AIRWAY MUCIN DYNAMICS IN INFECTION AND INFLAMMATION
Bethany Batson
A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor in Philosophy in the Department of Pathology and Laboratory Medicine in the School of Medicine.
Chapel Hill 2019
Approved by:
Claire Doerschuk
Mehmet Kesimer
Jonathon Homeister
Marianne Muhlebach
Wanda O’Neal
© 2019 Bethany Batson ALL RIGHTS RESERVED
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ABSTRACT
Bethany Batson: Airway Mucin Dynamics in Infection and Inflammation (Under the direction of Mehmet Kesimer)
Mucus plays a critical role in the innate immunity of the airways acting as the body’s first line of defense against pathogens. Mucus is a complex gel and much is still unknown regarding its constituents and how these cooperatively imbue mucus with its physiological and rheological properties. One such component, mucin, a large polymeric glycoprotein, is responsible for many of the rheological properties of mucus and its concentration has been linked to the development of several airway diseases. The importance of a properly functioning and mobile mucus layer is most evident when it becomes defective, a defining feature of the cystic fibrosis (CF) and asthmatic airway disease, which can have devastating consequences. A better understanding of why mucus becomes static and occludes airways or becomes a nidus for infection would not only clarify the pathogenesis of these diseases, but would inform future therapeutic advances. Using in vitro models and in vivo sputum samples, the following body of work aims to elucidate and characterize how the various mucus components including mucin, mucin interacting proteins, and regulatory exosomal miRNAs change in CF and asthmatic lung environments with the hypothesis that these qualitative and quantitative changes are key drivers in the development of pathologic mucus. We will show that the ratio of the two main gel-
iii forming mucins is unique to specific airway diseases as are the proteins with which theses mucin interact. A specific focus will be placed on IgGFc-binding protein
(FCGBP), a mucin interacting protein predicted to stabilize the colonic mucus layer, which we show is differentially secreted in CF and asthma lung environments.
Several exosomal miRNA were identified that target mucin and its glycosylation machinery which also show disease specificity in regards to their expression. These findings broaden our understanding of how mucins are altered and contribute to the function of mucus in health and disease.
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ACKNOWLEDGMENTS
Countless individuals have played a role in the creation, revision, and completion of this body of work. My committee members, Dr. Claire Doerschuk, Dr.
Marianne Muhlebach, Dr. Wanda O’Neal, Dr. Jonathon Homeister, and Dr. Mehmet
Kesimer provided the encouragement and input that facilitated the completion of my work and graduate studies.
I would like to dedicate this work to my mentor, Mehmet Kesimer, who nurtured my enthusiasm for research and gave me the opportunity to become an independent investigator. This work would not have been possible without his blessing and guidance. Not only did you push me scientifically, but your continued support while I struggled with a difficult pregnancy and a newborn made finishing my
PhD a reality. I have spent the past five years in the Kesimer lab, which have been filled with riveting scientific (and non-scientific) conversation, laughter, and collaboration, and exciting science. Each member of the lab has enhanced my graduate experience with their specific area of technical expertise but also in other less scientific ways. From Giorgia Radicioni, I have learned about Italy and scuba diving; from Boris Reidel, everything German and Star Wars; from Jerome
Carpenter, anything sports, PowerPoint animations, and what it means to be
“optihensive”, with Stephanie Livengood, I have shared my love for cats and dogs; and from Sabri Abdelwahab, I have learned about selfless generosity and developed
v a heightened appreciation for Baklava. I could never forget, the former lab manager and one of my best friends, Amina Ford, who taught me how to laugh through difficult times and that regardless of your title and position, with hard work you can achieve great things. I not only view these individuals as my labmates, but my friends and North Carolina family.
This work was truly a collaborative effort that involved numerous labs and core facilities within the CF center, and those both on and off UNCs’ campus. Within the Marsico Lung Institute and CF center I would like to acknowledge the following labs and cores: The CF Center Tissue Procurement and Cell Culture Core, The
Molecular Biology Core, The Histology Core, The Michael Hooker Microscopy Core, and really the entire 7th floor of Marsico Hall. Outside of the CF center, I would like to recognize Dr. Brenda Temple from The UNC Center for Structural Biology, the High
Throughput Genomic Sequencing Facility, Dr. Flavia Teles and Lynn Martin, and
The UNC Animal Histopathology Core. Outside of UNC, I would like to acknowledge the fruitful collaboration with Dr. Tiemeyer and Tadahiro Kumagai from University of
Georgia’s Complex Carbohydrate Research Center.
Lastly I would like to thank my family. My husband, Kellen Batson, has provided unending encouragement, unconditional love and support, which started with his willingness to move from the West Coast to North Carolina and continues even now as he encourages me to pursue my dream of becoming a medical doctor.
Life became a little bit more complicated when we decided to have Finley, but I wouldn’t choose to be married, parents, and best friends with anyone else! Finley
Marie Batson, even though you are unaware of what I have been up to these last
vi couple years, you have brought an incredible amount of joy, laughter, and smiles into my life. I work hard for you, my little cupcake, and hopefully one day you will be as proud of me as I already am of you.
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TABLE OF CONTENTS
LIST OF FIGURES ...... xiv
LIST OF TABLES ...... xix
LIST OF ABBREVIATIONS ...... xxi
INTRODUCTION: MUCUS & MUCIN ...... 1
CHAPTER 1. PART A: COMPREHENSIVE CHARACTERIZATION OF MUCINS WITHIN THE CF AIRWAY USING IN-VITRO MODELS ...... 7
Introduction: Cystic Fibrosis ...... 7
Methods ...... 11
Cell Culture ...... 11
CF cell culture models ...... 11
Mucin Isolation and Static Light Scattering ...... 12
Isolation and Analysis of Stored Gel Forming Mucins ...... 13
Whole Mount Immunohistochemistry (IHC) ...... 13
Agarose Gel Electrophoresis ...... 14
Mass Spectrometry ...... 15
Proteomic Semi-tryptic peptide analysis ...... 17
Rate Zonal Centrifugation ...... 17
Exosome Isolation ...... 18
MUC5B and MUC5AC Standard In-Vitro Experimental Design ...... 18
Results: ...... 20
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CF Cell Culture Models: Immunohistochemistry and MUC5B and MUC5AC concentration quantitation ...... 20
SMM: MUC5AC and MUC5B concentration quantitation ...... 21
CF cell culture models: Macromolecular characterization of secreted and intracellular gel forming mucins ...... 21
Ps.a. cell culture model: MUC5B Semi-tryptic peptide analysis ...... 23
CF cell culture models: Conformation analysis of the secreted gel forming mucins ...... 24
SMM cell culture model: Proteomic pathway analysis of secreted proteins ...... 24
Ps.a. cell culture model: Proteomic pathway analysis of secreted proteins ...... 25
Proteomic comparison of secretions from SMM and Ps.a. CF cell culture models ...... 26
CF Cell culture models: Proteomic analysis of mucin interacting proteins in secretions ...... 26
Ps.a. CF cell culture model: Pathway analysis of differentially expressed exosomal miRNA ...... 27
Ps.a. CF cell culture model: In silico MUC5B activity prediction based on differentially expressed exosomal miRNA ...... 28
Antibody based MUC5B bacteria degradation timecourse ...... 29
Light scattering determination of MUC5B concentration and macromolecular structure during bacteria degradation timecourse ...... 29
Antibody based MUC5AC bacteria degradation timecourse ...... 31
Light scattering determination of MUC5AC concentration and macromolecular structure during bacteria degradation timecourse ...... 31
Bacterial incubation control: antibody and light scattering measurements of MUC5B and MUC5AC ...... 33
Discussion: ...... 34
Chapter 1 Part A Figures ...... 46
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CHAPTER 1. PART B: COMPREHENSIVE CHARACTERIZATION OF MUCINS WITHIN THE CF AIRWAY: A COMPARATIVE ANALYSIS BETWEEN NON-DISEASE AND CF SPUTUM ...... 68
Introduction ...... 68
Methods ...... 70
Study Population ...... 70
Total Mucin Concentrations ...... 70
MUC5AC and MUC5B Concentrations ...... 70
Proteomic Semi-tryptic peptide analysis ...... 71
Purification of Gel-Forming Mucins ...... 71
Characterization of the Gel-Forming Mucins...... 72
Glycomics ...... 72
Microbiome ...... 72
Statistics ...... 73
Reagents ...... 73
Results ...... 74
Demographics ...... 74
Total mucin concentrations in CF vs. normal subjects ...... 74
Absolute MUC5B and MUC5AC concentrations in CF vs. normal subjects ...... 75
Macromolecular characterization of purified gel-forming mucins in CF vs. normal...... 76
Quantity, coverage, and localization of MUC5B and MUC5AC Semi-tryptic peptides in mucins from CF sputum and control sputum ...... 77
Sputum mucin concentrations and the CF sputum microbiome ...... 78
Glycomic analysis of gel forming mucins in CF vs. normal ...... 79
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Discussion ...... 80
Chapter 1 Part B Figures ...... 88
CHAPTER 2: COMPREHENSIVE CHARACTERIZATION OF MUCINS WITHIN THE TH2 ASTHMATIC AIRWAYS ...... 105
Introduction: Asthma ...... 105
Methods ...... 109
Cell Culture ...... 109
Asthma model: ...... 109
Mucin Isolation and Static Light Scattering ...... 110
Isolation and Analysis of Stored Gel Forming Mucins ...... 111
Exosome Isolation: ...... 111
Whole Mount Immunohistochemistry ...... 112
Agarose Gel Electrophoresis ...... 113
Mucin interactome isolation: ...... 114
Mass Spectrometry ...... 114
Rate Zonal Centrifugation ...... 116
Results ...... 117
Histological examination of IL-13 challenged and control cultures...... 117
IL-13 induced asthma cell culture models: antibody based MUC5B and MUC5AC concentration quantitation ...... 117
IL-13 induced asthma cell culture models: Absolute MUC5B and MUC5AC concentration quantitation ...... 118
IL-13 induced asthma cell culture models: Macromolecular characteriztion of the purified secreted and stored gel forming mucins ...... 119
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IL-13 induced asthma cell culture model: Proteomic pathway analysis of secreted proteins from non-asthmatic and asthmatic cultures ...... 120
IL-13 induced asthma cell culture model: Proteomic analysis of the mucin interactome from non-disease cultures ...... 122
IL-13 induced asthma cell culture model: Pathway analysis of differentially expressed exosomal miRNA from non-asthmatic and asthmatic cultures ...... 123
IL-13 induced asthma cell culture models: In silico MUC5B activity prediction based on differentially expressed exosomal miRNA from non asthmatic and asthmatic cultures ...... 124
Discussion ...... 125
CHAPTER 3: ISOLATION, PURIFICATION, AND CHARACTERIZATION OF FCGBP, A PUTATIVE MUCIN BINDING GLYCOPROTEIN ...... 156
Introduction: FCGBP ...... 156
Methods ...... 159
Ex-vivo cell culture challenges: Asthma and Cystic Fibrosis ...... 159
Human Sputum Samples: ...... 160
Mass Spectrometry Sample Preparation and Analysis ...... 160
Transfection: ...... 160
FGCBP Purification: ...... 161
Characterization: Light Scattering ...... 162
Characterization: Atomic force microscopy ...... 162
In-Gel Digestion……………………………………………………………………..163
Structural analysis ...... 164
Interaction analysis ...... 164
FCGBP BS3 Crosslinking ...... 166
Results: ...... 167
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Immunohistochemistry of FCGBP and MUC5AC during IL-13 challenge of non-asthmatic cultures ...... 167
Quantitation of FCGBP concentration in secretions of asthma and CF cell culture models and sputum using antibody and proteomic methodologies ...... 167
FCGBP purification ...... 168
FCGBP conformation and structure analysis by atomic force microscopy and light scattering ...... 169
FCGBP BS3 crosslinking analysis ...... 169
Proteomic analysis and localization of reduced FCGBP bands ...... 170
FCGBP computational analysis ...... 171
Semi tryptic peptide analysis of FCGBP GDPH cleavage sites ...... 171
Molecular weight and glycosylation predictions of FCGBP domains based on GDPH cleavage sites ...... 172
FCGBP layer properties and interactions with the MUC5B and MUC5AC standards ...... 173
Discussion ...... 174
Chapter 3 Figures ...... 181
SUMMARY AND FUTURE DIRECTIONS ...... 199
APPENDICES ...... 203
Appendix 1...... 203
Appendix 2...... 206
Appendix 3 ...... 235
REFERENCES ...... 280
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LIST OF FIGURES
Figure 1.1. CF cell culture models exhibit mucus hypersecretion and adhesion of mucus to surface...... 46
Figure 1.2. Comparison of MUC5AC in apical secretions after 120-hour SMM challenge and the SMM itself...... 47
Figure 1.3. Macromolecular characterization of secreted and stored mucins...... 48
Figure 1.4. Ps.a. CF cell culture model: MUC5B Semitryptic peptide analysis...... 49
Figure 1.5. Rate zonal conformation analysis of gel-forming mucins...... 50
Figure 1.6. Label free proteomic analysis of apical secretions...... 51
Figure 1.7. Shared significant differentially secreted proteins between the SMM and Ps.a. 120 hour cell culture challenge based on label free proteomic analysis...... 52
Figure 1.8. Changes in non-gel forming mucins and mucin interactome proteins in CF cell culture models...... 53
Figure 1.9. Changes in mucin interactome in CF cell culture models...... 54
Figure 1.10. Changes in exosome concentration size and miRNA cargo after 120 hour Ps.a challenge...... 56
Figure 1.11. Mucin type O-glycosylation genes targeted by significantly decreased exosomal miRNA after Ps.a. challenge...... 59
Figure 1.12. MUC5B gene predicted to be activated by differentially expressed miRNA after Ps.a. challenge...... 61
Figure 1.13. miR-6762-5p concentration negatively and significantly correlates with MUC5B concentration...... 62
Figure 1.14. Salivary MUC5B standard shows time dependent antibody loss after incubation with bacteria...... 63
Figure 1.15. Biophysical characterization of MUC5B after time course incubation with bacteria after re-purification via isopycnic centrifugation and analysis by SEC-MALS...... 64
Figure 1.16. A549 MUC5AC standard shows time and species dependent
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antibody loss after incubation with bacteria...... 65
Figure 1.17. Biophysical characterization of MUC5AC after time course incubation with bacteria after re-purification via isopycnic centrifugation and analysis by SEC-MALS...... 66
Figure 1.18. Gel forming mucin timepoint incubation with TSB...... 67
Figure 1.19. Total mucin concentration increases significantly in CF...... 89
Figure 1.20. Within CF cohort, total mucin concentration and the ratio of MUC5AC to MUC5B varies with human neutrophil elastase activity and age...... 90
Figure 1.21. Absolute MUC5B and MUC5AC concentrations, and the MUC5AC/MUC5B ratio are significantly elevated in CF ...... 91
Figure 1.22. Macromolecular properties of the purified gel forming mucins from sputum are not significantly different between healthy and CF...... 92
Figure 1.23. MUC5B and MUC5AC from CF sputum have significantly higher ratio of Semi-tryptic to full tryptic peptides...... 93
Figure 1.24. MUC5B and MUC5AC from CF and normal sputa show distinct signatures based on Semi-tryptic peptides and the mucin domains they map to...... 94
Figure 1.25. Specific domain localization of Semi-tryptic peptides differs between CF and normal...... 95
Figure 1.26. Relationship between microbiome and mucin concentrations...... 96
Figure 1.27. Purified gel forming mucins from control and CF sputum have significantly different glycan profiles...... 97
Supplementary Figure 1.28. Total mucin concentration does not significantly change with medication usage...... 99
Supplementary Figure 1.29. Visual representations of the Semi-tryptic peptide coverage of the gel-forming mucins reveal differences between normal and CF sputa ...... 100
Supplementary Figure 1.30. Relationship between microbiome and MUC5B and MUC5AC concentrations...... 101
Figure 1.31. Abundance of bacterial genera based on sputum microbiome
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sequence analysis shows stratification based on oral flora or classic pathogen classification...... 102
Figure 1.32. Relationship between microbiome and mucin concentrations...... 103
Figure 1.33. Significantly different non-sulfated glycans derived from gel forming mucins purified from healthy and CF sputum...... 104
Figure 2.1. Asthma cell culture models exhibit goblet cell hyperplasia and increased storage of intracellular mucins...... 134
Figure 2.2. Asthma cell culture models exhibit mucus hypersecretion which is adherent to the apical surface...... 135
Figure 2.3. Asthma cell culture models shows a disproportionate increase in MUC5AC during 20 day challenge with IL-13 ...... 136
Figure 2.4. Asthma cell culture models exhibit mucus hypersecretion dominated by MUC5AC...... 137
Figure 2.5. Macromolecular characterization of secreted gel-forming mucins from non-disease and asthmatic HBE cultures after 20 day challenge with IL-13...... 138
Figure 2.6. Macromolecular characterization of stored gel forming mucins isolated from asthma cell culture model...... 139
Figure 2.7 Label free proteomic analysis of apical secretions based on uniquely identified proteins and ingenuity pathway analysis of significantly changing proteins following 20 day IL-13 challenge ...... 140
Figure 2.8. Secretion pattern of non gel-forming mucins as measured by label free LC-MS/MS in apical secretions during Il-13 challenge ...... 141
Figure 2.9. Secretion pattern of increasing probable mucin interacting proteins as measured by label free LC-MS/MS during lL-13 challenge ...... 142
Figure 2.10. Secretion pattern of decreasing probable mucin interacting proteins as measured by label free LC-MS/MS during Il-13 challenge ...... 143
Figure 2.11. Changes in exosome concentration size and miRNA cargo after IL-13 challenge ...... 144
Figure 2.12. Mucin type O-glycosylation genes targeted by significantly decreased exosomal miRNA after IL-13...... 149
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Figure 2.13. Differentially expressed miRNA at baseline after IL-13 treatment ...... 150
Figure 2.14. MUC5B gene predicted to be down regulated by differentially expressed miRNA after 20 day IL-13 treatment ...... 152
Supplement Figure 2.15. Secretion pattern of probable mucin interacting proteins as measured by label free LC-MS/MS during Il-13 challenge ...... 153
Supplemental Figure 2.16. Secretion pattern of probable mucin interacting proteins as measured by label free LC-MS/MS during IL-13 challenge ...... 154
Supplemental Figure 2.17. Comparative analysis of canonical pathways enriched by differentially expressed proteins ...... 155
Figure 3.1. Secretion of FCGBP significantly increases in inflammation and infection ...... 181
Figure 3.2. Signficantly decreased miRNA predicted to increase FCGBP expression...... 182
Figure 3.3. FCGBP purification strategy and characterization ...... 183
Figure 3.4. Unreduced SDS-Page of 293F clones’ cell lysates following transfection, single cell selection, and expansion...... 184
Figure 3.5. Macromolecular characterization of purified FCGBP...... 185
Figure 3.6. BS3 identified cross-linked peptides from FCGBP with peptide fragmentation chromatogram...... 186
Figure 3.7.Band pattern of purified, unreduced, and reduced FCGBP and localization of reduced bands based on Semi-tryptic LC-MS/MS analysis ..... 187
Figure 3.8. Homology and repeated domain pattern of FCGBP ...... 188
Figure 3.9 Diagram of FCGBP protein backbone and domain structure ...... 189
Figure 3.10. Computational FCGBP structural prediction of 1 of the 12 repeated domains based on multiple sequence analysis...... 190
Figure 3.11. Predicted molecular weight of FCGBP domains based on GD/PH cleavage sites...... 193
Figure 3.12. FCGBP layer properties based on viscoelastic broadfit model of QCM-D frequency and dissipation measurements ...... 195
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Figure 3.13. FCGBP and mucin interactions as measured by quartz crystal microbalance ...... 196
Supplementary Figure3.14. Localization of FCGBP in CF lung tissue by RNAscope...... 198
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LIST OF TABLES
Chapter 1 Table 1 Shared differentially expressed proteins identified in apical secretions after 120 hours of SMM or Ps.a treatment ...... 55
Chapter 1 Table 2. Top 20 significantly decreasing and increasing miRNA isolated from apically secreted exosome like vesicles after 120 hour challenge with Ps.a. or control ...... 57
Chapter 1 Table 3. Significant KEGG pathways based on significantly increasing miRNA isolated from apically secreted exosomes after 120 hour challenge with Ps.a. or control ...... 58
Chapter 1 Table 4. Decreasing miRNA isolated from apically secreted exosomes following 120hour treatment with Ps.a or TSB (control) predicted to affect mucin type O glycan biosynthesis based on Diana miRpath analysis...... 60
Chapter 1. Table 5. Study Cohort Characteristics for non-disease controls and CF patient populations ...... 88
Chapter 1 Table 6. Significant Spearman r correlations between specific glycan core structures and microbiome composition ...... 98
Chapter 2, Table 1. Top 20 significantly decreasing and increasing miRNA isolated from apically secreted exosome like vesicles after 20 IL-13 challenge of non-disease HBE cultures ...... 145
Chapter 2 Table 2. Significant KEGG pathways based on significantly increasing miRNA isolated from apically secreted exosomes after 20 day IL-13 challenge or control of non disease cultures ...... 146
Chapter 3 Table 3. Top significantly decreasing and increasing miRNA isolated from apically secreted exosome like vesicles after 20 IL-13 challenge of asthmatic HBE cultures ...... 147
Chapter 3 Table 4. Significant KEGG pathways based on significantly increasing miRNA isolated from apically secreted exosomes after 20 day IL-13 challenge or control of asthmatic HBE cultures ...... 148
Chapter 2 Table 5. Significantly different miRNA at baseline ...... 151
Chapter 3 Table 1. Abundance of Semitryptic peptides at GDPH cleavage sites identified by LC-MS/MS after SDS-PAGE in-gel digestion ...... 191
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Chapter 3 Table 2. Predicted O and N linked glycosylation sites based on in silico prediction ...... ……192
Chapter 3 Table 3. Predicted molecular weight (kDa) based on protein backbone and various glycan core structures of full length FGCBP and specific domains representative of clusters A,B, & C domains and termini ...... 194
Chapter 3 Table 4 Proteomic results of DDk or QCM-D chip pull down...... 197
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LIST OF ABBREVIATIONS
AB-PAS Alcian Blue-Periodic Acid Schiff
AFM Atomic force microscopy
ALI Air liquid interface
ATP Adenosine triphosphate
BAL: Bronchial alveolar lavage
BSA Bovine serum albumin
CF Cystic Fibrosis
CFTR Cystic fibrosis transmembrane regulator
CREB cAMP response element-binding protein
CsCl Cesium Chloride
Cys Cysteine
DMBT-1 Deleted in malignant brain tumor -1
DTT Dithiothreitol
EGFR Epidermal growth factor receptor
ELISA Enzyme linked immunosorbent assay
EMT Epithelial mesenchymal transition
ENaC Epithelial sodium channel
ERK Extracellular-signal-regulated kinase
FASP Filter aided sample preparation
FBS Fetal bovine serum
FCGBP IgGFc-binding protein
FEV1% Forced expiratory volume in 1 second as a percent of the normal population FEV1
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Fra-2 Fos-related antigen 2
FXR-RXR Farnesoid X Receptor-Retinoid X Receptor
GalNAc N-Acetylgalactosamine
GuHCl Guanidine Hydrochloride
HBE Human bronchial epithelial
HLA Human leukocyte antigen
HNE Human neutrophil elastase
IgE Immunoglobulin E
IGF-1 Insulin like growth factor 1
IgG Immunoglobulin G
IHC Immunohistochemistry
IL-# Interleukin-#
IPA Ingenuity Pathway Analysis
IR Infrared
JAK Janus Activated Kinases
LC-MS/MS Liquid chromatography Tandem Mass Spectrometry
LPLUNC Long palate, lung and nasal epithelial clone
LXR-RXR Liver X receptor-Retinoid X Receptor
MAPK Mitogen activated protein kinase
MARCKS Myristoylated Alanine-Rich C Kinase Substrate
MEM Minimum essential media miRNA microRNA mRNA messenger RNA
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MSA Multiple sequence alignment
MW Molecular weight
NF-κB Nuclear factor-kappa B
NRF-2 Nuclear factor erythroid 2–related factor 2
ORF Open reading frame
ORMDL Orosomucoid-like proteins
OVA Ovalbumin
P2Y2 Purinoceptor 2
PARS Protease activated receptors
PBS Phosphate buffered saline
PCL Periciliary layer
PNS Post nuclear supernatant
Ps.a. Pseudomonas aeruginosa culture filtrate
QCM-D Quartz crystal microbalance with dissipation
RhoGDI Rho GDP-dissociation inhibitor
Rpm Revolutions per minute
RT Room temperature
SCC Saline-sodium citrate
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SDS sodium dodecyl sulfate
SEC-MALS Size exclusion chromatography-Multi angle light scattering
SMM Supernatant of mucopurulent material
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SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein receptor
Sp1 Transcription factor Sp1
SPLUNC Short palate, lung and nasal epithelial clone
STAT6 Signal transducer and activator of transcription 6
TAE Tris acetate, ethylenedinitrilotetraacetic acid
TBS Tris Buffered Saline
TGF-B Transforming growth factor beta
TH2 T-helper cell type 2
TIL Trypsin Inhibitor like cysteine rich domain
TNF-α Tumor necrosis factor alpha
TYK2 Tyrosine kinase 2
VNTR Variable number tandem repeat vWF von Willebrand factor
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INTRODUCTION: MUCUS & MUCIN
Mucus is a viscoelastic gel that covers and protects the body’s non- keratinized epithelial surfaces. The components that make up mucus vary by location and function though regardless of the location, the general function of mucus is innate immune defense [1]. In the respiratory tract mucus is responsible for coating the epithelium, entrapping inhaled particles and pathogens, and transporting them up and out of the airway maintaining a quasi-sterile lung environment. In the lung, the mucus layer is made up of two distinct layers; the pericilliary layer and the mobile mucus layer [2]. The pericilliary layer (PCL) is composed of mesh consisting of membrane tethered macromolecules including:
MUC1, MUC4, and MUC16 which maintains the separation between the two layers and also helps control the hydration of the mucus layer [2-4]. The mobile mucus layer contains secreted ions, water, antimicrobial peptides and globular proteins, and the gel forming secreted mucins [5]. The two main gel-forming mucins in the lung are MUC5B, which is secreted from both the superficial epithelium and the submucosal glands, and MUC5AC, which is secreted only from the superficial epithelium [6]. The importance of these molecules has been highlighted in recent studies, which used a MUC5B knockout mouse model to show that MUC5B is indispensible for mucocilliary transport whereas MUC5AC plays an important role in allergic inflammation and in response to specific stimuli, such as helminthes [7-9].
1 MUC5B and MUC5AC are large highly glycosylated polymeric proteins.
Though the number of repeats is variable, each of the gel forming mucins contain internal mucin domains that are rich in serine threonine and proline and are the sites of o-linked glycosylation [10]. Interspersed in these regions are cysteine rich domains. Both the N and C termini have cysteine rich regions, which share similarities to the von Willebrand factor (vWF) D and C domains. These terminal cysteines are critical for the polymerization of the mucin monomers through disulfide bond formation. This polymerization is initiated in the endoplasmic reticulum where the C termini dimerization occurs in addition to intramolecular disulfide bonds that promote proper folding of the domains [10, 11]. The mucin dimers are then transported to the Golgi where they glycosylated and then multimerized through disulfide bonding at the N termini [12]. Numerous enzymes have been implicated in the glycosylation of mucins which starts with the addition of N-Acetylgalactosamine
(GalNAc) and through the stepwise addition of sugars creates the different core structures that are can be further modified by addition of functional groups such as sialic acid and sulfate [13]. Mucins are extensively glycosylated and over 70%of their mass can be attributed to these carbohydrate decorations. The structural consequence of this are an added degree of stiffness in these highly glycosylated regions due to the increased volume they occupy and the resulting charge repulsion between sugar chains and a much larger molecular weight than would be predicted by just the protein backbone [11]. Functionally these sugars help allow mucin to form the viscous mesh like network in mucus.
2 The significant size of mucin multimers make the organization and packaging of these macromolecules into small secretory granules an impressive feat and one that is not completely understood though calcium charge shielding and the formation of non covalent calcium mediated crosslinking have been reported to be involved
[14, 15]. After packaging into smaller granules it is proposed that these granules likely undergo lateral fusion to create larger granules, which can be stored until secreted.
After packaging there are several classes of proteins that are required for the exocytosis of the mucin granule. The first step of exocytosis requires moving the mucin granule from the Golgi to the plasma membrane. This is facilitated by a trimeric protein complex made up of MARCKS (Myristoylated Alanine-Rich C Kinase
Substrate), Heat Shock Protein 70 and Cysteine String Protein, which interact with the cytoskeleton [16, 17]. After reaching the plasma membrane the granule is loosely tethered by Rab GTPase protein present on the granule surface [18]. Tight docking to the plasma membrane and preparation for exocytosis occurs through interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and Munc proteins, which both provide scaffolding and also interact with second messengers to stimulate exocytosis [18].
The exocytosis and secretion of mucins occurs at different rates, basal and stimulated thus allowing the mucus to “respond” to insult as part of the innate immune response. Though not completely understood, the different modes of secretions are both regulated and though some proteins of the exocytotic process are shared between them, each pathway also has unique protein members with
3 which it interacts. For example Munc13 is involved in both processes whereas
Munc18b, Synaptotagmin 2, and VAMP8 are thought to only be involved with stimulated secretion as their deficiency results in lack of secretions after exposure to traditional secretagogues [18-21]. Activation of the exocytotic machinery can occur through several pathways involving different G-coupled receptors such as purinoceptor 2 (P2Y2) and protease activated receptors (PARS) that when bound ligand, ATP and serine protease such as neutrophil elastase, respectively, activate a signaling cascade involving phospholipase-Cβ, diacylglyceral and inositol triphosphate [18, 22, 23]. The basal and stimulated secretion rates differ significantly with the stimulated rate occurring in two phases: a very rapid short 10second phase following by a slower (though still several fold higher than the basal rate) and longer plateau phase.
After exocytosis of the granules contents, there is an exchange of sodium and calcium ions leading to a rapid expansion of the mucin molecules as they undergo transition from a condensed state within the granule to a hydrated state forming the mucus gel [24]. Though mucins are the main component of mucus and important in determining the rheological properties of the mucus, they do not act alone [25-27].
Raynal et al showed that concentrated, purified MUC5B do not recapitulate the properties of mucus [28]. Additionally several publications have highlighted the role calcium and other globular proteins that interact with mucin play in changing the mucus properties [5, 29].
In addition to proteins and mucins, the apical secretions also contain exosomes. Exosomes are small membrane bound vesicles of late endosome origin
4 that carry protein, RNA, DNA, and lipid cargo reflecting to some degree the parental cell they derived from. Since first discovery in reticulocytes, exosomes have been identified in numerous other body fluids including plasma, saliva, bronchial alveolar lavage, urine and also in the secretions from human bronchial epithelial cell cultures
[30-33]. They have been proposed to have utility as a therapeutic, prognostic and disease biomarker in a diverse set of diseases including cancer, neurodegenerative, immune and infectious [34-38]. Their role in intercellular communication was also recently shown in an airway cell culture model where exosomes were shown to be taken up by other cell types and change the protein and miRNA expression of the recipient cell including that of the gel forming mucins [39] demonstrating their important regulatory role on mucin secretion by the epithelium.
As discussed previously, protecting the airways by entrapping, neutralizing, and moving foreign pathogens and particles out of the lung is the key function of mucus, but inherent to this is the need for the mucus to be mobile. In a healthy lung, mucus is transported both by tidal breathing, coordinated cilia beating, and cough clearance [1]. Increased mucin concentration has been shown to reduce the effectiveness of these clearance modalities through altering the osmotic pressure leading to compression of the cilia within the PCL and changing the adhesive and cohesive properties of the mucus to the epithelial surface [2, 25, 40, 41]. Therefore it is unsurprising that defects affecting mucus and mucus clearance lead to a variety of lung diseases such as asthma and cystic fibrosis (CF) which despite having very different etiologies are both characterized by pathologic mucus with altered properties. If and how the mucins are specifically altered in these diseases is not
5 well understood and the following body of work will examine how the mucins macromolecular properties and their interactions with other secreted proteins change in addition to how mucin regulation by exosomal miRNA is altered in these two airway diseases.
6 CHAPTER 1. PART A: COMPREHENSIVE CHARACTERIZATION OF MUCINS WITHIN THE CF AIRWAY USING IN-VITRO MODELS Introduction: Cystic Fibrosis
Cystic fibrosis (CF) is a multi organ autosomal recessive genetic disorder that arises from mutations in the cystic fibrosis transmembrane regulator (CFTR) [42].
Though different areas of the body exhibit different symptoms due to this deficiency, most of the morbidity and mortality of CF is associated with respiratory complications and this is where my work will focus [43]. CFTR functions both in secretion of chloride but also in the regulation of epithelial sodium channel (ENaC), which is responsible for sodium absorption [44]. Thus in CF there is a decrease in chloride secretion combined with an increase in sodium absorption ultimately resulting in a dehydrated mucus layer [45]. As discussed in the introduction, the mucus layer consists of a periciliary layer and a mobile mucus layer [2, 4]. The uppermost mobile mucus layer is the first layer to lose water as it has the lower osmotic pressure. As this layer becomes more dehydrated the gel forming mucins come into contact with membrane bound mucins present in the PCL and exhibit adhesion [46]. Secondly there is compression of the cilia within the PCL preventing regular movement of the overlying mucus layer by ciliary beating all of which lead to the development of a non mobile stagnant mucus layer or plaque [2]. Oxygen gradients and areas of hypoxia can develop within these mucus plaques creating a prime location for bacteria colonization, which will be discussed further below [47]. A natural response to this
7 bacterial infection is the host immune response, which includes heavy neutrophilic infiltration, dysregulated cytokine signaling, and also mucus hypersecretion, thus feeding into the ongoing cycle of dehydration, infection, inflammation, and mucus hypersecretion and eventually progressing to tissue destruction and fibrosis [1, 45,
48].
Numerous bacterial product and cytokine-mediated pathways leading to the upregulation of the gel forming mucins in cystic fibrosis have been described [49,
50]. In regards to MUC5AC, NF-κB has been implicated with several different upstream signaling partners including TNF-α, and MAP kinase (ERK), which can also signal through the cyclic AMP–responsive element binding protein (CREB) to increase MUC5AC transcription [51-54]. This pathway is responsive to neutrophil elastase, bacterial products, and inflammatory cytokines such as IL-1β. The epithelial growth factor receptor (EGFR) pathway has been shown to increase
MUC5AC expression through downstream activation of the Sp1 and Fra-2 transcription factors and can be activated by NE and reactive oxygen species in addition to matrix metalloproteases all of which are present in the CF airways [55-
57]. MUC5B involves similar pathways as MUC5AC, though has been studied less extensively. MUC5B expression has been shown to be upregulated in response to inflammatory cytokines such as IL-6 and IL17 and signals through the MAPK pathway [58]. Nucleotide triphosphate signaling through P2Y2 receptor and ERK pathway has also been shown to increases transcription and secretion of MUC5B
[59].
8 The development of mucostasis and bacterial infection presents in early childhood and persist into adulthood and until recently most of the focus was upon classical pathogenic strains such as Pseudomonas aeruginosa and Staphylococcus aureus [60, 61]. Application of 16S sequencing technologies to the lung microbiome has since revealed increased diversity including genera typically found in the oral cavity, which likely colonized the lung during infancy following aspiration, along with the pathogenic genera [62-64]. Also it has been shown that there is a progressive change in the CF microbiome that correlates with the age and disease severity [65,
66]. In early childhood (2 years) it was shown that mainly microbes belonging to the oral flora community colonized the lung and that several years later this shifted to a community dominated by classic CF pathogens which was accompanied by more severe inflammation and tissue damage [65].
There have been conflicting positions regarding the role and nature of mucins in the CF airways. Though numerous studies have shown at the transcript level that
MUC5B and MUC5AC are increased, initial reports suggested that these mucins were degraded by serine proteases within the airway and thus were not important in determining the mucus properties [67, 68]. This was refuted by Henderson et al which showed a several fold increase in total mucin in CF sputum using biophysical methods rather than antibody based [40]. The large size and significant glycosylation of mucins complicates their measurement and quantitation necessitating the need for less traditional techniques such as agarose gel electrophoresis western blot, multi-angle laser light scattering coupled with size exclusion chromatography to accurately measure concentration, and isopycnic density gradient ultra-
9 centrifugation to isolate and purify these macromolecules based on their buoyant density [69, 70]. At the other end of the spectrum, there are others that have reported that the mucins within the CF airways are cross-linked and this is responsible for the rheological changes of CF mucus [71]. Thus the question remains, in the highly proteolytic environment how is the macromolecular structure of the gel forming mucins altered and with what other proteins do they interact to create a pathologic CF mucus.
There are also many different hypotheses regarding the interaction and relationship between the microbiome and mucins. Some have suggested that the oral anaerobes are required to degrade mucins and provide metabolites for aerobes to use whereas others view mucin as a scaffold to separate different bacteria from each other and can alter their pathogenicity [72-74]. By combining comprehensive mucin characterization with microbiome data we hope to elucidate this relationship and also focus on specific bacterial genera and their effect on mucin.
10 Methods
Cell Culture
Primary human bronchial epithelial (HBE) cells passage 1 or 2 derived from non-cystic fibrosis and cystic fibrosis donors were grown at air liquid interface (ALI) and maintained according to previously established protocol [75]. Basal media was changed on alternating days and the apical surface washed with 37°C PBS 2-3 times each week. Challenges were performed on cultures once fully differentiated
(21-28 days after confluence), which was verified by the presence of ciliation and mucus production. For the following experiments, treatments and controls were performed on cells derived from the same donor lung allowing for a paired statistical analysis.
CF cell culture models
To emulate the CF lung environment, two different reagents were used: culture filtrate from Pseudomonas aeruginosa (Ps.a.) or supernatant of mucopurulent material (SMM), which were applied to the apical surface daily for 5 days. Ps.a. was prepared as previously described, 0.22μm filtered and diluted 1:5 in
MEM [76]. SMM was isolated and prepared according to Ribiero et al and the resulting material pooled from 5 CF donors in order to generate sufficient volume for the challenges and reduce interpersonal variability [77]. For the large 30mm collagen coated inserts, 100 μL of Ps.a. or SMM was added to the apical surface and for the smaller 12mm cultures, 40 μL was added. As controls, tripticase soy broth (TSB), the culture media used to grow the Pseudomonas cultures, or PBS
11 were used for the Ps.a. or SMM challenges, respectively. The apical surface was washed with warmed PBS prior to the start of and each day during the challenge.
The large inserts were washed with 1mL of PBS and the small cultures with 350uL.
These washings were stored at 4°C after addition of 8M GuHCl to achieve a final concentration of 4M in order to prevent further degradation. Basal media was collected at baseline, before start of treatment, and each day thereafter for cytokine analysis.
Mucin Isolation and Static Light Scattering
Isopycnic density gradient centrifugation was performed to isolate the gel forming mucins using 4mL of pooled apical secretions in 4M GuHCl at a starting density of 1.45g/ml CsCl in 4M GuHCl spun at 50,000 rpm in a fixed angle rotor
(70.1 TI) for 60-70 hours at 14°C [78]. A slot blot with vacuum filtration was performed on the resulting twelve 750μL fractions, which were then probed with polyclonal MUC5B antibody and monoclonal MUC16 (CA125) antibody to identify the peak with the highest concentration of MUC5B and minimal contamination by membrane bound mucins. These fractions were pooled and subjected to CL2B size exclusion chromatography (2 x 5mL) coupled with matrix assisted laser light scattering (Dawn Heleos II, Wyatt Technology) and refractometry (Optilab T-rEx,
Wyatt Technology) (SEC-MALS/dRi) at a flow rate of 0.5mL/min to determine concentration and macromolecular properties’ of the gel forming mucins, as described previously [40]. Files were analyzed with ASTRA software (Version 7.1.2).
12 Isolation and Analysis of Stored Gel Forming Mucins
After completion of the 5-day challenge, one large 30mm insert from treatment and control for each donor was used for post nuclear supernatant isolation
(PNS). After sequential gentle washings with prewarmed PBS minimizing mechanical stimulation, freshly prepared homogenization buffer (20mM HEPES,
130mM glutamic acid, 0.1mM CaCl, 3mM EGTA, 10nM N-Ethylmaleimide, Turbo
DNase reaction buffer/DNAse (according to manufacturer’s instructions, ambion), and cOmplete mini protease inhibitor tablets (according to manufacturer’s instructions, Roche), pH 7.2) was added to the apical surface of the cell cultures.
Cells were removed/scraped from the insert and homogenized with 50 strokes of the dounce homogenizer while on ice. An equal volume of 8M GuHCl was added to the resulting homogenate and then centrifuged at 200g for 10 minutes at 4°C to pellet any remaining cell debris. The supernatant was removed and subjected to isopycnic density gradient centrifugation at a starting density of 1.45g/ml CsCl in 4M GuHCl for
60-70 hours at 50,000 rpm with a fixed angle rotor (70.1 TI) [78]. Eighteen 500μL fractions were taken per gradient and analyzed for MUC5B and MUC16 reactively following slot blot with vacuum transfer. The MUC5B rich fractions were further analyzed by SEC-MALs/dRi as described above.
Whole Mount Immunohistochemistry (IHC)
After completion of challenge, the apical surface was washed gently and thoroughly with 37°C PBS prior to fixation with Carnoy’s Solution (60% Ethanol, 30%
Chloroform, and 10% glacial Acetic Acid) applied to both basal and apical compartment for 30 minutes at room temperature. Permeablilizeation was performed
13 with 0.2% Triton X in Tris Buffered Saline (TBS) for 30 minutes at room temperature and then the cultures were blocked overnight at 4°C with a solution of 1% BSA, 1%
Fish Gelatin, 0.1% Triton X and 5% normal donkey serum in 1 X TBS. Primary antibodies against MUC5B (1:500, polyclonal), MUC5AC (4ug/mL, 45M1), and x- tubulin (3ug/mL) were prepared in blocking buffer and applied to apical and basal surfaces overnight at 4°C. After washing cultures with blocking buffer diluted 1:10, secondary antibodies diluted (1:1000) were added to both culture surfaces and incubated overnight at 4°C protected from light. The following day, the cultures were washed and counterstained with Hoechst, according to manufacturer’s instructions.
The membrane was excised from the plastic insert and mounted on a slide with the apical surface facing upward. This was allowed to dry overnight before sealing and imaging.
Agarose Gel Electrophoresis
Daily apical secretions dialyzed into 6M urea both reduced (10mM DTT x 10 minutes at 95°C) and unreduced were loaded into a 0.7% (w/v) agarose gel using
6X bromophenol loading dye and subjected to electrophoresis until dye front was within ½ inch of lane end, using 1X Tris-acetate-EDTA (1xTAE) buffer with 1% SDS.
To facilitate detection of the unreduced samples, the gel was incubated at room temp for 10 minutes in a solution of 10 mM DTT prior to transfer [40, 79]. This was followed by a 60-minute vacuum transfer (50mbar) onto a nitrocellulose membrane while submerged in 4× saline-sodium citrate (4xSSC) buffer. The membrane was blocked with 1% milk and probed with monoclonal and polyclonal antibodies against
MUC5AC and MUC5B [40, 80-82]. Secondary IR-dye conjugated antibodies against
14 rabbit and mouse primary antibodies were applied and the resulting signal measured using Licor Odyssey Scanner and quantified via densitometric analysis using the software provided by manufacturer (Version 3.0.30). For each challenge and code, the intensity values were normalized to the highest value prior to statistical analysis to measure the change in mucin secretion and account for donor-to-donor variability.
Mass Spectrometry
Equal volumes (450μL) of daily apical secretion for treatment and control in
4M GuHCl were prepared for Liquid Chromatography Tandem Mass Spectrometry using a modified filter aided sample preparation (FASP) method [83]. Specifically, each sample solubilized in 4M GuHCL was reduced with DTT using a final concentration of 20 mM for 1 hour and 65°C and then alkylated with 50 mM iodoacetamide for 1 hour at 25 °C protected from the light. The samples were centrifuged at 14,000g for 10 minutes and the 10kDA filter washed twice with 4M
GuHCl and then an additional three times using 50mM ammonium hydrogen carbonate (NH4HCO3). The filter was placed in a new collection tube and 0.5 ug modified proteomic grade trypsin (Sigma) added and the samples were incubated in a humidified chamber for 18 hours at 37°C. The peptides were centrifuged and eluted from filter and then concentrated using vacuum centrifugation (Heto-vac). The peptides were then dissolved in 30 uL of 0.1% formic acid analyzed by liquid chromatography-tandem mass spectrometry (data dependent analysis) using a
Dionex ultimate 3000 RSLCnano system 6µl of samples were loaded in a trap column Acclaim PepMap 100, 100 µm x 2 cm, nanoViper C18 5 µm 100 Å, at 5
µL/min with aqueous solution containing 0.1 % (v/v) trifluoroacetic acid and 2 %
15 acetonitrile, while the column used for peptides separation is a Acclaim PepMap
RSLC, 75 µm x 15 cm, nanoViper C18 2 µm 100 Å) coupled to a hybrid quadrupole orbitrap mass spectrometer with a Nano spray source (Q-Exactive, Thermo Fisher,
Bremen, Germany).
Proteins were identified by searching against most current human database
(Proteome Discoverer 1.4) and were quantified based on total precursor intensity using Scaffold, Version 4 (Proteome Software Inc). A paired students T-test was used to compare the treatment and controls from each donor.
