS-biotin chains do not bind to the column and instead elute with the 10 mM sodium chloride wash

(Figure 5.2, left blot). The Siglec-8-Fc overlay shows two peaks of intense Siglec-8- binding, the first peak eluted with 150 mM sodium chloride, and the second peak eluted with 1.5 M sodium chloride. Compared to the streptavidin overlay, there appear to be relatively few KS-biotin chains in the high salt-eluted fractions where the Siglec-8- binding peaks are located. Siglec-8-binding KS chains are a low proportion (<1%) of the

total KS chains purified from human airway samples and affinity separation is a crucial

and successful step in isolating these Siglec-8-binding KS chains.

Siglec-8-Fc binding to the streptavidin-coated plate recapitulates the findings observed by dot-blot analysis (Figure 5.3). The 6-sulfo sialyl LacNAc biotin negative control (-) shows no Siglec-8-Fc binding, which validates Siglec-8-Fc specificity. The

6’-sulfo sialyl LacNAc biotin positive control (+) shows strong binding, which validates

the Siglec-8-Fc probe used in this analysis. The observed binding profile matches the

PVDF dot-blot analysis, with the majority of the Siglec-8 specific KS-biotin chains eluting in two peaks, the first with 150 mM salt and the second with 1.5 M salt. These

data reveal a separation of KS chains into Siglec-8-binding chains of either low or high

affinity based on salt elution and a removal of non-binding KS chains in a low-salt wash.

Interestingly, nasal lavage KS showed more robust binding to the Siglec-8 affinity

column than the tracheobronchial KS ligand (Figure 5.5). All of the Siglec-8-binding KS

chains remained bound to the affinity column during the 150 mM sodium chloride wash

and most, but maybe not all of the bound KS eluted with 1.5 M sodium chloride. This

observation is not unexpected because we previously found the nasal lavage intact ligand

(DMBT1S8) to be bound so tightly to the Siglec-8-COMP affinity column that 1 M

sodium chloride could not elute it, though the tracheobronchial ligands were successfully

eluted with the same buffer. Similar to the airway tissue purified KS, nasal lavage

Siglec-8-binding KS chains represent a minor species of the total airway KS chains and

the majority of KS chains are washed through the Siglec-8-COMP column in the first 12

fractions (Figure 5.4).

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four standard peaks (figure 5.7 A) and set up an equation (figure 5.7 B) to predict the

molecular weights of the KS-biotin samples.

The KS-biotin chains showed an absorbance profile that indicated a high- molecular weight species of KS that had a peak elution around 80 minutes. After transferring each fraction to a streptavidin-coated microwell plate and overlaying with

Siglec-8-Fc, we confirmed that the Siglec-8-binding KS was of an approximate molecular weight of 44 kDa, which roughly corresponds to 80 disaccharide repeats on a single KS chains and is much larger than any reported human KS chains (Figure 5.8).23 Anti-KS

(5D4) overlay showed some overlap with the Siglec-8-binding data but also indicated a

sub-population of highly sulfated KS chains that did not bind Siglec-8 but were of high

molecular weight as well (>20 kDa).

If the KS-biotin chains were pre-treated with keratanase I to produce cleavage products, tagged with hydrazine-biotin for subsequent analysis, and size-separated, we observed a shift of Siglec-8-binding KS-fragments down to ~2 kDa (Figure 5.9). This size would correspond roughly to a heptasaccharide, which retains the glycans and modifications necessary to bind to Siglec-8 in a format of bound KS to streptavidin- coated wells. Since keratanase I only cuts mono-sulfated disaccharide regions (Galβ1-

4GlcNAc(6S)) and all of the Siglec-8-binding KS shifted to this tight peak after enzymatic treatment. Anti-KS (5D4) overlay of keratanase I-treated KS-chains did not match the Siglec-8 overlay and instead showed large molecular weight KS chains that did not show a drastic shift upon enzymatic treatment. We hypothesize that the large KS chains showing 5D4-binding are composed of highly sulfated regions of the KS chains, which are not susceptible to keratanase I cleavage. An alternative explanation for the

drastic shift of Siglec-8-binding KS chains is that these chains are attached to a

glycopeptide rather than single amino acids, which was unsusceptible to initial

proteolysis.

The potential for KS-glycopeptides that were incompletely proteolyzed in our

analyzed sample was addressed by incubating the KS chains in 20 mM dithiothreitol

(DTT), thiol groups blocked with iodoacetamide, and KS chains treated with or without

pronase enzyme (20 h, 37°C). We did not observe a shift in Siglec-8-binding or 5D4-

binding with either treatment compared to untreated KS chains (data not shown). We

also purchased an endopeptidase (O-sialoglycoprotein endopeptidase, Cedarlane,

CLE100) specific for sialylated O-linked glycans and highly anionic glycans, which

similarly did not shift the elution time for Siglec-8-binding or 5D4-binding KS chains on

size exclusion chromatography (data not shown). KS chains are known to bind divalent

cations with their highly sulfated glycans,23 and so we incubated KS-biotin chains with 1

mM EDTA to chelate any divalent cations and again resolved the KS chains by size

exclusion chromatography followed by Siglec-8-Fc overlay of these fractions bound to

streptavidin-coated microwells and did not observe a shift in the elution times of these

KS-biotin chains (data not shown). These data implicate a population of high molecular weight, single KS chains that bind Siglec-8.

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82 that these Siglec-8-binding KS chains are a minor sub-population of the total KS chains and that this is an effective technique for purifying only the KS chains that bind Siglec-8.

Even though these immobilized KS chains bind Siglec-8, these same samples showed no significant apoptosis for any of the eluted fractions, nor for the synthetic positive control.

This may indicate an apoptosis mechanism dependent on internalization of the Siglec-8 ligand which is impossible with the effectively immobilized KS-biotin chains.

Alternatively, the KS-biotin chains may be acting like a “glue-trap” which keeps the eosinophils stuck to the KS chains and does not allow for effective cross-linking of the

Siglec-8 receptors which is necessary to induce apoptosis. An alternative multivalent display may be able to unravel these requirements for Siglec-8 induced eosinophil apoptosis.

Nasal lavage KS-biotin chains bound more strongly to the affinity column than the tracheobronchial extracted KS chains, which has also been observed for intact ligands from these respective samples. Similar to tissue derived KS chains, nasal lavage KS chains that bind Siglec-8 are a minor species of the total KS chains and this technique successfully isolates the Siglec-8-binding KS chains from those that do not bind Siglec-8.

Keratan sulfate chains from human tissues are typically expected to be in the range of 3-40 disaccharide repeats, not the ~80 disaccharide repeats that we estimate for

Siglec-8-binding KS chains. These Siglec-8-binding KS chains are eluting quickly off of the size exclusion column and based on a set of heparin sulfate molecular weight standards, these tracheobronchial KS chains are approximately 44 kDa. Human aggrecan

KS chains have been estimated to be between 4 kDa and 9 kDa by compositional analysis and size exclusion chromatography.18,24

To validate that these KS chains were not actually glycopeptides, which had resisted the initial protease digestion, or were in some way expanding their hydrodynamic radius through covalent or ionic interactions, we subjected the KS chains to proteolysis, reduction, and cation chelation. Neither further proteolysis with the nonspecific enzyme pronase, chemical reduction with DTT, nor divalent cation chelation with EDTA reduced the observed elution times for these KS chains off of the size exclusion column. With these data, we suggest that Siglec-8-binding KS chains are of high molecular weight and that upon keratanase I treatment, these KS chains shift to a more manageable 2 kDa oligosaccharide that retains Siglec-8-binding. Further analytical studies will be required to resolve the structural relationships between these Siglec-8 binding species.

Chapter 6: Revisiting Siglec-8 and Siglec-F Ligands

This thesis has investigated the sialylated KS chains extracted from human airways which bind to Siglec-8. An ongoing question in the field is whether Siglec-F, the mouse functional paralog of Siglec-8, has overlapping specificity to endogenously expressed airway ligands similar to the overlap observed for glycan microarray printed structures. Siglec-8 is an immune receptor strictly expressed by hominids. Although

Siglec-8 and Siglec-F perform similar functional roles in regulating allergic inflammation, mice do not produce a Siglec-8 ligand in their airways, whereas human airways do produce a Siglec-F ligand (Chapter 1: Figure 1.5). Kiwamoto et al (2015) identified a subset of the mucin glycoprotein Muc5b to be the glycoprotein carrier of

Siglec-F sialoglycan ligands, specifically expressed in the mucosal glands.25 Their data suggest multiple mucin glycoprotein carriers of Siglec-F ligands, with the mucosal gland

Siglec-F ligand primarily carried on a subset of Muc5b with the glycosylation necessary

for Siglec-F recognition. This subset of mouse Muc5b carries sialylated O-linked

glycans required for Siglec-F binding, similar to the observed ligands for Siglec-8 but

present on different carrier proteins. Although Siglec-F shows binding to sulfated

sialylated glycans on microarrays, multiple publications indicate that sulfation of mouse

airway Siglec-F ligands is not a requirement for recognition, whereas Siglec-8 requires a

sulfated galactose to bind.25,26

To determine the cross-reactivity of Siglec-F binding to Siglec-8 human airway

endogenous ligands, we used a Siglec-F-Fc probe to overlay the trachea tissue purified

aggrecanS8 ligands by western blot (Figure 6.1). Also, from our observation that Siglec-8

human airway ligands are each sensitive to keratanase enzymes, human trachea tissue

sections were pre-treated with keratanase enzymes prior to Siglec-F-Fc overlay to determine keratanase sensitivity of Siglec-F ligands in human airways (Figure 6.2). Mice do not express Siglec-8 ligands in their airways, so there was no need to observe Siglec-8 overlay of mouse trachea histological sections, however, we did observe keratanase sensitivity of endogenous Siglec-F ligands in mouse airways.

6.1 Materials and Methods

6.1.1 Siglec-F Overlay of Siglec-8 Ligands

Previously purified human tracheal Siglec-8 ligands (S8-1M, -600K, and -250K)

were electrophoretically resolved on 2% agarose/1.5% acrylamide composite gels,

transferred to a PVDF membrane, blot cut in half, each half blocked with 5% nonfat dry

milk in 0.1% Tween-20 supplemented Dulbecco’s phosphate buffered saline (PBST), and

overlaid with Siglec-8-Fc or Siglec-F-Fc pre-complexed with HRP-conjugated goat anti-

human IgG secondary antibody and incubated on the blots for 16 h at 4°C. The blots

were then washed 3 times with PBST and visualized by enhanced chemiluminescence

reagents (Figure 6.1).