For MUC5AC and MUC5B absolute quantification, an internal standard was prepared by spiking 3 heavy labeled internal peptide standards from each protein achieving a final concentration of 100 fmol /µl. All raw files obtained from tSIM-DIA analyses of sputum digest samples were processed by Skyline (version v1.4). For each peptide the ratio between the corresponding endogenous and internal standard peak areas of each precursor (MS) and top 3 most intensity product ions (MS/MS) was calculated. Ratios from the three peptides were averaged and MUC5B and
MUC5AC concentrations were calculated with the following equation:
Protein concentration = [L/H × C × a/b * c/d]
Where L/H is the average area ratio between light and heavy peptides, C is the concentration of injected internal standard, a is the volume used to resuspend the peptides, b is the samples starting volume, c/d is the dilution factor for mixing sample and internal standard (10/8) [84].
16 Proteomic Semi-tryptic peptide analysis
The label free proteomic spectrum files were uploaded and searched against the human database using Scaffold (Version 4) allowing for Semi-tryptic and fully tryptic cleavage sites. The unique MUC5B and MUC5AC Semi-tryptic peptides from the apical secretions following 120 hour challenge with Ps.a or TSB (control) were identified and aligned to the full mucin protein backbone to generate a percent coverage and localize the Semi-tryptic peptides to specific UniProt annotated regions of MUC5B (Q9HC84) or MUC5AC (P98088). The frequency of each type of non-tryptic cleavage site was also calculated for MUC5AC and MUC5B for each sample.
Rate Zonal Centrifugation
Rate zonal centrifugation was performed using 200μL of apical secretions in
4M GuHCl layered on top of a 12 mL 6-8M GuHCl gradient spun at 40,000 rpm in swing out bucket rotor (SW40 Ti) for 2.5 hours at 14°C [85]. The 200μL of secretion was obtained through pooling 50μL per day of apical secretions challenge for each individual donor. The twelve 1 mL fractions were analyzed by slot blot with vacuum filtration and probed with antibodies against MUC5B. The MUC5B intensity was quantified using the Licor Odyssey scanner and software (version 3.0.30). The intensities of each individual gradient’s fractions were normalized to the highest value and plotted according to fraction number with 1 being the uppermost fraction and 12 the bottom.
17 Exosome Isolation
Exosomes were isolated from equal volumes of apical secretions by differential centrifugation. Briefly the raw secretions were spun down at 3000g x 20 minutes at 4°C using a swing out bucket rotor (SW40 Ti) after which the supernatant was kept and centrifuged at 10,000 rpm x 110 minutes at 14°C. The supernatant was removed and centrifuged for a third time at 19,000 rpm x 1.5 hours at 14°C.
After this step, the supernatant was discarded and the remaining pellet was washed with 10 mL of PBS prior to the final centrifugation at 25,000 rpm x 60 minutes at
14°C. The supernatant was removed and the remaining pellet was resuspended in
100mL of PBS. The freshly isolated exosomes were diluted 1:500 - 1:1000 in
0.22uM filtered PBS and analyzed by nanoparticle tracking analysis as described previously [33]. Based on the resulting particle concentration measurements, volumes from each sample containing an equal number of particles were submitted for miRNA sequencing using the HTG EdgeSeq platform, the details for which have been described previously [39].
MUC5B and MUC5AC Standard In-Vitro Experimental Design
MUC5B and MUC5AC standards derived from healthy donor salivary secretions and apical washings from an A549 MUC5B knockdown cell line, a gift from Dr. Babu Subramani (UNC) respectively; that were subjected to two- dimensional isopycnic centrifugation using a starting density of 1.35g/ml were dialyzed into PBS. Ps.a. or TSB were diluted 1:5 in MEM and 0.22um filtered before adding to standards in a 1:4 ratio of Ps.a/TSB to standard. Standards were incubated at 37°C for 24 hours with rotation. Two aliquots were taken at 0, 10, 60,
18 120 minutes, and 24 hours after addition of the Ps. a. or TSB. For each of these timepoints, the first aliquot was added directly to premeasured urea to achieve a final concentration of 6M in preparation for agarose gel electrophoresis western blot using both monoclonal and polyclonal antibodies against the gel forming mucins.
The second aliquot was added to an equal volume of 8M GuHCl in order to repurifiy the gel forming mucins by isopycnic density-gradient centrifugation under dissociative conditions (4M GuHCl) using a starting density of 1.45 g/mL CsCl for further analysis by SEC-MALS.
19 Results:
CF Cell Culture Models: Immunohistochemistry and MUC5B and MUC5AC concentration quantitation
Cell cultures models of CF lung disease revealed significant hypersecretion of the gel forming mucins. MUC5AC and to a lesser extent MUC5B remained adherent to the apical surface after exposure to CF airway stimuli (Ps.a. and SMM) despite extensive washing as measured by whole mount immunohistochemistry (Figure
1.1A). Interestingly this increase in MUC5AC and MUC5B was not evident when the apical secretions of the Ps.a. treated cultures were analyzed by immunoblotting with antibodies against MUC5AC and MUC5B after agarose gel electrophoresis (Figure
1.1B and C, right panels) though MUC5AC and MUC5B did increase after the SMM challenge (Figure 1.1C, left panels).
In contrast to this, the proteomic analysis using absolute quantitative methods (Figure1.1D) showed a significant increase in mean (±SE) MUC5B in both
Ps.a. (7.481 ± 0.59 vs. 3.450 ± 0.92 pmol/mL) and SMM (32.70 ± 6.69 vs. 0.17 ±
0.03 pmol/mL) treated cultures as compared to their respective controls, TSB and
PBS. This same trend was evident with label free LC-MS/MS analysis (Figure 1.1E) measuring the total precursor intensity (TPI) of MUC5B (±SE) for Ps.a (1.19x1011 ±
3.17x1010 vs. 5.49x1010 ± 1.5x1010 TPI) and SMM (6.9x1010 ± 1.45x1010 vs.
8.01x108 ± 4.04x108 TPI).
MUC5AC did not increase after Ps.a. treatment when measured by (Figure
1.1D) absolute quantitation (0.49 ± 0.10 vs. 0.3 ± 0.09 pmol/mL) or when measured by (Figure 1.1E) label free proteomics (4 .98x109 ± 1.53x109 vs. 5.03x109 ± 2.32x109
20 TPI). In contrast, MUC5AC did significantly increase after SMM treatment by both absolute quantitation (2.11 ± 0.29 vs. 0.03 ± 0.006 pmol/mL) and label free
(1.21x1010 ± 1.28x109 vs. 0 TPI, where 0 indicates below limit of detection). The ratio of MUC5AC to MUC5B decreased slightly but not significantly in both Ps.a. (0.06 ±
0.01 vs. 0.11 ± 0.03) and SMM (0.1 ± 0.04 vs. 0.16 ± 0.015).
SMM: MUC5AC and MUC5B concentration quantitation
Analysis of the 0.22um filtered SMM after diluting to the concentration present in the daily apical washings revealed a surprising amount of MUC5AC by both agarose gel electrophoresis and absolute quantitative LC-MS/MS as compared to the apical secretions from the 24 and 120 hour SMM challenge timepoints (3.025 vs.
1.6 ± 0.13 vs. 2.11 ± 0.29 pmol/mL). Minimal staining by antibody was present for
MUC5B in the SMM after agarose gel electrophoresis and absolute quantitation showed the MUC5B concentration in the SMM to be equivalent to the amount found in apical secretions at the 24 hour SMM treatment timepoint, but was far less than the apical secretion MUC5B concentration after 120 hours of treatment (6.01 vs.
6.23 ± 0.96 vs. 32.7 ± 6.9 pmol/mL). Lane analysis of the agarose gel MUC5AC staining pattern (Figure 1.2) indicated that the lower MUC5AC band, which was evident after 24 hours in the apical secretions, aligns with the MUC5AC positive band present in the SMM.
CF cell culture models: Macromolecular characterization of secreted and intracellular gel forming mucins
Analysis of the secreted and stored gel forming mucins by SEC-MALS after purification by isopycnic density centrifugation (Figure 1.3) corroborated the increase
21 in concentration of the gel forming mucins, though this method does not discriminate between MUC5B and MUC5AC. After 120 hours of Ps.a. treatment the cultures exhibited a significant increase in mean (±SE) concentration of gel forming mucins in the apical secretions (47.38 ± 15.53 vs. 33.03 vs.11.38 ug/mL), as did the apical secretions from the SMM treated cultures (54.71 ± 4.90 vs. 12.08 ± 3.17 ug/mL)
(Figure 1.3A). In regards to the mean (±SE) concentration of the intracellular, stored gel forming mucins, the Ps.a. treated cultures exhibited a significant increase (2.70 ±
0.40 vs. 1.42 ± 0.34 ug/mL) whereas though the intracellular gel forming mucins isolated from the SMM treated cultures also increased in concentration (3.34 ± 0.93 vs. 1.32 ± 0.21 ug/mL), it did not reach significance (p=0.07) due to high donor variability (Figure 1.3D).
In general the molecular weight of both the apically secreted and intracellular gel forming mucins in both treatments followed a decreasing trend with the exception of the apically secreted mucins after the Ps.a. challenge, which remained unchanged (Figure 1.3 B&E). In the SMM treated cultures, the mean (±SE) MW of the apically secreted mucins significantly decreased (3.82x107 ± 3.50x106 vs.
1.21x108 ± 1.92 x107 g/mol), as did the MW of the gel forming mucins (3.26x108 ±
6.08x107 vs. 7.03x108 ± 5.90 x107 g/mol). Similar to the SMM, the mean (±SE) MW of the intracellular gel forming mucins decreased after Ps.a. treatment (1.45x108 ±
2.99x107 vs. 3.81x108 ± 5.86 x107g/mol), though the MW of the apically secreted mucins remained unchanged (2.51x107 ± 3.8ex106 vs. 2.49x107 ± 4.37x106 g/mol).
Following Ps.a. challenge, the mean (±SE) radius of gyration of the secreted mucins
(165.5 ± 5.91 vs. 165.1 ± 5.47 nm) and of the intracellular mucins (209.5 ± 18.91 vs.
22 209.1 ± 8.24nm) was unchanged (Figure 1.3C). This was a consistent finding for the
SMM treated cultures for both the apically secreted (191.1 ± 3.85 vs. 180.1 ± 5.53 nm) and intracellular gel forming mucins (234.3 ± 11.89 vs. 216.5 ± 7.92 nm) (Figure
1.3F).
Ps.a. cell culture model: MUC5B Semi-tryptic peptide analysis
Analysis of the MUC5B Semi-tryptic peptides following 120-hour challenge with Ps.a. or TSB (control) revealed no significant differences in the number of unique Semi-tryptic peptides to full tryptic peptides or in the domains to which the
Semi-tryptic peptides are localized along the MUC5B backbone. Semi-tryptic peptides provide insight into cleavage events, whether autocatalytic or bacterial mediated, that occur in the cell culture model prior to collection of the apical secretions and processing for LC-MS/MS analysis. Despite exposure to bacterial proteases, the MUC5B from apical secretions following 120 hour of Ps.a. exposure did not significantly differ from apically secreted MUC5B after 120 hour TSB exposure. The percent of Semi-tryptic peptides localized to the Cys-Rich domains, vWF-D domains and Interdomain regions was not significantly different between the
Ps.a. and TSB treatment groups (Figure 1.4 A-C). Also though the mean (±SE) ratio of Semi-tryptic peptides to full tryptic peptides was higher in the apically secreted
MUC5B after Ps.a. challenge (0.20 ± 0.05), it was not significant as compared to the
TSB (0.11 ± 0.01) (Figure 1.4D).
23 CF cell culture models: Conformation analysis of the secreted gel forming mucins
Conformational analysis of the apical secretions by rate zonal centrifugation revealed that the MUC5B peak shifted towards the lower fractions indicating a more compact conformation after SMM treatment (Figure 1.5A). This trend was not as prominent after the Ps.a. treatment, where the MUC5B peak spanned a broad range of fractions including both compact and linear conformations (Figure 1.5B). In both treatments, the MUC5AC peak appeared in a more bimodal distribution with an early sharp peak at fractions 2&3 and a later, broader peak at fractions 7-9 (Figure
1.5C&D).
SMM cell culture model: Proteomic pathway analysis of secreted proteins
Analysis of the apical secretions by label free proteomics revealed hundreds of proteins that significantly changed after treatment with SMM and Ps.a. as compared to their respective controls, PBS and TSB. Specifically after the SMM treatment, there were over 1600 proteins identified with 450 being differentially expressed/secreted. Among those 450 differentially expressed proteins, 295 proteins significantly decreased whereas 155 increased. Ingenuity Pathway Analysis
(IPA) (Figure 1.6B) of the significantly changing proteins revealed the top canonical pathways (p value, direction) to be: Actin Cytoskeleton Signaling (1.34x10-22, downregulated), Acute Phase Response Signaling (1.84x10-21, upregulated) LXR-
RXR Activation (9.57x10-21, upregulated), Integrin Signaling (1.95 x10-17, downregulated), and Epithelial Adherens Junction Signaling (3.59x10 -16, no prediction). When analyzing these proteins based on disease and biological
24 functions (Figure 1.6C) the most significantly enriched categories were inflammatory response, cellular compromise, cellular movement, immune cell trafficking, and cell death and survival. Numerous proteins (481) were identified in the SMM that was applied to the apical surface when challenging the cells, and 380 of those proteins were shared between the SMM itself, the apical secretions after 24 hours of SMM treatment and the apical secretions after 120 hours of treatment (Figure 1.6D).
Taking this overlap into account, when looking at the differences between Day1 and
Day5 of SMM treatment as a measurement of initial vs. sustained cell response, there were numerous (140) significantly changing proteins. This number was narrowed down to 48 after excluding proteins also present in the SMM itself due to uncertainty regarding the source of these proteins (SMM itself vs. HBE in response to SMM). Interestingly among those 48 proteins, 45 decreased when comparing Day
5 to Day1 and only 3 Increased. These proteins were significantly enriched in
Organismal Injury and Abnormalities, Developmental Disorders, Dermatological
Disease and conditions, Skeletal and Muscular Disorders, and Immunological
Disease. A complete table of all unique and differentially expressed proteins when comparing SMM to PBS is provided in Appendix 2.
Ps.a. cell culture model: Proteomic pathway analysis of secreted proteins
In regards to the Ps.a. challenge, there were approximately 600 proteins identified in the apical secretions with only 51 proteins significantly changing when comparing Ps.a. to TSB. Among those significantly changing proteins, only 8 decreased and 43 increased. The IPA analysis showed significant enrichment (p- value, direction of change) in the following canonical pathways: Acute Phase
25 Response Signaling (4.23 x10-6, upregulated), LXR/RXR Activation (6.06 x10-6, no prediction), RhoGDI Signaling (4.9 x10-5, no prediction), IGF-1 Signaling (6.47 x10-5, no prediction), and FXR/RXR Activation (1.08 x10-4, no prediction) (Figure 1.6F). In regards to the top disease and biological functions, Cellular Compromise,
Inflammatory Response, Cellular Movement, Dermatological Disease and
Conditions, and Organismal Injury and Abnormalities were enriched (Figure 1.6G). A complete table of all unique and differentially expressed proteins when comparing
Ps.a. to TSB is provided in Appendix 1.
Proteomic comparison of secretions from SMM and Ps.a. CF cell culture models
When comparing the significantly changing apically secreted proteins in both the SMM and Ps.a. cell culture challenges, only 14 proteins were shared between them (Figure 1.7A). Furthermore the direction of change was only similar for four of the identified proteins including MUC5B, Gelsolin, Secretoglobin family 3A member
1, and Vacuolar protein sorting associated protein 28 (Figure 1.7B-D and table 1).
Among the numerous identified proteins based on label free proteomics, other mucins including MUC1 and MUC4 significantly decreased after SMM challenge but not after the Ps.a. challenge. MUC16 did not significantly change in either conditions
(Figure 1.8A-C).
CF Cell culture models: Proteomic analysis of mucin interacting proteins in secretions
Numerous potential mucin interacting proteins were identified in the apical secretions in the CF cell culture models. Among those proteins, galectin 3, galectin 3
26 binding protein, and glutathione S transferase significantly decreased after SMM challenge (Figure 1.8D-F). Another category of proteins that increased in the apical secretions after both the SMM and Ps.a. challenges were those with immune modulatory effects. Several proteins within this group have been hypothesized to interact with mucin such as lysozyme C and Leukocyte elastase inhibitor, which both increased in the SMM and Ps.a challenges, and dipeptidyl peptidase 1 which significantly decreased after SMM challenge (Figure 1.8G-I). Another category of mucin interacting proteins that have calcium-binding functions including: Protein
S100, Annexin A2, and Calcyphosin, significantly changed after SMM challenge
(Figure 1.9A-C). Though numerous apically secreted proteins significantly changed in these two CF cell culture model systems, several proteins including: DMBT-1,
SPLUNC and LPLUNC, that have been implicated in CF or inflammation and predicted to associate with mucus, did not change (Figure 1.9D-F).
Ps.a. CF cell culture model: Pathway analysis of differentially expressed exosomal miRNA
MiRNA exosomal cargo, isolated from the apical secretions during the Ps.a. challenge, were sequenced and analyzed in order to obtain a glimpse of what is occurring within the HBE cells in the CF airway environment and to evaluate any mucin regulatory function. Of the 2280 miRNA identified, 187 miRNA were differentially expressed after the Ps.a. challenge. Among those differentially expressed apically secreted exosomal miRNA, 152 were downregulated and 35 were upregulated in response to the Ps.a. challenge, as demonstrated in the volcano plot (Figure 1.10B). The top 20 significantly, differentially expressed
27 exosomal miRNA that exhibited the greatest decrease and increase, as measured by log2Fold change, are listed in Table 2. In silico pathway analysis of the significantly increasing and decreasing miRNA revealed numerous, diverse pathways predicted to be affected. The top 20 most significantly changing pathways based on the differentially expressed miRNA are listed in Table 3. Interestingly
Mucin type O-glycosylation (p value=0.007) (Figure 1.11) was among the pathways predicted to be affected by the decreasing miRNA and 40 of the miRNA (Table 4) identified in our analysis were predicted to target specific genes involved in the glycosylation of the mucin backbone including: polypeptide N- acetylgalactosaminyltransferase 4, Alpha-N-acetylgalactosaminide alpha-2,6- sialyltransferase 1, CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3- sialyltransferase 1, Beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N- acetylglucosaminyltransferase, and Alpha-N-acetylgalactosaminide alpha-2,6- sialyltransferase 1.
Ps.a. CF cell culture model: In silico MUC5B activity prediction based on differentially expressed exosomal miRNA
In an attempt to identify miRNA that directly affect the muc5b gene expression, an IPA network analysis was performed highlighting the gel forming mucins and overlaying the miRNA expression data from the Ps.a cell culture challenge. Though no miRNA were predicted to target MUC5AC, nine miRNA were identified from our dataset that were predicted to target MUC5B. Among those miRNA, two increased significantly in our experimental system and 7 significantly decreased (Figure 1.12). This culminated in an overall predicted activation of
MUC5B. To verity these predictions, the concentrations of these miRNA were
28 correlated with the absolute MUC5B protein concentration measured in the apical secretions by LC-MS/MS. This revealed that miR 6767-5p was significantly
(p=0.022) negatively correlated (Pearson r=-0.71) with MUC5B protein concentrations, thus corroborating the IPA analysis findings (Figure 1.13).
Antibody based MUC5B bacteria degradation timecourse
MUC5B time-course incubation with three different bacteria genera known to be members of the CF lung microbiome, Pseudomonas aeruginosa (Ps.a),
Streptococcus sanguinis, and Prevotella melaninogenica showed loss of both polyclonal and monoclonal antibody reactivity by 24 hours (Figure 1.14).
Specifically, compared to the control at 0 minutes, there was a 100% and 98% loss of antibody signal for the polyclonal and monoclonal, respectively, after 24 hours of
Ps.a. incubation. The intermediate timepoints of the Ps.a. incubation (10, 60, and
120 minutes) showed a moderate loss of monoclonal and polyclonal signal intensity, approximately 45%. Streptococcus and Prevotella were less pronounced in their effect at 24 hours but still showed a 58% and 61% loss in antibody reactivity for the polyclonal antibody and a 68% and 63% loss for the monoclonal antibody, respectively.
Light scattering determination of MUC5B concentration and macromolecular structure during bacteria degradation timecourse
Interestingly when using the biophysical SEC-MALS method to measure the mean (±SE) concentration of high molecular weight species in the V0 region, which would include MUC5B, there was only a 35% decrease in concentration after 24 hours incubation with Pseudomonas (18.36 ± 2.1 vs. 28.26 ± 3.1 ug/mL). A similar
29 trend in the concentration measurements was found with both Streptococcus, which decreased by 29% after 24 hours incubation (20.28 ± 3.05 vs. 28.44 ± 1.4 ug/mL) and with Prevotella, which decreased by 35% after 24 hours (18.15 ± 4.5 vs. 27.86 ±
0.8 ug/mL). None of these decreases in concentration were significant (Figure
1.15A). The molecular weight (Figure 1.15B) did not significantly change at the 120- minute timepoint for Pseudomonas (1.79x107 ± 5.75x105 vs. 2.05x107 ± 1.49x106 g/mol), Streptococcus (1.81x107 ± 1.17x106 vs. 2.12x107 ± 3.08x106 g/mol), and
Prevotella (1.89x107 ± 1.19x109 vs. 1.96x107 ± 1.18x106 g/mol). The 24-hour timepoint revealed a large increase in mean (±SE) molecular weight, which was also accompanied by high variation among the repeats for each bacteria genera tested.
Specifically at 24 hours, MUC5B incubated with Pseudomonas increased in mean molecular weight by 2.7 fold, measuring 5.59x107 ± 3.7x107 g/mol, MUC5B incubated with Streptococcus increased 1.4 fold, measuring 3.05x107 ± 9.17x106 g/mol, and MUC5B incubated with Prevotella increased by 7.3 fold, measuring
1.43x108 ± 1.09x108 g/mol (Figure 1.15B). The trends seen in molecular weight were reflected in the measurement of the mean (±SE) radius of gyration (Figure 1.15C).
Though slightly reduced, the radius of gyration after 120 minutes incubation did not significantly change for Pseudomonas (180.6 ± 5.57 vs. 193.7 ± 4.7 nm),
Streptococcus (180.4 ± 7.2 vs. 191.5 ± 10.15 nm), or Prevotella (182.3 ± 5.6 vs. 186
± 4.9 nm) (Figure 1.15C). Similar to the molecular weight measurements, at the 24- hour timepoint both the variability among repeats and mean radius of gyration increased for Pseudomonas (243.5 ± 51.74 nm) and Prevotella (276.2 ± 73.55 nm).
30 The 24-hour timepoint after Streptococcus incubation was unchanged (189.7 ±
19.23 nm) as compared to the control at 0 minutes.
Antibody based MUC5AC bacteria degradation timecourse
When looking at the polyclonal and monoclonal antibody intensity of
MUC5AC after 0, 10, 60,120 minutes and 24-hour incubation with Pseudomonas,
Streptococcus, and Prevotella, there was a significant decrease in antibody signal intensity by 24 hours (Figure1.16). Specifically after the 24-hour incubation with
Pseudomonas, there was a 60% and 78% loss of polyclonal and monoclonal antibody signal, respectively, as compared to the 0 minute control. After 24-hour incubation with Streptococcus, there was a 53% and 65% loss of polyclonal and monoclonal antibody reactivity, respectively. The 24-hour Prevotella incubation revealed the most dramatic polyclonal and monoclonal antibody signal loss of 94% and 98%, respectively. Also the Prevotella incubation displayed a stepwise pattern of antibody reactivity loss throughout the timecourse as evidenced by the progressive 32%, 47%, and 81% loss of polyclonal antibody reactivity and a similar
45%, 66%, and 78% loss of monoclonal antibody reactivity at the 10, 60, and 120- minute incubation timepoints, respectively.
Light scattering determination of MUC5AC concentration and macromolecular structure during bacteria degradation timecourse
When analyzed by light scattering, the mean (±SE) concentration of the high molecular weight species in the V0 region (Figure 1.17A) decreased slightly and insignificantly after 24 hours incubation with Pseudomonas (3.26 ± 0.82 vs. 4.3 ±
0.27 ug/mL). In contrast, the concentration of MUC5AC did significantly decrease
31 after 24-hour incubation with Streptococcus (1.94 ± 0.65 vs. 4.5 ± 0.62 ug/mL) and
Prevotella (0.15 ± 0.07 vs. 4.615 ± 0.14 ug/mL) as compared to the 0-minute timepoint control. Also throughout the Prevotella incubation, the mean MUC5AC concentration decreased in a stepwise manner at the 10, 60 and 120-minute timepoints (3.42 ± 0.52 vs. 1.21 ± 0.37 vs. 0.75 ± 0.25 ug/mL) mirroring the pattern of antibody reactivity loss (Figure 1.17A, right panel). The mean (±SE) molecular weight decreased in the Pseudomonas (1.85x107 ± 3.15x106 vs. 3.09x107 ±
5.52x106 g/mol) and Streptococcus (1.78x107 ± 7.55x105 vs. 2.12x107 ± 4.24x106 g/mol) timecourse incubations until 120 hours (Figure 1.17B). Similar to the MUC5B incubation, at the 24-hour timepoint, the molecular weight of MUC5AC after the
Pseudomonas incubation increased 4.33 fold, measuring 1.34x108 ± 1.15x108 g/mol and after the Streptococcus incubation increased 1.4 fold, measuring 2.9x107 ±
1.28x106 g/mol (Figure 1.17B). In regards to the Prevotella incubation, the mean molecular weight of MUC5AC remained relatively unchanged at the 10 and 60 minute timepoints but then increased 1.9 and 5.3 fold at the 120 minute and 24 hour timepoints, respectively (Figure 1.17B). The mean (±SE) radius of gyration was more variable across the different bacterial genera analyzed (Figure 1.17C). The
Pseudomonas incubation showed a progressive decrease until the 120 minute timepoint (182 ± 12.53 vs. 194.8 ± 8.6 nm) which was followed by an increase to
220.7 ± 66.58 nm at 24-hours as compared to the 0-minute control. A similar pattern was seen in the radius of MUC5AC during the Prevotella timecourse incubation, which decreased until 120 minutes (188 ± 24.19 vs. 239.3 ± 34.86 nm) and then increased at 24 hours to 213.6 ± 21.44nm, though this final radius measurement
32 was still smaller than the starting radius at 0 minutes. The Streptococcus incubation showed a progressive decrease in the MUC5AC radius of gyration (178.8 ± 23.15 vs. 199.9 ±11.38 nm), though this did not reach statistical significance due to the high variability between repeats (Figure 1.17C).
Bacterial incubation control: antibody and light scattering measurements of MUC5B and MUC5AC
As a control for this study, TSB was incubated with the same MUC5B and
MUCAC standards in the same manner as the bacteria culture filtrates. There was neither a loss of antibody signal intensity nor a significant change in the macromolecular properties of the gel forming mucins after 120-minutes and 24-hour incubation with TSB (Figure 1.18).
33 Discussion:
Mucostasis with chronic infection and inflammation are hallmarks CF lung disease. While it is accepted that mucus hypersecretion and hyperconcentration occurs, the specific contribution of the individual gel forming mucins, their macromolecular conformation and structure in this highly proteolytic environment, and the disease specific differentially expressed proteins they interact with are not known. Bacterial infection and the resulting host immune response is a critical driver in CF pathogenesis. Recent technological advances have elucidated the complex nature of the CF microbiome which encompasses numerous different genera including those traditionally considered pathogenic and those belonging to the oral flora community including anaerobes. Still the relationship and interactions between these microbes and the mucin rich environment they inhabit is not well understood.
We address these questions using apical secretions collected from two well- established in vitro models of CF. One model focuses more narrowly on the effect of
Pseudomonas aeruginosa while the other offers a more complete view by using
SMM which bacterial products in addition to host immune cell products and a broad range of cytokines as well [86]. The apical secretions collected during these challenges were subjected to a battery of biophysical and biochemical analyses to accurately characterize the gel forming mucins, their interacting proteins and to evaluate whether exosomal miRNA are involved in mucin regulation. In order to focus on the specific effects different bacteria have on MUC5B and MUC5AC, an in vitro timecourse bacterial incubation was performed using culture filtrates from
34 Pseudomonas aeruginosa, Streptococcus sanguinis, and Prevotella melaninogenica.
Historically, there has been a debate in the field regarding the contribution of mucins to mucostasis with some reports stating that mucin concentrations in the CF airways are reduced as compared to normal [67, 68]. These studies primarily relied upon antibody based methods and Henderson et al showed that these methods, which are sensitive to epitope loss, do not accurately depict what is occurring in the
CF airway and instead demonstrated the utility of biophysical methods in quantifying mucin [40]. In this study both methodologies were used and compared. We showed using light scattering, label free and isotope labeled mass spectrometry that the concentration of the gel forming mucins increases in two CF cell culture models whereas by agarose gel western blot they did not. This discrepancy in concentration measurements based on antibody dependent and independent methodologies supports the previous findings of Henderson et al, refutes the idea that mucins are reduced in CF, and also questions the validity of using antibody based methods in such a highly proteolytic environment.
Though MUC5B increased significantly in both systems, MUC5AC only increased in the SMM model. By using absolute measurement of MUC5AC, it became clear that the increase in MUC5AC in the apical secretions was likely due to its presence within the SMM, which was applied to the apical surface. MUC5B was also identified in the SMM but the response from the cell culture surpassed the concentration present in the challenge reagent. The notion that SMM was the source of MUC5AC was further supported by the agarose gel western blot band pattern,
35 which revealed that the lower MUC5AC positive band that was present after 24 hours of SMM treatment aligned with the MUC5AC band from the SMM itself. In contrast, there was no detectable MUC5B signal in the SMM. Thus care should be taken when using the SMM model to account for the presence of mucin and other proteins that are derived from the SMM itself. Taking this into account, MUC5B was the dominant gel forming mucin found in the apical secretions from the epithelial cells after challenging with Ps.a. and SMM, but whole mount immunohistochemistry revealed that there was significant MUC5AC staining on the apical surface of the cells that was adherent and not able to be removed by the thorough PBS washings.
This suggests there is a difference in the adherence of the two gel forming mucins which has been suggested in other disease systems [87]. The abundance of
MUC5AC adherent to the cell surface, yet not in the apical secretions after Ps.a. challenge, combined with its presence in the SMM, which is derived from CF airways, raises the question of how and why MUC5AC is present at such high levels in the SMM. One hypothesis reflects the different clearance modalities that are present in the cell culture system and within the lung. The cell culture model primarily relies upon cilia beating to move mucus on the apical surface whereas in the lung both cilia beating and cough clearance occur. Thus perhaps MUC5AC is best cleared through cough clearance explaining its presence in the SMM and absence in the apical secretions following Ps.a. challenge. Also the Ps.a. model is an isolated system and lacks the ongoing inflammatory and immune response that is present in vivo. Therefore we propose that the MUC5B increase may be the epithelium’s response to the pathogen, as shown in the Ps.a challenge, but it is the
36 subsequent immune response that stimulates the MUC5AC hypersecretion to reach the level where it is also abundant within the secretions. A possible candidate for this is neutrophil elastase, which is highly prevalent within the CF airways as well as in
SMM, and has been shown to induce MUC5AC expression [55, 86, 88].
The fate of the gel forming mucins following secretion is still unknown. Some have suggested that they are degraded in this highly proteolytic environment and others that they are cross-linked together as a result of reactive oxygen species [67,
68, 71]. In order to answer this questions, gel forming mucins were purified from the apical secretions through isopycnic density gradient centrifugation and the concentration, molecular weight, and radius were measured by SEC-MALS.
Corresponding to the proteomic data, the concentration of apically secreted high molecular weight gel forming mucins did increase significantly in both the SMM and
Ps.a. models. Though the radius was unchanged in both treatments, the molecular weight decreased after the SMM challenge, yet remained constant after the Ps.a. challenge. While this discrepancy in the direction of molecular weight change could be due to other proteases in the SMM, such as elastase, it is important to note that the resulting decreased MW after SMM challenge was within the normal range for intact gel forming mucins. Interestingly, in both models, the intracellular/stored gel forming mucins did show a significant and consistent decrease in molecular weight after challenge, in addition to a significant increase in concentration and minimal change in radius. As the intracellular gel forming mucins are isolated from the proteolytic extracellular environment, this decrease in size likely reflects a change in the packaging or production, including glycosylation, of the gel forming mucins.
37 The conformational analysis of the apically secreted gel forming mucins by rate zonal centrifugation indicated that after SMM treatment the MUC5B had a more compact structure. This shift in conformation is similar to the published change seen in basally accumulated MUC5B from primary CF cell cultures [89]. After Ps.a. treatment, the MUC5B exhibited a more broad distribution extending to both linear and compact areas in the gradient. The MUC5AC tended to exhibit a shift toward the earlier more linear fractions in both treatments.
To assess the global response of the epithelial cells to the CF environment, pathway analysis of all the differentially secreted proteins following SMM or Ps.a. challenge was performed. Analysis of the apical secretions following SMM challenge proved complicated as the SMM, which was analyzed separately, contains hundreds of proteins, many of which are also found in the apical secretions. These shared proteins represent what is secreted and within the airway lumen in vivo and should not be dismissed, though this does present a challenge when delineating the epithelial cells’ specific response to SMM from what is already present within the
SMM. This issue presents itself again when comparing the number of differentially expressed proteins between the two different CF models. In the SMM model there are hundreds of significantly changing proteins whereas in the Ps.a. there are many fewer. This could be attributed to several factors: the Ps.a. challenge reflects a more limited view of the epithelium’s response to a specific pathogen and thus lacks the additional inflammatory stimuli, such as neutrophil elastase and cytokines, present within the SMM and also that the SMM is a source of numerous additional proteins that are not present in the Ps.a. reagent. Despite this difficulty, the pathway analysis
38 showed similar results for the different CF culture models. Interesting the acute phase response pathway was increased in both of the challenges. This pathway refers to an organism’s systemic innate immune response to different stimuli including infection and inflammation and functions to promote healing and re- establish homeostasis [90]. Interestingly these are typically blood-associated proteins such as C-reactive protein and previous publications have shown that this protein is elevated in CF serum and correlates with disease severity [91-93].
Additionally several of the signaling pathways (NK-κB, TNF-α, IL6-, IL-1, and IL-8) that generate the acute phase response pathway have been shown to be increased and important in CF pathogenesis and present within the SMM [48, 86, 94, 95]. This finding supports that these culture models do in fact replicate to some extent the CF lung environment heavy with infection and inflammation. Another pathway present in both CF cell culture models was the LXR and FXR/RXR pathways which are involved in fatty acid and cholesterol metabolism but also have been implicated in resolution of inflammatory response [96]. The actin cytoskeleton remodeling was the most significantly enriched canonical pathway in the SMM CF cell culture model.
Actin remodeling is an important step in the exocytosis of mucin granules and the enrichment of this pathway suggests that it is likely connected to the mucin hypersecretion evident in this model [97]. Thus a common and not unexpected finding is the enrichment in pathways associated with inflammation and infection both in the canonical pathways and the biological functions and disease categories after challenging with SMM and Ps.a.
39 A closer look at the specific proteins changing after SMM and Ps.a. challenge reveals a handful of shared proteins and still fewer that change in a similar direction.
MUC5B was present among these, as was gelsolin which functions in severing actin filaments and as discussed above, the remodeling of the actin cytoskeleton is important in mucin granule exocytosis. Also Secretoglobin family 3A member 1, which is immune regulatory [98], and vacuolar sorting associated protein 28 which functions in vesicle exocytosis were present and down regulated in both models [99].
These shared proteins may provide insight into the response of the epithelium to
Pseudomonas as its products are likely also present in the SMM as it is one of the most prevalent CF pathogens.
Mucins have been shown to interact with many other proteins from various functional classifications though all are unified in the innate immune response [5].
Mucin interacting proteins that function in immune regulation/modulation and calcium binding showed differential secretion patterns when challenged and yet other mucin associated proteins such as DMBT-1 and SPLUNC did not change. This reflects the dynamic nature of mucus, including the mucins and mucin interacting proteins that compose it, and how, by altering these components, the properties and function of mucus can be tailored to specific stimuli.
Another component of apical secretions that was analyzed in this study were exosomes. Exosomes and their miRNA cargo have important regulatory and intercellular communication functions and have been associated with a diverse collection or biological process including immune regulation, cancer progression and initiation, and neurodegenerative diseases [100]. This diversity in functionality is
40 represented in our pathway analysis of the significantly increasing and decreasing miRNA after Ps.a. challenge. In the context of mucin, we found that the mucin type-
O glycosylation pathway was predicted to be targeted by the decreasing miRNA.
Because mucins are heavily decorated in sugars, which account for 70-80% of their mass, changes in the glycosylation machinery could have significant effects on the overall properties of the mucus layer [10]. Another in silico analysis using IPA, focused on the direct effects of the miRNA on the MUC5B gene and found numerous candidate miRNA that target MUC5B and their cumulative effect was predicted to increase MUC5B expression. This is consistent with our proteomic results. As further support of the in silico analysis, one of the decreasing miRNA, miR6762-5p, significantly correlated with the absolute concentration of MUC5B measured from the apical secretions after challenge.
The CF lung microbiome is diverse, complex, and in contact with the mucus layer overlying the epithelial surface. The previously described experiments have shown that MUC5B is secreted in response to Ps.a. and SMM and though MUC5AC is also present, it appears adherent to the apical surface. There is also evidence that
MUC5AC is secreted in vivo as it was abundance in the SMM. In order to determine the direct effect that specific bacterial genera enzymes have upon the gel forming mucins, we performed a bacterial timecourse incubation using purified MUC5AC and
MUC5B standards and bacterial culture filtrates from Pseudomonas aeruginosa,
Streptococcus sanguinis and Prevotella melaninogenica. The monoclonal and polyclonal antibodies used to probe each timepoint target different mucin domains and were used to analyze whether specific regions of each mucin were more
41 susceptible to bacterial degradation. Specifically the monoclonal MUC5B antibody detects the repeated cysteine rich regions and the polyclonal detects the N terminus.
For MUC5B, the pattern of antibody reactivity loss was not different between the two different antibodies for any of the bacterial filtrates tested. Interestingly each genera exhibited a loss of epitope that was most dramatic at the 24-hour timepoint. Yet when compared to the non-antibody based light scattering measurements, these same timepoints still contained a significant amount of high molecular weight material that did not significantly decrease when compared to the 0 minute timepoint. This further supports the use of biophysical measurements to analyze mucin when exposed to a highly proteolytic environment. The small changes in molecular weight and radius of gyration at the 120 minute timepoint suggest that either the mucin multimers are not being degraded or cleaved by the bacterial enzymes or that the cysteine bonds holding the mucin monomers together are sufficient to keep the molecule intact. At 24 hours, the molecular weight and radius surprisingly increased, this was accompanied by high variability among the 5 repeats and thus was not statistically significant. Potential explanations for this include that the bacterial cleavage caused aggregation of the mucin into larger complexes or that the smaller mucin molecules are more easily cleaved and degraded and thus shift out of the V0 area, leaving behind the larger mucin multimers which results in an increased MW and radius. The later explanation is less likely as the polydispersity of the initial mucin standard was very close to 1.0, suggesting that there is little diversity based on the molecular weight that could account for the large shift evident at 24 hours.
42 Though the MUC5AC showed a similar degradation pattern to the MUC5B standard, there were several distinct differences regarding the specific bacteria. As with MUC5B, different MUC5AC antibodies targeting specific protein domains were utilized. The MUC5AC C terminus was recognized by the monoclonal antibody and the polyclonal antibody recognized the internal cysteine rich regions. In the three bacterial genera tested, there was significant loss in the reactivity of both antibodies after the 24 hour incubation. Interestingly, Prevotella exhibited a more consistent decrease in epitope loss throughout the timecourse. This loss of epitope was accompanied by a decrease in concentration of the high molecular weight species from the V0 region, indicating that Prevotella does produce and secrete enzymes capable of degrading MUC5AC. In contrast, the loss of MUC5AC antibody signal after incubation with Pseudomonas and Streptococcus was not accompanied by a significant decrease in concentration indicating that the antibody epitope was likely lost without macromolecular degradation. The molecular weight and radius measurements of MUC5AC remained relatively consistent up until the 24-hour timepoint, at which point the MUC5AC incubated with Pseudomonas and Prevotella increased dramatically in both of these parameters.
The inability of pseudomonas to break down and utilize mucin is supported by a recent publication by Flynn et al which showed that pseudomonas could not survive on a mucin substrate unless in the presence of other anaerobic bacterial that were capable of degrading mucin and releasing metabolites such as fatty acids and amino acids [74]. Additionally it has been published that Prevotella can and does utilize mucins in the gut as a fuel source and does so by expressing sulfatases [101,
43 102]. Streptococcus sanguinis is found in the oral cavity and though in the presence of MUC5B, cannot survive if MUC5B is the only fuel source available [72]. Another earlier study reported that Streptococcus sanguinis exhibited moderate-low growth when provided pig gastric mucin (predominately MUC5AC) as the sole carbon source, but when grown in co-culture with a fucosidase expressing bacteria, exhibited significantly higher growth [103]. The lack of degradation in our study by
Pseudomonas and Streptococcus is consistent with these findings, as is the ability of
Prevotella to degrade MUC5AC, which shares some similarity to MUC2 (48% amino acid identity) found in the gut.
It should be noted that there are several drawbacks to this study, including: 1) only secreted bacteria products and enzymes were used in this analysis, rather than live bacteria, 2) only one bacteria genera was used at a time rather than in combination, the later scenario would better replicate the CF lung environment, 3) the airway is a dynamic environment and secretes both anti-proteases and proteases, and lastly 4) mucin secretion is an ongoing process within the airways, and thus the kinetics of our experimental design do not truly represent the in vivo environment. Despite these drawbacks, our study examines the capability of different bacterial products to degrade specific gel forming mucins as determined by antibody and light scattering methods. When compared to the cell culture model using Ps.a. the results are consistent in that the molecular weight does not appear to be dramatically reduced by this bacteria’s enzymes and rather than degrade the mucin, Ps.a. stimulates more MUC5B secretion from the cell culture system.
44 Possible future directions that could expand upon the current study and address these drawbacks would be to repeat these experiments using a combination of the different bacterial products or live bacteria to determine if this creates a synergistic effect and promotes mucin degradation. Also the light scattering measurements in this study were performed on unreduced mucin. It is possible, as suggested by Henderson et al, that the disulfide bonds keep the mucin molecule intact even if cleaved by bacteria. Thus by reduction and alkylation, the effect and extent of cleavage may be more evident by light scattering.
The CF airway involves a complex interplay between bacteria and host defenses including the mucus layer which functions in innate immunity. As discussed above, each model system has limitations and drawbacks therefore the following section will analyze the mucin from in vivo human sputum samples from
CF and healthy individuals. The characterization of the gel forming mucins will be combined with microbiome data to elucidate the dynamic relationship and interplay between mucin and bacteria within the CF lung.
45 Chapter 1 Part A Figures
Figure 1.1. CF cell culture models exhibit mucus hypersecretion and adhesion of mucus to surface. A) Representative 3D renderings of whole mount IHC following 120 hours of treatment with PBS (control), SMM and Ps.a. Cilia, white; MUC5AC, red; MUC5B, green; Nuclei, blue. Representative agarose gel electrophoresis western blot of apical secretions following treatment with SMM (left) and Ps.a. (right) probed for (B) MUC5B and (C) MUC5AC, with densitometric quantitation below. D) Absolute quantitation of MUC5B and MUC5AC after SMM (left) and Ps.a. (Right) challenge using LC-MS/MS and heavy isotope labeled peptide standards. E) Label free LC-MS/MS analysis of MUC5B and MUC5AC concentrations based on total precursor intensity after challenge with SMM (left) and Ps.a. (right). Repeated measure ANOVA used for SMM analysis and paired t-test for Ps.a. No correction was made for multiple comparisons. Data represented as mean (±SE) in bar graphs or as Tukey box and whisker plots. P values: *≤0.05, **≤0.01, ***≤0.001.
46
Figure 1.2. Comparison of MUC5AC in apical secretions after 120-hour SMM challenge and the SMM itself. Representative western blot of apical secretions following 5 days of SMM challenge and raw SMM diluted 1:10 in PBS with lane profile analysis showing average MUC5AC intensity based on pixel location of upper (yellow) and lower (red) band.
47
Figure 1.3. Macromolecular characterization of secreted and stored mucins. After 120 hour challenge with SMM, Ps.a. or control (PBS and TSB, respectively), gel forming mucins were purified from apical secretions and whole cell lysate and analyzed by SEC-MALS/dRI. A) Concentration, (B) molecular weight, and (C) radius of gyration of secreted gel forming mucins after 120 hour SMM (left) and Ps.a. (right) challenge. The (D) concentration, (E) molecular weight, and (F) radius of gyration of the stored gel forming mucins purified from the whole cell lysate after SMM (left) and Ps.a. (right) challenge. Data represented in Tukey box and whisker plots. Paired Student t-tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001.
48
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e Control Ps.a. S Figure 1.4. Ps.a. CF cell culture model: MUC5B Semitryptic peptide analysis. MUC5B Semitryptic peptide localization is not significantly different between control and Ps.a. challenged cultures. A) Specific domain localization of unique Semitryptic MUC5B peptides in control (TSB) and Ps.a. challenged cultures. B) Control and (C) Ps.a. pie charts showing localization of Semi-trpytic peptides based on generation domain classification. D) Ratio of unique Semi- tryptic peptides to fully tryptic peptides per sample. Control, n=5; Ps.a, n=5. Paired Student t-test was performed.
49
Figure 1.5. Rate zonal conformation analysis of gel-forming mucins. Rate zonal centrifugation fractions of apical secretions following 120 hour of SMM (n=5) or control (PBS; n=5) treatment with immunoblotting for (A) MUC5B and (C) MUC5AC. Panel B and C employs the same method using apical secretions after 120 hours exposure to PSA (n=5) or TSB (n=5) to show the conformational change of (B) MUC5B and (D) MUC5AC. Density and concentration of GuHCl increases with higher fraction number. Linear conformations are typically found in the early fractions whereas more compact are found in the later (higher number) fractions. Paired Student t-tests were performed with the following p values: “=0.06, *≤0.05.
50
and d upregulatedor downregulated proteins (log2 fold change oftreatment vs. control)within specificpathways. Biological functi canonicaland (G) biological and disease process.Activation scoresz are calculated basedon the ratio of significantly Among proteins,these thoseshowing differential expression value<0.1)(p wereanalyzed by IPA revealingenriched the (F) treatment.The Venndiagram in E.shows theunique and sharedproteins between Ps.aand control 120after hours challenge. unique and shared proteinsthe in challenge reagentSMM and the apical secretionsafter and24 120 h activation (orange=upregulatedand blue expressed(p value<0.05) were analyzed by IPAshowing enriched the (B) canonical unique the andshared proteins betweenSMM and controlafter 120 hours challenge.Among these proteins differentially the (B apicallysecreted proteins control in and treatment groups ofCF cell culture modelswith additional ingenuitypathway analys Figure 1.6.Label proteomic free analysis ofapical s
-
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- downregulated)and (C) biological and disease process. Figure 1.6D) shows the
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nge with SMM (n=6)and Ps.a (n=5). Figure 1.6A) shows
VennDiagrams (A, D, &E) show unique and shared
pathways includingdirection the of
oursof SMM
ons
es
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Figure 1.7. Shared significant differentially secreted proteins between the SMM and Ps.a. 120 hour cell culture challenge based on label free proteomic analysis. Of the 14 shared proteins the three non-mucin proteins that significantly changed in the same direction are represented in panels B-D with SMM (n=6) on the left and Ps.a. (n=5). Data represented as tukey box and paired Student t-tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001.
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Figure 1.8. Changes in non-gel forming mucins and mucin interactome proteins in CF cell culture models. Secretion pattern of additional non gel-forming mucins and mucin interacting proteins as measured by label free LC-MS/MS in apical secretions after challenge with SMM (left; n=6) or Ps.a. (right; n=5). A-C) Total precursor intensity (TPI) of non gel forming mucins D-F) TPI of mucin interacting proteins. G-I) TPI of mucin interacting proteins with immune/inflammation modulating functions. Data represented in Tukey box and whisker plots. Paired Student t-tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001
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Figure 1.9. Changes in mucin interactome in CF cell culture models. Secretion pattern of mucin interacting proteins as measured by label free LC-MS/MS in apical secretions after challenge with SMM (left; n=6) or Ps.a. (right; n=5). A-C) Total precursor intensity (TPI) of calcium binding proteins D-F) TPI of mucin interacting proteins with immune and inflammatory functions that did not significantly change with challenge. Paired Student t- tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001
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Chapter 1 Table 1 Shared differentially expressed proteins identified in apical secretions after 120 hours of SMM or Ps.a treatment with respective log2 fold changes comparing challenge to control. P values of all shown proteins <0.05 by paired Student’s T test of total precursor intensity as measured by label free LC-MS/MS. * designates proteins with same direction of change in both model systems. Color indicates direction of change: increase (red) and decrease (blue) Log2 Fold Change Protein Name SMM vs. PBS Ps.a. vs. TSB Lysozyme C 6.71 -0.82 Mucin-5B* 6.42 1.11 Chloride intracellular channel protein 1 -1.64 0.66 Polymeric immunoglobulin receptor -3.06 0.31 Na+-dependent phosphate transport protein 2B -1.75 1.77 Gelsolin* -1.75 -0.46 Cathepsin D -3.40 0.43 Chloride intracellular channel protein 6 -3.98 0.75 Vacuolar protein sorting-associated protein 28* -∞ -0.84 Secretoglobin family 3A member 1* -4.09 -1.32 Na+/K+ transporting ATPase subunit beta-1 -1.42 0.54 Urokinase-type plasminogen activator -5.00 1.21 K-transporting ATPase alpha chain 2 -2.29 1.27 Fructose-bisphosphate aldolase C -3.07 0.28
55
A B. .
Figure 1.10. Changes in exosome concentration size and miRNA cargo after 120 hour Ps.a challenge. A) Change in concentration (particles/mL) and size (nm) of the exosomes following 120 hour challenge with Ps.a or control (TSB). B). Volcano plot of differentially expressed miRNA sequenced on HTG EdgeSeq platform isolated from exosome like vesicles secreted into the apical secretions during 120 hour challenge with Ps.a. or control (TSB; n=5). Statistics was performed on log2 normalized sequence reads using an adjusted p value of <0.1 to determine significance. Blue dots represent specific miRNA that decreased with Ps.a. treatment and red dots the specifically identified miRNA that increased after Ps.a. challenge. Data represented in Figure 1.10A as Tukey box and whisker plots. Paired Student t-tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001
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Chapter 1 Table 2. Top 20 significantly decreasing and increasing miRNA isolated from apically secreted exosome like vesicles after 120 hour challenge with Ps.a. or control (TSB; n=5). Adjusted p value cutoff for significance was <0.1 Decreasing miRNA Increasing miRNA ID Fold Change ID Fold Change miR-3940-5p -1.99 miR-6813-5p 1.98 miR-4497 -1.60 miR-4443 1.44 miR-6752-5p -1.58 miR-146a-5p 1.43 miR-5787 -1.58 miR-1306-3p 1.42 miR-149-3p -1.55 miR-4632-3p 1.38 miR-762 -1.55 miR-6726-3p 1.37 miR-6780b-5p -1.52 miR-6825-3p 1.36 miR-6087 -1.46 miR-1199-3p 1.34 miR-6088 -1.41 miR-1538 1.23 miR-8078 -1.37 miR-4448 1.18 miR-4792 -1.37 miR-661 1.09 miR-6803-5p -1.37 miR-1909-3p 1.07 miR-638 -1.37 miR-146b-5p 1.06 miR-1273f -1.36 miR-6068 1.00 miR-1273h-5p -1.34 miR-3663-5p 0.99 miR-3960 -1.33 miR-6861-3p 0.99 miR-5010-5p -1.32 miR-4783-3p 0.98 miR-1273d -1.32 miR-6131 0.84 miR-6089 -1.31 miR-4274 0.83 miR-4461 -1.30 miR-572 0.77
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Chapter 1 Table 3. Significant KEGG pathways based on significantly increasing miRNA isolated from apically secreted exosomes after 120 hour challenge with Ps.a. or control (TSB) (n=5). Number of genes in each pathway predicted to be affected by the miRNA and number of miRNA within the dataset targeting each specific pathway is listed. In silico predictions made using the Diana MirPath version 3 using the modified Fisher’s exact test with a p value cut off of 0.05 and Benjamini Hochberg correction. Increasing miRNA Decreasing miRNA KEGG pathway p-value #genes #miRNA KEGG pathway p-value #genes #miRNA Prion diseases 1.73E-08 8 9 Thyroid hormone signaling pathway 3.19E-07 96 72 Glycosphingolipid biosynthesis - 6.05E-07 10 11 Lysine degradation 4.46E-05 38 55 lacto and neolacto series TGF-beta signaling pathway 8.70E-05 30 19 Morphine addiction 4.72E-05 73 59 Proteoglycans in cancer 9.81E-04 67 21 Axon guidance 4.72E-05 99 66 Signaling pathways regulating Phosphatidylinositol signaling 1.72E-03 51 22 7.51E-04 58 56 pluripotency of stem cells system ErbB signaling pathway 2.38E-03 37 22 MAPK signaling pathway 7.87E-04 192 87 Glioma 4.35E-03 26 19 Sphingolipid signaling pathway 1.39E-03 88 64 Glycerophospholipid metabolism 6.23E-03 35 19 ErbB signaling pathway 1.84E-03 68 61
58 Phosphatidylinositol signaling 6.23E-03 32 19 Primary bile acid biosynthesis 4.21E-03 14 16
system Protein processing in endoplasmic 6.23E-03 59 25 Pancreatic cancer 4.95E-03 51 52 reticulum Vitamin digestion and absorption 1.14E-02 10 9 Hippo signaling pathway 4.95E-03 104 66 Pancreatic cancer 1.36E-02 26 17 Glutamatergic synapse 4.95E-03 82 67 MAPK signaling pathway 1.36E-02 87 23 Endocytosis 7.07E-03 146 73 Acute myeloid leukemia 1.51E-02 24 16 GABAergic synapse 8.16E-03 64 63 Choline metabolism in cancer 1.57E-02 38 21 mTOR signaling pathway 1.48E-02 48 59 Estrogen signaling pathway 1.72E-02 36 19 Dopaminergic synapse 1.48E-02 94 65 Hepatitis B 2.05E-02 46 23 Renal cell carcinoma 1.53E-02 50 59 Oxytocin signaling pathway 2.34E-02 56 21 Focal adhesion 1.75E-02 145 71 Neurotrophin signaling pathway 4.05E-02 44 21 cAMP signaling pathway 1.75E-02 141 72 Ras signaling pathway 4.05E-02 70 22 Prion diseases 1.94E-02 19 39
. moremiRNA. yellowpredictedare beto targetedby one miRNAwithin thelist. Genes in orangeare predicted toaffe be implementing the modified Fisher’sexact testwith a p value cut off of 0.05 and BenjaminiHochberg correction. miRNAimplicated in this pathway are listedin TableKEGG 6. pathway was determined us p value<0.1), decreasing miRNAisolated fromexosomes following 120 hour challenge with Ps.a. as comparedto control. challenge. Figure 1.11. Mucintype O
Specificgenes within the Mucin type O
- glycosylationgenes targeted by significantly decreasedexosomal miRNA afterPs.a.
- glycosylationpathway p
redicted beto affected by significantly (adjusted
ingDiana miRpath version3
ctedby two or
Genesin
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Chapter 1 Table 4. Decreasing miRNA isolated from apically secreted exosomes following 120hour treatment with Ps.a or TSB (control) predicted to affect mucin type O glycan biosynthesis based on Diana miRpath analysis. miR-548e-5p miR-6510-5p miR-616-3p miR-4271 miR-6124 miR-1207-5p miR-4496 miR-4459 miR-5591-3p miR-6756-5p miR-3934-5p miR-8085 miR-548ax miR-6738-5p miR-4722-5p miR-1303 miR-3127-3p miR-4421 miR-6812-5p miR-4516 miR-6893-5p miR-548ay-5p miR-937-5p miR-3674 miR-4284 miR-548d-5p miR-4466 miR-6808-5p miR-7847-3p miR-4502 miR-6165 miR-8069 miR-6816-5p miR-3652 miR-1225-3p miR-4430 miR-6870-5p miR-6799-5p miR-765 miR-574-5p
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Figure 1.12. MUC5B gene predicted to be activated by differentially expressed miRNA after Ps.a. challenge. Differentially expressed miRNA isolated from exosomes were used to create an overlay using the Ingenuity Pathway Analysis (IPA) My Pathways Grow function to predict upstream and downstream molecules then the Molecular Activity Predictor was applied to determine predicted activation direction for MUC5B. All miRNA were filtered based on adjusted p value <0.01.
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Figure 1.13. miR-6762-5p concentration negatively and significantly correlates with MUC5B concentration. miRNA isolated from apically secreted exosomes show differential expression in response to Ps.a. challenge which was correlated to the absolute MUC5B concentration measured from the same apical secretions by isotope labeled LC-MS.MS. Pearson correlation was performed, r=-0.71 and p value=0.022. Purple squares represent miR and MUC5B concentration after Ps.a. treatment and the teal squares after TSB (control).
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representedas bar charts with mean ±SE Repeated measureANOVA was performedwith the following p values:*≤0.05, **≤0.01, ***≤0.001. hours (laneAntibody 5). intensitynormalized to the highest value melaninogenica0 for minutes (lane 1), 10 minutes(lane 2),60 minutes(lane 3),120 minutes (lane 4),and 24 MUC5Bantibody after i bacteria. Figure 1.14.Salivary MUC5Bstandard shows time dependentantibody loss after incubation with
Representativeagarose gel western probedblots with a (panelA) polyclonal and (panelB) monoclonal
ncubationwith Pseudomonasaeruginosa, Streptococcus sanguinis, and Prevotella
within each repeat (n=5)is represented below.
Data
63
values:*≤0.05, **≤0.01, ***≤0.001. Data representedas bar charts with mean ± SE. (lane120 3), minutes (laneand 4), hours24 (laneRepeated 5). measure ANOVAwas performed with thefollowing p St (C) radius ofgyration of highmolecular weight MUC5Bin V0, after incubation with Pseudomonasaeruginosa, purificationvia isopycnic centrifugation and analysisbySEC Figure 1.15.Biophysical characterization of MUC5B aftertime cours
reptococcussanguinis, and Prevotella melaninogenica0 for minutes(lane 10 1), minutes (lane 2),60 mi
- MALS.
eincubation with bacteria afterre
A)Concentration (B) molecularweight and
nutes
-
64
followingp values:*≤0.05, **≤0.01, ***≤0.001. Data representedas bar charts with mean ±SE. valuewithin each repeat (n=5)is representedbelow. Repeated measure ANOVA minutes(lane 3),120 minutes (lane 4), and 24hours (laneAntibody 5). intensitynormalized to the highest Streptococcussanguinis, and Pr and (panelB) monoclonalMUC5AC antibody after incubation with Pseudomonas aeruginosa, incubation bacteria.with Figure 1.16.A549 MUC5ACstandard shows time and sp
Representative agarose gelwestern blotsprobed with a(panel A) polyclonal
evotella melaninogenica0 for minutes(lane 10 1), minutes (lane 2),60
eciesdependent antibody loss after
was performedwith the
65
values:*≤0.05, **≤0.01,***≤0.001. Data representedas bar charts with mean ± SE 120 minutes (laneand 4), hours 24 (lane 5).Repeated measureANOVA was performedwith the following p Streptococcussanguinis, and Prevotella melaninogenica0 for minutes( radius ofgyration ofhigh molecular weight MUC5ACin V0, after incubation with Pseudomonasaeruginosa, purificationvia isopycnic centri Figure 1.17.Biophysical characterization of MUC5AC aftertime course incubation with bacteria after re
fugationand analysisbySEC
-
MALS.
lane1),10 minutes(lane 2), minutes60 (lane 3),
A)Concentration (B) molecularweight and (C)
-
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Figure 1.18. Gel forming mucin timepoint incubation with TSB. Saliva MUC5B (left panel Chapter 1 Figures A-D) and A549 MUC5AC (right panel, Chapter 1 Figures E-H) are not degraded by TSB incubated for 120 minutes and 24 hours based on (A&E) agarose gel western blot and antibody detection using both polyclonal (left) and monoclonal (right) antibodies (n=2) and SEC-MALS concentration measurement (B&F) of the large high molecular weight species located in the V0 region. Molecular weight of both MUC5B (C) and MUC5AC (G) appear unchanged by the incubation with TSB. The radius of gyration is also unaffected for MUC5B (D) and MUC5AC (H).
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CHAPTER 1. PART B: COMPREHENSIVE CHARACTERIZATION OF MUCINS WITHIN THE CF AIRWAY: A COMPARATIVE ANALYSIS BETWEEN NON- DISEASE AND CF SPUTUM
Introduction
Cystic Fibrosis is an autosomal recessive genetic disorder caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR) [42]. Though systemic in nature, most of CF-associated morbidity and mortality is due to respiratory disease that arises from ineffective mucus clearance and, ultimately, mucus stasis that is associated with chronic inflammation and infection [43]. The large, highly glycosylated polymeric mucins are the major macromolecules in the airway mucus layer [10]. The two most abundant secreted airway mucins are
MUC5B secreted from both submucosal glands and superficial airway epithelium and MUC5AC secreted from superficial airway epithelium. [4, 6, 104]. The function of these two mucins differs, and their relative concentrations differ in muco- obstructive diseases, e.g., COPD, vs. asthma [84, 105] The mucus stasis that characterizes CF reflects defective clearance of the mucus layer by both cilia and cough-dependent mechanisms, largely raised mucin concentrations, increased mucus layer osmotic pressure and ciliary compression and increased adhesive interactions between the mucus layer and epithelial surfaces [2, 25, 40, 41, 46].
Static mucus may initiate and interact with bacterial infection in CF. Recent in vivo studies have shown that mucins can be degraded by bacterial saccharidases and utilized as energy sources by anaerobic bacteria aspirated into the lung from the
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oral cavity [68, 74]. The complexity and diversity of the microbial communities in the
CF lung has been elucidated by 16-S technologies which have revealed a succession from anaerobic oral flora species to classic CF pathogens, e.g.,
Pseudomonas aeruginosa, in the progression of CF lung disease [62, 64, 65].
Proteases secreted by bacteria and host inflammatory cells create a highly proteolytic CF airway luminal environment, which overwhelms the lung’s anti- protease activity [106-108]. This high proteolytic activity also has the capacity to degrade mucin protein backbones and alter its rheological properties, as well as provide amino acids for bacterial metabolism [109].
In this study, sputum samples from CF subjects and healthy controls were obtained to quantitate and characterize the mucins within the CF lung. Using quantitative mass spectroscopy methods, the concentrations of the MUC5AC and
MUC5B gel-forming mucins were measured and characterized with respect to their macromolecular properties based on proteolysis and glycomic structure. These findings were related to clinical and quantitative microbiome data to provide insight into the pathogenesis of bacterial-mucin interactions in the CF lung. In parallel, a controlled ex-vivo model system in which primary cell cultures were challenged with
Pseudomonas aeruginosa (Ps. a) was utilized to identify specific roles for this CF pathogen in the development of pathological CF mucus.
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Methods Study Population
Spontaneously expectorated or induced sputum samples were collected from
CF subjects (n=44) during periods of self-reported stable and exacerbation states
[65]. Demographics and clinical data, including spirometry measurements, were obtained at each sample collection. Frozen sputum samples were thawed on ice and solubilized in 4M guanidine hydrochloride. Control sputum samples from non- smokers with normal lung function were obtained from the SPIROMICS and TCORS cohorts. Demographics and clinical data from the subjects are provided in
Supplemental data (Table 1). FEV1 % predicted quantiles for the CF cohort were designate as follows: tercile1: <32.25%, tercile 2: 32.35-62.25%, and tercile 3:
>62.25%.
Total Mucin Concentrations
Total sputum mucin concentrations were measured using size exclusion chromatography coupled to laser photometry (DAWN HELEOS II, Wyatt
Technology) and refractometery (Optilab T-rEX, Wyatt Technology) after treatment with DNAse (Ambion, Life Technologies) [40]. Data were analyzed with the Wyatt
Technology ASTRA software, version7.1.2.
MUC5AC and MUC5B Concentrations
Samples were prepared for label free and internal label proteomic analysis in similar manners using a modified filter aided sample preparation (FASP) method
[83, 84]. Briefly, equal starting volumes of raw sputum (10uL), solubilized in
4MGuHCl, were reduced, alkylated, desalted with 10k cutoff centrifugal filters
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(Microcon-10), and then digested overnight with trypsin. The peptides produced by trypsin digestion were eluted, freeze dried, and analyzed by liquid chromatography- tandem mass spectrometry (a hybrid quadrupole Orbitrap mass spectrometer with a nanospray source, Q Exactive, Thermo Fisher Scientific) using data dependent analysis. Proteins were identified by searching against current human databases and quantified using Scaffold (Version 4) (Proteome Software Inc.) In addition, after rehydration of the peptides, 3 heavy labeled internal peptide standards for MUC5AC and MUC5B were spiked into each sample, which was then subjected to targeted selected-ion monitoring–data-independent acquisition analysis.
Proteomic Semi-tryptic peptide analysis
The label free proteomic spectrum files were uploaded and searched against the human database using Scaffold (Version 4) allowing for Semi-tryptic and fully tryptic cleavage sites. The unique MUC5B and MUC5AC Semi-tryptic peptides for the CF and control sputum were identified and aligned to the full mucin protein backbone to generate a percent coverage and localize the Semi-tryptic peptides to specific UniProt annotated regions of MUC5B (Q9HC84) or MUC5AC (P98088). The frequency of each type of non-tryptic cleavage site was also calculated for MUC5AC and MUC5B for each sputum sample.
Purification of Gel-Forming Mucins
A two-step isopycnic centrifugation method was employed to separate the gel-forming mucins from globular proteins and DNA. Briefly, equal volumes of solubilized sputum were subjected to isopycnic density gradient centrifugation at a starting density of 1.35g/ml in 4M GuHCl for 65 hours at 14°C at 100,000 rpm. The
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mucin rich fractions were identified by antibody reactivity, pooled, and dialyzed into light scattering buffer. The pooled mucin rich fractions were subjected to a second step of associative CsCl centrifugation with a starting density of 1.45g/mL and the mucin rich fractions identified by MUC5B antibody reactivity.
Characterization of the Gel-Forming Mucins
Molecular weight and radius of gyration of the purified gel-forming mucins were measured using size exclusion chromatography coupled with laser photometry
(DAWN HELEOS II, Wyatt Technology) and refractometery (Optilab T-rEX, Wyatt
Technology) as described previously [40].
Glycomics
The purified mucins were dialyzed into PBS and prepared for glycomic analysis. Briefly, lipids were extracted, followed by acetone protein precipitation and reductive beta elimination. This preparation was followed by Dowex ion exchange,
C-18 desalting, and graphite carbon chromatography prior to permethylation of samples and analysis by nanospray ionization MS using negative and positive ion mode to detect sulfated and non-sulfated glycans, respectively.
Microbiome
Aliquots of sputum from each CF patient were used for microbiome analysis using both clinical culture and microbiome sequencing methods as detailed previously [65]. In brief the modified universal primers directed against the V4 region of 16s rRNA gene were used for amplification and subsequently bead-cleaned and sequenced using the Illumina MiSeq platform. In a similar manner as described
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previously [65], the sputum samples were classified based on 23 taxa found to be most abundant, which accounted for > 95% of the sequences in the CF BAL microbiome study [62].
Statistics
Mixed models were used to account for the longitudinal sputum samples that were collected from the same individual at both stable and exacerbation timepoints.
Additionally gender and age were accounted for as covariates. Scaled Bray-Curtis distance non-metric multi-dimensional scaling (NMDS) analyses were used to determine if significant segregation occurred between the CF and control sputum based on various parameters.
Reagents
Reagents were obtained from Sigma Aldrich with the exception of the following: Mouse monoclonal MUC5AC, 45M1 (Thermo Scientific), Rabbit polyclonal
MUC5B prepared in house, Donkey serum and all secondary antibodies (Jackson
Immuno Research), Hoechst 33342 (Invitrogen). Primary HBE cells and ALI media were acquired from the CF Center Tissue Procurement and Cell Culture Core. Ps.a. filtrate was a gift from the lab of Dr. Scott Randell.
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Results Demographics
Comprehensive Sputum Analysis Study Design: Induced expectorated sputum was obtained from non-smoking, healthy subjects enrolled in the
SPIROMICS or TCORS studies according to reported protocols [110]. Spontaneous or induced sputum was collected from CF subjects at Dublin and Belfast research sites according to published protocols [62]. Demographics were recorded at the beginning of the study and time of sputum collection (Chapter 1. Table 5).
Total mucin concentrations in CF vs. normal subjects
To measure the total mucin concentrations of healthy vs. CF sputum, SEC-
MALS studies after DNase treatment were performed. Total mucin concentrations were raised in CF subjects under basal and exacerbating conditions as compared to control subjects (Figure 1.19A). Specifically, stable CF subjects (n=19) exhibited a ~
3.7 fold increase in mean total mucin concentrations (±SE) as compared to non- disease controls (n=10) (5477 ± 687.9 vs. 1468 ± 400.4 ug/mL sputum) (Figure
1.19A). CF subjects who self-reported exacerbations at the time of sputum collection
(exacerbating CF subjects) (n=24) also exhibited elevated total mucin concentration as compared to healthy controls (5817 ± 5 76.4 ug/mL). Total mucin concentration also did not differ significantly among the CF subjects based on FEV1% predicted quartiles (5460 ± 786.6 vs. 5967 ± 643.5 vs. 6067 ± 1041 ug/mL) (Figure 1.19B).
Total mucin concentrations also did not correlate with FEV1% predicted (Figure
1.19C) in this CF cohort (Pearson r = 0.124, ns) nor vary significantly based on use of hypertonic saline, inhaled corticosteroids, or antibiotics (Supplementary Figure
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1.28). Interestingly, there was a significant correlation between the age of the patient within the CF cohort and the total mucin concentration (Pearson r=0.430, p value=0.006; Figure1.20A) and also between sputum human neutrophil elastase
(HNE) activity and total mucin concentration (Pearson r=0.552, p value=0.006)
(Figure 1.20C).
Absolute MUC5B and MUC5AC concentrations in CF vs. normal subjects
The absolute MUC5B and MUC5AC concentrations in sputum from stable CF subjects (n=17) were also increased significantly compared to control subject (n=19) sputum (Figure 1.21A). Specifically, the absolute MUC5B concentration in the stable
CF cohort increased 8.5-9 fold (927.4 ± 179.3, CF, vs. 108.2 ± 20.38, controls, picomol/ml). MUC5AC started at lower levels than MUC5B but was increased 28-30 fold (288.7 ± 84.4 vs. 9.514 ± 3.74 picomol/mL as compared to control subjects. The ratio of MUC5AC to MUC5B also significantly increased when stable CF subjects were compared to normals (0.44 ± 0.114 vs. 0.10 ± 0.036) (1.21B). As with the total mucin concentration, there was a strong and significant correlation (Pearson r=0.546, p value=0.00015) between the MUC5AC to MUC5B ratio and the age of the
CF subjects (Figure 1.20D). The ratio of MUC5AC to MUC5B, however, did not significantly correlate with HNE levels in sputum (p value=0.244) (Figure 1.20B).
Few differences in mucins in CF subjects were observed during periods of exacerbation. Neither absolute concentrations of MUC5B (976.4 ± 103.3 picomol/mL), MUC5AC (267.8 ± 28.19 picomol/mL) (Figure 1.21A) nor the ratio of
MUC5AC to MUC5B (0.37 ± 0.06) (Figure 1.21B) were different in sputum from exacerbating (n=24) vs. stable CF (n=17) subjects. Similar findings were observed
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when the CF cohort was separated based upon FEV1% predicted quartiles for
MUC5B (881.7 ± 155.7 vs. 909.3 ± 104.3 vs. 1212 ± 282.8 pmol/mL), MUC5AC
(255.2 ± 38.63 vs. 225.7 ± 26.02 vs. 429.9 ±136.9 pmol/mL) and the ratio between them (0.3675 ± 0.07 vs. 0.3565 ± 0.07 vs. 0.4976 ± 0.19) (Figure 1.21C).
Macromolecular characterization of purified gel-forming mucins in CF vs. normal
The macromolecular properties of the gel-forming mucins purified from healthy controls and CF sputum: Two dimensional isopycnic purification followed by
SEC-MALS was performed to estimate the molecular weight and radius of the gel- forming mucins within control and CF sputum. The mean (±SE) molecular weights of mucins in stable (n= 15) CF vs. healthy control sputum were similar (n=10) (1.97x107
± 2.18x106 vs. 2.65x107 ± 2.88x106 g/mol) (Figure 1.22A). The radius of gyration of the mucins isolated from stable CF sputum (182.8 ± 7.07 vs. 195.0 ± 8.93 nm) was also not different than control samples (Figure 1.22B). As with the concentration measurements, molecular weight did not differentiate CF sputum based on patient
7 6 reported disease status (exacerbation, n=15) (1.97x10 ± 1.84x10 g/mol) or FEV1 % predicted quartile (1.87x107 ± 3.2x106 vs. 1.87x107 ± 1.7x106 vs. 2.19x107 ± 3.1x106 g/mol) (Figure 1.22C). These findings were consistent with measurements of radius of gyration, which for exacerbation CF sputum (n=24) were 190.0 ± 7.91 nm and within each FEV1 % predicted quantiles were 185.3 ± 10.53 vs. 185.4 ± 9.53 vs.
204.6 ± 21.23 nm (Figure 1.22C).
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Quantity, coverage, and localization of MUC5B and MUC5AC Semi-tryptic peptides in mucins from CF sputum and control sputum
Peptides identified for LC-MS/MS analysis are typically fully tryptic having been cleaved at both the N and C terminal by trypsin as part of the sample preparation process. If other enzymes are present and have cleaved a protein then one terminal may be tryptic while the other is not. These Semi-tryptic peptides reflect the native proteolytic environment. To elucidate the effects of the highly proteolytic
CF lung milieu on the mucin protein backbone a tryptic/Semi-tryptic mapping analysis was performed. This technique permits the detection of previously proteolyzed fragments. The ratio of Semi-tryptic peptides to full peptides between
CF (n=41) and health control (n=10) sputum was significantly greater for both
MUC5B (1.13 ± 0.06 vs. 0.51 ± 0.09) and MUC5AC (0.71 ± 0.07 vs. 0.07 ± 0.02)
(Figure 1.23A). The increased ratio of Semi/full tryptic peptides corresponded to significant increase in coverage of MUC5B (10.85% ± 0.54 vs. 5.19% ± 0.73) and
MUC5AC (6.21% ± 0.56 vs. 0.56% ± 0.23) in stable CF vs. healthy controls (Figure
1.23B). There were no significant differences between stable and exacerbation CF sputum based on the Semi-tryptic/full tryptic peptide ratio nor the Semi-tryptic peptide coverage for MUC5B and MUC5AC (data not shown).
To further understand the differences in mucin backbone proteolysis between
CF and healthy controls, a non-metric multidimensional scaling analysis of the unique Semi-tryptic peptides and the domains to which each peptide was mapped was performed. This analysis revealed a significant segregation of the control and
CF groups for both MUC5B (Figure 1.24A&C) and MUC5AC (Figure 1.24B&D). A visual representation of the Semi-tryptic peptide localization along the protein
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backbone for control and CF MUC5B and MUC5AC highlights differences in domains between normal and CF peptide coverage (Supplementary Figure 1.29).
This result corresponds to significant differences in the percent of Semi-tryptic peptides localized to specific mucin domains per sample for MUC5B (A) and
MUC5AC (D) in the control and CF group (Figure 1.25). In general, there is a greater percent of Semi-tryptic peptides localized to the vWFD domains in the control sputum as compared to CF. In contrast, CF shows a greater percent of peptides within the TIL, Cys-Rich, interdomain and Ser/Thr rich regions. This finding is further demonstrated in the pie charts showing overall peptide localization based on a general domain designations for MUC5B (Figure 1.25B-C) and MUC5AC (Figure
1.25E-F). Thus the CF gel-forming mucins segregate from the normal based on the number Semi-tryptic peptide and their domain localization.
Sputum mucin concentrations and the CF sputum microbiome
To identify the microbial communities within the CF sputum, sequencing of the bacterial 16S rRNA was performed and identified genera classified into the categories of oral flora, pathogens, or other (Supplementary Figure 1.31).
Stratification of the CF sputum samples was based on the following properties: 1) low pathogen/high oral flora; 2) high pathogen/low oral flora; and 3) those with approximately equal proportions of each group. Large but not significant differences were found between pathogen abundance and mucin measurements. Specifically, there were positive trends between pathogen abundance and total mucin (Figure
1.26A) (Pearson r=0.42) and (Figure 1.26B) the MUC5AC/MUC5B ratio (Pearson r=0.44), which was mirrored in the negative correlations found between pathogen
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abundance and MUC5B concentration (Pearson r=-0.33) and the positive correlation between pathogen abundance and MUC5AC concentration (Pearson r=0.32)
(Supplementary Figure 1.30A). To test for effects of salivary contamination, a comparison between MUC5B concentration and salivary amylase (Supplementary
Figure 1.30B) was performed and no significant relationship was found (Pearson r=
0.008, ns).
Glycomic analysis of gel forming mucins in CF vs. normal
Glycan profiles of control and CF gel-forming mucins and correlation to microbiome: Purified mucins were subjected to glycomic analysis to analyze the glycans structures of CF vs. control gel-forming mucins. Within the CF cohort, glycomic data were compared to the microbiome based on stratification by pathogen abundance. CF (n=10) gel-forming mucins exhibited significantly more sialylated O- glycans (14.65 ± 4.05 vs. 3.32 ± 0.67 pmol/ug mucin) and less sulfated O- glycans
(values) than the purified healthy control (n=7) gel-forming mucins (Figure 1.27A).
Significantly altered specific glycan cores and sialylated glycans are represented in
Supplementary Chapter 2 Figure 6 panels A-C. Among the CF samples, the sum of sialylated O-glycans normalized to mucin concentration as measured by SEC-MALS significantly correlated with the % abundance of pathogens as determined in microbiome analysis (Spearman r=0.82, p value=0.001) (Figure 1.27B). Other CF mucin sialylated glycan cores with significant correlations to the microbiome are listed in Chapter 1Table 6.
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Discussion
Defective mucus clearance with mucus stasis subsequent inflammation and infection are hallmarks of CF lung disease. Mucus layer hyperconcentration via osmotic interactions with PCL has been shown to be critical in initiating mucus stasis. However, recent data suggest MUC5AC and MUC5B have different properties and may differentially contribute another component to mucus status.
Neither the contribution of the individual MUC5B and MUC5AC gel-forming mucins nor how their macromolecular properties are modified by the inflammatory and infectious milieu of the CF lung in the context of mucus transport vs. stasis are known.
To study these processes, a comprehensive sputum characterization was performed using sputum from CF vs. normal healthy non-smoking subjects. Because of the difficulty in quantifying and characterizing mucins from an in vivo proteolytic environment [40, 104, 109], physical and proteomic rather than immunologic techniques were used to measure total mucin and individual mucin concentrations.
Henderson et al, using biophysical mucin measurements, were the first to show that total mucin concentrations were increased 3-4 fold in CF vs. control sputum [40].
Our current study, using a larger cohort confirmed that observation by again showing that total mucin concentrations were increased 3-4 fold in CF vs. control sputum.
Our studies extended these findings in several directions. First, total mucin concentrations were positively correlated with both age of the patient and sputum
HNE activity. Second, despite the heavy proteolytic burden within the CF airways, the molecular weight of mucins isolated from CF was not significantly different from
80
healthy subjects, and did not vary by disease status, FEV1 % predicted, or microbiome composition (data not shown). Third, the detailed Semi-tryptic peptide analysis of the MUC5B and MUC5AC protein backbone identified unique cleavage patterns between CF and normal and showed that both the amount and location of the cleavage sites within specific mucins domains were significantly different. Fourth, important differences in the glycan composition of CF vs. control gel-forming mucins were identified and significantly more sialylation than sulfation on the gel-forming mucins purified was observed in mucins from the CF sputum compared to controls.
To date, there are no published data describing the absolute concentrations of the individual gel-forming mucins, MUC5B and MUC5AC, to the increased total mucin concentration. Our data indicates that MUC5B and MUC5AC increased dramatically, ~ 9- and 30-fold, respectively, in CF vs. control sputum, and the
MUC5AC/MUC5B ratio rose to ~ 0.4 in CF vs. 0.1 in controls. Neither MUC5AC,
MUC5B, nor MUC5AC/MUC5B ratios were related to disease status (stable or exacerbation) or severity as measured by FEV1% predicted, although they did strongly correlate with age. When compared to other mucoobstructive diseases such as COPD, the concentrations of the MUC5B and MUC5AC in CF are significantly elevated (3.2 and 2.6 fold, respectively) though interestingly the ratio of MUC5AC to
MUC5B in CF and COPD sputum are similar (0.4 ± 0.08 vs. 0.5 ± 0.1) [84]. These data suggest that the same pathobiological processes may be operating in both mucoobstructive diseases but that the hypersecretion/hyperconcentration in CF is more pronounced, which appears to mirror the relative incidences of bronchiectasis and Pseudomonas in the two diseases.
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The data from the physical characterization of the gel-forming mucins purified from CF vs. control sputum revealed that the molecular weight and radius were not different from controls. To understand in more detail how the proteolytic CF airway environment might affect mucin structure but not mass, a mass spectrometry-based proteomics analysis of the coverage metric of MUC5AC and MUC5B mucins was performed. Analyses of Semi-tryptic peptides provided insight into the in vivo proteolytic cleavage of mucins and thus was used to identify sites of proteolysis in the gel-forming mucins. More Semi-tryptic peptides were detected in CF vs. control sputum mucins as analyzed by label free LC-MS/MS. Not only were the quantities of peptides greater in CF, but the localization of the Semi-tryptic peptides created a unique signature that discriminated CF vs. normal for both MUC5B and MUC5AC.
While both CF and control mucins yielded peptides that localized to the main non- glycosylated protein domains, CF sputum mucins had a significantly greater percentage of the Semi-tryptic peptides map to the interdomain regions and highly glycosylated domains. These findings suggest that glycosidase enzymes were present in the CF environment that removed the protective glycans in the variable number tandem region (VNTR) regions and allowed proteases to cleave the underlying mucin protein backbone. These inferences are consistent with our glycomic data (see below). It is interesting that while numerous potential non-tryptic cleavage sites were identified in CF mucins, the mucins remained relatively intact based upon molecular weight and radius measurements. Henderson et al suggested that the intactness of CF mucins in vivo likely reflected the presence of intra-mucin disulfide bonds that maintained the structure of the mucin polymer despite sections
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of the backbone being cleaved/removed. Such a structure may make CF mucins a favorable target for S-S bond reducing agents as therapeutics.
The glycomic analysis of the purified mucins suggested that mucins isolated from CF sputum had significantly altered glycan compositions. The CF mucins glycan side chains were more sialylated and less sulfated than control mucin glycans. Within the CF study population, the quantity of sialylated glycan strongly correlated with the microbiome composition. Specifically, the sputum with the highest abundance of oral flora/lowest abundance of pathogens had the lowest quantity of sialylated glycans decorating the mucin (0.58 pmol/ug mucin), whereas the sputum with the lowest abundance of oral flora but the highest abundance of pathogens had the highest amount of sialylated glycans per ug of purified mucin
(37.9 pmol/ug mucin). Specifically, there were significant increases with lower anaerobe/higher pathogen abundance in the sialylated glycans, including sialylated cores sialyl-Tn, sialyl-core 1 and sialyl-core2. Although there was an overall decrease in sulfation among the CF mucins, one of the sulfated glycans, sulfo sialo lewis X, increased significantly. This latter finding, has been reported by other groups and suggests a connection between mucin, the microbiome, and immune modulation [111, 112]. How these glycan changes arise is not known. Two possible explanations are that the bacteria glycosidases remove and utilize specific glycans or also that the epithelium responds to the presence of specific bacteria and alters the glycosylation intracellularly.
In addition to changes in the regulation of intra-cellular glycotransferases within the CF airway environment, there have been numerous glycan studies of CF
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airway secretions using various methodologies that have produced conflicting results
[111, 113-116]. To our knowledge this is the first study that has performed glycomics on purified gel-forming mucins from CF and control sputum.
The correlations of mucins with the microbiome composition provide an interesting/unknown connection between mucins and the different bacterial communities colonizing the CF airways. The paucity of sialylated glycans in the oral flora-dominated samples supports the hypothesis of Flynn et al that oral flora contain the enzymes necessary to degrade mucin to: 1) provide an energy source for themselves and 2) provide metabolic products that can be used as energy sources for other genera less adept at metabolizing mucins [74]. Thus the lack of sialylated glycans decorating the gel-forming mucins in sputum with a predominance of oral flora may provide in vivo evidence that these microbes do in fact cleave and utilize the sugars decorating the mucins backbone Robinson et al [117] suggested that clinical pseudomonas strains removed the sulfur from mucins though likely did so for non-growth related purposes. This notion is consistent with our data showing that the greatest amount of non-sulfated O-glycans were found in the sputum samples with the highest abundance of classic CF pathogens, including Pseudomonas.
Lastly, previously reported alterations in mucin glycosylation have been hypothesized to reflect defective acidification of intracellular organelles secondary to
CFTR mutations [118, 119]. The variability of glycosylation within our CF cohort that strongly correlated with the microbiome suggests that extracellular environmental factors may dominate the observed glycomics findings
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When comparing the in vivo sputum data to the in vitro Ps.a CF cell culture model described previously in Chapter 1A, there were several notable differences in the experimental variables tested and the results. The in vivo CF sputum samples are derived from an environment heavy with both bacterial and host immune cell proteases whereas the Ps.a. CF cell culture model contain primarily bacterial proteases. Interestingly, by whole mount IHC, both gel forming mucins increase in the Ps.a. model, though a majority of the MUC5AC remains adherent to the surface and not significantly increased in the apical secretions. In contrast the in vivo CF sputum showed a significant increase in the MUC5AC concentration (30 fold), suggesting that though MUC5AC creates an adherent mucus layer it can be cleared via spontaneous or induced cough clearance in vivo. The MUC5B Semi-tryptic peptide data after Ps.a. challenge reveals that while there is slightly more, though not significant, mucin proteolysis based on the increase in the ratio of Semi- tryptic/full tryptic peptides, the domain localization is similar between the treatment and control (TSB). This is consistent with the Ps.a. standard timecourse incubation which showed minimal degradation of the gel forming mucins. These finding when compared to the semi-tryptic in vivo CF sputum data highlights the combinatorial effect of the host immune proteases (i.e. elastase) and bacterial proteases from both oral flora and pathogenic sources that are present and active in the in-vivo environment. Another interesting finding apparent when comparing the in vitro and in vivo results was the ability of the Prevotella culture filtrate to degrade the MUC5AC standard compared with the negative correlation between the oral flora dominated sputum and absolute MUC5AC protein concentration. Though it is unknown whether
85
the change in the gel forming mucins is a direct effect of the bacteria or the epithelial response to specific bacteria, this data supports that Prevotella may be capable of utilizing MUC5AC which could account for the lower concentration of MUC5AC in the CF sputum samples with high Prevotella abundance.
Numerous therapies and treatments are used clinically to facilitate the clearance of mucus from the CF lung by directly targeting the mucus itself, e.g., hypertonic saline, or more indirectly by targeting the inflammatory and infectious stimuli producing mucus hypersecretion, e.g., corticosteroids and antibiotics, respectively. Despite their use in this CF population, no differences in total mucin concentration as a function of treatment were observed. It is possible that the results of these therapies may be more evident at an earlier timepoint in disease progression. Thus future work would involve expanding this study to include samples obtained from individuals with different degrees of disease severity in order to determine how the concentrations and ratio of the gel-forming mucins change with disease progression and in response to treatment.
In summary, our study expands on the contributions that gel-forming mucin concentrations make to CF airway disease pathogenesis. Our data confirmed that total mucin concentrations were 3-4-fold higher in CF than normal sputum and showed that this increase reflected increases in both MUC5B and MUC5AC. Like
COPD, MUC5AC increased disproportionally more than MUC5B, but given the lower basal levels of MUC5AC vs. MUC5B, MUC5B dominates in CF airways. Both
MUC5AC and MUC5B in CF are subject to enzymatic cleavage by a combination of glycosidases (in part from anaerobes) and proteases (in part from neutrophils).
86
These enzymatic modifications alter little the molecular weight of mucins but provide both sugar substrates for bacteria and render CF mucins susceptible to large molecular weight reductions by mucolytics targeting S-S bonds.
Thus far the focus of these mucin studies have been in the context of CF lung disease, which is heavily laden with infectious and inflammatory stimuli. Our studies have analyzed the effect that specific bacteria, found in the CF lung airways, have upon the gel forming mucins using in vitro models in combination with human sputum data, which more accurately reflects the dynamic and complex in vivo environment that the gel forming mucins are exposed to. CF is not the only airway disease characterized by pathologic mucus and thus the following chapter will study mucins in the asthmatic lung environment, which though characterized by mucus obstruction and inflammation, lacks the infectious stimuli which is a hallmark of CF.
In analyzing these two respiratory diseases with different etiologies, we hope to gain a better understanding of how the gel forming mucins and the proteins they interact with are altered and contribute to aberrant mucus properties.
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Chapter 1 Part B Figures
Chapter 1. Table 5. Study Cohort Characteristics for non-disease controls and CF patient populations Non-Disease Controls Cystic Fibrosis Subjects 29 43 Age, mean (SD) 49.4 (16.7) 27 (10) Gender, N (%) Male 10 (34.5%) 23(52.3%) Female 19 (65.5%) 21(47.7%) BMI, mean (SD) 27.1 (5.2) 21.4( 3.0) FEV1%, predicted, mean (SD) 96.3 (18.4) 52.2( 19.9) Disease Status at time of collection, N (%) Stable N/A 18 (42) Exacerbation N/A 25 (58) Δ508 status, N (%) Homozygous N/A 25 (57) Heterozygous N/A 14(32) Other N/A 5(11)
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Figure 1.19. Total mucin concentration increases significantly in CF. SEC-MALS of sputum treated with DNase separated by (a) disease status and within the CF population by (b-c) FEV1% predicted (normal, n=10; CF stable, n=14; CF exacerbation, n=24. FEV1% predicted categories: 1: values ≤36% (n=10), 2: values >36% but ≤65% (n=17), 3: values>65% (n=10). CF total mucin concentration is significantly higher than normal but does not discriminate between stable and exacerbation disease status as annotated at time of collection or based on FEV1% category. No significant correlation was observed between total mucin and FEV1% predicted (Pearson r=0.124, n=37). Data displayed as boxplots.