6.1.2 Sensitivity of Siglec-F Binding to Keratanase Enzymes

Post-mortem human trachea and mouse trachea histological tissue sections were analyzed similar to the protocol described in Chapter 2, however, prior to blocking, tissue slides were incubated with either keratanase I (AmsBio, 1.7 mU/mL) or keratanase II

(produced in house from an expression plasmid, 10 mU/mL) in 10 mM sodium acetate, pH

6.0 at 37°C for 24 h. Siglec-F-Fc (5 μg/ml) was pre-complexed in PBST supplemented with

1% BSA with alkaline phosphatase (AP)-conjugated goat anti-human IgG and IgM heavy and light chains (2 μg/ml, Jackson Immunoresearch, West Grove, PA) for 30 minutes at

4°C. Incubation of pre-complexed Siglec on the slides was for 16 h at 4°C. Slides were washed with PBST, equilibrated with 100 mM Tris-HCl (pH 8.3) supplemented with 0.1%

Tween 20 for 10 min, bound lectin conjugate detected with Vector Red alkaline phosphate substrate (Vector Laboratories, Burlingame, CA), counterstained with Hematoxylin QS

(Vector Laboratories), slides dehydrated, mounted in Krystalon (EMD Millipore) and imaged using a Nikon Eclipse 90i microscope (Figure 6.2).

6.2 Siglec Cross-reactivity Analysis

6.2.1 Siglec-8 Tracheal Ligands Cross-reactivity with Siglec-F

Although Siglec-8 and Siglec-F share overlapping binding specificity on a glycan

microarray,15 they do not share endogenous airway ligand binding specificity. Siglec-F

overlay of Siglec-8 purified human tracheal ligands of the three size classes (S8-1M, S8-

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6.2.2 Siglec-F Ligands Sensitivity to Keratanase Enzymes

To expand upon our observation that Siglec-F does not bind to Siglec-8 human

airway ligands, we pre-treated human and mouse trachea histological sections with

keratanase I enzyme. On keratanase I treated human trachea sections, Siglec-F showed

an enhanced intensity of binding to mucosal glands and connective tissue but still did not

bind cartilaginous tissue (Figure 6.2). Keratanase II treatment showed increased intensity

of Siglec-F binding that was not as intense as keratanase I, but now expands into the

cartilaginous tissue. Siglec-8 binding is reduced upon keratanase I treatment and

completely eliminated with keratanase II treatment, which is consistent with previous

experiments (Chapter 1, Figure 1.11). The human airway tissue ligands for these

paralogs appear to be completely divergent, with keratanase enzymatic treatment

eliminating Siglec-8 binding but increasing Siglec-F binding. This implies that keratanase digestion removes glycan structures that block accessibility of Siglec-F for its preferred ligand on human airways.

Siglec-F shows robust binding to untreated mouse trachea epithelial layer, connective tissue, and mucosal glands, but no binding to cartilage. Upon keratanase I or

II treatment, Siglec-F binding decreases in mouse airway connective tissue and mucosal glands but shows no reduction on the epithelial layer and still does not bind cartilage.

These data are interesting because keratanase sensitivity of Siglec-F-binding to some mouse airway mucosal ligands suggests a role for sulfation on the glycan structures, which has been previously ruled out as a requirement for Siglec-F binding to those ligands based on genetic studies.26 Those published data indicate that sulfation is not

required for binding, but do not address whether removal of sulfated (KS) glycans

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3’-sialyl-N-acetyllactosamine termini. Siglec-F does not bind as robustly to any of the

human trachea tissue purified Siglec-8 ligands. Despite the overlap of Siglec-8 and

Siglec-F binding specificity on glycan arrays, Siglec-8 and Siglec-F have different preferred sialoglycan ligand structures.

Both human and mouse airway ligands are sialidase sensitive, however, Siglec-8 ligands appear to be keratanase sensitive and thus sulfated, and only a small portion of

Siglec-F ligands are keratanase sensitive and only in mouse airway connective tissue and mucosal glands. Siglec-F binding to mouse airway ligands persists even after chemical desulfation of affinity-purified ligands or knockdown of key glyco-sulfotransferase enzymes, which suggests that sulfation is not necessary for Siglec-F binding. Although these data show keratanase sensitivity that implicates sulfation, this does not conclusively assign the sulfated glycans to the sialylated terminus where Siglec-F is expected to bind.

It is entirely possible that on mouse airway tissue sections, keratanase enzymes are cleaving a sulfated glycan segment of a polysaccharide that terminates in a 3’-sialyl-

LacNAc devoid of a sulfate, which is consistent with current published data.