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Figure 1.20. Within CF cohort, total mucin concentration and the ratio of MUC5AC to MUC5B varies with human neutrophil elastase activity and age. A) Total mucin concentration as a function of HNE (n=23) (Pearson r=0.552, p value=0.006). B) The trend between MUC5AC/MUC5B ratio and HNE (n=23) (Pearson r=0.247, p value=0.244). C) Correlation between total mucin concentration and age (n=40) (Pearson r=0.430, p value =0.006). D) Correlation of the ratio of MUC5AC to MUC5B and age (n=43) (Pearson r=0.546, p value =0.00015).
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Figure 1.21. Absolute MUC5B and MUC5AC concentrations, and the MUC5AC/MUC5B ratio are significantly elevated in CF. LC-MS/MS using isotope labeled peptides of (a) MUC5B and MUC5AC and (b) the ratio of MUC5AC/MUC5B separated by disease status and within the CF population by (c) FEV1% predicted (healthy, n=19; CF stable, n=17; CF exacerbation, n=24. FEV1% predicted categories: 1: values ≤36% (n=10), 2: values >36% but ≤65% (n=20), 3: values>65% (n=10). Though significantly higher than healthy controls, the concentrations of MUC5B, MUC5AC, and MUC5AC/MUC5B are not different based on disease status or FEV1% among the CF cohort. Data displayed as boxplots.
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Figure 1.22. Macromolecular properties of the purified gel forming mucins from sputum are not significantly different between healthy and CF. SEC- MALS measurement of (a) molecular weight and (b) radius of gyration of mucins purified by isopycnic centrifugation from sputum (healthy, n=10; CF stable, n=15; CF exacerbation, n=24. FEV1% predicted categories: 1: values ≤36% (n=10), 2: values >36% but ≤65% (n=17), 3: values>65% (n=10). Panel (c) shows no significant differences in molecular weight and radius of gyration within the CF cohort based on FEV1% predicted category.
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Figure 1.23. MUC5B and MUC5AC from CF sputum have significantly higher ratio of Semi-tryptic to full tryptic peptides. Label free LC-MS/MS analysis allowing (a) identification of unique Semi-tryptic and fully tryptic peptides per sputum sample for MUC5B and MUC5AC and (b) the percent of the mucin protein backbone covered by Semi-tryptic peptides (healthy, n=10; CF, n=41) The ratio of Semi-tryptic to full tryptic peptide and the coverage by Semi-tryptic peptides is significantly higher in CF samples.
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A B
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Figure 1.24. MUC5B and MUC5AC from CF and normal sputa show distinct signatures based on Semi-tryptic peptides and the mucin domains they map to. Non-metric multidimensional scaling analysis of unique (a) MUC5B and (b) MUC5AC Semi-tryptic peptide sequences per sample from healthy and CF sputa and of their domain signature generated by mapping each Semi-tryptic peptide to the (c) MUC5B or (d) MUC5AC backbone. There is significant separation between the CF and normal samples based on PERMANOVA (p<001) (MUC5B: normal, n=11; CF, n=44; MUC5AC: normal, n=7; CF, n=44.) Samples were excluded from analysis if no Semi- tryptic peptides were identified.
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Figure 1.25. Specific domain localization of Semi-tryptic peptides differs between CF and normal. Percent of unique Semi-tryptic peptides per sample mapped to individual Uniprot annotated protein domains for (a) MUC5B and (d) MUC5AC with pie charts showing localization based on region type for (b) normal and (c) CF for MUC5B and (d) normal and (e) CF for MUC5AC.
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Figure 1.26. Relationship between microbiome and mucin concentrations. (A) Total mucin concentration as determined by SEC-MALS after vigorous DNase treatment compared to abundance of genera classified as either oral flora or pathogen. Absolute quantitative LS-MS/MS using labeled peptides was used to determine MUC5AC and MUC5B concentrations and the ratio between them (B.) which was compared to microbiome data. Each pair of blue and red bars represents one CF sputum sample (n=17), which were arranged by increasing pathogen abundance from left to right. Total mucin vs. pathogen abundance, Pearson r= 0.42; MUC5AC/MUC5B ratio vs. pathogen abundance, Pearson r=0.44). Correlation values were not statistically significant.
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80 30 70 25 60 20 50 15 40 10 Abundance % 30 20 5
10 0 0 -5 CF Sputa
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Figure 1.27. Purified gel forming mucins from control and CF sputum have significantly different glycan profiles. (A) Negative and positive ion MS analysis of sulfated and non-sulfated (sialylated) glycans of purified gel forming mucins isolated via isopycnic centrifugation from healthy and CF sputa (healthy, n=7; CF, n=10). Significant positive correlation (b) between pathogen abundance and cumulative non-sulfated glycan sum normalized per mg mucin (Spearman r =0.82).
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Chapter 1 Table 6. Significant Spearman r correlations between specific glycan core structures and microbiome composition based on abundance of genera classified as pathogen or oral flora Glycan Cores Glycan Pathogen Oral Flora 2 0.721212 -0.57576 Core 1 4 0.709091 -0.6 sTn 17 0.806061 -0.81818 18 0.745455 -0.75758 Sialo-Core 1 19 0.793939 -0.7697 20 0.709091 -0.67273 21 0.781818 -0.68485
22 0.757576 -0.74545
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24 0.733333 -0.66061 Sialo-Core 2 25 0.684848 -0.58788 26 0.733333 -0.61212 Sialo-Core 4 39 0.769697 -0.66061 Glycan Sum 0.818182 -0.70909
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Supplementary Figure 1.28. Total mucin concentration does not significantly change with medication usage. Changes in total mucin concentration as a function of whether or not the patient was using (A) hypertonic saline (No; n=21, Yes; n=18), (B) inhaled Corticosteroids (No; n=21, Yes; n=18), and with (C) specific antibiotics (None; n=5, Tobramycin; n=20, Colistin; n=15).
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PatternLab forProteomics software ( Semi normal,n=11; CF, n=44; MUC5AC:normal, n=7; n=44.)CF, Samples were excluded analysis from noif boxesindicate Uniprotannotated protein domains MUC5Bfrom andMUC5AC, respectively. (MUC5B: x arranged(bottom to top) basedlocalizationon to MUC5BMUC5AC or proteinbackbone represented on the peptidesamong all healthy andsputaCF MUC5B for (A forming mucinsreveal differencesbetween normal andCF sputa Supplementary Figure Visual1.29. representations of the
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Figure 1.31. Abundance of bacterial genera based on sputum microbiome sequence analysis shows stratification based on oral flora or classic pathogen classification. 16s rRNA sequencing identified microbiome composition of subset of CF sputum samples (n=20). Each column represents one sputum sample. Cool toned bars (blue/green) indicate the genera is commonly identified as part of the oral flora community and warm (red/orange) toned bars indicate microbe is typically identified as a CF lung pathogen.
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Figure 1.32. Relationship between microbiome and mucin concentrations. (A) Total mucin concentration as determined by SEC- MALS and vigorous DNase treatment. Absolute quantitative LS-MS/MS using labeled peptides was used to determine MUC5AC and MUC5B concentrations, which was compared to microbiome data (b) as was the ratio between them (c). There were no significant correlations between the mucin parameters measured and microbiome. Pearson r value: total mucin vs. pathogen abundance, 0.42; Absolute MUC5B vs. pathogen abundance, - 0.33, Absolute MUC5AC vs. pathogen abundance, 0.32; Ratio D.
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Figure 1.33. Significantly different non-sulfated glycans derived from gel forming mucins purified from healthy and CF sputum. Positive ion MS detection of non-sulfated glycans represented in panels A-C. Specific absolute glycan measurements (pmol/ug mucin) were normalized to total amount of glycan present per sample to give relative abundance of each glycan per sample.
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CHAPTER 2: COMPREHENSIVE CHARACTERIZATION OF MUCINS WITHIN THE TH2 ASTHMATIC AIRWAYS Introduction: Asthma
Asthma is a heterogeneous chronic inflammatory respiratory disease. It is characterized by airflow limitations due to a combination of factors including: airway hyper-responsiveness/smooth muscle contraction and chronic inflammation leading to airway remodeling which includes thickening of the underlying basement membrane and goblet cell hyperplasia accompanied by mucus hypersecretion.
Asthma encompasses a wide range of phenotypes from mild to fatal, each of which have unique pathogenesis and risk factors. Recent work characterizing the different pathophysiological mechanisms behind different phenotypes has led to their classification as endotypes. Numerous genetic and environmental factors such as pollutants, allergens and smoking have been implicated in the development of asthma [120, 121]. Additionally specific loci have been identified that increase risk of asthma including HLA genes, receptors for IL-1 and 18, thymic stromal lymphopoietin, and most significantly ORMDL sphingolipid biosynthesis regulator 3
[122].
Distinguishing between different asthma endotypes has proven to be a complex task due to significant variation in cellular and molecular signaling [123] though the general classifications of TH2 cytokine high/eosinophilic or TH2 cytokine low/ neutrophilic are commonly used [124]. Several different signaling pathways have been shown to play a role in asthma especially in regards to the different
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endotypes. For simplicity this overview will focus on the allergic asthmatic airway response and will highlight specific characteristic of the most severe asthma endotype as primary cells from these patients will be used in the current study.
The allergic asthmatic airway response occurs in different phases; sensitization, early and late response. The sensitization occurs at first exposure with the allergen and leads to the production of IgE, which upon repeated exposure interacts/crosslink with the mast cell surface antibodies leading to release of prostaglandins, leukotriene, and histamines causing bronchial constriction [125]. The later response is characterized by the influx of the immune cells including, eosinophils, basophils, neutrophils, and also helper T cells [126]. Eosinophilia is a characteristic finding in TH2 mediated asthma and has been linked to atopy in patients [127]. Studies have also shown that eosinophil cell count and their secreted products have been correlated with worsened disease and lung function [128].
Interestingly high levels of neutrophils have been reported in sputum from asthmatic patients experiencing an acute exacerbation and also in sputum from the most severe asthma and some reports have suggested that neutrophil abundance may be a better predictor of lung function [129-131]. The function and role of this high neutrophilic infiltrate is not yet understood.
Helper T cells play an important role in asthma pathogenesis through the generation of TH2 cytokines (IL-3, 4, 5, 13) and in fact many current therapies target those specific cytokine products to help reduce the severity of and/or prevent
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asthma exacerbation and have shown efficacy when given to patients with poorly controlled asthma [132-134]. It is a complex signaling cascade of cytokines in vivo that creates the asthmatic response but recent studies have shown that IL-13 is a central mediator in asthma and plays a role both in the survival and recruitment of inflammatory cells and also can independently induce many of the characteristic features of asthma including: recruitment of inflammatory cells, increased IgE production, thickening of the basement membrane, goblet cell hyperplasia and mucus hypersecretion, and smooth muscle hyperactivity [135-137]. IL-13 has been shown to bind to the IL-13Rα1 receptor that forms a heterodimer with the IL-4Rα1 receptor [138, 139]. This leads to binding of specific Janus Activated Kinases to the receptors, JAK1 binds to IL-4R and JAK2 or TYK2 binds to IL-13R. This promotes the docking of STAT6 which is phosphorylated, dimerizes, and then translocate to the nucleus altering the transcription of IL-13 dependent genes [139].
In general the airflow alterations during an asthmatic exacerbation are triggered by environment or viral insults but are reversible [140]. In contrast the more severe endotypes frequently presents with irreversible airflow restriction likely due to extensive airway remodeling coupled with airway hyper-responsiveness [141]. The importance of mucus is evident in post mortem studies of lungs from fatal asthma and non-fatal asthma individuals where in addition to thickened basement membranes and smooth muscle shortening, there was significant enlargement of submucosal glands and increased mucus plugging of airways [142]. A second study concluded that the luminal occlusion by mucus and cells, which was significantly higher than controls, was a major contributing factor to fatal asthma [143]. Another
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distinguishing feature of severe asthma is the resistance to inhaled corticosteroids and short/long term β2 agonist which are typically used and effective at reducing inflammation and relaxing the airway smooth muscle surrounding the airways, respectively [141].
Additionally several studies have compared epithelial cells derived from asthmatic patients to non-asthmatics in regards to wound healing/repair, proliferation, inflammation, and differentiation [144-146]. Several methods have been established to re-create the asthmatic lung environment in vitro including the use of
IL-13 (10ng/mL) applied to the basolateral compartment of HBE cultures grown at air liquid interface and also mouse model challenged with intranasal IL-13 or sensitized and challenged with ovalbumin (OVA) [147-149]. In regards to mucin, studies of the mouse asthma model have shown that MUC5AC mRNA was increased in both IL-13 and OVA mouse model lung tissue and accompanied goblet cell hyperplasia [150].
Cell culture models of asthma have shown an increase in MUC5AC staining through immunohistochemistry that remains tethered to the surface when washed whereas the MUC5B was washable. [87] Additionally mucin concentration measured via
ELISA from sputum and endotracheal biopsies of asthma patients has been shown to increase in mucin as did the MUC5AC mRNA, though MUC5B mRNA decreased
[151]. Lastly a recent study measuring MUC5AC and MUC5B concentration by western blot in sputum from children with acute and stable asthma compared to healthy controls reported a disproportionate increase in MUCAC to MUC5B and that
MUC5B was produced as a low charge glycoform in acute asthmatics [105].
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Methods
Cell Culture
Primary human bronchial epithelial cells passage 1 or 2 from non-asthmatic and asthmatic donors were grown at air liquid interface and maintained according to previously established protocol [75]. Basal media was changed on alternating days and the apical surface washed with 37°C PBS 2-3 times during the week.
Challenges were performed on cultures once fully differentiated (21-28 days after confluence) and was verified by the presence of ciliation and mucus production. For the following experiments, treatment and control inserts were used from the same donor allowing for a paired statistical analysis.
Asthma model:
To stimulate an asthmatic lung environment polarized fully differentiated cultures from non-asthmatic and asthmatic donors were grown at air liquid interface
(ALI) and challenged daily for 20 days with 10 ng/mL IL-13 added to the basal media compartment (human recombinant IL-13, Peprotech). Prior to challenging, the lyophilized IL-13 powder was re-suspended in 0.05% BSA according to manufactures instructions and stored at -30°C until needed. The apical surfaces of the cultures were washed prior to the start of treatment after five days of mucus accumulation and every 5 days throughout the IL-13 challenge. The apical surface wash was performed with pre-warmed 37°C PBS which was allowed to incubate on the surface for 20 minutes twice before collection. Paired cultures from the same donor were treated in a similar fashion with the exception of 0.05% BSA was added
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to the basal media in the same volume as the IL-13. After the 20 day IL-13 challenge, the cultures were maintained during a 20-day recovery period with apical washings collected every 4-5 days. Two large 30mm Transwell-Clear inserts (one treatment and one control) per code were set aside for collection of stored intracellular mucins after the 20 days of IL-13 treatment or control. This challenge was performed on both non-disease control cells and also cells from individuals who reportedly died from asthma.
Mucin Isolation and Static Light Scattering
Isopycnic density gradient centrifugation was performed to isolate the gel forming mucins using 4mL of pooled apical secretions in 4M GuHCl at a starting density of 1.45g/ml CsCl in 4M GuHCl spun at 50,000 rpm in fixed angle rotor () for
60-70 hours at 14°C [78]. A slot blot with vacuum filtration was performed on the resulting twelve 750μL fractions, which were then probed with polyclonal MUC5AC antibody and monoclonal MUC16 (CA125) antibody to identify the peak with the highest concentration of MUC5AC and minimal contamination by membrane bound mucins. These fractions were pooled and subjected to CL2B size exclusion chromatography (2 x5mL) coupled with matrix assisted laser light scattering (Dawn
Heleos II, Wyatt Technology) and refractometry (Optilab T-rEx, Wyatt Technology)
(SEC-MALS/dRi) at a flow rate of 0.5mL/min to determine concentration and macromolecular properties’ of the gel forming mucins, as described previously [40].
Files were analyzed with ASTRA software (Version 7.1.2).
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Isolation and Analysis of Stored Gel Forming Mucins
After completion of the 20 day challenge, one large 30mm insert from treatment and control for each non-disease donor was used for post nuclear supernatant isolation (PNS). After sequential gentle washings with prewarmed PBS minimizing mechanical stimulation, freshly prepared homogenization buffer (20mM
HEPES, 130mM glutamic acid, 0.1mM CaCl, 3mM EGTA,10nM N-Ethylmaleimide,
Turbo DNase reaction buffer/DNAse (according to manufacturer’s instructions, ambion), and cOmplete mini protease inhibitor tablets (according to manufacturer’s instructions , Roche), pH 7.2) was added to the apical surface of the cell cultures.
Cells were removed/scraped from the insert and homogenized with 50 strokes of dounce homogenizer, while on ice. An equal volume of 8M GuHCl was added to the resulting homogenate to achieve a final concentration of 4M and then centrifuged at
200g x10 minutes at 4°C to pellet any remaining cell debris. The supernatant was removed and subjected to isopycnic density gradient centrifugation at a starting density of 1.45g/ml CsCl in 4M GuHCl for 60-70 hours at 50,000 rpm with a fixed angle rotor [78]. Eighteen 500μL fractions were taken per gradient and analyzed for
MUC5AC and MUC16 reactively following slot blot with vacuum transfer. The
MUC5AC rich fractions were further analyzed by SEC-MALs/dRi as described above.
Exosome Isolation:
Exosomes were isolated from equal volumes of apical secretions by differential centrifugation. Briefly the raw secretions were spun down at 3000G x 20 minutes at 4°C using a swing out bucket rotor (SW40 Ti) after which the supernatant
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was kept and centrifuged at 10,000 rpm x 110 minutes at 14°C. The supernatant was removed and centrifuged for a 3rd time at 19,000 rpm x1.5 hours at 14°C. After this step, the supernatant was discarded and the remaining pellet was washed with
10 mL of PBS prior to the final centrifugation at 25,000 rpm x 60 minutes at 14°C.
The supernatant was removed and the remained pellet resuspended in 100mL of
PBS. The freshly isolated exosomes were diluted 1:500-1:1000 in 0.22uM filtered
PBS and analyzed by nanoparticle tracking analysis as described previously [33].
Based on the resulting particle concentrations measurements, volumes from each sample containing an equal number of particles were taken and submitted for miRNA sequencing using the HTG EdgeSeq platform, the details for which have been described previously [39].
Whole Mount Immunohistochemistry
After completion of day 5, 10, 20 of IL-13challenge, the apical surface was washed gently and thoroughly with 37°C PBS prior to fixation of Carnoy’s Solution
(60% Ethanol, 30% Chloroform, and 10% glacial Acetic Acid) applied to both basal and apical surfaces at room temperature for 30 minutes. Permeablilizeation was performed with 0.2% Triton X in Tris Buffered Saline (TBS) for 30 minutes at RT and then the cultures were blocked overnight at 4°C with a solution of 1% BSA, 1% Fish
Gelatin, 0.1% Triton X and 5% normal donkey serum in 1 X TBS. Primary antibodies against MUC5B (1:500), MUC5AC (4ug/mL), and x-tubulin (3ug/mL) were prepared in blocking buffer and applied to apical and basal surfaces overnight at 4°C. After washing, cultures with blocking buffer diluted 1:10, secondary antibodies diluted
(1:1000) were added to the both culture surfaces and incubated overnight at 4°C
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protected from the light. The following day, the cultures were washed, counterstained with Hoechst according to manufacturer’s instructions. The membrane was excised from the plastic insert and mounted on slide with apical surface facing upward. This was allowed to dry overnight before sealing and imaging.
Agarose Gel Electrophoresis
Apical secretions collected every 5 days, dialyzed into 6M urea both reduced
(10mM DTT x 10 minutes at 95°C) and unreduced were loaded into a 0.7% (w/v) agarose gel using 6X bromophenol loading dye and subjected to electrophoresis until dye front was within ½ inch of lane end, using 1X Tris-acetate-EDTA (1xTAE) buffer with 1% SDS. To facilitate detection of the unreduced samples, the gel was incubated at room temp for 10 minutes in a solution of 10 mM DTT prior to transfer
[40, 79]. This was followed by a 60-minute vacuum transfer (50mbar) onto a nitrocellulose membrane while submerged in 4× saline-sodium citrate (4xSSC) buffer. The membrane was blocked with 1% milk and probed with monoclonal and polyclonal antibodies against MUC5AC and MUC5B [40, 80-82]. Secondary IR-dye conjugated antibodies against rabbit and mouse primary antibodies were applied and the resulting signal measured using Licor Odyssey Scanner and quantified via densitometric analysis using the software provided by manufacturer (Version
3.0.30). For each challenge and code, the intensity values were normalized to the highest value prior to statistical analysis to measure the change in mucin secretion and account for donor-to-donor variability.
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Mucin interactome isolation:
CL2B size exclusion chromatography was used to isolate the mucin rich fractions from apical secretions from control and IL-13 challenged non-disease HBE cultures. These fractions were subjected to proteomic analysis as detailed below to identify which other smaller globular proteins were interacting with mucin and thus present in the V0 region when they would otherwise be present in the included region based on their respective molecular weights. In order to maintain these interactions, 2 mL of apical secretions from days 15 and 20 of treatment and control were maintained in PBS until injected at a flow rate of 300uL/mL with collection of
1mL fractions. The fractions were probed for MUC5AC antibody reactivity after slot blot and vacuum transfer. The positive fractions were concentrated by vacuum centrifugation (Hetovac) and re-suspended in 4M GuHCl for mass spectrometry sample preparation as detailed below.
Mass Spectrometry
Equal volumes (450μL) of apical secretion collected at baseline and every 5 days during treatment and recovery, diluted 1:1 in 4M GuHCl, were prepared for
Liquid Chromatography Tandem Mass Spectrometry using a modified filter aided sample preparation (FASP) method [83]. Specifically, each sample solubilized in 4M
GuHCL was reduced with DTT using a final concentration of 20 mM for 1 hour at
65°C and then alkylated with 50 mM iodoacetamide for 1 hour at 25 °C protected from the light. The samples were centrifuged at 14,000g for 10 minutes and then washed twice with 4M GuHCl, followed by three additional washes with 50mM ammonium hydrogen carbonate (NH4HCO3). The 10 kDa filter unit was placed in a
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new collection tube and 0.5 ug modified proteomic grade trypsin (Sigma) was added.
The samples were incubated in a humidified chamber for 18 hours at 37°C. The peptides were centrifuged and eluted from the filter and then concentrated using vacuum centrifugation (Heto-vac). The peptides were then dissolved in 30 uL of
0.1% formic acid analyzed by liquid chromatography-tandem mass spectrometry
(data dependent analysis) using a Dionex ultimate 3000 RSLCnano system 6µl of samples were loaded in a trap column Acclaim PepMap 100, 100 µm x 2 cm, nanoViper C18 5 µm 100 Å, at 5 µL/min with aqueous solution containing 0.1 % (v/v) trifluoroacetic acid and 2 % acetonitrile, while the column used for peptides separation is a Acclaim PepMap RSLC, 75 µm x 15 cm, nanoViper C18 2 µm 100 Å) coupled to a hybrid quadrupole orbitrap mass spectrometer with a Nano spray source (Q-Exactive, Thermo Fisher, Bremen, Germany).
Proteins were identified by searching against the most current human database (Proteome Discoverer 1.4) and quantified using Scaffold, Version 4
(Proteome Software Inc.), using the total precursor intensity. A paired students T- test was used to compare the treatment and controls from each donor.
For MUC5AC and MUC5B absolute quantification, an internal standard was prepared by spiking 3 heavy labeled internal peptide standards from each protein achieving a final concentration of 100 fmol /µl. All raw files obtained from tSIM-DIA analyses of sputum digest samples were processed by Skyline (version v1.4). For each peptide the ratio between the corresponding endogenous and internal standard peak areas of each precursor (MS) and top 3 most intensity product ions (MS/MS)
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was calculated. Ratios from the three peptides were averaged and MUC5B and
MUC5AC concentrations were calculated with the following equation:
Protein concentration = [L/H × C × a/b * c/d]
Where L/H is the average area ratio between light and heavy peptides, C is the concentration of injected internal standard, a is the volume used to resuspend the peptides, b is the samples starting volume, c/d is the dilution factor for mixing sample and internal standard (10/8).
Rate Zonal Centrifugation
Rate zonal centrifugation was performed using 200μL of apical secretions diluted 1:1 with 8M GuHCl from day 20 of Il-13 treatment and control layered on top of a 12 mL 6-8M GuHCl gradient. The gradient and sample was spun at 40,000 rpm using a swing out bucket rotor (SW40 Ti) for 2.5 hours at 14°C [85]. The gradient was separated into twelve 1 mL fractions which were analyzed by slot blot with vacuum transfer and probed with antibodies against MUC5AC and MUC5B. The secondary IR-dye antibody intensity was quantified using the Licor Odyssey scanner and software. The antibody intensities of the gradient fractions from each donor were normalized to the highest value within that gradient and plotted according to fraction number with 1 being the uppermost fraction and 12 the bottom.
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Results Histological examination of IL-13 challenged and control cultures
Goblet cell hyperplasia was evident after the 20 day IL-13 challenge of non- disease HBE cultures in addition to significant morphological changes such as a cyst-like formations on the apical surface and invaginations of the thickened epithelium (Figure 2.1B-D). The goblet cells of both control (Figure 2.1A) and IL-13 treated cultures contain AB-PAS positive granules, though there is an increased number of goblet cells in the IL-13 treated cultures. The mucin layer was visualized with whole mount IHC after Carnoy fixation and though intracellular mucin was present after 5 days of IL-13 treatment there was a dramatic increase in the amount of MUC5AC and MUC5B coating the epithelium after 20 days of treatment (Figure
2.2).
IL-13 induced asthma cell culture models: antibody based MUC5B and MUC5AC concentration quantitation
The amount of gel forming mucin present in the apical secretions from non- disease HBE cultures and asthmatic HBE cultures during the IL-13 treatment was determined by agarose gel electrophoreses western blot. Both cell culture types showed a significant increase (94.7% in non-disease and 98.8% in asthmatic) in
MUC5AC staining by day 20, which become prominent after 10 days of IL-13 treatment (Figure 2.3A&B). There was very little baseline secretion of MUC5AC in either group. Interestingly at the end of the recovery period, day 40, the non-disease cultures returned to baseline levels of MUC5AC antibody intensity, whereas the asthmatic cell cultures remained slightly (14%) though not significantly elevated as
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compared to the baseline levels. In regards to MUC5B, the non–disease cultures exhibited higher baseline antibody signal which rapidly dropped off, by approximately 56.5%, after day 5 of IL-13 treatment. This decrease was more prominent in the asthmatic cultures and at day 5 of IL-13 treatment the MUC5B antibody signal was undetectable. Both culture types exhibited an increased in
MUC5B secretions around day 15 (Figure 2.3C&D). During the recovery phase, after stopping IL-13, both mucins exhibited a decrease in MUC5B staining intensity with the non-disease cultures returning to 29% and the asthmatic cultures to 54% of the baseline MUC5B antibody intensity.
IL-13 induced asthma cell culture models: Absolute MUC5B and MUC5AC concentration quantitation
Absolute quantitation by isotope labeled LC-MS/MS of MUC5AC showed a significant increase in the mean ± SE concentration by day 20 of IL-13 treatment for both the non-disease (16.22 ± 3.75 vs. 2.95 ± 1.54 pmol/mL) and asthmatic cultures
(46.8 ± 21.49 vs. 0.13 ± 0.067 pmol/mL) representing a 5.5 and 356 fold increase, respectively (Figure 2.4A). Interestingly, unlike the asthmatic cultures which increased in MUC5AC throughout the treatment, the non-disease cultures had a higher amount of MUC5AC at baseline and exhibited a small decrease at the onset of IL-13 challenge, before increasing significantly. The MUC5B concentration lower in the non-disease cultures as compared to the asthmatic cultures (Figure 2.4B).
Despite this, both culture types showed a similar response to IL-13. Both exhibited an initial drop in MUC5B after the onset of IL-13 treatment, which then slowly increased throughout the 20 days. Interestingly when compared to the western blot
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results, the increase in MUC5B during the latter half of the IL-13 challenge was less dramatic when using absolute quantitative methods and did not reach significance in either culture type. When looking at the ratio of MUC5AC to MUC5B throughout the
IL-13 challenge there was a significant, 10 fold, increase in the non-disease cultures
(17.32 ± 6.32 vs. 1.60 ± 0.81) and a 983 fold increase in the asthmatic cultures after
IL-13 challenge (5.68 ± 0.92 vs. 0.006 ± 0.002) reflecting the very low concentration of MUC5AC present at baseline in the asthmatic cultures (Figure 2.4C).
IL-13 induced asthma cell culture models: Macromolecular characteriztion of the purified secreted and stored gel forming mucins
Characterization of the gel forming mucins from the apical secretions by SEC-
MALS after purification by isopycnic centrifugation showed that the concentration of gel forming mucins increased by 1.83 fold in the non-disease cultures in response to
IL-13 (43.40 ± 5.82 vs. 23.70 ± 3.095 ug/mL), whereas it did not in the asthmatic cultures (32.91 ± 6.25 vs. 32.39 ± 7.64 ug/mL) (Figure 2.5A). Analysis of the macromolecular properties of the gel forming mucins revealed that the molecular weight (3.54x107 ± 2.16x106 vs. 6.08 x 107 ± 2.47x107 g/mol) and the radius (148.9 ±
6.3 vs. 146.3 ± 6.9nm) were unchanged for the non-disease cultures in response to
IL-13 Figure 2.5B&C, left panels). In contrast, the gel forming mucins from the asthmatic cultures were much larger at baseline than the non-disease cultures but decreased significantly after IL-13 treatment in terms of molecular weight (1.12x108
± 1.03x107 vs. 1.99x108 ± 6.11 x106 g/mol) and radius of gyration (155.5 ± 2.07 vs.
183.4 ± 4.28 nm)(Figure 2.5B&C, right panels). Even after this decrease, the gel
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forming mucins were still significantly larger, 3 fold, in molecular weight than those isolated from the non-disease cultures (Figure 2.5B).
In regards to the stored intracellular gel forming mucins which were isolated and purified from non-disease HBE cultures, the concentration increased 3 fold (9.77
± 1.58 vs. 2.87 ± 0.59 ug/mL) whereas the molecular weight (7.44x107 ±5.10x106 vs.
3.62x108 ± 9.14x107 gram/mol) and radius (161.9 ± 5.58 vs. 183.5 ± 12.03 nm) both decreased significantly after 20 days of IL-13 treatment (Figure 2.6A-C). Due to the scarcity of the asthmatic cells, the intracellular gel forming mucins were not isolated as this required sacrificing the cultures at day 20.
IL-13 induced asthma cell culture model: Proteomic pathway analysis of secreted proteins from non-asthmatic and asthmatic cultures
In addition to the gel forming mucins, all of the secreted proteins were analyzed by label free LC-MS/MS in order to provide a global view of how the secretome changes in response to IL-13. For both the non-disease and asthmatic cultures, there were approximately 1000 proteins identified. For the non-disease cultures, 174 of those proteins were found to be unique to the IL-13 challenge group and 432 to the control. When comparing secretions from day 20 of IL-13 treatment to baseline there were 286 significantly changing proteins, of which 219 decreased significantly and 67 increased (Figure 2.7A). Pathway analysis of the differentially secreted proteins revealed several different canonical pathways predicted to be affected (p-value, direction of change) including: Remodeling of Epithelial Adherens junctions (2.52x10-16, decreased), RhoGDI signaling (5.54x10-16, increased),
Phagosome Maturation (3.88x10-14, no prediction), Epithelial Adherens Junction
Signaling (4.39x10-14, no prediction), and Ephrin receptor signaling (9.64x10-14,
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decreased)(Figure 2.7B). The significant disease and biological function pathways included; cellular compromise, inflammatory response, cellular movement, cellular assembly and organization, and cellular function and maintenance (Figure 2.7C). A complete table of all unique and differentially expressed proteins when comparing
IL-13 to baseline for non-asthmatic cultures is provided in Appendix 3.
For the asthmatic cultures, there were 126 significantly changing proteins, 44 of which decreased significantly and 82 increased. (Figure 2.7D). The significantly changing proteins were enriched in the following canonical pathways (p-value, direction): Mitochondrial dysfunction (2.08x10-9, no prediction), Sirtuin Signaling
Pathway (5.74x10-8, increased), Oxidative Phosphorylation (2.01x10-6, increased),
NRF2-mediated oxidative stress response (1.10x10-4, increased), Phagosome maturation (1.47x10-4, no prediction) (Figure 2.7E). The top disease and biological functions that were enriched after IL-13 treatment were cellular compromise, inflammatory response, free radical scavenging, cancer, and organismal injury and abnormalities (Figure 2.7F).
In addition to the gel forming mucins, the membrane bound mucins, MUC1
(Figure 2.8A&B), MUC4 (C&D), and MUC16 (E&F) were analyzed by label free LC-
MS/MS and in both non-disease cultures (left panels) and asthmatic cultures (right panels). All three of these mucins decreased after IL-13 treatment. This was significant in all comparisons, except MUC1 from the asthmatic cultures apical secretions which decreased, but did not reach significance. A complete table of all unique and differentially expressed proteins when comparing IL-13 to baseline for asthmatic cultures is provided in Appendix 4.
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IL-13 induced asthma cell culture model: Proteomic analysis of the mucin interactome from non-disease cultures
In order to focus on the proteins likely interacting with mucin (the mucin interactome), size exclusion chromatography was performed on secretions from day
20 of IL-13 treatment and control and the resulting V0, mucin rich, fractions analyzed by LC-MS/MS. The changes seen in the interactome were compared to the global changes in the apical secretions to determine specific IL-13 induced changes.
Certain proteins that were increased in the secretion were also present and significantly increased after size exclusion such as FCGBP, Serpin b2, Dipeptidyl peptidase 4 and Gelsolin (Figure 2.9A, B, D, & F). Interestingly a handful of proteins such as LPLUNC-1 and Dipeptidyl peptidase 1, which significantly increased in the mucin rich fractions after size exclusion, were not found to be increased in the apical secretions during the IL-13 challenge (Figure 2.9C&E). Similarly there were proteins such as S100-A2 and Complement C3 found to be significantly decreased after size exclusion that were also significantly decreased in the apical secretions after the IL-
13 treatment (Figure 2.10 A&B). More interesting were the proteins, including
DMBT-1 and Galectin-3, which remained unchanged in the apical secretions during the IL-13 challenge, yet were significantly decreased after size exclusion chromatography (Figure 2.10 C&D).
Sufficient volume was not available to perform proteomics on the secretions with and without size exclusion for the asthmatic cultures. Therefore the overall secretion changes in the mucin interacting proteins highlighted and analyzed above are provided in Supplementary Figure 2.14 and Supplementary Figure 2.15. The apically secreted proteins from the asthmatic culture showed a similar secretion
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pattern with the exception of dipeptidyl peptidase 1, which increased slightly (but not significantly) in the non-disease cultures but decreased in the asthmatic cultures during Il-13 challenge.
IL-13 induced asthma cell culture model: Pathway analysis of differentially expressed exosomal miRNA from non-asthmatic and asthmatic cultures
Exosome like vesicles were also isolated from the apical secretions collected on days 15 and 20 for the control and IL-13 treatment using both non-disease and asthmatic cultures. Both culture types showed an increase in exosome concentration after IL-13 treatment, which did not reach significance (Figure 2.11A&C, left panels).
The size of the vesicles did not significantly change with treatment (Figure 2.11A&C, right panels). There were numerous differentially expressed miRNA sequenced from the isolated exosomes from the non-disease cultures after IL-13 treatment.
Interestingly, only a handful of miRNA significantly changed after IL-13 treatment of the asthmatic cultures (Figure 2.11B&D). The top significantly decreasing and increasing miRNA and the pathways they are predicted to affect are listed in Chapter
2 Tables 1 and 2 for the non-disease cultures and Chapter 2 Tables 3 and 4 for the asthmatic cultures. Among the pathways predicted to be targeted by the decreasing miRNA after IL-13 challenge of non-asthmatic cultures, was mucin type-O glycosylation (Figure 2.12). Specific genes targeted included: Polypeptide N- acetylgalactosaminyltransferase, Glycoprotein-N-acetylgalactosamine 3-beta- galactosyltransferase 1, CMP-N-acetylneuraminate-beta-galactosamide-alpha-2, 3- sialyltransferase 1, and beta-1, 4-galactosyltransferase 5 (Figure 2.12).
When comparing the different culture types at baseline, prior to IL-13 treatment, there were many significantly different miRNA. This is evident in the heat
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map which shows a distinction between the different cell culture types (asthmatic vs. non-disease) at baseline and after IL-13 based on their miRNA profiles. This distinction is reinforced by the volcano plot displaying the differentially expressed miRNAs at baseline when comparing the asthmatic cultures to non-disease (Figure
2.13 A&B). The differentially expressed miRNAs at baseline are listed in Chapter 2
Table 5. It is interesting to note, that while few miRNA significantly changed after IL-
13 challenge in the asthmatic culture system, many of the miRNA found to be significantly increased at baseline as compared to non-diseased were later found to be differentially expressed (increased) in the non-disease cultures after IL-13 challenge.
IL-13 induced asthma cell culture models: In silico MUC5B activity prediction based on differentially expressed exosomal miRNA from non asthmatic and asthmatic cultures
Additionally in silico activity prediction of the gel forming mucin genes based on the experimentally determined, significantly changing miRNA after IL-13 revealed several different miRNA in both culture types predicted to target MUC5B. The cumulative effect of these miRNAs was predicted to down regulate MUC5B gene expression. No miRNA within our dataset were identified as targeting MUC5AC
(Figure 2.14A&B).
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Discussion
Asthma is a chronic airway disease associated with mucus hypersecretion and in severe cases obstruction [152]. The IL-13 model of asthma shows that by antibody based western blot and absolute proteomic measurements MUC5AC is the predominant secreted mucin in these conditions. It is important to note that IL-13, as a TH2 cytokine, only represents a subset of the asthmatic phenotypes and therefore these results may not be applicable to all types of asthma [124, 129]. By using a cell cultures from individuals who died due to asthma complications and comparing these to non-disease cultures, we attempted to understand the differential response of epithelial cells to the same stimulus and how that leads to varying degrees of disease severity. There were several commonalities among the two different cell types. The first of which was the increased MUC5AC and the significantly altered ratio of MUC5AC to MUC5B. This appears to be a disease specific change, which is most evident when viewed in comparison to the ratio of MUC5AC to MUC5B in the
CF cell culture models, which are also characterized by mucus hypersecretion. In the asthma model, the ratio of 5AC to 5B increases because MUC5AC increases disproportionately. In contrast, in CF this ratio decreases because MUC5B remains the dominant gel forming mucin. This ratio remains consistent even when analyzing
CF sputum samples in which 5AC does increase significantly and to a greater extent than MUC5B, yet still MUC5B remains the dominant gel forming mucin. An interestingly point when comparing these two models is that IL-13 is produced primarily by the host immune helper T cells and is elevated in asthma models [132,
148, 153]. In contrast, in the Ps.a CF model, only an increase in MUC5B was
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evident; though when inflammatory products from the SMM or from the in vivo sputum samples were present, MUC5AC increased. This supports the conclusion that MUC5AC hypersecretion may be host immune cell mediated whereas MUC5B may be epithelial cell driven.
The macromolecular characterization of the gel forming mucins revealed significant differences between the different culture types. While the non-disease gel forming mucins increased in concentration, the molecular weight and radius were unchanged. In contrast the asthmatic gel forming mucins were several fold (3) larger than the non-disease gel forming mucins at baseline and remained significantly larger, even after significantly decreasing in molecular weight after IL-13. Though cell cultures derived from only 3 different asthmatic donors were tested, the response among those three was remarkably consistent. There are several potential explanations for this larger size. One group suggests that the increased MUC5AC is cross-linked through oxidative mechanisms creating a stiffened mucus gel [71].
Though crosslinking could potentially account for a larger molecular weight, both cell culture types would be affected by oxidative stress during the IL-13 challenge, yet they do not both contain mucin with higher than expected molecular weights. Also if
IL-13 induced oxidative stress did lead to an increased crosslinking, then we would expect a further increase in the molecular weight as compared to baseline, rather than a significant decrease. Another hypothesis is that the larger size could arise from an alteration in the multimerization process within the asthmatic cells. It is not well understood how this process is regulated, though in other biological systems using proteins that share homologous domains with mucins (von Willebrand Factor),
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it has been shown that disulfide isomerase activity can affect the multimerization
[154, 155]. Within this study’s proteomic dataset there were no significant differences between the non-disease and asthmatic cultures at baseline in terms of proteins with isomerase functionality. It is important to note that this analysis only involved secreted proteins, not intracellular and thus may not be best suited to evaluate changes in proteins involved in intracellular mucin multimerization.
Alternatively, because the size of the secreted gel forming mucins from the asthmatic cultures are similar to the size of the stored, intracellular gel forming mucins isolated from the non-disease cultures, the altered size could reflect a defect in unpacking from the mucin granule. Defective mucin unpacking has been proposed in other mucoobstructive disease such as CF [89]. Unfortunately, the intracellular mucins were not isolated from the asthmatic cultures as this could have helped identify an unpacking defect, which could be plausible if the intracellular and secreted gel forming mucins were the same size in the asthmatic cells. The difference in size is not likely due to electrostatic or hydrophobic interacting proteins creating larger complexes, as the isolation and purification of the gel forming mucins was performed under dissociative conditions (4M GuHCl) [5]. Very strongly bonded proteins, such as through covalent bonds, may not be disrupted by chaotropic agents and thus a proteomic analysis of the mucin rich fractions following isopycnic centrifugation and size exclusion chromatography may be useful in identifying those potential cross linking proteins that are present in the asthmatic cultures but not the non-disease.
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In an attempt to determine which proteins are differentially secreted and interacting with mucin during the IL-13 challenge, the secretome, containing all the apically secreted proteins, was compared to the interactome. The secretome and interactome were both derived from the same raw source material but were subjected to slightly different downstream processing. Both were analyzed by LC-
MS/MS, but prior to this, the mucin interactome was separated from whole secretions by size exclusion chromatography. This isolated a set of proteins that co- eluted with the mucins in the Void volume (V0) rather than in the included volume, as would be expected based on their predicted molecular weights, thus suggesting an interaction with mucin [5].
The results from these experiments support that the interacting proteins are disease specific. Though there were examples where the secreted proteins increased both in the apical secretion and the interactome, there were others that were enriched in the interactome without increasing in the secretion. This was the same pattern seen with the decreased interactome proteins. It is not surprisingly that when the overall apical secretion of the protein is decreased (i.e. downregulated by
IL-13), it would also be decreased in the void. Alternatively there were proteins, such as DMBT-1, that remained stable in concentration in the apical secretions during the challenge, but decreased significantly in the interactome. Given the previous data regarding the absolute concentrations of MUC5B and MUC5AC, there are several possible explanations for the mucin interactome changes, including: (A) the interacting proteins are secreted differentially based on the IL-13 stimulation, and
(B) the mucin interacting proteins may interact with one specific mucin and thus the
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change in the dominant mucin within the V0 volume (from MUC5B to MUC5AC) also corresponds to a change in the associated interacting proteins. The changes in the interacting proteins again highlight the role of mucus in innate immunity. For example, Serpinb2 has been shown to increase in the OVA mouse asthma model and also after parasitic (schistosome) and viral infection, while also having been shown in vivo to regulate the Th1 and Th2 immune response [156-158]. Similarly
LPLUNC-1 and the serine proteases, Dipeptidyl peptidase 1 and 4, have also been implicated in the host innate immune response [5, 159]. Gelsolin is involved in mucin secretion and facilitates the actin remodeling that occurs during exocytosis of the mucin granule [97]. DMBT-1 also has immune functionality but was decreased in the
V0 after the IL-13 challenge. These particular changes suggest that the immune properties of mucus can be specifically tailored by mucus secretion and protein secretion to respond to specific injury or insult [86]. The data above is based on the secretions from non-disease cultures. Unfortunately, the volume of apical secretions collected from the asthmatic cultures was insufficient for the interactome analysis, though the secretome was analyzed via label free LC-MS/MS. In general, the asthmatic cells responded in a similar fashion as the non-disease cultures in regards to the secretion pattern of the highlighted mucin interacting proteins. It would be interesting to repeat this study and to perform the interactome analysis using the asthmatic apical secretions in order to determine if other interacting proteins are present or enriched that could account for the severe disease phenotype.
Global overall changes in the secretome as determined by ingenuity pathway analysis revealed many pathways including: epithelial junction or cytoskeletal
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remodeling, oxidation and reduction, and host of difference metabolic pathways.
Many of these pathways have been reported in the literature as playing a role in asthma pathogenesis [160-162]. In the non-disease cultures, the most significantly enriched canonical pathways were RhoGDI signaling and ephrin signaling pathways; whereas in the asthmatic cultures, mitochondrial dysfunction, sirtuin signaling, and oxidative pathways were the top pathways. When viewed in the context of airway inflammation and asthma there appears to be a logical connection between these pathways. The ephrin signaling pathway has been noted to down regulate TH2 cell response in asthma. Similarly increased oxidative stress, due to an imbalance of oxidants and anti-oxidants, is a well-documented feature of the asthmatic pathogenesis as is mitochondrial dysfunction [163-165]. Sirtuin signaling has also been attributed to the development of airway hyper-responsiveness and allergic airway diseases and is associated with the influx of inflammatory cells and altered cytokine signaling [166, 167]. The similarity in signaling pathways between the non- disease and asthmatic cultures is evident in the summary canonical pathway heat map (Supplemental Figure 2.17) which also reveals a prominent difference between the two cell cultures. At the 20 day IL-13 time point, the asthmatic cultures show a significant increase and enrichment in the nuclear factor erythroid 2–related factor
2 (NRF-2) mediated oxidative stress pathway. This has been shown to be an important pathway in determining susceptibility to and severity of allergen induced asthma by altering the influx of immune cells and goblet cell hyperplasia [168].