In human airways, Siglec-F ligands are not keratanase sensitive, and surprisingly

Siglec-F binding to histological sections increases upon keratanase treatment, indicating increased accessibility after removal of keratan sulfate chains. This highlights a clear disconnect in binding specificity between these two eosinophil Siglecs that has not been discovered through binding studies of immobilized glycans on arrays. Further analysis of the endogenous sialoglycan ligands for Siglec-F would be required for an in depth comparison of Siglec-8 and Siglec-F binding specificities, but with these and other published data it appears that Siglec-F and Siglec-8 have diverged in their preferred

endogenous ligands. This is consistent with hypotheses concerning the evolutionary

divergence of Siglecs due to selective pressure by pathogens.27

Chapter 7: Conclusions and Discussion

We report that sialylated keratan sulfate chains are endogenous human airway ligands for the immune regulatory receptor Siglec-8. These sialylated keratan sulfate chains may be carried on large proteoglycans, however, Siglec-8 recognition is embodied by the glycans. Siglec-8-specific KS chains from human airways are represented by a

minor fraction of large molecular weight KS chains, which can be modified semi-

synthetically and isolated through a multi-step purification procedure.

The dynamic language between glycan structures written by glycosyltransferases

and their recognition by glycan-binding proteins is a cornerstone of the human

inflammatory process, from detection of pathogens, trafficking to the sites of

inflammation, activation of the immune response, and resolution of inflammation.28 The

interplay of these “writers” and “readers” is rapidly evolving as pathogens and hosts

compete for biological success. The Siglec family of sialic acid-binding lectins, most of

which are expressed on subsets of immune cells and are immune regulatory, have been

hypothesized to have rapidly diverged through selective pressure from pathogens. This

thesis also places some emphasis on the evolutionary divergence between the human

Siglec-8 and mouse Siglec-F receptors, which are empirically-determined functional

paralogs. Engagement of these Siglecs on the surface of eosinophils induces apoptosis of

these activated immune cells leading to resolution of ongoing inflammation. These and

similar findings targeting Siglec-F in mouse models of induced airway inflammation

91 validate Siglec-8 as a target for therapeutic resolution of human airway allergic inflammation.11, 12, 13 This thesis further characterized the endogenous Siglec-8 ligands in human airway tissue and airway lavage and examined the cross-reactivity between these ligands and endogenous Siglec-F ligands expressed in mouse airways.

We identified Siglec-8 ligands in human airway tissue extracts as high molecular weight proteoglycans carrying sialylated keratan sulfate chains that engage Siglec-8.

Upon further analysis, we determined that the tissue-extracted ligands carried on the large proteoglycan aggrecan were not likely the physiologically relevant ligands due to their expression in cartilage tissue, which is not easily accessible to airway-infiltrating and cytokine-primed eosinophils. Though we do observe Siglec-8 binding to submucosal glands on human airway histological sections, we did not observe expression of the tissue-extracted proteoglycan ligand, aggrecanS8, in submucosal glands. This finding prompted us to explore human airway secretions in the form of isotonic salt nasal irrigation samples (nasal lavage) for a Siglec-8 ligand that could engage airway- infiltrating eosinophils. We identified a similar high molecular weight carrier protein,

DMBT1S8, as the nasal lavage ligand, which again carried sialylated keratan sulfate chains as the Siglec-8-specific binding partner. DMBT1 is a mucin-like glycoprotein, typically found on mucosal surfaces, which has been described as an immune regulatory molecule with tissue-dependent functions.29 Sialylated keratan sulfate chains have not been previously identified on DMBT1, however, our predictive O-linked glycosylation profile of DMBT1 indicates interspersed regions of the peptide sequence that may contain sialylated KS chains in a pattern that could be optimal for surface receptor cross- linking. In comparison, aggrecan’s core protein contains short clustered regions of KS

chains, which are not expected to engage surface receptors in a manner optimal for cross-

linking. Given the structural complexity of these proteoglycans and the heterogeneity of

keratan sulfate chains along the protein backbone, we focused our efforts on the isolation

of Siglec-8-specific KS chains from airway tissue and nasal lavage by extensive

proteolysis to discard the protein component and leave intact KS chains attached

presumably to a single amino acid.

KS chains were isolated from human samples by extensive proteolysis, separation

by anion exchange chromatography, methanol precipitation of the eluted KS chains, and

enzymatic depolymerization of non-KS glycosaminoglycans followed by removal by centrifugal filtration. KS chains were semi-quantifiable by a differential keratanase I and keratanase II reducing sugar colorimetric assay and a fluorescent sialic acid quantification assay. A more instructive carbohydrate analysis was performed on a

DIONEX ICS 6000 after enzymatic or acid hydrolysis of the KS chains. Carbohydrate analysis of human tracheobronchial extract and human nasal lavage KS chains revealed differences in KS carbohydrate composition and chain length. Tracheobronchial KS chains appeared to be longer (5-6 disaccharides) and more likely to be fucosylated and sialylated than the nasal lavage KS chains (2-3 disaccharides). These isolated KS chains, which contain a single amino acid or small peptide, were chemically tagged with an azido-functionalized linker followed by “click” chemistry attachment to a synthesized cycloalkyne-phosphatidylethanolamine to generate KS neoglycolipids. Although these semi-synthetic KS neoglycolipids demonstrated Siglec-8-binding by dot-blot analysis on

PVDF membranes, their amphipathic properties limited solubility in aqueous buffers and

adsorption onto microwell plates for glycolipid ELISA binding assays. To overcome

these challenges, the KS chains were instead biotinylated on the remaining amino acid.

Siglec-8-binding KS-biotin chains were isolated from heterogeneous populations

of KS chains by affinity chromatography using a pentameric Siglec-8 construct

immobilized on Ni-Sepharose beads. The majority of the KS chains from human airway

tissue extract and nasal lavage did not bind to the affinity column and instead flowed

through in a low-salt wash buffer. A small portion of tracheobronchial KS chains bound

to the affinity column and were eluted first with a salt-buffer containing 150 mM sodium

chloride and the rest of the KS chains eluted with a high-salt buffer containing 1.5 M

sodium chloride. The majority of nasal lavage KS chains also flowed through the affinity

column with only a small portion of Siglec-8-specific KS chains eluting in high-salt

buffer and remaining bound through the 150 mM salt wash, implicating a higher affinity

interaction compared to tracheobronchial Siglec-8-specific KS chains. The Siglec-8-

binding fractions off of the affinity column were pooled, concentrated, and resolved on a

Superdex 200 increase 10/300 size exclusion column to approximate the molecular

weight of the KS-biotin chains. A tight peak of Siglec-8-binding KS-biotin chains was

observed for these size-excluded fractions and the molecular weight was approximated at

~44 kDa by comparison with a set of heparan sulfate molecular weight standards run in

the same conditions. A portion of the Siglec-8-binding KS-biotin chains were pre-treated

with keratanase I enzyme, the reducing end cleavage products tagged with hydrazine- biotin prior to size-exclusion, and subsequent fractions immobilized on streptavidin- coated microwells for binding analysis. Siglec-8 overlay revealed an approximate molecular weight shift to ~2 kDa which retained robust binding to Siglec-8.

Functional analysis of the intact aggrecanS8 incubated with primary IL-5-actived

human blood purified eosinophils indicates a modest but significant increase in apoptosis

compared to an oxidized aggrecanS8 control.17 Similar analysis with the DMBT1S8 nasal

lavage ligand is underway. To achieve the multi-valency and solubility required for

cross-linking Siglec-8 receptors on the surface of eosinophils, the isolated Siglec-8-

binding KS chains, currently hypothesized to be monovalent, will likely need to be

chemically attached to a multivalent carrier that allows for proper engagement of

eosinophils. Immobilized KS chains on streptavidin-coated microwells were unable to

induce eosinophil apoptosis, despite robust Siglec-8 binding. Intact Siglec-8-specific KS

chains along with keratanase I cleaved KS chains need to be compared for Siglec-8-

binding affinity as well as functional ability.

Finally, Siglec-8’s binding specificity to human airway tissue extracts and

histological sections was compared to its mouse functional paralog receptor Siglec-F.

Siglec-8 is found exclusively in Hominidae (human, chimpanzee, and orangutan) but not

in rodents where Siglec-F is expressed.27 Despite overlap of Siglec specificity on glycan

printed arrays, Siglec-F bound modestly to Siglec-8 tracheal extract purified ligands

which showed intense Siglec-8 binding. Siglec-8 binding to human trachea histological

sections is keratanase sensitive, whereas Siglec-F binding intensity to similar sections is increased following pre-treatment of sections with keratanase enzymes indicating that removal of KS chains allows for easier access of Siglec-F to its ligands in human airways. In mouse tracheal sections, which do not express a Siglec-8 ligand, keratanase pre-treatment of the sections decreases a portion of Siglec-F binding found in connective

tissue and mucosal glands but had no effect on binding to ligands expressed on the

epithelial layer. These and other published data indicate an evolutionary divergence

between the Siglec-F and Siglec-8 receptors and imposes a requirement for human tissue

samples to acquire Siglec-8 ligands and a limitation in the use of mouse models for

Siglec-8 targeted resolution of allergic airway inflammation.15, 26

We hypothesize that sialylated KS chains with the terminal epitope “Neu5Acα2-

3[6S]Galβ1-4[6S]GlcNAc” are the endogenous Siglec-8 ligands in both human airway tissue extracts and in human nasal lavage. A more detailed structural analysis of the

Siglec-8-binding KS chains would better determine the requirements for Siglec-8 recognition and would enhance synthesis of similar therapeutics targeting Siglec-8 for the

resolution of allergic airway inflammation. Siglec-8 is also expressed on the surface of

mast cells, where engagement inhibits mediator release from activated mast cells.9 These

findings need to be expanded to explore the resolution of allergic inflammation due to

chronic mast cell activation in human airways as well as other tissues such as skin, where

chronic mast cell activation leads to diseases like eczema and psoriasis. We conclude

that a minor portion of high molecular weight keratan sulfate chains in human airways

are terminally sialylated ligands for Siglec-8 that may function to regulate allergic airway inflammation mediated by activated eosinophils.

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18. Brown, G.M., Huckerby, T.N., Bayliss, M.T. & Nieduszynski, I.A. Human aggrecan keratan sulfate undergoes structural changes during adolescent development. J Biol Chem 273, 26408-26414 (1998).

19. Lauder, R.M., Huckerby, T.N. & Nieduszynski, I.A. The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage. Glycoconj J 14, 651-660 (1997).

20. Weyers, A. et al. Isolation of bovine corneal keratan sulfate and its growth factor and morphogen binding. FEBS J 280, 2285-2293 (2013).