Additional studies have shown that different NRF2 polymorphisms predispose individuals to oxidant induced acute lung injury [169]. Thus this significantly
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changing pathway in conjunction with the disease progression known for these individuals provides an intriguing connection between molecular signaling and disease severity.
It is interesting to note the number of changing proteins in both cell culture models in response to IL-13. The non-disease cultures tend to show more dramatic changes and have more unique proteins as compared to the controls. Whereas the asthmatic cultures in response to IL-13, had fewer than 20 unique proteins and despite having identified approximately the same number of total proteins as the non-disease cultures, there were almost half as many significantly changing proteins. This was a similar trend seen in the miRNA analysis that were sequenced from the exosome like vesicles. The non-asthmatic cultures showed numerous differentially expressed miRNA after IL-13 treatment, whereas the asthmatic cultures showed very few. Interestingly, among the pathways associated with significantly decreasing miRNA was Mucin-type O glycosylation, in which 11 genes were predicted to be targeted by three differentially expressed miRNA: miR 23b-3p, 27b-
3p, and 106a-5p. These miRNA were different from those identified in the CF cell cultures model. This could indicate that there is disease specificity to the miRNA regulation of mucin glycosylation or that the glycosylation of a particular gel forming mucin (MUC5AC vs. MUC5B) requires specific regulation. It is possible that the differences in glycosylation could account for the narrow range of measurements in the secreted mucins after IL-13 as determined by light scattering.
Additionally using IPA to predict the activation status of MUC5B or MUC5AC based on the differentially expressed miRNA after IL-13 challenge in both non-
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disease and asthmatic cultures, there were several miRNA identified as target
MUC5B. Using the molecular activity predictor function, IPA predicted that the cumulative effect of those miRNA based on their experimental log fold changes in both culture types would be a decrease MUC5B expression. Two of the miRNA
(miR-149-3p and miR-6781-5p) that were predicted to target MUC5B were differentially expressed and shared between the two cultures indicating that this is a disease (IL-13) specific and not culture type specific regulation. This is further supported through a comparison with the Ps.a CF cell culture miRNA analysis which revealed a subset of the differentially expressed miRNAs that were predicted to target MUC5B were also shared between the CF and asthma models. Intriguingly the same miRNA predicted to downregulate MUC5B in the IL-13 model, changed in the opposite direction in the CF model leading to a predicted activation of MUC5B which correlated to the increase in MUC5B protein concentration in this model.
Returning to the fact that there were very few miRNA differentially expressed in the asthmatic cell-culture IL-13 model, a comparison between the differentially expressed miRNA between the two different cell types at baseline revealed significant differences. Interestingly the miRNA differences found at baseline in the asthmatic cultures were also present in the non-disease cultures but only after IL-13 challenge. This supports the idea that the asthmatic cells though far removed from the source of inflammation may still have altered genetic regulation that increases susceptibly to or severity of asthmatic disease. Lastly, though few significant differences related to the gel forming mucins were found between the two different culture systems when exposed to chronic IL-13 (with the molecular weight and
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radius of gyration being the notable exceptions) that could explain these individuals’ severe asthma endotype, asthma is a multifactorial disease and likely it is a combinatorial effect of mucus with altered properties, airway hyper-responsiveness and other factors that contribute to a specific asthmatic endotype. That being said, understanding the properties of mucus and how alterations lead to mucus plugging and obstruction could prove invaluable in the development of therapeutics that could be broadly applicable to all mucoobstructive conditions including asthma and CF.
Just as mucin hypersecretion is a unifying feature between CF and asthmatic lung disease, we identified another significantly changing, mucin interacting protein that is altered in both airway diseases, FCGBP. The following chapter will investigate the role of FCGBP in health and disease to better understand the role it plays in the pathogenesis of muco-obstructive lung disease.
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Chapter 2 Figures:
Figure 2.1. Asthma cell culture models exhibit goblet cell hyperplasia and increased storage of intracellular mucins. Panel A shows representative non- disease un-stimulated HBE ALI cultures with hematoxylin and eosin (H&E) staining on the left and Ab-PAS on the right. Panel B-D show HBE cultures after 20 days of IL-13 challenge with B showing the cyst-like formation on the apical surface and C exemplifies the goblet cell hyperplasia containing mucin granules which stain strongly by ab-PAS. Panel D shows the gland land formations present after Il-13 treatment
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Figure 2.2. Asthma cell culture models exhibit mucus hypersecretion which is adherent to the apical surface. Representative 3D renderings of whole mount IHC following 5 days, 10 days, and 20 days of daily IL-13 treatment added to the basolateral compartment shows increasing amount of MUC5B and MUC5AC staining on the apical surface that was not able to be removed by thorough and repeated PBS washings. Cilia, white; MUC5AC, red; MUC5B, green; Nuclei, blue.
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Figure 2.3. Asthma cell culture models shows a disproportionate increase in MUC5AC during 20 day challenge with IL-13 using non-disease (n=5) (A&C on left) and asthmatic (n=3) (B&D on right) cell cultures. Representative agarose gel electrophoresis western blot of apical secretions throughout 20 day IL-13 and 20 day recovery period collected every five days starting at baseline prior to start of the challenge. Samples shown were reduced with 10 mM DTT and probed for (A & B) polyclonal MUC5AC and (C&D) monoclonal MUC5B with densitometric quantitation below. Bar graphs represent the mean ±SE of the intensity normalized to the highest value within each repeat. Repeated measure non-parametric Anova without correction for multiple comparisons comparing each day of treatment with baseline intensity measurement was performed with the following p values: *≤0.05, **≤0.01, ***≤0.001.
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values::*≤0.05, **≤0.01,***≤0.001. comparingday each of withplots Baseline asthmatic the responseison the right(triangles). Eachcollection day isrepresented by different a color: 13 diseaseHBE cells (n=5) andasthmatic HBE cells collected(n=3) ever MUC5AC, MUC5B(B) the (C)and ratio of MUC5AC/MUC5BMUC5AC in apical secretions nonfrom culturesexhibit mucus hypersecretion which dominated is by MUC5AC. Absolute quantitationof (A) Asthmacell culture modelsusing both nondisease (left sample set)and asthmatic (rightsample set Asthma2.4. Figure cellculture modelsexhibit mucus hypersecretion dominated by MUC5AC. . Thenon
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Figure 2.5. Macromolecular characterization of secreted gel-forming mucins from non-disease and asthmatic HBE cultures after 20 day challenge with IL-13. After the 20 day challenge with IL-13 applied to the basolateral compartment, the gel forming mucins were purified from the apical secretions and analyzed by SEC-MALS/dRI. A) Concentration, (B) molecular weight, and (C) radius of gyration of secreted gel forming mucins after IL-13 challenge with non-disease on the left and asthmatic cultures on the right of each panel. Gold bars designate controls and purple the IL-13 treatment groups. Data represented in Tukey box and whisker plots. Paired Student t-tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001.
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Figure 2.6. Macromolecular characterization of stored gel forming mucins isolated from asthma cell culture model. After the 20 day challenge with IL-13 applied to the basolateral compartment, the gel forming mucins were purified from the whole cell lysate after extensive washing, while also taking care to minimize mechanical stimulation, and analyzed by SEC-MALS/dRI. A) Concentration, (B) molecular weight, and (C) radius of gyration of the stored gel forming mucins purified from the whole cell lysate after the 20 day IL-13 challenge. Data represented in Tukey box and whisker plots. Paired Student t-tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001.
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Biologicalandfunctions diseases arebased numberon of significant associated proteins. significantlyupregulated downregulatedor proteins (log2 fold changeof treatment vs. control) within specific enriched the (E)canonical and (F)biological and disease process.Activation scoresz are calculated basedon the ratio of challenge.Among these proteins,those showing differentialexpression (p value<0.0.05) were analy showsthe unique andshared proteins between IL direction ofactivation (orange=upregulatedand blue=downregulated) and (C) b expressed(p value<0.05) proteins were analyzedby IPAwhich revealed theenriched (B)canonical pathways including the IL and asthmatic(n=3; bottom) HBE cell cultures. Figure A showsa Venn diagramof the unique andshared proteins between pathwayanalysis ofsignificant Figure 2.7 Labelfree proteomic analysisof apical secretions based on uniquely identifiedproteins and ingenuity
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Figure 2.8. Secretion pattern of non gel-forming mucins as measured by label free LC-MS/MS in apical secretions during Il-13 challenge of non-disease HBE (left; n=5) or asthmatic HBE cultures (right; n=3). A Data represented in tukey box and whisker plots or dot plot with SE. Paired Student t-tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001
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*≤0.05,**≤0.01, ***≤0.001 secretiondata (left) and pairedStudent t comparisoncorrection comparing each Datarepresented in tukeyandbox whisker plots. Repeated measurenon chromatographyusing secretionsfrom days and15 of20 the IL MS/MSduring lL Figure2.9. Secretion pattern of increasing probable mucininteracting proteins as measured by labelfree LC
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Figure 2.11. Changes in exosome concentration size and miRNA cargo after IL-13 challenge of non-disease HBE cultures (n=5) and asthmatic cultures (n=3). Change in concentration (particles/mL) and size (nm) of the exosomes following 20 day challenge with IL-13 (purple bars) or control (gold bars) isolated from non-disease (A) and asthmatic (B) cell cultures. Volcano plot of differentially expressed miRNA sequenced on HTG EdgeSeq platform isolated from exosome like vesicles secreted into the apical secretions during IL-13 challenge from (C) non-disease and (D) asthmatic cell cultures. Statistics were performed on log2 normalized sequence reads using an adjusted p value of <0.1 to determine significance. Blue dots represent specific miRNA that decreased with IL-13 treatment and red dots the specifically identified miRNA that increased after IL- 13. challenge. Data represented in figure A as Tukey box and whisker plots for non-disease HBE data and as scatter plots with mean ± SE. Paired Student t- tests were performed with the following p values: *≤0.05, **≤0.01, ***≤0.001
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Chapter 2, Table 1. Top 20 significantly decreasing and increasing miRNA isolated from apically secreted exosome like vesicles after 20 IL-13 challenge of non- disease HBE cultures (n=5). Adjusted p value cutoff for significance was <0.1 Decreasing Log2 Fold Increasing Log2 Fold miRNA Change miRNA Change miR-138-5p -1.42 miR-6126 2.72 miR-934 -1.38 miR-6780b-5p 2.50 miR-455-3p -1.33 miR-6124 2.35 miR-138-1-3p -1.29 miR-6780a-5p 2.12 miR-449c-5p -1.24 miR-149-3p 2.07 miR-449b-3p -1.20 miR-6088 2.02 miR-193b-3p -1.13 miR-6875-5p 1.83 miR-181d-5p -1.08 miR-211-3p 1.82 miR-449b-5p -1.02 miR-3197 1.76 miR-181b-5p -1.01 miR-1244 1.73 miR-20a-3p -0.98 miR-375 1.68 miR-23c -0.98 miR-4728-5p 1.68 miR-449a -0.96 miR-1255b-2-3p 1.62 miR-17-3p -0.93 miR-3175 1.58 miR-365a-3p -0.92 miR-7111-5p 1.57 miR-31-5p -0.90 miR-7107-5p 1.56 miR-192-5p -0.90 miR-5196-5p 1.55 miR-20a-5p -0.90 miR-6880-5p 1.52 miR-205-5p -0.88 miR-654-5p 1.42 miR-181c-5p -0.88 miR-1285-5p 1.41
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Chapter 2 Table 2. Significant KEGG pathways based on significantly increasing miRNA isolated from apically secreted exosomes after 20 day IL-13 challenge or control of non-disease cultures (n=5). Number of genes in each pathway predicted to be affected by the miRNA and number of miRNA within the dataset targeting each specific pathway is listed. In silico predictions made using the Diana MirPath version 3 using the modified Fisher’s exact test with a p value cut off of 0.05 and Benjamini Hochberg correction. Decreasing miRNA Increasing miRNA KEGG pathway p-value #genes #miRNA KEGG pathway p-value #genes #miRNAs TGF-beta signaling pathway 2.33E-07 35 10 Proteoglycans in cancer 2.00E-07 117 43 Proteoglycans in cancer 2.33E-07 82 12 Axon guidance 9.59E-07 84 41 Glutamatergic synapse 1.30E-05 49 11 Endocytosis 6.07E-06 133 44 Endocytosis 4.79E-05 84 13 Pancreatic cancer 1.37E-04 48 34 Morphine addiction 6.71E-05 38 11 Thyroid hormone signaling pathway 1.37E-04 77 45 Nicotine addiction 8.05E-05 19 11 Ras signaling pathway 1.41E-04 136 44 Amphetamine addiction 1.58E-04 30 12 Glutamatergic synapse 2.94E-04 74 41
146 Wnt signaling pathway 4.53E-04 57 13 Prion diseases 6.89E-04 16 23 ErbB signaling pathway 5.12E-04 41 12 FoxO signaling pathway 6.89E-04 84 34
Hippo signaling pathway 5.12E-04 54 12 Adherens junction 6.89E-04 48 39 Axon guidance 5.45E-04 52 12 Estrogen signaling pathway 1.04E-03 58 36
Retrograde endocannabinoid signaling 6.87E-04 42 11 MAPK signaling pathway 1.50E-03 150 46
cAMP signaling pathway 1.18E-03 77 12 cGMP-PKG signaling pathway 1.56E-03 104 45 Circadian entrainment 1.26E-03 42 12 Platelet activation 2.27E-03 81 39 Cholinergic synapse 1.38E-03 46 10 Focal adhesion 2.27E-03 125 42 FoxO signaling pathway 1.38E-03 55 11 cAMP signaling pathway 3.27E-03 120 42 Glioma 1.44E-03 27 10 Renal cell carcinoma 3.80E-03 45 34 GABAergic synapse 1.44E-03 34 11 Long-term depression 3.80E-03 40 36 Mucin type O-Glycan biosynthesis 1.84E-03 11 3 Hepatitis B 3.80E-03 82 37 Protein processing in endoplasmic 2.51E-03 60 12 Prostate cancer 3.80E-03 59 37 reticulum Signaling pathways regulating 3.12E-03 53 13 Thyroid hormone synthesis 3.80E-03 44 38 pluripotency of stem cells
Chapter 3 Table 3. Top significantly decreasing and increasing miRNA isolated from apically secreted exosome like vesicles after 20 IL-13 challenge of asthmatic HBE cultures (n=5). Adjusted p value cutoff for significance was <0.1 Decreasing miRNA Log2 Fold Change Increasing miRNA Log2 Fold Change miR-211-3p -5.52 miR-375 1.85 miR-1343-5p -3.03 miR-6780b-5p 1.49 miR-1228-3p -2.14 miR-6741-5p 1.38 miR-1910-3p -1.90 miR-6803-5p 1.34 miR-4304 -1.85 miR-149-3p 1.33 miR-6791-5p -1.79 miR-1273d -1.26
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Chapter 3 Table 4. Significant KEGG pathways based on significantly increasing miRNA isolated from apically secreted exosomes after 20 day IL-13 challenge or control of asthmatic HBE cultures (n=3). Number of genes in each pathway predicted to be affected by the miRNA and number of miRNA within the dataset targeting each specific pathway is listed. In silico predictions made using the Diana MirPath version 3 using the modified Fisher’s exact test with a p value cut off of 0.05 and Benjamini Hochberg correction. Decreasing miRNA Increasing miRNA KEGG pathway p-value #genes #miRNAs KEGG pathway p-value #genes #miRNAs Unsaturated fatty acids biosynthesis 1.15E-02 4 3 Thyroid hormone signaling pathway 1.76E-04 25 5 Morphine addiction 1.15E-02 18 6 Biotin metabolism 3.40E-04 1 1 Transcriptional misregulation in cancer 1.61E-02 33 5 SNARE interactions in vesicular transport 1.96E-02 9 4 ErbB signaling pathway 4.88E-02 15 4 Synaptic vesicle cycle 1.96E-02 14 4 Glycerophospholipid metabolism 4.88E-02 20 6 ErbB signaling pathway 1.96E-02 16 5 Axon guidance 1.96E-02 24 5 Regulation of actin cytoskeleton 1.96E-02 40 5 Prostate cancer 1.96E-02 20 5 MAPK signaling pathway 3.29E-02 42 5 Glutamatergic synapse 3.29E-02 20 5 Long-term depression 3.29E-02 15 5
148 Proteoglycans in cancer 3.29E-02 31 5
Figure 2.12. Mucin type O-glycosylation genes targeted by significantly decreased exosomal miRNA after IL-13. Specific genes within the Mucin type O- glycosylation pathway predicted to be affected by significantly (adjusted p value <0.1)decreasing miRNA isolated from exosomes following 20 day challenge with IL-13 as compared to control. KEGG pathway was determined using Diana miRpath version 3 implementing the modified Fisher’s exact test with a p value cut off of 0.05 and Benjamini Hochberg correction. Genes in yellow are predicted to be targeted by one miRNA within the list. Genes in orange are predicted to be affected by two or more miRNA.
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Figure 2.13. Differentially expressed miRNA at baseline after IL-13 treatment in asthmatic and non-disease HBE cultures represented as a heat map (A) and volcano plot (B). An adjusted p value cut off of 0.1 was used to determine significance. Red indicates increased expression and blue decreased. The Green horizontal bars on the heat map represent the baseline group of both asthmatic and non-disease cultures whereas the yellow bar represents the IL-13 challenged asthmatic (n=3) and non-disease cultures (n=5). The volcano plot shows the significantly changing miRNA in asthmatic cultures at baseline as compared to non-disease.
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Chapter 2 Table 5. Significantly different miRNA at baseline (without IL-13 challenge) when comparing asthmatic cultures (n=3) to non-disease HBE cultures (n=5) based on an adjusted p value of <0.1. Decreasing Log2Fold Increasing Log2Fold miRNA Change miRNA Change miR-138-5p -1.41899 miR-6126 2.722349 miR-934 -1.38195 miR-6780b-5p 2.496285 miR-455-3p -1.32575 miR-6124 2.352388 miR-138-1-3p -1.29091 miR-6780a-5p 2.118945 miR-449c-5p -1.24203 miR-149-3p 2.066972 miR-449b-3p -1.20009 miR-6088 2.018258 miR-193b-3p -1.13127 miR-6875-5p 1.827787 miR-181d-5p -1.07529 miR-211-3p 1.82312 miR-449b-5p -1.01835 miR-3197 1.762913 miR-181b-5p -1.00733 miR-1244 1.734615 miR-20a-3p -0.98106 miR-375 1.680459 miR-23c -0.97621 miR-4728-5p 1.67593 miR-449a -0.95849 miR-1255b-2-3p 1.620281 miR-17-3p -0.92672 miR-3175 1.581484 miR-365a-3p -0.92438 miR-7111-5p 1.570177 miR-31-5p -0.89966 miR-7107-5p 1.563114 miR-192-5p -0.89695 miR-5196-5p 1.55303 miR-20a-5p -0.89673 miR-6880-5p 1.515652 miR-205-5p -0.88439 miR-654-5p 1.41969
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Figure 2.14. MUC5B gene predicted to be down regulated by differentially expressed miRNA after 20 day IL-13 treatment of non-disease (n=5) and asthmatic (n=3) HBE cultures.. Differentially expressed miRNA isolated from exosomes were used to create an overlay using the Ingenuity Pathway Analysis (IPA) My Pathways Grow function to predict upstream and downstream molecules. Then the Molecular Activity Predictor was applied to determine predicted direction of regulation of MUC5B based on the experimentally determined log2Fold changes of specific miRNA. All miRNA were filtered based on adjusted p value <0.01.
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Supplement Figure 2.15. Secretion pattern of probable mucin interacting proteins as measured by label free LC-MS/MS during Il-13 challenge of asthmatic HBE cultures apical secretions (n=3). Data represented as scatter plots with SE.. Repeated measure non-parametric ANOVA without multiple comparison correction comparing each timepoint during the IL-13 challenge to baseline was performed on the secretion data with the following p values: *≤0.05, **≤0.01, ***≤0.001
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Supplemental Figure 2.16. Secretion pattern of probable mucin interacting proteins as measured by label free LC-MS/MS during IL-13 challenge of asthmatic HBE cultures apical secretions (n=3). Data represented as scatter plots with SE.. Repeated measure non-parametric ANOVA without multiple comparison correction comparing each timepoint during the IL-13 challenge to baseline was performed on the secretion data with the following p values: *≤0.05, **≤0.01, ***≤0.001
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Supplemental Figure 2.17. Comparative analysis of canonical pathways enriched by differentially expressed proteins at days 5 and 20 of IL-13 treatment in non-disease( n=5;top two rows) and asthmatic cultures (n=3; bottom two rows). Blue indicates down regulation and orange up regulation of a specific pathway. * designates the NRF2 mediated oxidative stress pathway
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CHAPTER 3: ISOLATION, PURIFICATION, AND CHARACTERIZATION OF FCGBP, A PUTATIVE MUCIN BINDING GLYCOPROTEIN
Introduction: FCGBP
Pathological mucus leading to obstruction is a hallmark shared by both cystic fibrosis and asthma [152, 170]. Mucin though an important component in mucus and responsible for many of its rheological properties, does not act alone and is associated with a host of other proteins [5, 28, 41]. One such protein is IgG Fc gamma binding protein, also known as FCGBP. It was originally identified within the secretory granules of goblet cells in the colon and small intestine and as indicated by its nomenclature was proposed to bind to the Fc gamma region of IgG [171, 172].
These preliminary studies showed that FCGBP was strongly associated with the cytoskeletal elements, as it was not able to be separated from colonocyte cell homogenates despite use of three strong detergents [171]. This finding was to some extent replicated when the chaotropic agent, GuHCl, was used to isolate MUC2 from colonic mucus along with its strongly associated/bound proteins [173, 174]. In this way, FCGBP was identified as covalently bound to MUC2 and proposed to act as a crosslinking protein and to stabilize the mucin get network [174]. The crosslink was proposed to form through reactive anhydride chemistry after autocatalytic cleavage of the GDPH site found 11 times within the FCGBP protein [174, 175]. The cleavage of this sequence, which has also be identified in other proteins, was reported to be dependent on pH which varied by different tissue
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(i.e. in the colon, acidic pH was required whereas in the stomach, it occurred at neutral ph [174]. Though the initial studies showed that FCGBP bound with high affinity and specificity to both monomeric and aggregated IgG, this result has not been reproducible in our hands (data not shown) and by other groups [171, 172,
174]. While it has been suggested to the function in the immunologic defense of the gut, studies verifying this are lacking.
The conformation and structural analysis of FCGBP have been limited. It is known that the internal region has several repeats homologous to the von
Willebrand Factor D domains (vWF-D) and trypsin inhibitory regions (TIL and TILa).
Also though some of the individual FCGBP domains are present in early species, the combination of the termini with the repeated internal domains characteristic of
FCGBP and other mucin like proteins only occurred in vertebrates [175]. Also an evolutionary analysis of the N terminal portion of this protein, which was reported to confer the protein with its IgG binding capacity, found this domain to be present in bacterial proteins from species generally characterized by non-flagella and non- ciliated “gliding” motility [175]. Lastly a study identifying FCGBP in colon, did so by first purifying the proteins that were α(1-6) fucosylated indicating that FCGBP is modified in this way [176].
Beyond the gut, FCGBP has been identified in placental tissue and also in various cancers affecting the colon, gallbladder, and head and neck squamous cell carcinomas [176-179]. It has been shown to be down regulated in thyroid, gall bladder and also colon cancer and when it was expressed, was associated with better survival [176, 177, 179]. In regards to head and neck squamous cell
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carcinoma, it was reported to be expressed at higher levels when the cancer was positive for human papillomavirus and associated with better prognosis [178]. Both the gallbladder and head and neck squamous cell carcinoma studies suggested
FCGBP was negatively associated with epithelial mesenchymal transition and was negatively regulated by TGF -β, a known mediator of EMT [178, 179]. Lastly,
FCGBP was found to be upregulated within the crypts of patients with ulcerative colitis. [177]
Information regarding FCGBP is limited especially in regards to its function in the respiratory system. The connection to the inflammatory environment is a repeated theme amongst these studies and in light of its predicted mucin binding ability, FCGBP is an intriguing protein to study in the context of inflammatory muco- obstructive airway diseases. We hypothesize that FCGBP is in fact a mucin interacting protein and when differentially secreted in airway diseases, contributes to the development of a mucus layer with altered properties that becomes pathologic and static.
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Methods Ex-vivo cell culture challenges: Asthma and Cystic Fibrosis
To emulate the chronic asthmatic lung environment, primary, fully differentiated, human bronchial epithelial (HBE) cells cultured at air liquid interface
(ALI) were challenged with 10ng/mL of recombinant human IL-13 (Peprotech) added daily to the basal media for 20 days. The apical surfaces of the cultures were washed before the start of treatment and every 5 days throughout treatment. A paired culture from the same donor was treated similarly without the addition of IL-
13. Instead an equal volume of 0.05% BSA, the diluent used to re-suspend the lyophilized IL-13 as per the manufactures instructions was added to the basal media daily. An aliquot of the apical secretions was subjected to 0.7% (w/v) agarose gel electrophoresis under reducing conditions and probed with antibodies against
FCGBP. An equal volume of 8M GuHCl was added to a second aliquot, which was prepared for proteomic analysis as detailed below.
The CF lung environment was simulated through challenging the apical surface of HBEs daily for five days with the supernatant of mucopurulent material
(SMM) or culture filtrate from Pseudomonas aeruginosa (Ps. a.). PBS and
Trypticase Soy Broth (TSB) were used as controls for each challenge respectively.
Apical secretions were collected at baseline and each day prior to application of treatment. To prevent further degradation, an equal volume of 8M GuHCl was added to the secretions after collection.
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Human Sputum Samples:
Frozen human sputum samples from non-disease controls, asthmatics, and cystic fibrosis were thawed on ice and solubilized with GuHCl, achieving a final concentration of 4M. These samples were prepped for mass spectrometry in the manner outlined below.
Mass Spectrometry Sample Preparation and Analysis
Apical secretions and sputum samples were prepared for LS-MS/MS using a modified filter assisted sample preparation (FASP) method as described previously.
Briefly samples were reduced, alkylated, digested overnight with trypsin and the eluted peptides freeze-dried the following morning in preparation for LC-MS/MS analysis. The resulting peptides were resuspended in 0.1% formic acid and analyzed by liquid chromatography-tandem mass spectrometry using the hybrid quadrupole
Orbitrap mass spectrometer with a nanospray source (Q Exactive, Thermo Fisher
Scientific) using data dependent analysis. Proteins were identified by searching against most current human database and quantified using Scaffold, version 4
(Proteome Software Inc.).
Transfection:
293F cells were transfected with the pCMV6-entry tagged cloning vector containing the FCGBP-myc-DDK ORF (RC222632, Origene) using the X- tremeGENE HP DNA Transfection Reagent (Roche) according to manufactures instructions. Briefly the transfection reagent diluted with Opti-MEM (Sigma) was warmed to 25°C and then combined and gently mixed with FCGBP plasmid for 20 minutes, which was added to a 10cm plate of 80% confluent 293F cells and
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incubated for 48 hours at 37°C. The cells were then switched to selection media containing 700μg/mL Geneticin (G418 Sulfate, Gibco) and single cone selection was performed on the surviving cells using serial dilution. Successful transfection and screening of individual clones was verified by co-localization of the anti-myc and anti-FCGBP antibodies after western blot using both cell lysate and expression culture media lacking FBS.
FGCBP Purification:
Clone 4 was grown to 70% confluence under selection and after gentle washing with 37C PBS was switched to expression media (without FBS) for 3 days.
The media was then collected and stored at 4°C after removal of cell debris. The media was concentrated 15X using 100kDa cutoff Amicon Ultra-15 centrifugal filter units. The concentrated media was rotated overnight at 4°C with Anti-DyKDDDDK
G1 Affinity Resin (GenScript) according to manufacturer’s instructions for batch binding. The resin and media were added to a PD-10 column (GE) with frit under gravitational flow. The resin was washed extensively under gravity flow using cold
Tris buffered saline (TBS). The bound FCGBP was eluted using competitive elution buffer (FLAG peptide at a concentration of 500μg/mL in TSB) following incubating at
25°C for 60 minutes. This was followed by an acid elution step (0.1M Glycine HCl, pH3.5), the flow through from which was immediately neutralized with 1M Tris, pH
9.0. The resin was regenerated according to manufacturer’s instructions for repeated use.
A 2mL aliquot of the eluent was subjected to size exclusion chromatography on a Superose 6 10/300 GL(30cm x 10mm, 24 mL bed volume, GE Healthcare Life
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Sciences) column at flow rate of 300μl min-1. The 1mL fractions were subjected to
FCGBP immunoblotting. The peak FCGBP fractions from repeated size exclusion runs were pooled and diluted 1:1 with buffer A (25mM Tris pH 7.4) to reduce the salt concentration in preparation for anion exchange. The diluted fractions (12-50mL) were loaded on to a 1 mL Resource Q column (GE Healthcare) using the Amersham
Biosciences, Ettan LC high-pressure chromatography system at a flow rate of
2ml/min with buffer A. Four addition column volumes of buffer A were used to wash the column prior to starting the 2 step gradient using buffer B (25 mM Tris, 1M NaCl, pH 7.4). The first gradient segment reached 50% of buffer B over 15 column volumes. The second segment increased buffer B to 100% for five column volumes to elute any remaining material. The 500μL fractions were subjected to FCGBP immunoblotting. Purity was assessed through mass spectrometry and GelCode blue staining of SDS-PAGE gel.
Characterization: Light Scattering
The FCGBP rich fractions from anion exchange were subjected to size- exclusion chromatography at a flow rate of 300μl min-1, which was coupled to a laser photometer (DAWN HELEOS II, Wyatt Technology) and refractometer (Optilab T- rEX, Wyatt Technology). The data was analyzed with the ASTRA software, version
7.1.2 (Wyatt Technology).
Characterization: Atomic force microscopy
A solution of Nickel Chloride (10mM, 0.22 filtered) was applied to a freshly cleaved mica disk for 1 minute and then rinsed with ddH2O. A solution of FCGBP diluted 1:3 with ddH20 was deposited on the mica for 3 minutes before repeated
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rinsing with ddH2O and drying with nitrogen gas. Serial images were taken of the
FCGBP using an ARROW-UHF AuD-20 cantilever (NanoWorld Innovative
Technologies) in non-contact mode with the Cypher Atomic Force Microscopy system (Asylum Research/Oxford Instruments). The resulting images were analyzed using AR SPM software (Asylum/Oxford Instruments). Images were first corrected for sample tilt (flatten 0) and a mask was applied to select any particles taller than
200pm (chosen by selecting a height greater than the average background of the mica substrate. Particles with a volume less than 40nm3 were excluded. Over 2000
FCGBP particles were analyzed in this way.
In-Gel Digestion
After electrophoresis of FCGBP, unreduced and reduced with 10mM DTT, on a 4-20% Mini-PROTEAN TGX Stain-Free precast polyacrylamide gel (BioRad) using Tris Glycine running buffer, the gel was washed with water for five minutes and fixed with 50% methanol and 7% acetic acid for 15 minutes. After fixation, the gel was washed and stained with GelCode Blue Protein Stain for 30 minutes and then de-stained overnight with water. The following day, the bands were cut out from the gel and cut into small 1mm cubes. The gel pieces were incubated twice in 25mM ammonium bicarbonate with 50% acetonitrile for 20 minutes at 37°C. The samples were reduced with 10mM DTT in 25 mM ammonium bicarbonate and alkylated with
50 mM iodoacetamide in 25 mM in the dark. After removing the iodoacetamide solution, the gel pieces were washed and shrunk with 100% acetonitrile and allowed to dry at room temperature. The gel pieces were then rehydrated with a solution of trypsin in 25mM ammonium bicarbonate and incubated overnight at 37°C. The
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solution containing the peptides was removed the following morning. Additional peptides were extracted from the gel pieces using two washes with 0.1% trifluoroacetic acid and 50% acetonitrile. The peptides were then concentrated using vacuum centrifugation (Heto-vac) and analyzed by LC-MS/MS as described previously.
Structural analysis
Multiple sequence alignment (MSA) was performed on the internal repeated domains of FCGBP from species ranging from upper mammals to reptiles using
Clustal X. The aligned sequence for each repeat was submitted for secondary structure analysis using the s2D method (Simultaneous Sequence-Based Prediction of the Statistical Populations of Ordered and Disordered Regions in Proteins) available from the Vendruscolo lab (Centre of Misfolding Disease, University of
Cambridge). Additionally an amino acid sequence logo of each conserved domain region was generated from the multiple sequence alignment using Weblogo 3 to display the frequency of amino acids at specific sequence positions. N and O linked glycosylation sites were predicted using the NetOGLycan 4.0 server and
NetNGlycan 1.0 server. The location of these predicted glycosylation sites were input into the Prot Pi Protein tool along with different glycosylation structures (Tn,
Core1 Core2, Core 3 and Core4) and were used to predict molecular weight changes based on these modifications.
Interaction analysis
Quartz crystal microbalance with dissipation (QCM-D) was used to determine if the gel forming mucins and FCGBP interact as described previously [5]. Briefly a
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solution of FCGBP or mucin standard previously dialyzed into PBS was flowed across the gold plated quartz chip, after reaching a stable layer, any open gold surface was blocked with 0.05% BSA diluted in PBS. After achieving a stable layer and washing with PBS, a second solution was flowed across the surface of the first layer. Interactions were determined based on changes in frequency and dissipation.
A shift in frequency correlates with mass deposition onto the surface where as the dissipation shift provides information regarding how compact the layer is. This was performed with FCGBP, MUC5B standard made from Saliva and A549 cell line, and
MUC5AC from an A549 cell line in various orientations (i.e. e. baseline layer made with mucins + FCGBP applied second or the baseline layer made with FCGBP + mucins applied second.) The enriched saliva 5B standard was also fractionated to further purify the gel forming mucins from other proteins using CL2B size exclusion chromatography and the different fractions were applied to a baseline layer of
FCGBP to analyze interactions by QCMD.
The interactions were corroborated using an on chip digestion of the interacting proteins. Briefly the interacting layer that was formed and measured during the QCM-D experiment was washed and incubated overnight with trypsin.
The resulting peptides were freeze dried and prepared for LC-MS/MS analysis. Also a DDK tag bead pull down was performed after binding the myc tagged FCGBP to the beads, blocking with 0.05% BSA and then incubating with different Saliva 5B standard fractions. FCGBP was eluted by competition from the DDK beads using commercial concentrated 3XFLAG tag protein solution. The controls for both the
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chip and DDK bead pull down experiments included, FCGBP alone, FCGBP followed by 0.05% BSA, and 0.05% BSA followed by the different standard fractions.
FCGBP BS3 Crosslinking
To identify locations within FCGBP that interact with mucin, apical secretions from day 20 of the IL-13 challenge from three separate non-diseased cell culture lines were subjected to size exclusion chromatography and then treated with 5mM
BS3 cross linker according to manufacturer’s instructions. Also an aliquot of secretions was treated with BS3 cross linker prior to size exclusion chromatography.
All samples were then prepared for LC-MS/MS analysis as described previously.
The Stavrox software was used to identify cross-linked peptides.
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Results:
Immunohistochemistry of FCGBP and MUC5AC during IL-13 challenge of non- asthmatic cultures
FCGBP increased significantly during IL-13 challenge in a time dependent manner. The agarose gel western blot antibody staining of FCGBP showed a significant 94% increase in intensity (Figure 3.1A). This finding was consistent with the label free proteomic analysis which showed that the total precursor intensity
(±SE) increased 36 fold by day 15 (5.49x1011 ± 8.1x1010 vs. 1.52x1010 ± 1.19x1010
TPI) and then slightly decreased, resulting in a 32 fold increase by day 20 (4.87x1011
± 5.15x1010 vs. 1.52x1010 ± 1.19x1010 TPI) as compared to baseline FCGBP measurements (Figure 3.1B). Whole mount IHC staining of MUC5AC, FCGBP, Cilia and DAPI showed an IL-13 dependent increase in FCGBP, though it remained intracellular and by day 20 appeared to pool within the epithelial layer (Figure 3.1C).
This was different from the gel forming mucin, MUC5AC which also showed an increase in production/secretion but remained more adherent to the apical surface despite extensive washing (Figure 2.2).
Quantitation of FCGBP concentration in secretions of asthma and CF cell culture models and sputum using antibody and proteomic methodologies
This increase was not specific to IL-13 as both CF cell culture models
(detailed in Chapter 1) also showed significant increase in the total precursor of
FCGBP after 5 days challenge with SMM (7.56x108 ± 2.34x108 vs. 1.18x107 ±
9.52x106 TPI) or Ps.a (1.04x109 ± 3.48x108 vs. 6.61x107 ± 6.02x107 TPI) as compared to control (Figure 3.1D). Exosomal miRNA analysis from the Ps.a. and IL-
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13 cell cultures models revealed several differentially expressed miRNA that were predicted to affect the expression of the FCGBP gene (Figure 3.2A-C). These predictions matched the increase in FCGBP as measured by label free LC-MS/MS
(Figure 3.1B).
To validate this in vitro increase, FCGBP was measured in human sputum samples collected from non-disease controls, asthmatics, and cystic fibrosis patients. FCGBP was normalized to the total precursor intensity of Zymogen granule protein 16B which is produce by the salivary glands and was found to be relatively consistent across all subjects. Both CF sputum and asthma showed a significant increase in FCGBP as compared to non-disease controls, but were not significantly different from each other. Specifically the CF sputum increased approximately 10 fold in normalized total precursor intensity (±SE) (0.504 ± 0.14 vs. 0.051 ± 0.013
TPI) and the asthma sputum increased 26 fold (1.33 ± 0.55 vs. 0.051 ± 0.013 TPI)
(Figure 3.1E).
FCGBP purification
In an attempt to further understand the role of FCGBP in health and disease, a plasmid was constructed containing FCGBP with a c-myc tag and a neomycin resistance gene cassette, which used to transfect 293F cells (Figure 3.3C). Colonies derived from the single cell clones 4 and 11 showed strong FCGBP signal which co- localized with the c-myc antibody signal (Figure 3.4) and were used for further purification as outlined in figure 4A. Cell lysate and expression media from clone 4 and 11 were further purified by DDK immunoprecipitation. Clone 4 showed the strongest FCGBP signal co-localizing with the c-myc antibody signal at a band size
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of approximately 500+kDa and was chosen for further purification using size exclusion chromatography and anion exchange (Figure 3.3B). There was a prominent peak based on UV intensity on both the size exclusion and anion exchange chromatograms which after slot blot and immune detection proved to be
FCGBP positive peaks (Figure 3.3D&E). Purity of the final anion exchange product was verified by proteomics and SDS-Page and coomassie staining (Figure 3.7).
FCGBP conformation and structure analysis by atomic force microscopy and light scattering
Atomic force microscopy of the purified FCGBP protein revealed numerous crinkled appearing structures (images in Figure 3.5 A-C) that when quantitated showed a dominant peak at a volume of 1300nm3 followed by a smaller, though still pronounced peak at 2600nm3. At the volume of 3800-4100nm3 there appears another very slight elevation containing a low population of large FCGBP species
(Figure 3.5D). There were also two distinct populations based upon molecular weight (MW) and radius of gyration as measured by size exclusion chromatography coupled with multi angle light scattering. Specifically the larger peak (peak 1) containing a higher concentration of FCGBP had a mean MW (±SD) of 1400 ± 100 kDa and a mean radius (±SD) of 22.53 ± 2.26 nm. The smaller, lower concentration peak (peak 2) had a molecular weight of 4466.67 ± 378.6 and radius of 47.9 ±.12.8 nm (Figure 3.5E).
FCGBP BS3 crosslinking analysis
Due to the suggestion of multimerization, BS3 cross linking was performed on
IL-13 apical secretions that had high levels of FCGBP followed by size exclusion
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prior to proteomic analysis. In apical secretions from three different donor cells treated with IL-13, a cross link between 4951NMVLQTTKFCGBP4958 and
4951NMVLQTTKGLR4961 was identified. A second cross link was also identified in all three samples between 5318VDLPAEKLASVSVSR5332 and
5017APGSSKGBGEGBGPQGBPVBLAEETAPYESNEABGQLR5054 though had slightly higher error (Figure 3.6A&B).
Proteomic analysis and localization of reduced FCGBP bands
The band staining pattern in coomassie showed one prominent band in the unreduced sample which was above the highest ladder marker (460kDa) and thus approximated to be around 500 kDa. Of note, there was significant material at the top of the well that showed minimal migration into the gel likely due to size (Figure
3.7A, unreduced). Interestingly, the reduced sample showed 3 prominent bands at
118kDa, 66kDa, and 51 kDa (Figure 3.7A, reduced). To understand how FCGBP was cleaved after reduction into three asymmetrical bands, and in gel digestion followed by LC-MS/MS was performed on the three excised bands. The three bands contained 50-200 unique tryptic and Semi-tryptic peptides that when mapped to the protein backbone showed a distinct pattern. Band 118kDa contained peptides predominately from the C terminal whereas the 51kDa band covered the N terminal and also 3 repeating regions in the interior of the protein. The 66kDa band also contained peptides from 3 repeating internal regions unique from those identified from the 51kDa band (Figure 3.7B).
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FCGBP computational analysis
Computation analysis using a multiple sequence alignment containing
FCGBP sequences from xenopus to human revealed 3 sets of 3 unique repeating domains within the interior of FCGBP. It is interesting to note that though each type of domain was present in all genera analyzed, the 3 sets of 3 repeats was unique to upper primates. This repeating pattern is diagramed in Figure 3.8A and the sequence similarity of the different domains is exemplified in the dendrogram (Figure
3.8B). Interestingly 11 of the 12 repeated regions contain a GDPH cleavage site
(grey arrows in Figure 3.8A) which is highly conserved evolutionarily (Figure 3.8C).
The predicted domains (vWF-D, TIL, and vWF-C domain) within each repeating region are exemplified in Figure 3.9. Further computational analysis and secondary structure analysis of the specific domains revealed that each domain began with a strong helix, C8 region that contains conserved Cysteines as is evident in the height of the Web logo below (Figure 3.10). This is followed by a weak strand region that is the TIL domain and then by a strong strand region which begins the vWFC/TILa domain and continues on into the vWFD domain (Figure 3.10). Each repeat ends in another region of strong helix. Though this analysis was performed on individual domains, the domains are continuous with one another, thus though the figure shows the domain starting and ending with a region of strong helix, in the native molecule this likely represents one continuous region of helix.
Semi tryptic peptide analysis of FCGBP GDPH cleavage sites
A GDPH cleavage site was also found to be highly conserved and repeated in
11 out of the 12 domains (Figure 3.8C). Because of this regions predicted activity,
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we assessed if within our purified FCGBP this particular site was found cleaved. The semi-tryptic analysis of the in gel digestion of the reduced and unreduced samples revealed that this region was in fact found in its cleaved form several hundred times and made up a significant percentage of the peptides identified (Chapter 3 Table 1).
Predominately peptides at the C terminal side of this cleavage site, beginning with
PH- were identified. Identification of this cleavage site, led us to redefine the domain regions that could account for the different band sizes.
Molecular weight and glycosylation predictions of FCGBP domains based on GDPH cleavage sites
Dividing FCGBP into domains based on the GD/PH cleavage sites resulted in 12 domains. After molecular weight prediction, the protein backbone of the C terminal and domain 12 had a molecular weight of approximately 90 kDa (Figure
3.11), whereas the interior repeated domains fell into two categories predicted to be approximately 40kDa and 45kDa (Figure 3.11). To determine if glycosylation could account for the differences between the predicted molecular weights based on
GD/PH cleavage sites and those measured in the SDS-PAGE, an in silico prediction of O glycosylation and N glycosylation sites was performed (Chapter 3 Table 2).
Because the glycosylation of FCGBP is not known, five simple core models (Tn and cores1-4) without any further/ more complex modification were used to predict the molecular weight. In doing this, the MW of the domains increased nearing those that were predicted by the SDS-PAGE (Chapter 3 Table 3).
The cleaved GDPH sites were also found in the sputum and cell culture models. Interestingly in contrast to the purified protein, only the N terminal portion of the cleavage site, ending with -GD was identified.
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FCGBP layer properties and interactions with the MUC5B and MUC5AC standards
QCM-D analysis of FCGBP protein alone revealed that it could form a mucin like layer with an average thickness of 28.6 ± 0.912 nm, viscosity of 1.869 ± 0.041 cP and an elasticity of 9771 ± 434.8 Pa (Figure 3.12 A-C). Interestingly when adding
FCGBP to a pre-formed mucin layer of MUC5B or MUC5AC, there was little binding based on the shift in dissipation or frequency (Figure 3.13 A-C). When adding the gel forming mucins to a pre-formed FCGBP layer, MUC5AC did not bind but there was significant binding of MUC5B standard (Figure 3.13 D-F) This binding only occurred with MUC5B enriched from saliva and not when purified from A549 5AC knockdown cell lines (Figure 3.13 D vs. E). To determine what other proteins in the
MUC5B standard could be binding to FCGBP, size exclusion was performed on the
MUC5B standard to further purify the gel forming mucins from other smaller globular proteins/small mucins. It was evident that the gel forming mucin within the V0 did not bind to FCGBP (Figure 3.13G) but a protein in the included fractions 17-20 and 21-
24 did bind (Figure 3.13H&I). Pull downs using two different methods, DDK immunoprecipitation and QCM-D on-chip digestion (Chapter 3 Table 4), revealed a small group of proteins that were only identified when FCGBP was first present on the bead or chip and thus did not exhibit non-specific binding to the pull down substrate. Cross linking analysis did not identify any cross links between these candidate proteins and FCGBP.