21. Macauley, M.S. et al. Antigenic liposomes displaying CD22 ligands induce antigen- specific B cell apoptosis. J. Clin. Invest 123, 3074-3083 (2013).

22. Lopez, P.H. & Schnaar, R.L. Determination of glycolipid-protein interaction specificity. Methods Enzymol 417, 205-220 (2006).

23. Caterson, B. & Melrose, J. Keratan Sulphate, a complex Glycosaminoglycan with Unique Functional Capability. Glycobiology 28, 182-206 (2018).

24. Plaas, A.H., West, L.A. & Midura, R.J. Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo-beta-galactosidase digestion products. Glycobiology 11, 779-790 (2001).

25. Kiwamoto, T. et al. Endogenous airway mucins carry glycans that bind Siglec-F and induce eosinophil apoptosis. J Allergy Clin Immunol 135, 1329-1340 (2015).

26. Patnode, M.L. et al. Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue. J. Biol. Chem 288, 26533-26545 (2013).

27. Angata, T., Margulies, E.H., Green, E.D. & Varki, A. Large-scale sequencing of the CD33- related Siglec gene cluster in five mammalian species reveals rapid evolution by multiple mechanisms. Proc. Natl. Acad. Sci. U. S. A 101, 13251-13256 (2004).

28. Schnaar, R.L. Glycobiology simplified: diverse roles of glycan recognition in inflammation. J. Leukoc. Biol 99, 825-838 (2016).

29. Madsen, J., Mollenhauer, J. & Holmskov, U. Review: Gp-340/DMBT1 in mucosal innate immunity. Innate Immun 16, 160-167 (2010).

Ryan N. Porell 725 North Wolfe Street, Baltimore, MD 21205 Cell: (443) 966.0671 Home: (410) 592.9802 [email protected]

Education

• Johns Hopkins University, Baltimore, MD August 2014-September 2018

Doctorate of Philosophy in Chemical Biology

• Johns Hopkins University, Baltimore, MD August 2014-May 2016

Master of Science in Chemical Biology

• University of Maryland, Baltimore County (UMBC) August 2010-May 2013

Bachelor of Science in Biochemistry, minor in psychology GPA: 3.55/4.0 Research Experience

• May 2015 – September 2018 Johns Hopkins University

Thesis advisor: Ronald L. Schnaar, Ph.D. Structure-function relationship studies between the natural sialylated keratan sulfate ligands from human airway tissue and synthetic ligands for the immuno-regulatory receptor Siglec-8.

• February 2015 – May 2015 Johns Hopkins University

Rotation advisor: Ronald L. Schnaar, Ph.D. Extraction, isolation, and characterization of endogenous human airway sialoglycan ligands for Siglec-8 and Siglec-9 immuno-regulatory receptors.

• November 2014 – February 2015 Johns Hopkins University

Rotation advisor: Philip Cole, Ph.D. Semi-synthesis of an adenosine 2A receptor small molecule antagonist – Fc protein fusion drug for immuno-enhancement as a cancer therapeutic.

• September 2014 – November 2014 Johns Hopkins University

Rotation Advisor: Yuan Chuan Lee, Ph.D. Conjugation of an activated L-Rhamnose to BSA for affinity and avidity analysis of a recombinant horseshoe crab plasma lectin.

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• August 2012 – May 2013 University of MD, Baltimore County (UMBC)

Organic synthesis and structure activity relationships of a small molecule inhibitor for a crucial hepatitis c virus replicase enzyme. Training Workshops

• September 25-29, 2017 Complex Carbohydrate Research Center (CCRC), Athens, GA

Trained in glycomics and glyco-proteomics mass spectrometry involving chemical preparation of samples, instrumentation, and software analysis of data.

• July 22-24, 2016 Northwestern Hospital, Chicago, IL

Trained in isolation of human leukocytes, generation of eosinophils from murine bone marrow, immune cell receptor endocytosis assays, and eosinophil apoptosis assays.

• July 2015 – August 2015 Johns Hopkins University, Baltimore, MD

Two-week intensive “Techniques in Glycobiology” hands-on laboratory course with lectures, experimentation, and seminars taught by current leaders in glycobiology.

• June 1-3, 2015 Scripps Research Institute, San Diego, CA

Trained in oligosaccharide chemoenzymatic synthesis, glycan microarray analysis, and cell targeting nanoliposome development. Teaching Experience

• May 2018 – August 2018 Johns Hopkins University

Summer Student Mentor Mentored a summer student working on culturing airway mucosal gland-like epithelial cells (Calu-3) to analyze and enhance their biosynthetic capacity to secrete sialylated keratan sulfate proteoglycans through over-expression of key biosynthetic enzymes.

• May 2017 – August 2017 Johns Hopkins University

Summer Student Mentor Mentored a summer student and directed them through the isolation, purification, and quantitative analysis of keratan sulfate glycan chains from bovine cornea tissue with subsequent attachment to different chemical scaffolds for lectin binding assays.

• November 2016 – February 2017 Johns Hopkins University

Rotation Student Mentor Mentored a rotation student and assisted them with the quantification of Siglec-9 binding properties to gangliosides decorated on silver nanocubes through localized surface plasmon resonance.

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• September 2015 – May 2016 Johns Hopkins University

Teacher Assistant Attended classes, held office hours, and conducted review sessions for the Graduate Chemical Biology I and II courses (two semesters).