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Discussion
The structure and function of FCGBP in health and disease within the airways is not known. We showed that FCGBP is differentially expressed in two different cell culture models of airway diseases, cystic fibrosis and asthma, both of which are characterized by inflammation and pathological mucus. We confirmed our in vitro findings with sputum from patients with these same respiratory diseases which also showed a significant increase in the FCGBP as measured by mass spectrometry.
These significant increases in the FCGBP protein could be mediated by miRNA regulation as we identified specific differentially expressed miRNA in the CF and asthma cell cultures models that were predicted to increase FCGBP gene activity paralleling the increase in protein levels. Though the regulation is likely more complex than a single miRNA, the identified miRNA warrant further validation to determine their effect on FCGBP and for potential use as a method for controlling
FCGBP expression in order to elucidate its function.
To address the lack of structural information relating to FCGBP and predict which regions are likely necessary for its proper functionality, we undertook a computational bioinformatics approach to create a multiple sequence alignment of the entire FCGBP protein and use this to identify conserved amino acids and secondary structure. Though homologs are present in earlier species, due to the high degree of divergence in these earlier species, the multiple sequence analysis was limited to FCGBP sequences from frog to human. This revealed that the internal repeats were evolutionarily conserved though the number of duplications was different across species. For example, only humans and upper primates showed the
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three clusters of the three different domains whereas earlier species had fever sets of the three repeats. It is common for duplications, whether driven by structural of functional factors, of several domains to occur at one time and to accumulate in higher species. It has been reported that proteins with repeated sequences of domains usually function in protein -protein interaction networks and complex assembly.
Each of the domains shared similar secondary structure including: region of helix, weak stand and strong strand and in addition to highly conserved, likely structural cysteines, and an autocatalytic GD/PH cleavage site was found in 11 of the 12 domains. This site has also been identified in the gel forming mucins, MUC2 and MUC5AC [174]. Once cleaved the aspartate is predicted to form a reactive anhydride capable of forming new covalent bonds and acting as a crosslink between different proteins [174]. The presence of the cleaved GD/PH site was evident in the purified FCGBP from 293F cells, the in vitro apical secretions, and the in vivo sputum as shown by an abundance of Semitryptic peptides with GD at the C terminal or PH at the N terminal. Interestingly, previous publications have suggested that it is the aspartate that participates in the new covalent bond [173-175]. Peptides representing both sides of the cleavage site were identified in the purified FCGBP from 293F cells, but interestingly in the cell culture and sputum samples, only the N terminal side of the GDPH cleavage site was identified and thus not covalently bound to another peptide/protein. In contrast, the C terminal peptide (beginning with
PH) was never identified in the cell culture and sputum samples and thus could be bound to another peptide and not identified by the current MS/MS method. The
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cleaved GD/PH sites were not only present after reduction, but were identified at a similar frequency within the unreduced 500kDa band suggesting that disulfide bonds among the highly conserved cysteines are responsible for maintaining the structure of FCGBP despite these cleavages.
Characterization of the purified FCGBP indicated that this protein likely forms multimers. This is supported by the atomic force microscopy volume distributions which showed a prominent peak at 1300 nm3 in addition to two other peaks found at multiples of this volume, 2600 and 3900 nm3. This same trend was evident in the light scattering determination of molecular weight which revealed two different peaks: one (Peak1) with higher concentration but lower molecular weight and radius and the second (peak 2) with three time higher molecular weight, double the radius, but lower in concentration. Several important conclusions were evident in these measurements. Firstly, the appearance of FCGBP on the AFM chip indicates that there is substantial glycosylation as it spread out in a similar manner as the gel forming mucins rather than as a discrete node typical of globular proteins. This likelihood of glycosylation was strengthened using in silico prediction of O glycosylation and N glycosylation sites of the FCGBP protein backbone, which returned numerous different sites listed in Chapter 3 Table 2. The second conclusion from these measurements is that FCGBP likely multimerizes. This was supported by the light scattering data, in which Peak 1 measured at molecular weight that was 2+ times greater than what would be predicted based on the protein backbone of
FCGBP. If one accounts for the glycosylation, then this MW could indicate that
FCGBP forms a dimer. This conclusion was further strengthened through the
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identification of cross-linked peptides located at the C terminus of FCGBP. The two cross-linked peptides were from the same peptide region and therefore it is unlikely that this represents an intraprotein crosslink. This multimeric form of FCGBP may also account for the larger MW material that exhibited minimal migration in the SDS-
PAGE gel and remained at the top of the well (Figure 3.7A).
Previously, studies have reported a similar asymmetrical band pattern after western blot of reduced FCGBP and suggested that some form of autocatalytic cleavage was occurring [171, 172, 174, 176]. Evidence of cleaved GDPH sites, glycosylation of the protein backbone, combined with the localization of the unique peptides identified from the different excised bands, supports the premise that the 66 kDa band represents the larger internal domains (Cluster B from Figure 3.11) based on predicted molecular weight whereas the lower more intense band at approximately 51 kDa represents the lower molecular weight, repeated, internal domains (Clusters A and C from Figure 3.11) and the N terminal. The largest band at approximately 118kDA likely represents the C terminal. This is based on the molecular weight prediction and also the colocalization and identification by LC-
MS/MS of the myc tag with this band. The molecular weights predicted for each domain cluster and the termini would result in smaller bands than is indicated by the
SDS PAGE gel but after adding in simple core sugars based on in silico predicted glycosylation sites, the predicted and actual molecular weights become more comparable. It should be noted that the glycosylation of this protein is not known and therefore the sugar decoration and modification could be more complex than these calculations represent.
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Measurement of the layer properties formed by purified FCGBP by QCM-D revealed that FCGBP could formed a ~30nm thick layer with an elasticity and viscosity similar to mucus. Interactions between pre-formed layers of the different gel forming mucins (MUC5B enriched from saliva and A549 MUC5AC knockout and
MUC5AC from A549 MUC5B knockdown cell line) and FCGBP revealed minimal interactions as measured by shifts in dissipation or frequency. Interestingly, when the order was reversed and the mucins were flowed over a pre-formed layer of
FCGBP, there was a dramatic shift in both frequency and dissipation with the
MUC5B saliva standard. The directionality of this interaction could have been attributed to accessibility of the proper binding location (i.e. the portion of MUC5B responsible for the FCGBP interaction was bound to the gold plated QCM-D chip in the first experimental set up and thus could not bind FCGBP, but this portion was accessible and able to bind when in solution and flowed across the FCGBP layer).
This was ruled out when no interaction was evident even after coating the gold chip with Poly L Lysine which would alter the charge and consequently what portion of the MUC5B bound to it. Thus a second explanation could be that FCGBP was interacting with a different protein within the enriched MUC5B standard. This indeed was the case as the fractioned MUC5B only showed a shift in frequency and dissipation when the non-gel forming mucin fractions 17-20 and 21-24 were flowed over the pre-formed FCGBP layer and not the V0 region containing MUC5B. In an attempt to determine which proteins were interacting with FCGBP, a pull down using the QCM-D chip and the DDK tagged beads was performed and the eluted fractions analyzed by label free LC-MS/MS. Unfortunately very few proteins were identified
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and among those many were common proteomic containments derived from keratin which likely occurred during the sample preparation process. There are several explanations for the lack of interaction. It is known that different cell types express different glycosylation machinery and thus it is likely that the FCGBP produced in the
293T cells exhibits different glycosylation [180]. If the interaction between FCGBP and mucin is mediated by their carbohydrate decoration, then an alteration in this could eliminate an interaction. Also the conditions may not be suitable for an interaction to occur. If the interaction between mucin and FCGBP occurs within the mucin granule and requires specific proteins, pH, or ions to facilitate this process then it is unlikely that we would be able to replicate this interaction using PBS [181].
This latter idea is supported by recent preliminary RNAscope images (Supplemental
Figure 3.14) that show strong expression of FCGBP within the submucosal glands thus supporting the hypothesis that the interaction with MUC5B, which is also produced in the submucosal glands, occurs intracellularly prior to secretion [6]. This also challenges the assumption that FCGBP is produced primarily by goblet cells, as little staining was evident in the goblet cells as compared to the submucosal glands.
The function of FCGBP remains unknown though its increase in expression and secretion is evident in a various inflammatory conditions including the airway diseases asthma and cystic fibrosis. Further work will be required to determine what areas of the FCGBP protein are required structurally and functionally such as site directed mutagenesis of the C terminal region identified by the cross linked peptides to determine if the multimerization of FCGBP is inhibited or of the GDPH cleavage sites to prevent their autocatalytic cleavage. To test the effect of these mutations on
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the function of FCGBP, a function must first be known. It is likely the function will be elucidated in future studies involved the FCGBP knockout mouse model, which is currently being generated. Though the story is not (or ever) complete, this work provides a framework and a purified FCGBP protein with which future experiments can build off of and utilize in order to determine the role of this large glycosylated protein in mucus both in health and disease.
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Chapter 3 Figures
Figure 3.1. Secretion of FCGBP significantly increases in inflammation and infection. A) Label free quantitation of FCGBP secreted during the 20 day IL-13 challenge (n=5) B) Representative agarose gel electrophoresis of FCGBP antibody intensity during 20 day IL-13 challenges (n=5) with quantitation below. Antibody intensity is normalized to highest value within each repeat and each line represents a different repeat(n=5) C) Whole mount IHC of non-disease cultures at Day 5 with and without IL-13 stimulation and at Day 10 and 20 of IL-13 challenge (MUC5AC: red, FCGBP: Green, Cilia: white, Nuclei: Blue). D) Comparison of FCGBP concentration in apical secretions as measured by LC-MS/MS in CF (Ps.a. and SMM) and Asthma (IL-13) cell culture models. FCGBP values represent concentration at endpoint of each treatment (CF models: 120 hours and Asthma: 20 days). E) Concentration of FCGBP in sputum from non-disease (controls), CF, and Asthma patients. To correct for differences in sputum starting volume, FCGBP was normalized to ZG16B a salivary protein found at equal concentrations among all groups. Repeat measure (without multiple comparison correction), traditional Anova and paired Student’s t-test were used to analyze these changes with the following p values: *≤0.05, **≤0.01, ***≤0.001. Unless otherwise noted, data is presented in Tukey box and whisker plots.
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Figure 3.2. Signficantly decreased miRNA predicted to increase FCGBP expression. Significantly differentially expressed miRNA isolated from exosome like vesicles following treatment with (A) Ps.a and IL-13 of (B) non-disease cultures and (C) asthmatic cultures. In silico interaction pathway and activity prediction was generated using the Ingenuity Pathway Analysis software Grow and Molecular Activity Predictor overlay functions. Only miRNA with an adjusted p value <0.1 were included in this analysis. Orange coloration designates predicted activation. Green coloration represents decreased log2Fold change of the individual miRNA determined experimentally.
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highestvalue (black)of slo (green),protein abundance as measuredby UV (magentaand overlay FCGBP antibody intensity normalized to the usingblotsize exclusion fractions.RepresentativeE) anionexchange chromatogram with concentrationsalt gradient measuredby UV signal (blue) and overlay FCGBPantibody intensitynormalized to the highest value (black) of sl and clone11. C) Plasmid construct.RepresentativeD) size exclusionchromatogram with proteinabundance immunostainedFCGBP for (red)and c clone and fromwhat source FCGBPwill purifiedbe from. B) Western blot from DDk the tag immunoprecipitation FCGBPof beginning with DDKtag immunop Figure 3.3.FCGBP purification strategy andcharacterization
t blot usingblot t size exclusionfractions.
-
myc(green) using celllysate (left lan
recipitation (IP)of the expanded single cellclones to determinewhich
.Panel Ashows the threesteps usedpurification in
es) and es) media (rightlanes) forclone 4
ot
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Figure 3.4. Unreduced SDS-Page of 293F clones’ cell lysates following transfection, single cell selection, and expansion. Blot was probed with monoclonal c-myc (green) and polyclonal FCGBP (red) with the (A) overlay of both channels and separate channels showing FCGBP (B) and c-myc (C) individually below. Lane designations are shown in the table on the right. White stars indicate clones (4&11) showing co-localization.
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Figure 3.5. Macromolecular characterization of purified FCGBP. Representative atomic force microscopy images of FCGBP with 100 nm scale bar(A-C) with the volume quantitation of 2000+ particles below showing frequency distribution of volumes (nm³)(D). E) Representative light scattering chromatograms of purified FGCBP with average molecular weight and radius of gyration (± SD) of the two main peaks as measured by SEC-MALS (n=3) in the table below.
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cross identifiedwas that assigned tothe specific crosslink and thecolor indicates the relative intensity. The peptides found links identifiedwere i chromatography isolateto theFCGBP rich populationfor furtheranalysis by label freeLC secretionsfollowing IL Figure 3.6.BS3 identifiedcross
-
linked inAand Bare located
dentified atleast once in allthreesamples analyzed. Eachcoloredbar indicates unique a peptide
-
13 treatmentwere incubated with BS3 crosslinker and thensubjected to size exclusion
-
linkedpeptides fromFCGBP peptidewith fragmentation chromatogram.
at C the terminusof FCGBP and are not found repeatedin otherregions
-
MS/MS. Eachof the cross
ofFCGBP.
Apical
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similarto the colored bars in thefigure (B)above. FCGBPprotein backbone arranged N terminalto Cterminal from left to right.The verticallines correlate withmap each verticalline indicating thelocation backboneamino acid sequence.The charts in panelCrepresent a consolidated version of coverage the representsa specific peptide identified in thatsample. The X axis representsthe FCGBP protein peptides unique 66kDato the bandand the blue uniqueare 51kDato the Eachband. dash on the geldigestion. The green indicatebars unique peptidesfound in 118 the kDa band,the orange are peptides identifiedin the specific weightladder is present in thetable below.B) FCGBP protein coverageby unique trypticand purifiedFCGBP unreduced (left) and reduced(right). Lane analysis and comparison tohigh molecular bands based on Figure 3.7.Bandpattern ofpurified, unreduced, and reduced FCGBP and localizationof reduced
Semi
-
trypticLC
FCGBPbands thatwere excised and analyzed by LC
-
MS/MSanalysis
ofa peptide unique to thatsample alignedto the
. A) Coomassie.A) blue stainingof S
DS
-
MS/MSafter in
-
PAGEgel of
Semi
Yaxis
tryptic
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alignment.The tallerthe letterthe more conservedis. it indicatesthe frequency at which isoccurs in the multiple sequence web logothe (C) where the heightof a letter at a specificlocation highconservat end ofeach vWF Greyarrow indicates the GDPHcleavage site locatedat N the terminal times(3,4,5) (6,7,8)and (9,10,11)within theFCGBP protein sequence. domains,a repeatingpattern of triplic begins with a vWF sequencesimilarity as represented in the dendrogram. (B) Eachdomain Diagramofgeneral FCGBP domains coloredbased degreeon of Figure 3.8.Homology andrepeated domain pat
ionwithin each domainfrom frog tohuman as shown in
-
Ddomain. Computational analysis of this regionshows
- Ddomain and withthe exception of the first two
ate domainsate becan seen three
ternof FCGBP
.A)
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My within each ofthe vWFC domains is aTILa region. Imagewas generatedusing Prostie sequenceof vWFD domains (green)TIL domains(blue) and vWF C domain (Orange.Not shown Figure 3.9 Diagramof FCGBP protein backbone domain and structure
Domainsimage creator.
shows repeating
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regionslikely play animportant structural and/or functional role. cleavage species..Based onthe logos, regionsof highly conserved amino acids are apparent.For FCGBP thisincludes theGDPH bottom.The heig domainssub are listed at the top of each paneand weblogo a generatedfrom the multiple sequence alignmentispresent at which repr analysis. sequence Figure 3.10.Computational FCGBP structural predictionof 1 of the 12 repeateddomains based on multiple
site and
esentareas of strong helix (tallblue bars),week strand (short green bars,)and strong strand (Tallgreen bars). The
htof theletter in
cysteines,w
Secondarystructure ofone repeated domain is indicatedby the blue and green verticalbars above
hich are likelyinvolved in disulfidebonds. The high degree of conservation indicatesthat these
thelogo
represents the frequencyat which occurs it at thatposition across hundreds of
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Chapter 3 Table 1. Abundance of Semitryptic peptides at GDPH cleavage sites identified by LC-MS/MS after SDS-PAGE in-gel digestion of reduced FCGBP bands (118, 66, 51 kDa) and single unreduced band (500kDa). Number of times each peptide was identified in the reduced and unreduced bands is presented with and without normalization to the total number of Semitryptic peptides found within each sample as a measure of relative abundance (%). # of identifications # of identifications normalized to total # C terminal N terminal Peptide Semi-tryptic peptides Peptide Reduced Unreduced Reduced Unreduced Bands Band Bands Band EGGEVSCEPSSCGPHETCRPSGGSLGCVAVGSTTCQASGD PHYTTFDGR/R 382 51 14.16% 6.06% EQGGQGVCLPNYEATCWLWGD PHYHSFDGR/K 96 161 3.56% 19.14% CGPGGGSLVCTPASCGLGEVCGLLPSGQHGCQPVSTAECQAWGD PHYVTLDGHR 457 102 16.94% 12.13% PHYVSFDGR/R 229 36 8.49% 4.28%
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Chapter 3 Table 2. Predicted O and N linked glycosylation sites based on in silico prediction software NetOGlyc 4.0 and NetNGlyc 1.0, respectively. Disulfide bond prediction site pairs are based on Uniprot annotations. GDPH cleavage sites obtained from alignment to FGCBP (Q9Y6R7) using NCBI blastp Modification Amino Acid Location 81, 435, 638, 661, 761, 769, 772, 798, 825, 834, 1036, 1043, 1053, 1196, 1432, 1438, 1444, 1466, 1550, 1553, 1641, 1649, 1708, 1836, 1843, 1860, 1863, 1885, 1891, 1961, 1966, 1969, 1974, 1975, 2245, 2250, 2253, 2259, 2272, 2356, 2428, 2429, 2435, 2439, 2442, 2627, 2634, 2639, 2645, 2667, 2669, 2674, O- 2751, 2754, 2850, 2909, 3037, 3044, 3061, 3064, 3086, 3092, Glycosylation 3167, 3170, 3175, 3176, 3446, 3451, 3454, 3460, 3473, 3557, 3636, 3643, 3828, 3835, 3840, 3846, 3868, 3870, 3952, 3955, 4043, 4051, 4110, 4238, 4245, 4262, 4265, 4284, 4285, 4287, 4293, 4368, 4371, 4376, 4377, 4647, 4652, 4655, 4661, 4674, 4755, 4758, 5007, 5020, 5021, 5062, 5113, 5125, 5128, 5137, 5142, 5145, 5148, 5149, 5253, 5254, 5255 N- 75, 91, 1405, 1497, 1743, 1830, 2111, 2698, 3312, 3899, 3926, Glycosylation 4145, 4232, 4414, 4513, 4540, 4992, 5001, 5186 494, 502, 886, 894, 1274, 1282, 1695, 1704, 2094, 2102, 2475, Disulfide bond 2483, 2896, 2905, 3295, 3303, 3676, 3684, 4097, 4106, 4496, 4504, 4878, 4886 476-479, 868-871,1256-1259, 1677-1680, 2076-2079, 2457- GDPH Cleavage 2460, 2878-2881, 3277-3280, 3658-3661, 4079-4082, 4478- Sites 4481
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Figure 3.11. Predicted molecular weight of FCGBP domains based on GD/PH cleavage sites. MWs were calculated based on amino acid sequence using the ExPASy compute pI/Mw tool. Domains were grouped based on similar molecular weights which results in three clusters designated as A, B, and C of the internal repeated domain regions.
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Chapter 3 Table 3. Predicted molecular weight (kDa) based on protein backbone and various glycan core structures of full length FGCBP and specific domains representative of clusters A, B, & C domains and termini 1 3 4 12 Full
Protein 50157.2 40726.5 45830.8 97481 572016.68
Tn 50969 41943 47860 102962 600,028 Core1 51293 42916 49157 106529 619,323 Core2 51700 44135 50783 110999 643503 Core3 51375 43162 49485 107432 624208 Core4 51782 44381 51111 111902 648388
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Figure 3.12. FCGBP layer properties based on viscoelastic broadfit model of QCM-D frequency and dissipation measurements (n=7). A: Plot of FCGBP layer thickness (B) Shear elasticity and (C) Viscosity. Each line represents an individual repeat. Summary tukey box and whisker graphs are provided below. Note the units between the plot and box and whisker graph differ for elastic modulus and viscosity to allow direct comparison to literature values of mucin layer properties.
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Figure 3.13. FCGBP and mucin interactions as measured by quartz crystal microbalance with dissipation showing shifts in frequency (F) (orange, right y-axis)) and dissipation (D)(blue, left y-axis)) after application of different proteins A-C: Represent orientation of Mucin standards applied first followed by BSA to block and then FCGBP (n=3 for each standard, left to right: Salivary MUC5b, A549 MUC5B, A549 MUC5AC). D- F: Represent orientation of FCGBP applied first followed by BSA to block and then the different mucin standards (n=3 for each standard, Left to right: Salivary MUC5b, A549 MUC5B, A549 MUC5AC). G-I: Interaction between FCGBP applied first and the different constituents present in the enriched Salivary 5B standard separated by size exclusion chromatography (left to right: Fractions 8-9 (gel forming mucins), Fractions 17-20, Fractions 21-24).
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Chapter 3 Table 4 Proteomic results of DDK or QCM-D chip pull down. Identified proteins were excluded if they bound to the substrate in the absence of FCGBP or if they were found as a contaminant in the FCGBP DDK Bead pulldown candidates QCM-D chip pulldown candidates Fractions 17-20 Fractions 21-24 Fractions 17-20 Fractions 21-24 Desmoplakin Desmoglein-1 Keratin, type I cytoskeletal 16 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 17 Desmocollin-1 Peroxiredoxin-1 Keratin, type II cytoskeletal 4 Desmoglein-1 Keratin, type II cytoskeletal 1b Elongation factor 1-alpha 1 Peroxiredoxin-1 BPI fold-containing family B Junction plakoglobin Caspase-14 member 1 Elongation factor 1-alpha 1 Galectin-7 Desmoplakin Hornerin Desmoplakin Keratin, type II cytoskeletal 6C Junction plakoglobin Lysozyme C Keratin, type I cytoskeletal 13
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in panelA. Quantitation isongoing. Image courtesyof Dr. Kenichi Okuda images showingFCGBP (red) localization proximal in CF lung tissue.B representsthe magnifiedgrey box SupplementaryFigure3.14. Localization of FCGBP in CF lung tissue byRNAscope.
PreliminaryRNAscope
edregion
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SUMMARY AND FUTURE DIRECTIONS
The mobile mucus layer lining the respiratory epithelium is a vital part of the lung’s innate immune defense, though if dysfunctional, can have detrimental effects.
Two etiologically diverse airway diseases characterized by pathologic mucus are cystic fibrosis and asthma. This body of work has examined the manner in which mucins and their interacting secreted proteins are altered in the heavily infected and inflammatory CF lung environment as compared to the predominately TH2 driven, inflammatory asthmatic environment. We showed that the two gel forming mucins,
MUC5AC and MUC5B, occur in drastically different ratios in each disease, with
MUC5B dominating in the CF environment and MUC5AC in the asthmatic.
Regardless of this change, the macromolecular structure of the secreted mucins is not significantly altered in either disease state, though there is a slight reduction in the molecular weight of the secreted mucins isolated from the in vivo and in vitro CF models. This decrease may be a result of glycosylation differences between normal and CF sputum. By comparing these differences in glycosylation to the microbiome data, we discovered a strong relationship between the sialylated glycans decorating the gel forming mucins and the predominant bacterial genera. Specifically, the bacteria commonly associated with the oral flora community are associated with the least amount of sialylated glycans. There are several potential explanations that warrant further investigation. The first is that specific groups of bacteria can utilize
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the sugars decorating the mucin, accounting for their decrease abundance on the isolated gel forming mucins. The second is that the epithelium responds to the presence of different bacteria and alters the glycosylation of the gel forming mucins accordingly. Analysis of the expression pattern of the glycosyltransferases in the presence of different bacteria, or glycomics of the intracellular gel forming mucins would shed light upon this question. Additionally, glycan analysis of the gel forming mucin standards after exposure to different bacteria genera would elucidate if the bacteria were truly cleaving the sugars. Not only does this study highlight the connection between the gel forming mucins and the microbiome, but the significant increase in sulfo sialyl lewis X epitope highlights an additional relationship between the gel forming mucins and the host inflammatory response. The implications of the increased abundance of this specific glycan have yet to be fully explained. Future work will also be required to understand the rheological consequences of altered mucin glycosylation.
An interesting similarity between the mucins isolated from the in vitro asthma and CF models, was a significantly decreased size of the intracellular/stored gel forming mucins. This commonality likely reflects an alteration in the production or packaging of the gel forming mucins that occurs in hyper-secretory states regardless of the stimuli (different types of inflammation and bacterial infection). The exact details of mucin production and secretion are not completely understood, but our work suggests there is a different pathway or mechanism at work that facilitates hypersecretion of mucin.
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In regards to the regulation of mucin, analysis of the apically secreted exosomal miRNA in the in vitro asthma and CF models revealed numerous candidate miRNA that are predicted to target the MUC5B gene and also the enzymes responsible for O-glycosylation. Though these regulatory effects are predicted and require further validation, they show promise as the predictions correlate with the measured MUC5B protein concentration changes in these models.
This work suggests that the epithelial cells have the capacity to not only regulate the expression of the gel forming mucins but can also refine and alter the glycans decorating them. As highly glycosylated proteins, these changes have the potential to significantly modify the mucins and resulting mucus properties.
The gel forming mucins act alongside numerous other secreted mucin interacting proteins to confer mucus with its rheological and functional properties.
We showed that many potential mucin interacting proteins are differentially expressed/secreted in both CF and asthmatic disease models. One of the most significantly changing proteins in both disease models was FCGBP, which we showed was also elevated in sputum from individuals with CF and asthma. To better understand the structure and mucin interacting functionality of this protein, we, for the first time, expressed, purified, and characterized FCGBP. Our preliminary studies showed that this protein is expressed as a glycosylated multimer predominately in the submucosal glands. Work is ongoing to understand if and how
FCGBP interacts with mucin and by doing so, how this interaction alters the mucus properties leading to a more adherent and static mucus layer.
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By characterizing the gel forming mucins, the changes in the mucin interacting proteins, and the regulation of the mucin genes and glycosylation machinery by exosomal miRNA, this work highlights disease specific changes that could potentially change the properties of the resulting mucus layer and be the impetus in the development of a pathologic mucus. Continued validation and further examination of the consequences of these changes is required, but importantly this work provides the foundation and springboard for these studies. Hopefully this work will help elucidate the pathogenesis of mucostasis and muco-obstruction and be useful in guiding and informing the development of more effective therapies to relieve the burden of these devastating airway diseases.
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APPENDICES
Appendix 1. Unique proteins identified in HBE apical secretions after 120 hours challenge with Ps.a (n=5) or TSB (Control) (n=5) and significantly (p<0.05, paired student t-test) changing proteins after challenge with associated log2 fold change of total precursor intensity per protein between Ps.a. and Control.
Log2 Significantly Changing Proteins Apical Secretions: Ps.a. 120 hours Fold Apical Secretions: Control 120 hours Chang Ps.a. vs. Control e
Pendrin Bone morphogenetic protein 3 IgGFc-binding protein 6.0
Ectonucleotide pyrophosphatase/phosphodiesterase family DPYSL3 protein Intercellular adhesion molecule 1 2.6 member 3
cDNA FLJ55549, highly similar to 3-ketoacyl-CoA Tyrosine-protein kinase receptor OS=Homo STE20-like serine/threonine-protein kinase 1.8 thiolase, peroxisomal (EC 2.3.1.16) sapiens GN=SLC34A2-ROS1 PE=2 SV=1
Fer-1-like protein 6 Golgi resident protein GCP60 cDNA FLJ53342, highly similar to Granulins 1.8
Protein S100-P Casemin kinase II subunit beta Sodium-dependent ph 1.8
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Tissue-type plasminogen activator Follistatin Chloride intracellular channel protein 1.6
cDNA FLJ38393 fis, clone FEBRA2007212 Coatomer subunit alpha Protein-lysine 6-oxidase 1.6
cDNA, FLJ94557, highly similar to Homo sapiens Neutrophil gelatinase-associated lipocalin 1.5 FK506 binding protein 4, 59kDa (FKBP4), mRNA
Far upstream element-binding protein 2 Serum amyloid A-1 protein 1.5
cDNA, FLJ95012, highly similar to Homo sapiens Uncharacterized protein UDP-glucose pyrophosphorylase 2 (UGP2), 1.5 mRNA
cDNA, FLJ94599, highly similar to Homo sapiens Alpha-1-antichymotrypsin 1.5 GDP-mannose 4,6-dehydratase (GMDS), mRNA
S-methyl-5'-thioadenosine phosphorylase Retinoic acid receptor responder protein 1 1.4
Protein-L-isoaspartate O-methyltransferase EH domain-containing protein 1 1.3
60S ribosomal protein L9 (Fragment) Insulin-like growth factor binding protein 3 1.3
Thymidine phosphorylase Potassium-transporting ATPase alpha chain 2 1.3
Golgi apparatus protein 1 Urokinase-type plasminogen activator 1.2
3'(2'),5'-bisphosphate nucleotidase 1 Mucin-5B 1.1
Heterogeneous nuclear ribonucleoprotein M Syntenin-1 1.0
V-type proton ATPase catalytic subunit A Synaptosomal-associated protein 23 1.0
Persulfide dioxygenase ETHE1, mitochondrial Protein-glutamine gamma-glutamyltransferase 2 1.0
cDNA, FLJ92996, highly similar to Homo sapiens CD109 antigen guanine nucleotide binding protein (G protein), 0.9 beta polypeptide 1 (GNB1), mRNA
cDNA FLJ45031 fis, clone BRAWH3018548, Placenta-specific 8, isoform CRA_b 0.9 highly similar to Vinculin Na(+)/H(+) exchange regulatory cofactor NHE- RF2 Epididymis luminal protein 189 0.9
cDNA FLJ76826, highly similar to Homo sapiens Alcohol dehydrogenase class 4 mu/sigma chain 0.8 ceruloplasmin (ferroxidase) (CP), mRNA
204 2,4-dienoyl-CoA reductase, mitochondrial Myristoylated alanine-rich C-kinase substrate 0.8 (Fragment)
60S ribosomal protein L18a cDNA FLJ52936, weakly similar to Tropomy 0.8
Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, Transmembrane channel-like protein 0.8 mitochondrial
Kallikrein-12 Chloride intracellular channel protein 6 0.8
Trifunctional enzyme subunit beta, mitochondrial Beta-actin-like protein 2 0.7
Lipolysis-stimulated lipoprotein receptor Complement C3 0.7
cDNA FLJ53963, highly similar to Leukocyte Sialic acid synthase 0.7 elastase inhibitor
Destrin Brain acid soluble protein 1 0.7
Heterogeneous nuclear ribonucleoprotein A1, Chloride intracellular channel protein 1 0.7 isoform CRA_a cDNA FLJ60299, highly similar to Rab GDP dissociation inhibitor beta 0.6
Cysteine-rich protein 2 0.6
Sodium/potassium-transporting ATPase subunit 0.5 beta-1
Actin, cytoplasmic 1 0.5 14-3-3 protein theta 0.5
Cathepsin D 0.4
14-3-3 protein gamma 0.4
14-3-3 protein beta/alpha 0.3
Polymeric immunoglobulin receptor 0.3
Fructose-bisphosphate aldolase C 0.3
cDNA FLJ52712, highly similar to Tubulin beta-6 -0.4 chain
205 Gelsolin -0.5
Lysozyme C -0.8
Vacuolar protein sorting-associated protein 28 -0.8 homolog
Prolactin-inducible protein -1.0
Clusterin -1.0
Secretoglobin family 3A member 1 -1.3
Ly-6/neurotoxin-like protein 1 -3.5 -inf Lipolysis-stimulated lipoprotein receptor
Appendix 2. Unique proteins identified in HBE apical secretions after 120 hours challenge with SMM (n=5)or PBS (Control) (n=5) and significantly (p<0.05, paired student t-test) changing proteins after challenge with associated log2 fold change of total precursor intensity per protein between SMM and Control. Within this group, protein names are bolded if found both in the apical secretion after 120 hour SMM treatment and in the neat SMM used to challenge the cells. Comprehensive list of proteins identified in the SMM (pooled from 6 CF donors), challenge reagent.
Significantly Changing Proteins Log2 Apical secretions: SMM 120 Apical Secretions: Control 120 Fold SMM (reagent alone) hours hours SMM vs. Control Change
Eukaryotic translation initiation Immunoglobulin heavy constant Intraflagellar transport protein 25 DnaJ homolog subfamily B member 6 11.2 factor 3 subunit J gamma 2 homolog
Protein OSCP1 Dynamin-1-like protein Alpha-2-macroglobulin 10.3 Coatomer subunit gamma-1
Dynein intermediate chain 2, Immunoglobulin lambda-like N(G),N(G)-dimethylarginine Serine protease 23 10.0 axonemal polypeptide 5 dimethylaminohydrolase 2
Beta-galactoside alpha-2,6- UPF0568 protein C14orf166 Alpha-1-acid glycoprotein 1 8.9 Alcohol dehydrogenase class-3 sialyltransferase 1
Glutamate--cysteine ligase 26S proteasome non-ATPase Immunoglobulin kappa constant 8.8 Gamma-glutamylcyclotransferase regulatory subunit regulatory subunit 12
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60S ribosomal protein L13 Regulator of nonsense transcripts 1 Alpha-1-antitrypsin 8.5 AP-2 complex subunit alpha-2
Intraflagellar transport protein 81 Immunoglobulin heavy constant Cytochrome b-245 heavy chain 8.2 AP-2 complex subunit alpha-1 homolog gamma 1 (Fragment)
Alpha-N-acetylgalactosaminide GMP synthase [glutamine- Echinoderm microtubule- Plastin-2 8.1 alpha-2,6-sialyltransferase 1 hydrolyzing] associated protein-like 2
X-ray repair cross- Histidine--tRNA ligase, cytoplasmic Legumain Phospholipase B-like 1 8.0 complementing protein 6
Immunoglobulin heavy constant Immunoglobulin heavy variable Phosphoserine aminotransferase FAD-linked sulfhydryl oxidase ALR 8.0 alpha 1 5-51
Ribokinase Poly [ADP-ribose] polymerase 4 Lactotransferrin 7.5 Dynactin subunit 2
Kunitz-type protease inhibitor 1 Caspase-7 Lysozyme C 6.7 Mucin-4 (Fragment)
Neural cell adhesion molecule L1-like Prostaglandin reductase 1 Twinfilin-2 6.4 Pseudouridine-5'-phosphatase protein
Transitional endoplasmic Protein PRRC1 Dynein asSEbly factor 5, axonemal Mucin-5B 6.4 reticulum ATPase
DNA dC->dU-editing enzyme Glutathione peroxidase Ribonuclease T2 (Fragment) 6.4 Matrin-3 APOBEC-3B
Bis(5'-nucleosyl)- Kynureninase Inositol-3-phosphate synthase 1 Plasma protease C1 inhibitor 5.7 tetraphosphatase [asymmetrical]
Disintegrin and metalloproteinase Pyridoxine-5'-phosphate oxidase Chitinase-3-like protein 1 5.5 Adenosine deaminase domain-containing protein 9
Glutamate--cysteine ligase catalytic Intraflagellar transport protein 20 Serum albumin 5.5 Signal-regulatory protein beta-1 subunit homolog
Rho guanine nucleotide DNA damage-binding protein 1 UPF0553 protein C9orf64 Cystatin-A 5.5 exchange factor 1
Eukaryotic translation initiation factor Annexin A6 Fructose-1,6-bisphosphatase 1 5.5 Ras-related protein Rab-2A 3 subunit D
Erythrocyte band 7 integral GDP-L-fucose synthase SH2 domain-containing protein 4A Protein S100-A8 5.5 membrane protein
LIM and cysteine-rich domains protein 207 Ribonuclease 4 Haptoglobin 5.4 Fatty acid synthase 1
Nascent polypeptide-associated Transcriptional activator protein Pur- complex subunit alpha, muscle- Coactosin-like protein 5.2 ADP-sugar pyrophosphatase alpha specific form
Titin Basigin Talin-1 4.8 Drebrin-like protein
Zymogen granule protein 16 Polypeptide N- Filamin-A 4.8 Translin homolog B acetylgalactosaminyltransferase 3
Fibroblast growth factor-binding Lamina-associated polypeptide 2, Serotransferrin 4.7 14-3-3 protein gamma protein 1 isoform alpha
Cleavage and polyadenylation Intraflagellar transport protein 80 Protein S100-A9 4.7 Protein disulfide-isomerase A6 specificity factor subunit 5 homolog
60S ribosomal protein L8 Cytoplasmic dynein 1 intermediate D-dopachrome decarboxylase 4.5 N-acetyl-D-glucosamine kinase (Fragment) chain 2
Polypeptide N- Glucose-6-phosphate isomerase Kallikrein-13 4.2 Ras-related protein Rab-5C acetylgalactosaminyltransferase 2 (Fragment)
Sodium- and chloride-dependent Peptidyl-prolyl cis-trans isomerase neutral and basic amino acid U2 small nuclear ribonucleoprotein A' 3.6 Ras suppressor protein 1 FKBP1A transporter B(0+)
Pendrin Bardet-Biedl syndrome 2 protein Matrix metalloproteinase-9 3.4 Heme-binding protein 1
Low molecular weight Cysteine and histidine-rich domain- Interleukin-19 Complement C4-B 3.3 phosphotyrosine protein containing protein 1 phosphatase
cAMP-dependent protein kinase Aquaporin-5 Paraspeckle component 1 Lamin-B1 3.2 type II-alpha regulatory subunit
Vitelline membrane outer layer Translationally-controlled tumor Protein-lysine 6-oxidase Glutaredoxin-1 3.2 protein 1 homolog protein
Ubiquitin carboxyl-terminal Haptoglobin-related protein Neurocalcin-delta Profilin-1 3.2 hydrolase 15
Adenylyl cyclase-associated protein Nucleoside diphosphate kinase A Sulfotransferase 1A1 3.1 Docking protein 3 1
208 Cytosolic 5'-nucleotidase 3A Purine nucleoside phosphorylase 3.1 Nucleoprotein TPR
Actin-like protein 6A Annexin A3 3.0 Fibronectin
Geranylgeranyl transferase type-2 Vinculin 3.0 Calcium-binding protein 39 subunit alpha
Capping protein (Actin filament) Mucin-7 2.8 Protein PBDC1 muscle Z-line, beta, isoform CRA_d
Haloacid dehalogenase-like Syntaxin-2 Zinc-alpha-2-glycoprotein 2.7 hydrolase domain-containing protein 2
N-acylethanolamine-hydrolyzing acid Neutrophil gelatinase-associated 2.6 Kininogen-1 amidase lipocalin
EF-hand domain-containing protein Sideroflexin-1 2.6 Sialic acid synthase D2
Redox-regulatory protein FAM213A Epididymal secretory protein E1 2.6 Ferritin light chain
Nuclear ubiquitous casemin and Aminopeptidase N 2.5 CD166 antigen cyclin-dependent kinase substrate 1
Cilia- and flagella-associated protein Serine/threonine-protein Protein FAM49B 2.4 45 phosphatase 2A activator
DnaJ homolog subfamily B member Catalase 2.4 Eukaryotic initiation factor 4A-I 13
Protein kinase C and casemin kinase substrate in neurons protein 3 Phosphoglycerate mutase 1 2.2 Glutathione peroxidase (Fragment)
Eukaryotic translation initiation factor Peptidyl-prolyl cis-trans Ubiquitin-conjugating enzyme E2 L3 2.1 2 subunit 2 isomerase FKBP4
Atlastin-3 Glutathione S-transferase omega-1 2.1 Xaa-Pro aminopeptidase 1
Cleavage and polyadenylation Growth factor receptor-bound 2.1 Prolyl endopeptidase specificity factor subunit 6 protein 2
Apoptosis-associated speck-like ATP-dependent RNA helicase DDX1 2.1 Glutathione synthetase protein containing a CARD
N-acetylgalactosamine-6- 209 Kinesin-like protein KIF21A Alpha-actinin-1 1.9 sulfatase
Intraflagellar transport protein 52 Leukocyte elastase inhibitor 1.9 Afamin homolog
Ubiquitin carboxyl-terminal Ubiquitin-conjugating enzyme E2 K Triosephosphate isomerase 1.9 hydrolase 14
Immunoglobulin kappa variable Disks large homolog 1 Rho GDP-dissociation inhibitor 1 1.8 3-20
Eukaryotic translation initiation factor Acetyl-CoA acetyltransferase, Macrophage-capping protein 1.8 3 subunit A cytosolic
Desmoglein-3 Calreticulin 1.8 Proteasome subunit alpha type-7
ProSAAS Myosin-9 1.7 Tumor protein D54
Protein-glutamine gamma- Myosin light polypeptide 6 1.6 N-acetylglucosamine-6-sulfatase glutamyltransferase 2
Polymerase I and transcript release Superoxide dismutase [Mn], 1.5 Protein AMBP factor mitochondrial
PHD finger-like domain-containing Rab GDP dissociation inhibitor beta 1.5 NAD kinase protein 5A
Alanine--tRNA ligase, 3-ketoacyl-CoA thiolase, peroxisomal Cystatin-B 1.5 cytoplasmic
Trypsin-1 NAD(P)H-hydrate epimerase 1.4 Enolase-phosphatase E1
F-actin-capping protein subunit Adenosylhomocysteinase 2 1.3 Di-N-acetylchitobiase alpha-1
Proteasome activator complex Uncharacterized protein Prolyl endopeptidase 1.3 subunit 1
Intraflagellar transport protein 46 Microtubule-associated protein Protein S100-P 1.2 homolog RP/EB family member 1
Acidic leucine-rich nuclear Cytoplasmic aconitate hydratase Ester hydrolase C11orf54 1.2 phosphoprotein 32 family member E
Rab GDP dissociation inhibitor 210 Pleiotropic regulator 1 1.1 Actin-related protein 2 alpha
Protein SET Neutral amino acid transporter B(0) 6-phosphogluconolactonase 1.1 Ras GTPase-activating-like protein MICOS complex subunit IQGAP1 1.0 Neutrophil cytosol factor 4
Calcium/calmodulin-dependent Glutathione reductase, 0.8 Keratin, type I cytoskeletal 18 protein kinase type II subunit delta mitochondrial
26S proteasome non-ATPase Peptidyl-prolyl cis-trans isomerase 0.7 Stress-induced-phosphoprotein 1 regulatory subunit 11 A
Cilia- and flagella-associated protein Attractin -1.1 Serum amyloid A-1 protein 57
Insulin-like growth factor-binding Proteasome activator complex Glutaredoxin-3 -1.1 protein 7 subunit 2
Serine hydroxymethyltransferase, Glyceraldehyde-3-phosphate CUGBP Elav-like family member -1.1 mitochondrial dehydrogenase 2
Protein-arginine deiminase type-1 Actin, cytoplasmic 2 -1.2 Alpha-2-antiplasmin
Stanniocalcin-2 Heat shock protein HSP 90-alpha -1.3 ATP-citrate synthase
Hypoxanthine-guanine Angiogenin Heat shock protein HSP 90-beta -1.3 phosphoribosyltransferase
BAG family molecular chaperone Echinoderm microtubule- AP-2 complex subunit alpha-1 -1.4 regulator 3 associated protein-like 4
Acid sphingomyelinase-like Sodium/potassium-transporting -1.4 Chitinase-3-like protein 1 phosphodiesterase 3b (Fragment) ATPase subunit beta-1
Proteasome activator complex subunit Gamma-glutamylcyclotransferase -1.5 14-3-3 protein eta 3
Nucleoplasmin-2 Mucin-1 -1.6 Calpain small subunit 1
Cysteine and glycine-rich protein 1 Kallikrein-10 -1.6 Glutathione peroxidase
Glycerol-3-phosphate dehydrogenase Chloride intracellular channel Glutaminyl-peptide -1.6 1-like protein protein 1 cyclotransferase
Interleukin-36 gamma Keratin, type II cytoskeletal 6A -1.7 Ras-related protein Rab-21
Polypeptide N- 211 Macrophage migration inhibitory factor -1.7 Transgelin acetylgalactosaminyltransferase 1
Basic leucine zipper and W2 domain- Glutathione synthetase -1.7 Osteoclast-stimulating factor 1 containing protein 1
Small glutamine-rich tetratricopeptide Gelsolin -1.8 Fibrinogen alpha chain repeat-containing protein alpha
Sodium-dependent phosphate Cellular retinoic acid-binding 40S ribosomal protein S24 -1.8 transport protein 2B protein 2
N-acetylmuramoyl-L-alanine Lysine--tRNA ligase Peroxiredoxin-2 -1.8 amidase
Na(+)/H(+) exchange regulatory Malate dehydrogenase, Alpha-2-macroglobulin-like protein 1 -1.8 cofactor NHE-RF1 mitochondrial
Casemin kinase II subunit alpha 3 Calpain-1 catalytic subunit -1.8 Prefoldin subunit 6
Hepatitis B virus x interacting Galectin Protein SET -1.9 protein
1-phosphatidylinositol 4,5- Fructose-bisphosphate aldolase bisphosphate phosphodiesterase Prominin-1 -1.9 C beta-4
V-type proton ATPase subunit S1 Guanine nucleotide-binding protein -1.9 Keratin, type II cytoskeletal 8 (Fragment) subunit alpha-11
Bifunctional epoxide hydrolase 2 Calmodulin -1.9 CD44 antigen
N(4)-(beta-N- GrpE protein homolog 1, Activated RNA polymerase II -1.9 acetylglucosaminyl)-L- mitochondrial transcriptional coactivator p15 asparaginase
Sepiapterin reductase Phosphoglucomutase-1 -1.9 Glutathione S-transferase Mu 4
Platelet-activating factor 26S protease regulatory subunit 7 Apolipoprotein A-I -2.0 acetylhydrolase IB subunit beta
Actin-related protein 2/3 complex Syntaxin-3 Peroxiredoxin-1 -2.0 subunit 5
Protein arginine N-methyltransferase Myeloid cell nuclear Kallikrein-11 -2.0 5 differentiation antigen
STE20-like serine/threonine-protein Destrin -2.0 Protein CutA kinase
212
Thioredoxin reductase 1, Malignant T-cell-amplified Exportin-1 -2.0 cytoplasmic sequence 1
CAP-Gly domain-containing linker Ezrin -2.0 Metalloproteinase inhibitor 1 protein 1
G-protein coupled receptor family C Galectin-3 -2.0 Dipeptidyl peptidase 3 group 5 member B
Rho GTPase-activating protein DnaJ homolog subfamily B member 1 Keratin, type II cytoskeletal 2 epidermal -2.0 25
Brain-specific angiogenesis inhibitor cAMP-dependent protein kinase Thioredoxin -2.1 1-associated protein 2-like protein 1 type I-alpha regulatory subunit
Hematopoietic lineage cell- Valine--tRNA ligase Keratin, type II cytoskeletal 5 -2.1 specific protein
WD40 repeat-containing protein Epidermal growth factor receptor Adenylosuccinate synthetase -2.1 SMU1 kinase substrate 8 isozyme 2
ATP-binding cassette sub-family A Ubiquitin carboxyl-terminal N-acetylglucosamine-6-sulfatase -2.2 member 13 hydrolase isozyme L3
Eukaryotic translation initiation factor Omega-amidase NIT2 -2.2 Ras-related protein Rab-11B 4 gamma 1
U5 small nuclear ribonucleoprotein Unconventional myosin-Ib -2.2 Cytochrome c (Fragment) 200 kDa helicase
Protease serine 2 preproprotein Transforming protein RhoA -2.2 Carbonyl reductase [NADPH] 1
Mitogen-activated protein kinase Crk-like protein Ras-related protein Rab-7a -2.2 1
Protein DEK (Fragment) Calcium-binding protein 39 -2.2 Perilipin-3
AP-1 complex subunit gamma-1 Tetraspanin-1 -2.2 ADP-ribosylation factor 3
Tripartite motif-containing protein 2 Complement decay-accelerating factor -2.3 Protein disulfide-isomerase A3 GTP:AMP phosphotransferase AK3, Tubulin polymerization-promoting mitochondrial Dipeptidyl peptidase 1 -2.3 protein family member 3
Potassium-transporting ATPase alpha Carboxypeptidase Q -2.3 Coronin-1C chain 2
21
3 WD repeat-containing protein 35 Inositol monophosphatase 1 -2.3 Costars family protein ABRACL
Testin Cofilin-1 -2.3 Annexin A1
Splicing factor 3A subunit 1 Tubulin beta-4B chain -2.3 Immunoglobulin J chain
Actin-related protein 2/3 complex Heterogeneous nuclear Prefoldin subunit 3 -2.3 subunit 1B ribonucleoprotein H3
Malate dehydrogenase, S-methyl-5'-thioadenosine Cysteine--tRNA ligase, cytoplasmic -2.3 mitochondrial phosphorylase
Mitochondrial 2-oxoglutarate/malate Aspartate aminotransferase, -2.3 Ras-related protein Rab-8B carrier protein cytoplasmic
Endoplasmic reticulum Peroxiredoxin-5, mitochondrial -2.4 Protein RCC2 aminopeptidase 1
Axonemal dynein light intermediate ADP-ribosylation factor 3 -2.4 Rho GTPase-activating protein 1 polypeptide 1
LDLR chaperone MESD Fructose-bisphosphate aldolase A -2.4 Wiskott-Aldrich syndrome protein
Activator of 90 kDa heat shock protein Core histone macro-H2A.1 -2.5 Omega-amidase NIT2 ATPase homolog 1
Heterogeneous nuclear Probable aminopeptidase NPEPL1 -2.5 Heat shock protein 105 kDa ribonucleoproteins C1/C2
DNA-(apurinic or apyrimidinic Importin subunit alpha-1 WD repeat-containing protein 1 -2.6 site) lyase
Insulin-degrading enzyme Galectin-3-binding protein -2.7 Creatine kinase B-type
Actin-related protein 2/3 complex Tubulin alpha-1A chain -2.7 Formin-binding protein 1 subunit 1A
Oxygen-dependent Heterogeneous nuclear coproporphyrinogen-III oxidase, Transmembrane channel-like protein 5 -2.7 ribonucleoprotein U mitochondrial
Protein-L-isoaspartate O- FACT complex subunit SSRP1 Mucin-4 -2.7 methyltransferase
Protein CYR61 Keratin, type I cytoskeletal 17 -2.7 Ribonuclease T2 (Fragment)
214
Glucosamine-6-phosphate isomerase Lysosome-associated membrane Acylamino-acid-releasing enzyme -2.8 2 glycoprotein 2
Acyl-protein thioesterase 1 Flap endonuclease 1 Keratin, type II cytoskeletal 1 -2.8 (Fragment)
Coiled-coil-helix-coiled-coil-helix Keratin, type I cytoskeletal 14 -2.8 Aldose 1-epimerase domain-containing protein 5
Programmed cell death 6-interacting Protein NDRG2 (Fragment) -2.8 Glia maturation factor gamma protein
Putative neutrophil cytosol factor HCG2044799 Pigment epithelium-derived factor -2.8 1B
Methionine aminopeptidase 2 Glutathione S-transferase P -2.8 Heat shock 70 kDa protein 4
Adenine Apolipoprotein A-II Calcium and integrin-binding protein 1 -2.8 phosphoribosyltransferase
Succinate--CoA ligase [GDP-forming] WAP four-disulfide core domain -2.9 14-3-3 protein theta subunit beta, mitochondrial protein 2
Guanine nucleotide-binding protein Growth factor receptor-bound Unconventional myosin-Ic -2.9 G(i) subunit alpha-2 protein 2
Heterogeneous nuclear Epidermal growth factor receptor -2.9 T-complex protein 1 subunit theta ribonucleoprotein U-like protein 1 kinase substrate 8-like protein 1
Heterogeneous nuclear Ribosomal protein L15 (Fragment) Annexin A2 -2.9 ribonucleoprotein F
BPI fold-containing family A Caprin-1 Beta-2-microglobulin -3.0 member 1
Charged multivesicular body protein S-formylglutathione hydrolase -3.0 DCC-interacting protein 13-alpha 2a
General vesicular transport factor Pyruvate kinase PKM -3.0 Angiotensinogen p115
Eukaryotic translation initiation factor Keratin, type I cytoskeletal 13 -3.1 Pigment epithelium-derived factor 3 subunit I
26S proteasome non-ATPase Heterogeneous nuclear 215 Polymeric immunoglobulin receptor -3.1 regulatory subunit 1 ribonucleoprotein L
cDNA FLJ60124, highly similar to Nuclear mitotic apparatus protein Fructose-bisphosphate aldolase C -3.1 Mitochondrial dicarboxylate carrier 1
Actin-related protein 2/3 complex Interstitial collagenase RNA-binding protein EWS -3.1 subunit 2
Far upstream element-binding protein Keratin, type I cytoskeletal 19 -3.2 Coactosin-like protein 2
Thioredoxin domain-containing Proliferation-associated protein Proteasome subunit alpha type-3 -3.2 protein 12 2G4
UTP--glucose-1-phosphate Involucrin -3.2 Complement factor H uridylyltransferase
Serine/threonine-protein Zinc finger protein 185 Phosphoglycerate kinase 1 -3.2 phosphatase CPPED1
Lysosome-associated membrane Clathrin light chain B Keratin, type I cytoskeletal 16 -3.2 glycoprotein 1
NADH dehydrogenase [ubiquinone] Heterogeneous nuclear -3.2 Glutathione S-transferase A2 flavoprotein 2, mitochondrial ribonucleoprotein K
Follistatin-related protein 3 Proteasome subunit alpha type-5 -3.2 Bridging integrator 2
Adenylate kinase 2, ATP-dependent RNA helicase DDX3X L-lactate dehydrogenase B chain -3.3 mitochondrial
Nuclear autoantigenic sperm protein Plectin -3.3 Cytochrome b5
Mitochondrial import inner membrane Interleukin enhancer-binding Unconventional myosin-Id -3.3 translocase subunit Tim13 factor 2
Importin-9 Creatine kinase B-type -3.3 Flavin reductase (NADPH)
Cell surface glycoprotein CD200 Guanine nucleotide-binding protein Aspartate aminotransferase, -3.3 receptor 1 G(I)/G(S)/G(T) subunit beta-1 cytoplasmic
ATPase family AAA domain- Acid ceramidase -3.4 Putative hydrolase RBBP9 containing protein 3A
Tryptophan--tRNA ligase, Sperm acrosome-associated protein 9 Cathepsin D -3.4 cytoplasmic
Mitotic checkpoint protein BUB3 216 Synaptosomal-associated protein 23 Dystroglycan -3.4 (Fragment)
RPS10-NUDT3 readthrough Peflin Niban-like protein 1 -3.4 (Fragment)
Lipolysis-stimulated lipoprotein Annexin A1 -3.4 Adenosylhomocysteinase receptor
Glutamine--tRNA ligase (Fragment) 14-3-3 protein epsilon -3.5 Integrin alpha-M
Cysteine-rich protein 2 Peroxiredoxin-6 -3.5 Protein unc-13 homolog D
Receptor-type tyrosine-protein Heterogeneous nuclear Hsc70-interacting protein -3.5 phosphatase zeta ribonucleoproteins C1/C2
Bis(5'-adenosyl)-triphosphatase Histone H4 -3.5 PDZ and LIM domain protein 5 ENPP4
Inter-alpha-trypsin inhibitor heavy Importin subunit alpha-3 Connective tissue growth factor -3.6 chain H1
Aldehyde dehydrogenase family 3 U1 small nuclear ribonucleoprotein A -3.6 Dysferlin member B1
Abl interactor 1 EH domain-containing protein 4 -3.6 Elongation factor 1-delta
Cytochrome b-c1 complex subunit Calpain small subunit 1 -3.6 Gamma-glutamyl hydrolase Rieske, mitochondrial
Glycine cleavage system H protein, Histone H2B type 1-B -3.7 Ras-related protein Rab-18 mitochondrial
Regulation of nuclear pre-mRNA Lysosomal alpha-mannosidase -3.7 Prostaglandin E synthase 3 domain-containing protein 1B
Proteasome activator complex Platelet-activating factor Protein TFG (Fragment) -3.7 subunit 1 acetylhydrolase IB subunit alpha
Outer dense fiber protein 3B T-complex protein 1 subunit gamma -3.7 Vitronectin
Enoyl-CoA delta isomerase 1, 1,4-alpha-glucan-branching Transmembrane protein 231 -3.7 mitochondrial enzyme
Na(+)/H(+) exchange regulatory Hydroxyacylglutathione Acyl-coenzyme A thioesterase 13 -3.8 cofactor NHE-RF2 hydrolase, mitochondrial
WAP four-disulfide core domain NADH-cytochrome b5 reductase 3 Mucin-4 -3.8 protein 2
217
Deoxyribose-phosphate aldolase Keratin, type I cytoskeletal 10 -3.8 Ribose-5-phosphate isomerase
V-set domain-containing T-cell Thioredoxin domain-containing Proteasome subunit beta type-1 -3.8 activation inhibitor 1 protein 17
ADP-ribosylation factor 6 Flavin reductase (NADPH) -3.9 Galectin-1 Ras-related protein Rab-14 Syntaxin-binding protein 1 Stress-induced-phosphoprotein 1 -3.9
Aldo-keto reductase family 1 member Phospholipid-transporting ATPase IC -3.9 Destrin C1
Suppressor of tumorigenicity 14 Proteasome subunit beta type-3 -4.0 Pyridoxal kinase protein
Vesicle-associated membrane protein NAD(P)H dehydrogenase Chloride intracellular channel protein 6 -4.0 8 [quinone] 1
Puromycin-sensitive FACT complex subunit SPT16 -4.0 Retinal dehydrogenase 1 aminopeptidase
Poly(U)-binding-splicing factor PUF60 Guanine nucleotide-binding protein GTP-binding nuclear protein Ran -4.1 (Fragment) G(I)/G(S)/G(T) subunit beta-2 (Fragment)
Glutathione S-transferase theta-1 Agrin -4.1 Syntaxin-binding protein 2
Pulmonary surfactant-associated Secretoglobin family 3A member 1 -4.1 Nuclear transport factor 2 protein B
Biotinidase Keratin, type II cytoskeletal 7 -4.1 Integrin beta
Aldehyde dehydrogenase family 16 Mitochondrial peptide methionine ADP-sugar pyrophosphatase -4.1 member A1 sulfoxide reductase
Bleomycin hydrolase Proteasome subunit alpha type-7 -4.2 Aminopeptidase N
Protein SEC13 homolog Growth/differentiation factor 15 -4.2 Neutrophil cytosol factor 2
Insulin-like growth factor-binding Heterogeneous nuclear Protein phosphatase 1G -4.2 protein 2 ribonucleoproteins A2/B1
C-1-tetrahydrofolate synthase, Sodium/potassium-transporting -4.2 Grancalcin cytoplasmic ATPase subunit alpha-1
Aldehyde dehydrogenase, dimeric Interleukin-18 -4.2 AP-2 complex subunit beta NADP-preferring
218
Ubiquitin-like modifier-activating Arrestin domain-containing protein 1 -4.2 Chitotriosidase-1 enzyme 1
Copper transport protein ATOX1 CD166 antigen -4.2 Thymosin beta-4
Complement C1q tumor necrosis Transitional endoplasmic reticulum -4.3 Dipeptidyl peptidase 1 factor-related protein 5 ATPase
Brain-specific angiogenesis inhibitor 1- Prefoldin subunit 4 -4.3 Importin subunit beta-1 associated protein 2
Eukaryotic translation initiation factor Tyrosine-protein kinase Lyn -4.3 Calcyphosin 4E
Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha Transmembrane channel-like protein -4.4 Elongation factor 1-gamma isoform
Uncharacterized protein 60S ribosomal protein L23 Cadherin-1 -4.4 (Fragment)
Density-regulated protein Annexin A5 -4.4 Guanylate-binding protein 1
Coiled-coil domain-containing protein Nicotinamide Proteasome subunit beta type -4.4 80 phosphoribosyltransferase
Small nuclear ribonucleoprotein Pregnancy zone protein Radixin -4.5 Sm D1
V-type proton ATPase subunit B, Epidermal growth factor receptor -4.5 Neutrophil defensin 1 brain isoform kinase substrate 8-like protein 2
5'-nucleotidase domain-containing Histidine triad nucleotide-binding Proteasome subunit beta type-4 -4.5 protein 1 protein 1
NHL repeat-containing protein 3 Ribonuclease inhibitor -4.5 Cathepsin D
Serine/threonine-protein phosphatase Sciellin -4.5 Inorganic pyrophosphatase PP1-beta catalytic subunit
Matrilysin Keratin, type I cytoskeletal 9 -4.7 Tubulin-specific chaperone A
EF-hand calcium-binding domain- EGF-containing fibulin-like extracellular -4.7 Ribonuclease inhibitor containing protein 1 matrix protein 1
219 Aldehyde dehydrogenase, Cytoskeleton-associated protein 4 Myosin-14 -4.7 dimeric NADP-preferring
Heterogeneous nuclear 40S ribosomal protein S20 Myoferlin -4.7 ribonucleoprotein A1 (Fragment)
Vesicle-associated membrane Glycine--tRNA ligase Monocyte differentiation antigen CD14 -4.7 protein-associated protein A
Guanine nucleotide-binding protein Eukaryotic translation initiation Beta-hexosaminidase subunit alpha -4.9 subunit alpha-14 factor 6
3'(2'),5'-bisphosphate Radial spoke head protein 9 homolog Heat shock 70 kDa protein 4 -4.9 nucleotidase 1
Kunitz-type protease inhibitor 1 Oxygen-regulated protein 1 -5.0 Fructose-1,6-bisphosphatase 1 (Fragment)
Toll-interacting protein Urokinase-type plasminogen activator -5.0 Thymidine phosphorylase
Vacuolar protein sorting-associated Annexin A11 -5.0 Copine-3 protein 4B
BUB3-interacting and GLEBS motif- Adenylate kinase 2, mitochondrial -5.0 Beta-2-glycoprotein 1 containing protein ZNF207
Interleukin-1 receptor accessory Tissue alpha-L-fucosidase -5.1 Elongation factor 2 protein
Eukaryotic translation initiation factor Glutamate--cysteine ligase catalytic -5.1 Eosinophil cationic protein 3 subunit K subunit
U1 small nuclear ribonucleoprotein 70 Calpain-2 catalytic subunit -5.2 ITIH4 protein kDa
Tetraspanin Retinal dehydrogenase 1 -5.2 Calpain-1 catalytic subunit
Ubiquitin-conjugating enzyme E2 Ropporin-1-like protein 60S ribosomal protein L10a -5.2 L3
Farnesyl pyrophosphate Glypican-4 Major vault protein -5.2 synthase
Cytochrome c oxidase asSEbly factor Proteasome subunit alpha type -5.3 Aldose reductase 6 homolog (Fragment)
Isocitrate dehydrogenase RNA-binding protein FUS Keratin, type II cytoskeletal 8 -5.3 [NADP] cytoplasmic
220
Cytochrome c oxidase subunit Annexin A4 -5.3 Tropomodulin-3 NDUFA4
Radial spoke head 1 homolog Filamin-B -5.4 Peroxiredoxin-6
NEDD8-MDP1 readthrough N(4)-(beta-N-acetylglucosaminyl)-L- -5.5 60S acidic ribosomal protein P2 (Fragment) asparaginase
Prefoldin subunit 2 Syntaxin-binding protein 2 -5.5 Heme-binding protein 2
Uridine diphosphate glucose Na(+)/H(+) exchange regulatory Keratin, type I cytoskeletal 18 -5.5 pyrophosphatase cofactor NHE-RF1
Plexin-B2 CD109 antigen -5.5 Ras-related protein Rab-7a
Tubulin polymerization-promoting Peptidyl-prolyl cis-trans ELAV-like protein 1 -5.5 protein family member 3 isomerase FKBP5
Cdc42-interacting protein 4 DNA damage-binding protein 1 -5.5 Triokinase/FMN cyclase
Ceroid-lipofuscinosis neuronal protein Calcyphosin -5.5 Elongation factor 1-alpha 1 5
Puromycin-sensitive Galectin Ras-related protein Rap-1A -5.6 aminopeptidase
Transformer-2 protein homolog beta Phospholipid transfer protein -5.8 Nuclear migration protein nudC
ATP synthase F(0) complex subunit Beta-hexosaminidase subunit beta -5.8 Myosin regulatory light chain 12A B1, mitochondrial
Receptor of activated protein C kinase Reticulocalbin-1 -5.9 Transthyretin 1
Plasma alpha-L-fucosidase Histone H2A type 2-C -6.0 14-3-3 protein beta/alpha
Protein phosphatase 1 regulatory Echinoderm microtubule-associated -6.1 Twinfilin-2 subunit 7 (Fragment) protein-like 2
Apoptosis-associated speck-like Ras-related protein Ral-B Prelamin-A/C -6.1 protein containing a CARD
60S ribosomal protein L36 Bile salt-activated lipase -6.5 Actin-related protein 3
Alcohol dehydrogenase class 4 Nicotinate 221 C-C motif chemokine 15 (Fragment) -7.0 mu/sigma chain phosphoribosyltransferase
Fatty acid-binding protein, Uncharacterized protein C6orf132 40S ribosomal protein SA -7.2 adipocyte
Mesothelin Cytosolic non-specific dipeptidase -7.3 Transcobalamin-1
60S ribosomal protein L17 (Fragment) Interleukin enhancer-binding factor 3 -7.4 Fermitin family homolog 3
60S ribosomal protein L27a Myeloblastin +inf Histone H2B type 1-B
Archain 1, isoform CRA_a Fibronectin +inf Protein FAM49B
Serine-threonine kinase receptor- Anterior gradient protein 2 CD177 antigen +inf associated protein homolog
Kinectin Fibrinogen gamma chain +inf Plasminogen
Bactericidal permeability- 40S ribosomal protein S6 Fibrinogen beta chain +inf increasing protein
Ras-related C3 botulinum toxin Sialidase-1 Alpha-1B-glycoprotein +inf substrate 1
Carcinoembryonic antigen-related 60S ribosomal protein L32 (Fragment) +inf Ezrin cell adhesion molecule 8
Inosine-5'-monophosphate Peptidoglycan recognition protein 1 +inf Phospholipase B-like 1 dehydrogenase 2 (Fragment)
Septin-2 Immunoglobulin heavy constant mu +inf WD repeat-containing protein 1
Protein arginine N-methyltransferase N(G),N(G)-dimethylarginine Fibrinogen alpha chain +inf 1 dimethylaminohydrolase 1
Immunoglobulin heavy constant Splicing factor U2AF 65 kDa subunit +inf Protein S100-P gamma 3
Immunoglobulin heavy constant Importin-7 +inf Biliverdin reductase A gamma 4
Carcinoembryonic antigen- Serine/threonine-protein kinase 26 Olfactomedin-4 +inf related cell adhesion molecule 8
Glyoxalase domain-containing Beta-1,4-glucuronyltransferase 1 Azurocidin +inf protein 4
222
ADP-ribosylation factor 4 Myeloperoxidase +inf Apolipoprotein B-100
Bifunctional purine biosynthesis Oligoribonuclease, mitochondrial Alpha-1-acid glycoprotein 2 +inf protein PURH
UDP-GlcNAc:betaGal beta-1,3-N- Maltase-glucoamylase, intestinal +inf Cathelicidin antimicrobial peptide acetylglucosaminyltransferase 7
Aflatoxin B1 aldehyde reductase Multivesicular body subunit 12A Corticosteroid-binding globulin +inf member 2
Charged multivesicular body protein 5 Cysteine-rich secretory protein 3 +inf Polyadenylate-binding protein 1
Exportin-2 Non-secretory ribonuclease +inf Maltase-glucoamylase, intestinal
14 kDa phosphohistidine Cornulin Hemopexin +inf phosphatase
Secernin-2 Alpha-2-HS-glycoprotein +inf Neutrophil elastase
Vacuolar protein sorting-associated Ribose-5-phosphate isomerase +inf Arginase-1 protein 28 homolog
60S ribosomal protein L18a Rho GDP-dissociation inhibitor 2 +inf Protein DJ-1
Vasodilator-stimulated 60S ribosomal protein L3 Angiotensinogen +inf phosphoprotein
Disintegrin and metalloproteinase Mucin-5AC +inf Lactoylglutathione lyase domain-containing protein 10
60S ribosomal protein L10 Vitamin D-binding protein +inf Keratin, type II cytoskeletal 1
Coatomer subunit epsilon Serpin B10 +inf Cathepsin S
ADP-ribosyl cyclase/cyclic ADP- Thioredoxin reductase 1, 60S ribosomal protein L14 +inf ribose hydrolase 2 cytoplasmic
Ran-specific GTPase-activating SH3 domain-binding glutamic Integrin beta +inf protein acid-rich-like protein 3
Alpha-aminoadipic Semialdehyde EF-hand domain-containing Folate receptor gamma +inf dehydrogenase (Fragment) protein D2
Dynein light chain 1, axonemal Coronin-1A +inf Galectin-3
Thrombospondin-1 Neutrophil collagenase +inf Clathrin interactor 1
223
Hsp90 co-chaperone Cdc37 Beta-2-glycoprotein 1 +inf Carbonic anhydrase 3
Septin-7 Cathelicidin antimicrobial peptide +inf Latexin
Intraflagellar transport protein 74 Ubiquitin-like modifier-activating Immunoglobulin lambda constant 2 +inf homolog enzyme 1
Delta(24)-sterol reductase Neutrophil elastase +inf 6-phosphogluconolactonase
Calumenin Resistin +inf 14-3-3 protein epsilon
Eukaryotic translation initiation factor Immunoglobulin heavy constant +inf Adenosine kinase 3 subunit L alpha 2 (Fragment)
Isopentenyl-diphosphate Delta- Cytidine deaminase +inf Antithrombin-III isomerase 1
60S ribosomal protein L27 Fumarylacetoacetase +inf Rho GDP-dissociation inhibitor 1
Mannose-1-phosphate Arginase-1 +inf Core histone macro-H2A.1 guanyltransferase beta
Methionine adenosyltransferase 2 ADP-ribosyl cyclase/cyclic ADP- Transthyretin +inf subunit beta ribose hydrolase 2
Intraflagellar transport protein 22 Programmed cell death 6- Protein S100-A7 +inf homolog interacting protein
Lysosome-associated membrane Low affinity immunoglobulin Ribonuclease pancreatic +inf glycoprotein 1 gamma Fc region receptor III-B Spectrin beta chain, non- Ras GTPase-activating protein- Antithrombin-III +inf erythrocytic 1 binding protein 1
Tyrosine-protein kinase FRK Carbonic anhydrase 1 +inf Gamma-enolase
Inter-alpha-trypsin inhibitor heavy DNA topoisomerase 1 +inf Apolipoprotein A-I chain H1
High mobility group protein B2 Immunoglobulin heavy variable 3-7 +inf Peroxiredoxin-2
HSPE1-MOB4 readthrough Neutrophil defensin 1 +inf Carbonic anhydrase 2
Ribosyldihydronicotinamide Centrin-2 +inf Inositol monophosphatase 1 dehydrogenase [quinone]
GTPase HRas ITIH4 protein +inf Keratin, type I cytoskeletal 10
224
Vasodilator-stimulated Immunoglobulin kappa variable Carboxypeptidase A4 +inf phosphoprotein 3-15
40S ribosomal protein S16 Vitronectin +inf RNA-binding protein EWS
Bifunctional glutamate/proline--tRNA Eosinophil cationic protein +inf Leucine-rich alpha-2-glycoprotein ligase
Acylphosphatase Protein S100-A12 +inf Peroxiredoxin-1
Nck-associated protein 1 Apolipoprotein B-100 +inf Alpha-2-HS-glycoprotein
Tyrosine-protein phosphatase non- Tubulointerstitial nephritis antigen-like +inf Phosphoglucomutase-1 receptor type substrate 1
Nicotinate Tripeptidyl-peptidase 1 +inf Nucleoside diphosphate kinase phosphoribosyltransferase
Glycogen phosphorylase, liver Cystatin-M Apolipoprotein(a) +inf form
von Willebrand factor A domain- Alcohol dehydrogenase Afamin +inf containing protein 7 [NADP(+)]
V-type proton ATPase catalytic Mitochondrial peptide methionine Heterogeneous nuclear +inf subunit A sulfoxide reductase ribonucleoprotein Q
Proteasome subunit beta type Kininogen-1 +inf Peroxiredoxin-5, mitochondrial
Apoptosis inhibitor 5 Hemoglobin subunit beta +inf Epididymal secretory protein E1
Regulator of chromosome Chitotriosidase-1 +inf Calponin condensation (Fragment)
Alpha-N-acetylglucosaminidase Alpha-2-antiplasmin +inf Plectin
Intraflagellar transport protein 172 Hemoglobin subunit alpha +inf Myotrophin homolog
cDNA FLJ55673, highly similar to ADP-ribosylation factor-like protein 3 Hemoglobin subunit delta +inf Complement factor B (EC 3.4.21.47)
DnaJ homolog subfamily A member 4 Transgelin +inf Folate receptor gamma
Neuropilin m7GpppX diphosphatase +inf Histone H4
225
High mobility group protein B3 Tyrosine-protein phosphatase Protein AMBP +inf (Fragment) non-receptor type 6
Acidic leucine-rich nuclear Bactericidal permeability-increasing Mucin-13 +inf phosphoprotein 32 family protein member A
Electron transfer flavoprotein subunit F-actin-capping protein subunit Immunoglobulin lambda variable 1-47 +inf beta alpha-2
Thimet oligopeptidase Plasminogen +inf Fumarylacetoacetase
Ras-related C3 botulinum toxin Cold-inducible RNA-binding protein C-reactive protein +inf substrate 2
Nucleosome asSEbly protein 1-like 4 Cytochrome b5 +inf Ester hydrolase C11orf54
Twinfilin-1 Immunoglobulin heavy variable 4-34 +inf Alpha-2-macroglobulin
Rab GDP dissociation inhibitor Calsyntenin-1 Integrin alpha-M +inf alpha
Low affinity immunoglobulin gamma Calpain-5 +inf Nucleophosmin Fc region receptor III-B
Glucose-6-phosphate 1- Importin-5 (Fragment) Bridging integrator 2 +inf dehydrogenase
D-3-phosphoglycerate Polymeric immunoglobulin Ubiquitin-conjugating enzyme E2 D3 +inf dehydrogenase receptor
Acetyl-CoA acetyltransferase, Superoxide dismutase [Mn], Immunoglobulin J chain +inf mitochondrial mitochondrial
Complement factor D Histidine-rich glycoprotein +inf Lamin-B1
Actin-related protein 2/3 complex 60S ribosomal protein L11 -inf Transforming protein RhoA subunit 4
Adenylosuccinate synthetase isozyme Actin-related protein 2/3 complex -inf Myeloperoxidase 1 subunit 3
Ras-related protein Rab-4A Actin-related protein 2 -inf CD177 antigen
Pleckstrin homology domain- Actin-related protein 2/3 complex Ras GTPase-activating-like -inf containing family S member 1 subunit 5-like protein protein IQGAP1
226 Deoxyuridine 5'-triphosphate CD59 glycoprotein -inf Neutrophil collagenase nucleotidohydrolase, mitochondrial
Guanine nucleotide-binding protein Synaptic vesicle membrane Uroplakin-3b-like protein -inf G(I)/G(S)/G(O) subunit gamma-12 protein VAT-1 homolog
Ras-related protein R-Ras2 Histone H2A.V -inf Serpin B3
Actin-related protein 2/3 complex Tetraspanin -inf Cystatin-A subunit 2
Cytoplasmic FMR1-interacting protein Ribosyldihydronicotinamide Proteasome subunit beta type-10 -inf 1 dehydrogenase [quinone]
Actin-related protein 2/3 complex Programmed cell death protein 10 Retinoic acid-induced protein 3 -inf subunit 1B
Junctional adhesion molecule A Tubulin-specific chaperone A -inf Complement C4-B
Mesencephalic astrocyte-derived Cytochrome c oxidase subunit 6B1 -inf Superoxide dismutase [Cu-Zn] neurotrophic factor
Desmoglein-2 Deoxyribonuclease-2-alpha -inf Selenium-binding protein 1
Ras-related C3 botulinum toxin Asparagine--tRNA ligase, cytoplasmic -inf Aminopeptidase B substrate 1
Putative small nuclear Beta-mannosidase -inf Cathepsin B ribonucleoprotein G-like protein 15
Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, Sialic acid synthase -inf Brain acid soluble protein 1 mitochondrial
Syndecan binding protein (Syntenin), Guanine nucleotide-binding Histone-binding protein RBBP4 -inf isoform CRA_a protein G(i) subunit alpha-2
Thioredoxin reductase 2, Calpastatin -inf 14-3-3 protein zeta/delta mitochondrial
KH domain-containing, RNA-binding, signal transduction-associated protein Glutathione peroxidase -inf Vinculin 1
Thioredoxin-like protein 1 (Fragment) Xaa-Pro aminopeptidase 1 -inf Macrophage-capping protein
227 F-actin-capping protein subunit Lysosomal acid phosphatase Histone H2B type 1-L -inf alpha-1
Aflatoxin B1 aldehyde reductase Calcyphosin-like protein -inf Protein S100-A12 member 2
Aminoacylase Nucleobindin-1 -inf Olfactomedin-4
NIF3-like protein 1 Coronin-1B -inf Zinc-alpha-2-glycoprotein
Beta-mannosidase CD82 antigen -inf Acyl-CoA-binding protein
Cytoplasmic FMR1-interacting protein Glucose 1,6-bisphosphate synthase -inf Ceruloplasmin 1
Urokinase plasminogen activator Fatty acid-binding protein, Non-specific lipid-transfer protein -inf surface receptor epidermal
G-protein-coupled receptor family C Protein kinase C delta type -inf Vitamin D-binding protein group 5 member C
Adipogenesis regulatory factor Ubiquitin thioesterase OTUB1 -inf L-lactate dehydrogenase B chain
Splicing factor 3B subunit 3 Cathepsin L1 -inf Phosphoglucomutase-2
BTB/POZ domain-containing protein BPI fold-containing family B Small nuclear ribonucleoprotein E -inf KCTD12 member 1
Cytosolic acyl coenzyme A thioester Cytochrome c (Fragment) -inf Vimentin hydrolase
Serine/threonine-protein phosphatase Phosphatidylethanolamine- Src substrate cortactin -inf (Fragment) binding protein 1
Kallikrein-6 Metalloreductase STEAP4 -inf Pro-cathepsin H
Pre-mRNA-processing factor 19 Nck-associated protein 1 -inf Plasma protease C1 inhibitor
Adapter molecule crk Proteasome subunit alpha type -inf Resistin
Chloride intracellular channel Desmocollin-3 Nucleobindin 2, isoform CRA_b -inf protein 1
Serine/arginine-rich-splicing factor 1 Polypyrimidine tract-binding protein 1 -inf Cytidine deaminase
Eukaryotic translation initiation factor 2 Peptidyl-prolyl cis-trans isomerase C -inf D-dopachrome decarboxylase subunit 1
228
Secernin-1 Lysosomal alpha-glucosidase -inf Tropomyosin alpha-4 chain
Immunoglobulin lambda-like Catenin beta-1 Peptidyl-prolyl cis-trans isomerase C -inf polypeptide 5
Enhancer of rudimentary homolog Dual oxidase 1 -inf Glycogenin-1
Keratin, type II cytoskeletal 80 PDZ and LIM domain protein 1 -inf Hexokinase-3
DnaJ homolog subfamily A member 1 Solute carrier family 15 member 2 -inf Protein S100-A11
Phosphatidylinositol transfer protein Pirin -inf Transgelin-2 beta isoform
Immunoglobulin heavy constant F-box only protein 50 Cell division control protein 42 homolog -inf mu
Charged multivesicular body protein von Willebrand factor A domain- SH3 domain-binding glutamic -inf 4b containing protein 7 acid-rich-like protein
Interleukin enhancer-binding factor Prostasin -inf Purine nucleoside phosphorylase 2
Tyrosine-protein phosphatase 40S ribosomal protein S5 Hexokinase-1 -inf non-receptor type substrate 1
Fructose-bisphosphate aldolase 5'-3' exoribonuclease 2 Sorcin -inf A
Intraflagellar transport protein 27 Copine-2 -inf Gelsolin homolog
2',3'-cyclic-nucleotide 3'- Carboxypeptidase E -inf Catalase phosphodiesterase
Intraflagellar transport protein 27 Rootletin -inf Protein disulfide-isomerase homolog
RNA-binding motif protein, X LIM and SH3 domain protein 1 -inf Coronin-1A chromosome
Ribosome-binding protein 1 Small nuclear ribonucleoprotein Sm D2 -inf Matrix metalloproteinase-9
Tyrosine-protein kinase Yes Follistatin-related protein 1 -inf Alpha-1B-glycoprotein
229 Nuclease-sensitive element-binding 6-phosphogluconate Multivesicular body subunit 12A -inf protein 1 dehydrogenase, decarboxylating
Cytochrome c oxidase subunit 5B, Immunoglobulin heavy constant RuvB-like 2 -inf mitochondrial alpha 1
Specifically androgen-regulated gene Catenin alpha-1 -inf Heat shock protein HSP 90-beta protein
Capping protein (Actin filament) 2',3'-cyclic-nucleotide 3'- Desmocollin-3 -inf muscle Z-line, beta, isoform phosphodiesterase CRA_d
Vacuolar protein sorting-associated Serine/arginine-rich-splicing factor 7 -inf 78 kDa glucose-regulated protein protein 28 homolog
Small proline-rich protein 2B 40S ribosomal protein S21 -inf Glutaredoxin-1
Alpha-mannosidase Adapter molecule crk -inf Fibrinogen beta chain
Argininosuccinate lyase Biotinidase -inf S-formylglutathione hydrolase
Leucine-rich repeat-containing protein Mammalian ependymin-related protein -inf Mucin-5B 59 1
Intraflagellar transport protein 25 SEC14-like protein 3 -inf Cystatin-B homolog
Spectrin alpha chain, non- Ribonuclease UK114 ATP-dependent RNA helicase A -inf erythrocytic 1
Rab GDP dissociation inhibitor 60S acidic ribosomal protein P1 Arylsulfatase A -inf beta
Follistatin-related protein 1 Ras-related protein R-Ras2 -inf Annexin A3
High mobility group protein HMG- Peptidoglycan recognition protein Alpha-N-acetylgalactosaminidase -inf I/HMG-Y 1
Malate dehydrogenase, Myoglobin Tyrosine-protein kinase FRK -inf cytoplasmic
Peroxiredoxin-4 Sialate O-acetylesterase -inf Moesin
KH domain-containing, RNA-binding, Ubiquitin-conjugating enzyme E2 signal transduction-associated protein -inf Calmodulin variant 2 1
230
Ras-related protein Ral-A Keratin, type I cytoskeletal 15 -inf Glutathione S-transferase P
Urokinase plasminogen activator Ras-related protein Ral-A -inf Thioredoxin surface receptor
Eukaryotic initiation factor 4A-II Choline transporter-like protein 2 -inf Transaldolase
Low molecular weight Heat shock 70 kDa protein 4L phosphotyrosine protein -inf Talin-1 phosphatase
3-hydroxybutyrate dehydrogenase Retinoid-inducible serine -inf Fibrinogen gamma chain type 2 carboxypeptidase
Choline transporter-like protein 2 S-phase kinase-associated protein 1 -inf Hemopexin
40S ribosomal protein S21 Aminoacylase -inf L-lactate dehydrogenase A chain
Proteasome subunit beta type-7 T-complex protein 1 subunit zeta -inf Rho GDP-dissociation inhibitor 2
2'-deoxynucleoside 5'-phosphate N- Immunoglobulin heavy constant Epithelial cell adhesion molecule -inf hydrolase 1 gamma 4
Peptidyl-glycine alpha-amidating Glutathione S-transferase Profilin -inf monooxygenase omega-1
Eukaryotic translation initiation factor S-adenosylmethionine synthase Glutathione reductase, -inf 2 subunit 1 isoform type-2 mitochondrial
Serine/arginine-rich-splicing factor 2 Glutamate-rich protein 3 -inf Pyruvate kinase PKM (Fragment)
Small nuclear ribonucleoprotein E Annexin A7 -inf Myosin light polypeptide 6
UPF0160 protein MYG1, 4-trimethylaminobutyraldehyde Ubiquitin-60S ribosomal protein -inf mitochondrial dehydrogenase L40
NAD-dependent malic enzyme, Dipeptidyl peptidase 2 -inf Lysozyme C mitochondrial
Tripartite motif-containing protein 29 Proteasome subunit alpha type-2 -inf Serpin B10
GMP reductase Sulfatase-modifying factor 2 -inf Tropomyosin alpha-3 chain
Heterogeneous nuclear 231 Threonine--tRNA ligase, cytoplasmic -inf Non-secretory ribonuclease ribonucleoprotein D0
Guanine nucleotide-binding protein Immunoglobulin heavy constant Alpha-N-acetylglucosaminidase -inf G(k) subunit alpha alpha 2 (Fragment)
Actin-related protein 2/3 complex 60S ribosomal protein L12 -inf Leukotriene A-4 hydrolase subunit 5-like protein
Cell division control protein 42 Adenylyl cyclase-associated DnaJ homolog subfamily A member 1 -inf homolog protein 1
Protein piccolo Ferritin heavy chain -inf Carbonic anhydrase 1
Peptidyl-prolyl cis-trans 60S ribosomal protein L12 Citrate synthase -inf isomerase FKBP1A
Proteasome subunit beta type-10 Alcohol dehydrogenase 1C -inf Heat shock protein HSP 90-alpha
Solute carrier family 15 member 2 Lysosomal protective protein -inf Azurocidin
ADP/ATP translocase 1 Ras-related protein Rap-1b -inf Alpha-1-acid glycoprotein 1
Serine/arginine-rich-splicing factor 2 Quinone oxidoreductase PIG3 -inf Alpha-actinin-4 (Fragment)
Alpha-galactosidase A Folate receptor alpha -inf Myosin-9
Heterogeneous nuclear Charged multivesicular body protein 4b -inf Complement C3 ribonucleoprotein A/B
Protein ABHD14B RuvB-like 1 -inf Phosphoglycerate mutase 1
Spondin-2 Golgi membrane protein 1 -inf Transketolase
Small nuclear ribonucleoprotein Sm Immunoglobulin heavy constant Spondin-2 -inf D2 gamma 3
Ras-related protein Rab-1A Nucleosome asSEbly protein 1-like 1 -inf Phosphoglycerate kinase 1
Peptidyl-prolyl cis-trans 40S ribosomal protein S28 Glucose 1,6-bisphosphate synthase -inf isomerase A
Aldo-keto reductase family 1 member CD82 antigen -inf Alpha-actinin-1 C3
Far upstream element-binding protein Calreticulin 1
232
Sulfatase-modifying factor 2 Alpha-1-acid glycoprotein 2
Profilin Cofilin-1
Guanine nucleotide-binding protein Mucin-5AC subunit alpha-13
Leucine zipper transcription factor-like Glucose-6-phosphate isomerase protein 1 (Fragment)
Heat shock cognate 71 kDa Glutamate-rich protein 3 protein
Citrate synthase Haptoglobin
60S ribosomal protein L5 Filamin-A
Myosin-10 Plastin-2
Lipopolysaccharide-responsive and Profilin-1 beige-like anchor protein
Palmitoyl-protein thioesterase 1 Heat shock 70 kDa protein 1B
Coronin-1B Alpha-enolase
Kallikrein-7 Lactotransferrin
Golgi-associated plant pathogenesis- Triosephosphate isomerase related protein 1
Golgi membrane protein 1 Leukocyte elastase inhibitor
Glyceraldehyde-3-phosphate Pirin dehydrogenase
Cartilage intermediate layer protein 1 Hemoglobin subunit delta
Keratin, type I cytoskeletal 24 Alpha-1-antitrypsin
Choline transporter-like protein 4 Serotransferrin
Immunoglobulin heavy constant Elongation factor 1-beta gamma 2
233 Nucleobindin-1 Hemoglobin subunit beta
Immunoglobulin heavy constant PDZ and LIM domain protein 1 gamma 1 (Fragment)
2'-deoxynucleoside 5'-phosphate N- Immunoglobulin kappa constant hydrolase 1
Protein S100-A13 Myeloblastin
Proteasome subunit beta type-6 Actin, alpha cardiac muscle 1
Keratin, type II cuticular Hb5 Hemoglobin subunit alpha
Ras-related protein Rap-1b Protein S100-A9
Neutrophil gelatinase-associated Histone H1.5 lipocalin
Calpastatin Actin, cytoplasmic 2
Tropomyosin alpha-4 chain Protein S100-A8 (Fragment) Beta-microSeminoprotein Serum albumin
4-trimethylaminobutyraldehyde dehydrogenase
10 kDa heat shock protein, mitochondrial
Growth-regulated alpha protein
Retinoic acid-induced protein 3
Keratin, type II cytoskeletal 1b
Tubulin alpha-4A chain
Annexin A7
Tubulin alpha chain
Sorcin
Keratin, type II cytoskeletal 6B
234 CD59 glycoprotein
Appendix 3. Unique proteins identified by label free LC-MS/MS in non asthmatic HBE apical secretions during Il-13 challenge or at baseline (control)(n=5) and significantly (p<0.05, paired student t-test) changing proteins after challenge with associated log2 fold change of total precursor intensity per protein comparing day 20 of IL-13 treatment to baseline.