• January 2016 – April 2016 Johns Hopkins University

Rotation Student Mentor Mentored a rotation student working on conjugating fluorescent tags onto the reducing end of chemically released glycosaminoglycan chains from proteoglycans.

• September 2012 – May 2013 UMBC, Catonsville MD

Chemistry and Mathematics Tutor Tutored colleagues and/or high school students in general chemistry, organic chemistry, and calculus I. Additional Professional Experience

• September 2018 Complex Carbohydrate Research Center (CCRC), Athens, GA

Collaborated with the CCRC to develop a mass spectrometric database for the analysis of keratan sulfate glycosaminoglycan chains with differential sialylation, sulfation, and fucosylation.

• October 2016 Johns Hopkins University

Reviewer for Essentials of Glycobiology, Third Edition Reviewed four chapters of the third edition of the textbook Essentials of Glycobiology. Regional and National Meetings Attended

• July 13, 2018 NIH Bethesda, MD

NIH Glycoday 2018

• May 5, 2018 University of Pennsylvania, Philadelphia, PA

11th annual Frontiers in Chemistry and Biology Interface Symposium

• March 10-11, 2018 San Diego, CA

InterPEG 2018

• March 8-10, 2018 San Diego, CA

San Diego Glycobiology Symposium

• June 25-30, 2017 West Dover, VT

Carbohydrates Gordon Research Conference

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• June 24-25, 2017 West Dover, VT

Carbohydrates Gordon Research Seminar

• April 12-13, 2017 Bethesda, MD

InterPEG 2017

• November 19-22, 2016 New Orleans, LA

Society for Glycobiology 2016

• June 29, 2016 NIH Bethesda, MD

NIH Glycoday 2016

• May 14, 2016 Johns Hopkins University, Baltimore, MD

9th annual Frontiers in Chemistry and Biology Interface Symposium

• April 20-21, 2016 Bethesda, MD

InterPEG 2016

• December 1-4, 2015 San Francisco, CA

Society for Glycobiology 2015

• May 28, 2015 NIH Bethesda, MD

NIH Glycoday 2015

• May 16, 2015 UMBC Catonsville, MD

8th annual Frontiers in Chemistry and Biology Interface Symposium Publications and Co-publications:

Journal Articles: 1. Gonzalez-Gil, A.1, Porell, R.N.1, Fernandes, S.M., Wei, Y., Yu, H., Carroll, D.J., McBride, R., Paulson, J.C., Tiemeyer, M., Aoki, K., Bochner, B.S., Schnaar, R.L. (2018) “Sialylated keratan sulfate proteoglycans are Siglec-8 ligands on human airways.” Glycobiology 28:10 p. 786-801 1Anabel and Ryan are co-first authors. Editor’s choice.

2. Yu, H., Gonzalez-Gil, A., Wei, Y., Fernandes S.M., Porell, R.N., Vajn, K., Paulson, J.C., Nycholat, C.M., Schnaar, R.L. (2017). "Siglec-8 and Siglec-9 binding specificities and endogenous airway ligand distributions and properties." Glycobiology 27:7 DOI: https://doi.org/10.1093/glycob/cwx026. On the cover.

Published Abstracts:

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3. Chen, Zi., Zhang, F., Tang, H., Porell, R.N., Schnaar, R.L., Kumagai, T., Tiemeyer, M., Bochner, B.S., Zhou, L., Zheng, T., Zhu, Z. (2017). “Anti-inflammatory functions of Siglec-E and Siglec-9 and alteration of their ligands in mouse airway inflammation and lung emphysema.” Glycobiology 27:12. Abstract 14

4. Fernandes, S.M., Porell, R.N., Gonzalez-Gil, A., Kurz, S., Aoki, K., Tiemeyer, M., Schnaar, R.L. (2017). “Siglec-9 recognizes sialylated keratan sulfate glycoproteins on human airways.” Glycobiology 27:12. Abstract 52

5. Gonzalez-Gil, A., Sil-Lee, H., Porell, R.N., Fernandes, S.M., Kim, J., Schnaar, R.L. (2017). “Siglec-8 ligands in human airway secretions.” Glycobiology 27:12. Abstract 119

6. Porell, R.N., Gonzalez-Gil, A., Fernandes, S.M., Vajn, K., Yu, H., Aoki, K., Kurz, S., Tiemeyer, M., Schnaar, R.L. (2016). “Different Airway Ligands for Human and Mouse Eosinophilic Siglecs.” Glycobiology 26:12 Abstract 275

7. Fernandes, S.M., Gonzalez-Gil, A., Porell, R.N., Aoki, K., Tiemeyer, M., Schnaar, R.L. (2016). “Ligands for siglecs in human airway exudates: comparison of Siglec-8, Siglec-9, Siglec-E, and Siglec-F binding patterns.” Glycobiology 26:12 Abstract 276

8. Gonzalez-Gil, A., Porell, R., Fernandes, S.M., Aoki, K., Tiemeyer, M., Schnaar, R.L. (2015). “Siglec-8 binds to sialylated keratan sulfate chains on aggrecan extracted from human airways.” Glycobiology 25:11 Abstract 66

Poster and Oral Presentations

• July 13, 2018: Poster Presentation "Siglec-8-Binding Keratan Sulfate Chains that Regulate Airway Inflammation" NIH Glycoscience Day 2018. Bethesda, MD • May 5, 2018: Poster Presentation “Isolating the Sulfated Glycans that Put the Brakes on Airway Inflammation”, 11th annual Frontiers in Chemistry and Biology Interface Symposium. University of Pennsylvania, Philadelphia, PA • April 9, 2018: 30 minute Presentation “Sulfated Glycans Put the Brakes on Airway Inflammation”, Chemical Biology Interface Program Research in Progress at JHU School of Medicine, Baltimore, MD • March 10-11, 2018: Poster Pitch, Poster and Oral Presentation “Characterization of Siglec-8-binding Keratan Sulfate Chains from Human Airways”, InterPEG 2018. San Diego, CA • March 8-9, 2018: Poster Presentation “Characterization of Siglec-8-binding Keratan Sulfate Chains from Human Airways”, San Diego Glycobiology Symposium 2018. San Diego, CA • September 23, 2017: Poster Presentation “Semi-Synthetic Sulfated Glycans to Regulate Airway Inflammation”, 2017 CBI Retreat. Conference Center at Sheppard Pratt, Towson, MD • June 25-30, 2017: Poster Presentation “Sialylated Keratan Sulfates are Ligands for Siglec-8 in Human Airways”, Carbohydrates Gordon Research Conference. West Dover, VT

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• June 24-25, 2017: Poster Presentation “Sialylated Keratan Sulfates are Ligands for Siglec-8 in Human Airways”, Carbohydrates Gordon Research Seminar. West Dover, VT • April 12-13, 2017: Poster Pitch, Poster and Oral Presentation “Different Airway Ligands for Human and Mouse Eosinophil Siglecs”, InterPEG 2017. Bethesda, MD • February 7, 2017: 30 minute Presentation “Siglec-8 Ligands in Human Airways: Targeting Airway Inflammatory Diseases”, Pharmacology Research in Progress at JHU School of Medicine, Baltimore, MD • September 24, 2016: Poster Presentation “Glycans that Regulate Lung Inflammation”, 2016 CBI Retreat. Conference Center at Sheppard Pratt, Towson, MD • September 11, 2016: Poster Presentation “Glycans that Regulate Lung Inflammation”, 2016 Pharmacology Retreat. Carriage House of the Belmont Manor in Elkridge, MD • June 29, 2016: Poster Pitch and Poster Presentation “Glycans that Regulate Airway Inflammation”, NIH Glycosciences Day 2016. Bethesda, MD • May 14, 2016: Poster Presentation “Glycans that Regulate Airway Inflammation”, 9th annual Frontiers in Chemistry and Biology Interface Symposium. Johns Hopkins University, Baltimore, MD • May 10, 2016: Poster Presentation “Sialylated Keratan Sulfate is a Ligand for Siglec-8 in Human Airways”, Baltimore/Washington Glycobiology Interest Group 5th Annual Poster Session. Johns Hopkins University, Baltimore, MD • April 20-21, 2016: Poster Pitch, Poster and Oral Presentation “Sialylated Keratan Sulfate is a Ligand for Siglec-8 in Human Airways”, InterPEG 2016. Bethesda, MD • March 8, 2016: Oral Presentation “Siglec-8 and -9 Sialoglycan Ligands in Human Airways”, Baltimore/Washington Glycobiology Interest Group. Johns Hopkins University, Baltimore, MD • September 19, 2015: Poster Presentation “Glycans that Regulate Lung Inflammation”, 2015 CBI Retreat. Conference Center at Sheppard Pratt, Towson, MD • May 28, 2015: Poster Presentation "Siglec-8 and Siglec-9 in human airways" NIH Glycosciences Day 2015. Bethesda, MD • May 16, 2015: Poster Presentation “Isolation and Purification of Siglec-8 Ligand in Human Lung”, 8th annual Frontiers in Chemistry and Biology Interface Symposium. UMBC, Catonsville, MD

Awards/Funding

• July 13, 2018 Award from NIH Glycoday 2018 Meeting

Outstanding Presentation Award for presentation titled: “Siglec-8-Binding Keratan Sulfate Chains that Regulate Airway Inflammation”

• April 19, 2017 Scheinberg Travel Award

Scheinberg Travel Award for Spring 2017, granted through the Department of Pharmacology at Johns Hopkins School of Medicine.

• April 13, 2017 Award from InterPEG 2017 Meeting

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Outstanding Presentation Award for presentation titled: “Different Airway Ligands for Human and Mouse Eosinophil Siglecs”

• September 14, 2016 Travel Award for SFG 2016 Meeting

Travel Award for the 2016 Meeting of the Society for Glycobiology, granted by the Society for Glycobiology Treasurer, Dr. Richard Steet.

• June 2016 – May 2018 National Heart, Lung, and Blood Institute

Supported by the Lung Inflammatory Disease Program of Excellence in Glycoscience (LIDPEG, P01HL107151, http://lidpeg.org). Schnaar lab funding for students working on LIDPEG project.

• September 2014 – May 2016 Johns Hopkins University

National Institute of Health Predoctoral Training Grant (T32; GM080189). Student stipend provided by the Chemical Biology Interface Program.

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