Significantly Changing Proteins Control Log2 Fold Control IL-13 vs. IL-13 Change
Farnesyltransferase, CAAX box, alpha, isoform Nucleoside diphosphate kinase 7 Flotillin-2 -INF CRA_a
cDNA, FLJ96923, highly similar to Homo sapiens cDNA FLJ58275, highly similar to Cadherin-1 -4.55 ribophorin II (RPN2), mRNA Keratin, type II cytoskeletal 4
Putative ciliary rootlet coiled-coil protein-like 3 Nucleosome asSEbly protein 1-like 1 ATP synthase subunit alpha, mitochondrial 4.77 protein (Fragment)
cDNA, FLJ95208, highly similar to Homo sapiens PYD and CARD domain IgGFc-binding protein Mannose-1-phosphate guanyltransferase beta 5.00 containing (PYCARD), transcript variant 1, mRNA
235
N-acetyllactosaminide beta-1,3-N- Synaptic vesicle membrane protein VAT-1
Spectrin beta chain, non-erythrocytic 1 -1.64 acetylglucosaminyltransferase 3 homolog
cDNA, FLJ94179, highly similar to Homo Complement C1q tumor necrosis factor- Adenylosuccinate synthetase isozyme 1 sapiens tektin 2 (testicular) (TEKT2), -2.95 related protein 5 mRNA
Plectin Tctex1 domain-containing protein 4 Major vault protein 2.46
Mitochondrial carrier homolog 2 variant 40S ribosomal protein S12 Beta-galactosidase 1.46 (Fragment)
ADP-sugar pyrophosphatase Nuclear transport factor 2 Anterior gradient protein 3 homolog 3.31
Serine/threonine-protein phosphatase 2A catalytic Ferritin light chain Histone H3 1.78 subunit alpha isoform Liver carboxylesterase 1 Splicing factor, proline- and glutamine-rich T-complex protein 1 subunit beta +INF
V-set and transmembrane domain- Trifunctional enzyme subunit alpha, ATP-binding cassette sub-family D member 3 4.35 containing protein 2-like protein mitochondrial +INF Stomatin-like protein 2, mitochondrial Spectrin alpha chain, non-erythrocytic 1 ATP synthase subunit d, mitochondrial
NADPH:adrenodoxin oxidoreductase, Carboxypeptidase D STK25 protein 2.40 mitochondrial
Alpha/beta hydrolase domain-containing protein Purine nucleoside phosphorylase Histone H2B type 1-N 1.59 11
CCT8 protein Ras-related protein Rab-5B ATP synthase subunit beta, mitochondrial 4.03
cDNA FLJ44920 fis, clone BRAMY3011501, T-complex protein 1 subunit gamma Protein disulfide-isomerase A4 highly similar to Heterogeneous nuclear +INF ribonucleoprotein U cDNA FLJ77057, highly similar to Homo sapiens doublecortex; lissencephaly, X-linked EF-hand domain-containing protein D2 ATP synthase subunit gamma, mitochondrial 3.00 (doublecortin) (DCX), transcript variant 2, mRNA
Latent-transforming growth factor beta- Ceroid-lipofuscinosis neuronal protein 5 ATP synthase subunit gamma -3.69 binding protein 3
G protein-coupled receptor, family C, group Glutathione S-transferase kappa 1 Proteasome subunit beta type-4 -4.97 5, member C, isoform CRA_d
Insulin-like growth factor binding protein
236 Testis-specific gene 10 protein (Fragment) Protein ARPC4-TTLL3 -INF 3
Ubiquitin-conjugating enzyme E2 L3 Monoglyceride lipase Beta-actin-like protein 2 -1.89
cDNA FLJ75185 Lactoylglutathione lyase Choline transporter-like protein 4 -2.19
ATP-dependent RNA helicase A Protein OSCP1 Galectin-3-binding protein -2.91
Type II 3a-hydroxysteroid Proteasome subunit alpha type-3 Keratin 1 -1.92 dehydrogenase variant
cDNA FLJ57133, highly similar to Bifunctional Proteasome subunit alpha type Tumor susceptibility gene 101 protein -6.34 purine biosynthesis protein PURH (Fragment) Tumor necrosis factor ligand superfamily Coiled-coil domain-containing protein 96 member 10 Stomatin-like protein 2, mitochondrial 3.81
Small nuclear ribonucleoprotein Sm D1 Septin 9, isoform CRA_a Calreticulin variant (Fragment) 2.28 Isochorismatase domain-containing protein 2, -1.80 mitochondrial NADH-cytochrome b5 reductase 14-3-3 protein gamma
cDNA, FLJ95650, highly similar to Homo sapiens 10 kDa heat shock protein, mitochondrial Retinoid-inducible serine carboxypeptidase -2.24 karyopherin (importin) beta 1 (KPNB1), mRNA Alcohol dehydrogenase class 4 mu/sigma SH3 and multiple ankyrin repeat domains protein ADP-ribosylation factor 6 chain -1.15 2
cDNA FLJ55506, highly similar to Puromycin- Pulmonary surfactant-associated protein Arachidonate 15-lipoxygenase 2.60 sensitive aminopeptidase (EC3.4.11.-) B
Dystroglycan 1 (Dystrophin-associated Dynein light chain 1, axonemal Sulfhydryl oxidase 1 -2.75 glycoprotein 1), isoform CRA_a
cDNA FLJ55534, highly similar to 4- Lysosome membrane protein 2 trimethylaminobutyraldehyde Adapter molecule crk -INF dehydrogenase (EC 1.2.1.47)
Methylcrotonoyl-CoA carboxylase beta chain, Alpha-N-acetylgalactosaminide alpha- EGF-containing fibulin-like extracellular -4.53 mitochondrial 2,6-sialyltransferase 1 matrix protein 1
Kinesin-like protein KIF19 Cytosol aminopeptidase Folate receptor alpha -2.19
Neural cell adhesion molecule L1-like Catenin delta-1 Protein S100-A6 -INF protein
Delta-1-pyrroline-5-carboxylate synthase Dynein intermediate chain 1, axonemal Cathepsin L1 -2.96
cDNA FLJ50594, highly similar to Deoxynucleoside triphosphate Chromosome 9 open reading frame 19, Pulmonary surfactant-associated protein -2.31 237 triphosphohydrolase SAMHD1 isoform CRA_a (Fragment) A2
Enoyl-CoA delta isomerase 1, Nuclear migration protein nudC Dopamine receptor interacting protein 4 -3.54 mitochondrial
cDNA FLJ78473, highly similar to Homo cDNA FLJ54547, highly similar to Alpha-1 catenin S-(hydroxymethyl)glutathione sapiens EF-hand domain (C-terminal) -1.93 (Cadherin-associated protein) dehydrogenase (Fragment) containing 2 (EFHC2), mRNA
cDNA, FLJ92608, highly similar to Homo Pyruvate dehydrogenase E1 component subunit sapiens aldehyde dehydrogenase 1 Transmembrane channel-like protein -3.28 beta, mitochondrial family, member A3 (ALDH1A3), mRNA
Inosine-5'-monophosphate dehydrogenase 2 Protein SET Transmembrane channel-like protein -4.45 (Fragment)
Tandem C2 domains nuclear protein Dihydrolipoyl dehydrogenase Metalloreductase STEAP4 -9.19
Nicotinamide phosphoribosyltransferase Trefoil factor 3 Transforming protein RhoA -3.30 cDNA, FLJ93591, highly similar to Homo sapiens transforming growth factor, beta 2 (TGFB2), Transmembrane protease serine 2 Superoxide dismutase [Cu-Zn] -1.56 mRNA (Fragment)
Cytochrome c oxidase subunit 5B, mitochondrial Heme-binding protein 2 Plasminogen activator inhibitor 2 3.26
Vacuolar protein sorting-associated Calmodulin ATP synthase subunit O, mitochondrial -1.63 protein 37B (Fragment)
cDNA FLJ54574, highly similar to Staphylococcal 5'-nucleotidase Protein S100-A2 -5.03 nuclease domain-containing protein 1
cDNA, FLJ94534, highly similar to Homo GTP-binding nuclear protein Ran sapiens capping protein (actin filament), Mucin-16 -3.78 gelsolin-like(CAPG), mRNA
GTP-binding nuclear protein Ran Anterior gradient protein 3 homolog Sialate O-acetylesterase -5.03 (Fragment)
Perilipin-3 Myotrophin Attractin -4.32
T-complex protein 1 subunit gamma Sodium-dependent phosphate transport TRAF3-interacting protein 1 -2.53 (Fragment) protein 2B
Amyloid beta (A4)-like protein 2, isoform CRA_a P37 AUF1 Citrate synthase +INF Serpin B5 Vacuolar-sorting protein SNF8 60 kDa heat shock protein, mitochondrial 5.58
238 Sarcoplasmic/endoplasmic reticulum calcium cDNA FLJ77178 40S ribosomal protein S8 +INF ATPase 2
Isocitrate dehydrogenase [NADP], Autoantigen La (Fragment) Citrate synthase 1.93 mitochondrial
cDNA PSEC0037 fis, clone NT2RP1000800, highly similar to N-acetyllactosaminidebeta-1,3-N- CGI-150 protein Plastin-3 -2.67 acetylglucosaminyltransferase
Eukaryotic translation initiation factor 5A- Myosin regulatory light chain 12A Mucin-16 -3.58 1
cDNA FLJ78452, highly similar to Homo sapiens CD63 antigen G-protein coupled receptor 126 -INF legumain (LGMN), transcript variant 2, mRNA
N(G),N(G)-dimethylarginine Dystroglycan Serine/threonine-protein kinase 26 -INF dimethylaminohydrolase 1
Epsin-1 Charged multivesicular body protein 5 BPI fold-containing family A member 1 -2.70
Glyoxalase domain-containing protein 4 Peroxiredoxin-4 Beta-galactosidase -1.00 cDNA FLJ51884, highly similar to Collagenase-3 splice variant COL3-9B-2 Dynein light chain roadblock-type 1 Mitochondrial inner membrane protein +INF
Apoptosis-inducing factor 1, mitochondrial Myeloid leukemia factor 1 Deleted in malignant brain tumors 1 protein -6.62
Zinc finger MYND domain-containing protein 10 KLK10 protein (Fragment) Poly(rC)-binding protein 1 -1.71
Platelet-activating factor acetylhydrolase IB cDNA FLJ51907, highly similar to Stress- Ubiquitin-40S ribosomal protein S27a -2.25 subunit alpha 70 protein, mitochondrial
Kinesin-like protein Charged multivesicular body protein 4b Histone H2A type 1-B/E 1.04
Transmembrane 9 superfamily member 2 Ferritin heavy chain Tetraspanin-1 -2.29
Neuroblast differentiation-associated protein Tumor susceptibility gene 101 protein Histone H4 1.07 AHNAK Prosaposin (Variant Gaucher disease and variant metachromatic Coiled-coil domain-containing protein 69 Tissue alpha-L-fucosidase -2.15 leukodystrophy) variant (Fragment)
Coiled-coil domain-containing protein 40 Puromycin-sensitive aminopeptidase Calcium and integrin-binding protein 1 -3.31
Gamma-glutamyltranspeptidase 1 (Fragment) Intraflagellar transport protein 74 -5.11 Adhesion G-protein coupled receptor F1 homolog
239 Inositol monophosphatase 1 (Fragment) Aspartate aminotransferase Prohibitin-2 2.68 cDNA, FLJ92996, highly similar to Homo
cDNA FLJ46245 fis, clone sapiens guanine nucleotide binding protein Laminin, gamma 1 (Formerly LAMB2), isoform TESTI4020596, highly similar to Homo (G protein), beta polypeptide 1 (GNB1), -2.76 CRA_a sapiens calpain 5 (CAPN5) mRNA
cDNA, FLJ94267, highly similar to Homo Platelet-activating factor acetylhydrolase IB sapiens glutathione S-transferase omega Calcium-binding protein 39 -5.65 subunit beta 1 (GSTO1), mRNA
UTP--glucose-1-phosphate Trifunctional enzyme subunit alpha, mitochondrial Deoxyribonuclease II (Fragment) -6.30 uridylyltransferase
Tumor-associated calcium signal transducer Malate dehydrogenase, mitochondrial Non-specific protein-tyrosine kinase 2.27 2
Armadillo repeat-containing protein 4 2',3'-cyclic-nucleotide 3'- cDNA FLJ14514 fis, clone NT2RM1000742, -4.74 phosphodiesterase highly similar to Prominin-1
Tenascin C (Hexabrachion), isoform Guanine nucleotide-binding protein 2,4-dienoyl-CoA reductase, mitochondrial -2.44 CRA_a G(I)/G(S)/G(T) subunit beta-2 Epididymis secretory protein Li 100 Tryptophan--tRNA ligase, cytoplasmic Fatty acid-binding protein, epidermal 1.72
X-ray repair cross-complementing protein 6 Protein disulfide-isomerase Epididymal secretory protein E1 -2.65
Asparagine--tRNA ligase, cytoplasmic Platelet glycoprotein 4 (Fragment) Ras-related protein Rab-5C -2.89
Epidermal growth factor receptor kinase Syntaxin-3 Dipeptidyl peptidase 3 -5.75 substrate 8-like protein 2
Protein cordon-bleu Proteasome subunit alpha type-5 Arrestin domain-containing protein 1 -2.73
cDNA FLJ57964, highly similar to Heterogeneous Neuropilin 2, isoform CRA_c WAP four-disulfide core domain protein 2 -2.64 nuclear ribonucleoprotein H'
Heterogeneous nuclear ribonucleoprotein H Thioredoxin reductase 1 Acyl-CoA-binding protein -1.23
Polypeptide N-acetylgalactosaminyltransferase Unconventional myosin-VI Cathepsin S -2.06
Cathepsin Z Histone H1.3 40S ribosomal protein S8 +INF
WD repeat-containing protein 34 Glutamate dehydrogenase Actin-related protein 3 -3.15 cDNA FLJ76072, highly similar to Homo sapiens GIPC PDZ domain containing family, cDNA FLJ54992, highly similar to TOM1-like 1 Small proline-rich protein 3 member 1 (GIPC1), transcript variant 1, -3.93 protein mRNA
240 Protein FAM92B Calpain-1 catalytic subunit Sorcin -1.61
Alpha-amylase AP-1 complex subunit beta-1 Epitheliasin -3.22
Laminin subunit beta-2 Argininosuccinate synthase Multivesicular body subunit 12A -INF
Vacuolar protein sorting-associated protein Ras-related protein Rab-2A ST13 protein (Fragment) -4.72 37B
S100 calcium binding protein A10 (Annexin II ligand, calpactin I, light Growth arrest-specific protein 8 Alpha-N-acetylglucosaminidase -2.26 polypeptide (P11)), isoform CRA_b (Fragment)
Acid sphingomyelinase-like Protein FAM177A1 UMP-CMP kinase -4.29 phosphodiesterase 3b
cDNA FLJ56337, highly similar to High mobility Ras-related protein Rab-1B HSPA9 protein (Fragment) 2.17 group protein B1
Adenylosuccinate synthetase isozyme 2 T-complex protein 1 subunit beta Protein disulfide-isomerase +INF
cDNA FLJ61383, highly similar to CD200R1 protein Aconitate hydratase, mitochondrial Serine/threonine-protein kinase 24 (EC -3.42 2.7.11.1)
Activator of 90 kDa heat shock protein ATPase N-acylsphingosine amidohydrolase (Acid Histone H2B type 3-B 0.93 homolog 1 ceramidase) 1, isoform CRA_c
S100 calcium binding protein A13, isoform CRA_a cDNA FLJ59142, highly similar to CD59 glycoprotein -1.61 (Fragment) Epididymal secretory protein E1
Interferon induced transmembrane protein 1 (9- Chromosome 9 open reading frame 88, 40S ribosomal protein SA +INF 27), isoform CRA_a isoform CRA_a
Solute carrier family 25 (Mitochondrial carrier Nitric oxide synthase, inducible Glucan , branching enzyme 1 variant (Fragment) phosphate carrier), member 3, isoform 3.00 CRA_a
Adenosylhomocysteinase Cornifin-B Peptidyl-prolyl cis-trans isomerase -1.23
cDNA FLJ58073, moderately similar to Tubulin polymerization-promoting protein -1.02 Katanin p80 subunit B 1 variant (Fragment) Cathepsin B (EC 3.4.22.1) family member 3
cDNA FLJ58863, highly similar to Protein Putative uncharacterized protein Galectin-7 -2.53 NipSnap3A DKFZp686J1169
Rho GDP-dissociation inhibitor 1 241 Cytochrome b5 Ceruloplasmin -4.90 (Fragment)
ATP synthase subunit gamma, mitochondrial F-actin-capping protein subunit beta BRO1 domain-containing protein BROX -3.81
cDNA FLJ54716, highly similar to Target of Myb Galactoside 3(4)-L-fucosyltransferase Syntaxin-binding protein 2 -3.80 protein 1 cDNA FLJ77316, highly similar to Homo sapiens interferon, gamma-inducible protein 30 (IFI30), Calpain 2, large [catalytic] subunit Keratin, type II cytoskeletal 2 epidermal -2.90 mRNA variant (Fragment)
Aflatoxin B1 aldehyde reductase member 2 RuvB-like 1 Ceruloplasmin -4.91 cDNA FLJ78217 WASF2 protein (Fragment) Histone H3.1 -2.91
Rhophilin-2 Phosphoglycerate mutase 1 (Brain) Ras-related protein Rab-14 -1.90
4-trimethylaminobutyraldehyde dehydrogenase Plastin-3 Cystatin-S +INF
Epididymis secretory protein Li 100 cDNA FLJ61580, highly similar to Glutathione S-transferase kappa 1 4.84 Calsyntenin-1
Hemoglobin alpha-1 globin chain variant cDNA, FLJ95457, highly similar to Homo CD2-associated protein -1.21 (Fragment) sapiens tubulin, beta, 4 (TUBB4), mRNA
Tubby-like protein Tetraspanin Ras-related protein R-Ras2 -3.43
Rho-related GTP-binding protein RhoC Carboxypeptidase M (Fragment) Heterogeneous nuclear ribonucleoprotein F +INF (Fragment)
Signal transducer and activator of transcription GNAI2 protein Lysozyme C 2.39
Prohibitin Malate dehydrogenase 14-3-3 protein theta -3.44
Isocitrate dehydrogenase [NADP] cDNA FLJ57046, highly similar to Lysosomal IQ and ubiquitin-like domain-containing protein -6.62 cytoplasmic alpha-glucosidase (EC 3.2.1.20)
STE20-like serine/threonine-protein kinase Epididymis luminal protein 211 Transcobalamin-1 -2.09
Transforming growth factor-beta-induced protein cDNA FLJ57602, highly similar to Moesin -2.49 ig-h3 Creatine kinase M-type (EC 2.7.3.2) Mucin-2 Netrin-4 Radixin -2.52
Alpha-mannosidase Beta-2-microglobulin Alpha-actinin-4 -2.56
TNF receptor-associated protein 1 TBC1 domain family, member 10A COL21A1 protein -4.19 variant (Fragment)
242 Alanine--tRNA ligase, cytoplasmic Histone H2B type 1-J 40S ribosomal protein S6 +INF
LIM and SH3 domain protein 1 TMSB4X protein (Fragment) ALDH3A1 protein (Fragment) -1.55
Unconventional myosin-Ie Fetuin-B MUC5AC (Fragment) 9.15
Obg-like ATPase 1 Beta-globin Protein S100-A11 -1.42
Solute carrier family 15 (H+/peptide cDNA FLJ55263 Acyl-CoA-binding protein -2.92 transporter), member 2
Aldehyde dehydrogenase, mitochondrial Transmembrane channel-like protein 4 Dipeptidyl peptidase 4 3.82
cDNA, FLJ79223, highly similar to Nucleosome Histone H2B Unconventional myosin-Ib -3.44 asSEbly protein 1-like 4
Heat shock 70kDa protein 4 isoform a variant HCG40889, isoform CRA_b Alpha-actinin-1 -2.16 (Fragment)
cDNA FLJ54755, highly similar to Vacuolar ATP Fructose-bisphosphate aldolase Kallikrein-11 -1.49 synthase subunit d (EC 3.6.3.14)
Ras-related C3 botulinum toxin substrate 1 Teneurin-4 Transketolase (Rho family, small GTP binding protein -2.52 Rac1)
Cationic amino acid transporter 2 Transferrin variant (Fragment) Actin, alpha cardiac muscle 1 -2.70
Proteasome subunit alpha type (Fragment) Tyrosine-protein kinase receptor Radial spoke head 1 homolog -3.24
cDNA FLJ53963, highly similar to Anterior gradient 2 homolog (Fragment) Tubulin beta chain -1.22 Leukocyte elastase inhibitor
Unconventional myosin-Vb WD repeat-containing protein 1 Intelectin-2 7.84
Hydroxyacylglutathione hydrolase, mitochondrial Guanine nucleotide-binding protein G(i) -2.56 Glutathione S-transferase A2 (Fragment) subunit alpha-1
High affinity copper uptake protein 1 Periostin Tropomyosin alpha-4 chain -1.33
Palmitoyl-protein thioesterase 1 14-3-3 protein epsilon Tubulin, beta 2C -1.16
Carcinoembryonic antigen-related cell Prohibitin-2 Annexin -2.36 adhesion molecule 5
Ras GTPase-activating-like protein Tetraspanin (Fragment) Mucin-5AC 7.19 IQGAP1
243 -3.21 Proteasome subunit beta type Peroxiredoxin-2 Ubiquitin-conjugating enzyme E2 D3
Macrophage colony-stimulating factor 1 Histone H2A type 1-J Choline transporter-like protein 2 isoform 2 -INF (Fragment)
EF-hand domain-containing protein 1 Fatty acid-binding protein, adipocyte Tubulin beta-4A chain -1.21
cDNA, FLJ93654, highly similar to Homo sapiens serpin peptidase inhibitor, clade Guanine nucleotide-binding protein subunit Catalase -3.31 B (ovalbumin), member 2 (SERPINB2), alpha-11 mRNA
cDNA FLJ76886, highly similar to Homo sapiens Carboxylic ester hydrolase (Fragment) Capping protein (Actin filament) muscle Z- loss of heterozygosity, 11, chromosomal region 2, -2.71 line, beta, isoform CRA_d gene A (LOH11CR2A), transcript variant 1, mRNA
Guanine nucleotide-binding protein G(i) Chloride intracellular channel protein (Fragment) Elongation factor 1-alpha 1 -2.58 subunit alpha-2
N(4)-(beta-N-acetylglucosaminyl)-L- -INF Pyridoxal kinase Heat shock protein HSP 90-beta asparaginase
Guanine nucleotide-binding protein G(q) Coronin Cofilin-2 -3.40 subunit alpha
Soluble calcium-activated nucleotidase 1 Cytochrome c Rab GDP dissociation inhibitor alpha -2.20
SCCA2/SCCA1 fusion protein isoform 1 SH3 domain-binding glutamic acid-rich-like Septin-9 -2.04 protein
Single-stranded DNA-binding protein CD133 isoform H TUBB6 protein -1.32
Eukaryotic initiation factor 4A-II Cofilin-1 (Fragment) Ras-related protein Rab-7a -3.96
cDNA FLJ46662 fis, clone TRACH3006800, highly similar to Homo Glutamine synthetase Neurexin-3 -INF sapiens mucin 16, cell surface associated (MUC16), mRNA
Pleckstrin homology domain-containing Elongation factor 1-gamma Calmodulin -4.86 family S member 1
Sulfide dehydrogenase like (Fragment) Fatty acid-binding protein, heart Ras-related protein Rab-10 -2.39
Ferritin Glutathione S-transferase pi (Fragment) Calpain small subunit 1 -1.00 Tubulointerstitial nephritis antigen-like -6.45 SCCA1/SCCA2 fusion protein Alpha-amylase 1
244
cDNA FLJ58182, highly similar to Protein CYR61 Albumin (Fragment) 14-3-3 protein beta/alpha -3.64
cDNA FLJ61415, highly similar to Protein kinase C and casemin kinase substratein neurons protein Mucin-4 (Fragment) Envoplakin, isoform CRA_a -INF 3
Brain-specific angiogenesis inhibitor 1- Amino acid transporter Dipeptidyl peptidase 1 -4.72 associated protein 2
Syntaxin-7 Tubulin beta-3 chain Ras-related protein Ral-B -INF Cytochrome b-c1 complex subunit 2, mitochondrial Tubulin alpha-4A chain 40S ribosomal protein S3 +INF
Cytoplasmic dynein 2 light intermediate chain 1 Myoglobin Connective tissue growth factor -4.00 cDNA FLJ75883, highly similar to Homo NADPH:adrenodoxin oxidoreductase, sapiens glucosamine (N-acetyl)-6-sulfatase Tubulin alpha-1B chain -4.53 mitochondrial (Sanfilippo disease IIID) (GNS), mRNA
Neurexin-3 Annexin Glutamate-rich protein 3 -2.04
cDNA FLJ75934, highly similar to Homo Glucose-6-phosphate 1-dehydrogenase Cystatin-S sapiens vacuolar protein sorting 4B (yeast) -INF (Fragment) (VPS4B), mRNA
Urokinase-type plasminogen activator Actin, cytoplasmic 2 Carbohydrate sulfotransferase 6 1.80
cDNA FLJ78635, highly similar to Homo Glutamate-cysteine ligase, modifier subunit, sapiens ATP synthase, H+ transporting, 2.32 cDNA, FLJ93674 isoform CRA_a mitochondrial F0 complex, subunit b, isoform 1 (ATP5F1), transcript variant 1, mRNA
Actin-related protein 2/3 complex subunit 3 Unconventional myosin-Id -4.33
Succinyl-CoA ligase [ADP/GDP-forming] subunit Dynein light chain 2, cytoplasmic -1.74 alpha, mitochondrial Ras-related protein Ral-A HSPA9 protein (Fragment) -INF
cDNA, FLJ95737, highly similar to Homo sapiens aconitase 2, mitochondrial (ACO2), Protein lin-7 homolog C +INF nuclear geneencoding mitochondrial protein, mRNA Intraflagellar transport protein 88 homolog Mucin-4 -3.29
Glutathione peroxidase Mucin-4 -3.23
245
Ribonuclease pancreatic Specifically androgen-regulated gene protein -INF
Guanine nucleotide-binding protein G(k) Meckelin -2.52 subunit alpha
Charged multivesicular body protein 6 Prosaposin variant (Fragment) -1.97
Epoxide hydrolase 1 DnaJ homolog subfamily B member 13 -4.46 -1.73 DPY30 domain-containing protein 2 (Fragment) Dynein light chain 1, cytoplasmic
cDNA, FLJ95704, highly similar to Homo sapiens serine protease inhibitor, Kunitz type 1 (SPINT1), Dihydrolipoyl dehydrogenase, mitochondrial 1.61 mRNA
Brain-specific angiogenesis inhibitor 1-associated L-lactate dehydrogenase B chain -0.96 protein 2-like protein 1 Protein disulfide-isomerase A4 S-phase kinase-associated protein 1 +INF
Alcohol dehydrogenase class-3 Elongation factor 1-gamma 2.44
Cadherin-related family member 4 Na(+)/H(+) exchange regulatory cofactor -2.71 NHE-RF1
Actin related protein 2/3 complex subunit 1A Electron transfer flavoprotein subunit beta +INF variant (Fragment)
cDNA FLJ58832, highly similar to Heterogeneous Anterior gradient 2 homolog (Fragment) 2.10 nuclear ribonucleoprotein A3
EF-hand calcium-binding domain-containing Chloride intracellular channel protein 6 -2.86 protein 7
Pleckstrin homology domain-containing family A -2.18 Destrin member 4
MAGUK p55 subfamily member 5 YWHAE/FAM22A fusion protein (Fragment) -2.63
Phosphoenolpyruvate carboxykinase [GTP], Cofilin-1 -2.17 mitochondrial
Thrombospondin 1, isoform CRA_a Ras-related protein Rab-5A -3.27
cDNA FLJ76817, highly similar to Homo sapiens ATP-dependent 6-phosphofructokinase, non-POU domain containing, octamer-binding 4.21 platelet type (NONO), mRNA
246 CD82 antigen Tetratricopeptide repeat protein 21A -INF
ADP/ATP translocase 1 Complement factor H -4.72 Intraflagellar transport protein 25 homolog Cathepsin D -1.91
Laminin subunit beta-3 Triosephosphate isomerase -0.88
Syndecan binding protein (Syntenin), isoform Tetraspanin -4.41 CRA_a
Calpastatin, isoform CRA_a Aminopeptidase N -INF
Glutaredoxin-1 Protein S100-A9 -1.40
WD repeat-containing protein 54 Monocyte differentiation antigen CD14 -4.73
Dual adapter for phosphotyrosine and 3- Alpha-enolase -1.08 phosphotyrosine and 3-phosphoinositide
cDNA FLJ75934, highly similar to Homo sapiens vacuolar protein sorting 4B (yeast) (VPS4B), Sorbitol dehydrogenase -5.93 mRNA
Dynamin-2 Cystatin-SN 12.18
Disintegrin and metalloproteinase domain- Aldehyde dehydrogenase family 3 member -2.63 containing protein 9 B1
Epidermal growth factor receptor kinase cDNA FLJ78449 -4.85 substrate 8
cDNA FLJ61465, highly similar to Homo sapiens Fructose-bisphosphate aldolase C -0.97 vacuolar protein sorting 37C (VPS37C), mRNA
Guanine nucleotide-binding protein subunit Acid sphingomyelinase-like phosphodiesterase 3b -3.17 alpha-14
Mitotic interactor and substrate of PLK1 RAB1B protein -2.09
Actin-related protein 2/3 complex subunit 5 Dynein light chain Tctex-type 1 -1.75
Proteasome subunit alpha type-7 Fetuin-B +INF
cDNA FLJ61465, highly similar to Homo Laminin subunit alpha-5 sapiens vacuolar protein sorting 37C -INF
247 (VPS37C), mRNA
Microsomal glutathione S-transferase 1 Beta actin variant (Fragment) -2.15
cDNA, FLJ92718, highly similar to Homo sapiens Beta-mannosidase -3.17 tripeptidyl peptidase I (TPP1), mRNA
cDNA FLJ31801 fis, clone NT2RI2009066, weakly Meckelin -INF similar to Restin
Anoctamin-6 Programmed cell death protein 10 -3.68
cDNA FLJ60397, highly similar to Lysosomal Dystroglycan 1 (Dystrophin-associated 3.32 protective protein (EC 3.4.16.5) glycoprotein 1), isoform CRA_a
Aspartate aminotransferase, mitochondrial Epidermal growth factor receptor kinase -3.58 substrate 8-like protein 1
cDNA FLJ53936, highly similar to Medium- Leucine zipper transcription factor-like protein 1 chain specific acyl-CoA dehydrogenase, +INF mitochondrial (EC 1.3.99.3)
Apoptosis-associated speck-like protein ADP-ribosylation factor-like protein 6 -5.09 containing a CARD
cDNA FLJ78473, highly similar to Homo sapiens EF-hand domain (C-terminal) containing 2 Ezrin -2.42 (EFHC2), mRNA 1.94 Flotillin-2 Soluble calcium-activated nucleotidase 1
Glucosidase, alpha acid (Pompe disease, Uncharacterized protein KIAA1211-like glycogen storage disease type II), isoform -4.70 CRA_a
Protein XRP2 CTSH protein -2.65
cDNA FLJ55176, highly similar to G-protein 14-3-3 protein zeta/delta -1.79 coupled receptor family C group 5 member B
Hydroxymethylglutaryl-CoA lyase, RuvB-like 1 (Fragment) +INF mitochondrial cDNA FLJ53342, highly similar to Granulins Intelectin 1 6.64
Glutamate dehydrogenase 1, mitochondrial Dynein heavy chain 5, axonemal -4.76
248 cDNA, FLJ78886, highly similar to Natural Midkine -INF resistance-associated macrophage protein 2
cDNA FLJ58539, highly similar to Keratin, type II Glypican-1 -3.43 cytoskeletal 4
Plexin-B2 PDZ and LIM domain protein 1 -INF
Ubiquitin-conjugating enzyme E2 D3 Plastin-1 -1.87 -1.74 Tetratricopeptide repeat protein 21B ADP-ribosylation factor 6
Eukaryotic translation initiation factor 5A Heat shock cognate 71 kDa protein -0.86 (Fragment)
Non-specific protein-tyrosine kinase Adapter molecule crk -6.07 (Fragment)
Laminin alpha-3 chain variant 1 Proteasome subunit alpha type -2.40
Phospholipid-transporting ATPase IC Intraflagellar transport protein 27 homolog -1.83 Chloride intracellular channel protein 1 Drebrin-like protein -2.69
ADP-ribosylation factor 6 Fatty aldehyde dehydrogenase -1.55
Mitochondrial carrier homolog 2 variant Dual adapter for phosphotyrosine and 3- (Fragment) -INF phosphotyrosine and 3-phosphoinositide
Programmed cell death protein 6 CD109 antigen -5.31
Vacuolar protein sorting-associated protein Phosphoglycerate mutase (Fragment) -8.23 28 homolog
WD repeat-containing protein 60 Ras-related protein Rap-1A -3.04
Calnexin Palmitoyl-protein thioesterase 1 -INF
N(4)-(beta-N-acetylglucosaminyl)-L-asparaginase ADP/ATP translocase 2 1.72 -3.58 RILP-like protein 2 Coiled-coil domain-containing protein 114
Syntaxin-binding protein 1 Unconventional myosin-Ic -4.10
Flavin reductase (NADPH) Radial spoke head 14 homolog -4.01
Aminopeptidase B Adenylate kinase 8 -INF
249 cDNA FLJ77316, highly similar to Homo
Rho-related GTP-binding protein RhoG sapiens interferon, gamma-inducible protein -5.11 30 (IFI30), mRNA Dynein light chain roadblock-type 2 Brain acid soluble protein 1 -2.07
Bardet-Biedl syndrome 1 protein Leucine-rich alpha-2-glycoprotein -2.66
Chromosome 11 open reading frame 60, isoform F-actin-capping protein subunit alpha-2 -INF CRA_a
cDNA, FLJ96627, highly similar to Homo Annexin (Fragment) sapiens calpain 1, (mu/I) large subunit -2.05 (CAPN1), mRNA
Guanine nucleotide-binding protein subunit Angiotensin-converting enzyme -2.68 alpha-13
F-actin-capping protein subunit alpha-2 Radial spoke head protein 3 homolog -4.68
cDNA, FLJ94213, highly similar to Homo sapiens cDNA FLJ60299, highly similar to Rab GDP -2.08 pregnancy-zone protein (PZP), mRNA dissociation inhibitor beta
Mannosyl (Alpha-1,3-)-glycoprotein beta-1,2- Spondin-2 N-acetylglucosaminyltransferase variant +INF (Fragment)
Testican-2 Serpin B10 +INF
cDNA, FLJ78886, highly similar to Natural Prohibitin 3.18 resistance-associated macrophage protein 2
cDNA FLJ76272, highly similar to Homo sapiens solute carrier family 23 (nucleobase transporters), Tyrosine-protein kinase FRK -INF member 1 (SLC23A1), transcript variant 2, mRNA
cDNA FLJ35079 fis, clone PLACE6005283, highly similar to Lysosome-associated membrane Protein S100-A16 -2.49 glycoprotein 1
Neurobeachin Keratin, type I cytoskeletal 17 -2.67
G-protein coupled receptor 126 Nucleoside diphosphate kinase homolog 5 -4.22
Cathepsin L1 Ester hydrolase C11orf54 -1.21
250
cDNA FLJ43948 fis, clone TESTI4014924, highly similar to Homo sapiens cytoplasmic -3.78 Guanine deaminase FMR1 interacting protein 1 (CYFIP1), transcript variant 1, mRNA
Fibronectin 1, isoform CRA_n Enoyl-CoA hydratase, mitochondrial +INF
Dihydrolipoyl dehydrogenase, mitochondrial Coronin-1B -INF Radial spoke head protein 4 homolog A Aldo-keto reductase family 1 member C3 -2.95
Actin-related protein 2/3 complex subunit 5 Bardet-Biedl syndrome 1 protein -INF Prominin 2, isoform CRA_a Heat shock 70 kDa protein 1B -1.20
Pleckstrin homology domain-containing family S Annexin A2 -1.78 member 1
Peroxidasin homolog Sulfide dehydrogenase like (Fragment) 2.59
Plasma protease C1 inhibitor Villin-like protein -3.30
Sushi repeat-containing protein SRPX2 Chloride intracellular channel protein 5 -3.34
cDNA FLJ77982, highly similar to Homo cDNA FLJ56155, highly similar to UTP--glucose- sapiens dynein, axonemal, intermediate -2.66 1-phosphate uridylyltransferase 2 (EC 2.7.7.9) polypeptide 1, mRNA
cDNA FLJ16254 fis, clone HLUNG2015418, Guanine nucleotide-binding protein G(s) -3.07 highly similar to Homo sapiens Cadherin subunit alpha isoforms XLas
Mesothelin Myoferlin -6.05
Protein CutA Neutrophil gelatinase-associated lipocalin -2.20 Keratin, type II cytoskeletal 7 Kalirin -0.66
Flotillin-1 60S ribosomal protein L14 +INF
Ras-related protein Ral-B Zymogen granule protein 16 homolog B Stromelysin-2 Profilin (Fragment) Ras-related protein Rab-5A
251 Unconventional myosin-Ic
SH2 domain-containing protein 4A
Dynein, axonemal, heavy chain 11
Biotinidase
Tetratricopeptide repeat protein 30A
Aldehyde dehydrogenase family 1 member A3
Tetratricopeptide repeat protein 30B
Epididymis secretory protein Li 106
Glutathione S-transferase omega-1
cDNA, FLJ95483, highly similar to Homo sapiens chitobiase, di-N-acetyl- (CTBS), mRNA
Complement factor properdin, isoform CRA_c
Ubiquitin domain-containing protein 1
Twinfilin-1
Histone H3
Coronin-1B
UMP-CMP kinase
cDNA FLJ36533 fis, clone TRACH2004428, highly similar to Lactotransferrin (EC 3.4.21.-) (Fragment)
Rho GDP-dissociation inhibitor 1
Envoplakin, isoform CRA_a
Latent-transforming growth factor beta-binding protein 3
cDNA FLJ77982, highly similar to Homo sapiens dynein, axonemal, intermediate polypeptide 1, mRNA
252
Cellular retinoic acid-binding protein 2
Peptidyl-glycine alpha-amidating monooxygenase
Histone H2B type 3-B
Intercellular adhesion molecule 1
Epitheliasin
Peptidyl-prolyl cis-trans isomerase
ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2
Beta-galactosidase
Ras-related protein Ral-A
Toll interacting protein variant (Fragment)
Uncharacterized protein C11orf52
Dipeptidylpeptidase III isoform 1 variant (Fragment)
Serine/threonine-protein kinase 26
Ectonucleotide pyrophosphatase/phosphodiesterase family member 3
Growth-regulated alpha protein
Fructose-bisphosphate aldolase C
Matrix metalloproteinase-9
Uncharacterized protein
Low-density lipoprotein receptor class A domain- containing protein 1
Laminin subunit gamma-2
253 cDNA FLJ78448, highly similar to Homo sapiens argininosuccinate synthetase (ASS), transcript
variant 1, mRNA
Beta-2-microglobulin
Protein kinase C and casemin kinase substrate in neurons protein 2
Cortactin isoform a variant (Fragment)
Niban-like protein 1
cDNA FLJ53687, highly similar to Hsc70- interacting protein D-3-phosphoglycerate dehydrogenase Calpain-2 catalytic subunit IFT74 protein (Fragment)
Voltage-dependent anion-selective channel protein 2
Intraflagellar transport protein 81 homolog
Periplakin
Calpain-5
Beta-mannosidase
Heterogeneous nuclear ribonucleoprotein A1
Creatine kinase B-type
Histone H2B type 1-N
cDNA, FLJ96627, highly similar to Homo sapiens calpain 1, (mu/I) large subunit (CAPN1), mRNA
Vacuolar protein sorting-associated protein 28 homolog
RAB13 protein (Fragment)
CEACAM1 protein (Fragment)
254 Fucosyltransferase
DnaJ homolog subfamily C member 5
COL21A1 protein
Tumor susceptibility gene 101 protein
Isocitrate dehydrogenase [NADP]
cDNA FLJ75883, highly similar to Homo sapiens glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) (GNS), mRNA
Unconventional myosin-VI
Glutathione S-transferase A1
2',3'-cyclic nucleotide 3' phosphodiesterase, isoform CRA_a
cDNA FLJ57046, highly similar to Lysosomal alpha-glucosidase (EC 3.2.1.20)
TIMP metallopeptidase inhibitor 2, isoform CRA_b
Non-specific protein-tyrosine kinase
Carcinoembryonic antigen-related cell adhesion molecule 5
Thioredoxin reductase 1, cytoplasmic
cDNA FLJ53025, highly similar to Complement C4-B
Alpha-1-antitrypsin
Aminopeptidase N
Uncharacterized protein C6orf132
Kallikrein-10
Sialate O-acetylesterase
255 Leukocyte elastase inhibitor
Vacuolar protein sorting-associated protein 37B
Follistatin-related protein 1
Prosaposin variant (Fragment)
Complement C1q tumor necrosis factor-related protein 5
Arrestin domain-containing protein 1
TNC protein
Plastin-3
Cystatin-M
Acyl-CoA-binding protein
CD82 antigen
Programmed cell death protein 10
cDNA FLJ51032, highly similar to CD9 antigen
RAB1B protein
EH domain-containing protein 1
Aldo-keto reductase family 1 member C1
Histone H2A type 1-B/E
Putative uncharacterized protein DKFZp686J1169
Chloride intracellular channel protein 5 Deoxyribonuclease II (Fragment) cDNA FLJ56912, highly similar to Fibulin-2
Capping protein (Actin filament) muscle Z-line, beta, isoform CRA_d
IQ motif containing GTPase activating protein 1
256 Cartilage intermediate layer protein 1
Apolipoprotein D (Fragment)
Transketolase (Fragment)
Heavy chain of factor I (Fragment)
Specifically androgen-regulated gene protein
cDNA FLJ54023, highly similar to Heat shock protein HSP 90-beta
Carcinoembryonic antigen-related cell adhesion molecule 6
Alpha-amylase 1
YWHAE/FAM22A fusion protein (Fragment)
Guanine nucleotide-binding protein G(i) subunit alpha-2
WD repeat domain 1, isoform CRA_a (Fragment)
Dynein light chain 2, cytoplasmic
cDNA FLJ60461, highly similar to Peroxiredoxin-2 (EC 1.11.1.15)
Fibulin-1
Complement C4-B
cDNA FLJ77858, highly similar to Homo sapiens N-acylsphingosine amidohydrolase (acid ceramidase) 1 (ASAH1), transcript variant 1, mRNA
cDNA FLJ53691, highly similar to Serotransferrin
Insulin-like growth factor-binding protein 3
Serotransferrin
Bile salt-activated lipase
257 Cathepsin B
Rootletin
Epididymal secretory protein E1
Elongation factor 1-alpha
Complement factor H
Cofilin-1
Sodium-dependent phosphate transport protein 2B
Calmodulin
Transmembrane channel-like protein
Serum albumin
cDNA, FLJ95457, highly similar to Homo sapiens tubulin, beta, 4 (TUBB4), mRNA
cDNA FLJ14514 fis, clone NT2RM1000742, highly similar to Prominin-1
cDNA FLJ32131 fis, clone PEBLM2000267, highly similar to Tubulin alpha-ubiquitous chain
Annexin A1
Mucin-4
Beta actin variant (Fragment)
258
Appendix 4. Unique proteins identified by label free LC-MS/MS in asthmatic HBE apical secretions during Il-13 challenge or at baseline (control)(n=5) and significantly (p<0.05, paired student t-test) changing proteins after challenge with associated log2 fold change of total precursor intensity per protein comparing day 20 of IL-13 treatment to baseline.
Significantly Changed Proteins: Log2 Control IL-13 Fold Control vs. IL-13 Change
Putative ciliary rootlet coiled-coil protein-like Saccharopine dehydrogenase-like Trefoil factor 3 +INF 3 protein oxidoreductase
AP-1 complex subunit beta-1 Ferritin light chain Tyrosine-protein kinase receptor -2.14
40S ribosomal protein S26 Septin-2 Citrate synthase 1.20
GTP-binding nuclear protein Ran 3-hydroxyacyl-CoA dehydrogenase type-2 Twinfilin-1 -1.28 (Fragment)
Succinyl-CoA ligase [GDP-forming] subunit 259 Cytoplasmic dynein 1 heavy chain 1 Mucin-16 -2.67 beta, mitochondrial
ATP-dependent RNA helicase DDX3X Tetratricopeptide repeat protein 25 Alpha-amylase -4.27
cDNA FLJ51818, highly similar to ADP-ribosylation factor-like protein 6 Mucin-16 -2.56 Phosphoglucomutase-1 (EC 5.4.2.2)
Solute carrier family 25 Acid sphingomyelinase-like (Mitochondrial carrier phosphate Aspartate--tRNA ligase, cytoplasmic 1.81 phosphodiesterase 3b carrier), member 3, isoform CRA_a
Tumor necrosis factor ligand Alpha-mannosidase Dipeptidyl peptidase 4 8.79 superfamily member 10
Methylcrotonoyl-CoA carboxylase beta Nitric oxide synthase, inducible IgGFc-binding protein 9.32 chain, mitochondrial
Ornithine aminotransferase, mitochondrial Angiogenin ATP synthase subunit gamma 2.04
tRNA-splicing ligase RtcB homolog ADP-ribosylation factor 6 40S ribosomal protein SA 3.42
Transcriptional activator protein Pur-alpha Platelet glycoprotein 4 (Fragment) Peroxiredoxin-2 0.58
Hydroxysteroid dehydrogenase-like protein Alkaline phosphatase, tissue- cDNA FLJ59142, highly similar to -2.33 2 nonspecific isozyme Epididymal secretory protein E1
Tyrosine-protein kinase receptor Glycine--tRNA ligase Cytochrome b5 1.98
Neuroblast differentiation-associated protein cDNA FLJ54752, highly similar to Fetuin-B 1.64 AHNAK Poly(rC)-binding protein 2
cDNA, FLJ95483, highly similar to Homo Methyltransferase-like protein 7A sapiens chitobiase, di-N-acetyl- (CTBS), Periostin 1.28 (Fragment) mRNA
Valine--tRNA ligase Intelectin-2 60S ribosomal protein L27a 2.75
T-complex protein 1 subunit theta Secretoglobin family 3A member 1 -5.54
Sodium/potassium-transporting Chloride intracellular channel protein 3 0.56 ATPase subunit beta-1
260 Chromosome 9 open reading 2'-deoxynucleoside 5'-phosphate N- frame 19, isoform CRA_a -4.62 hydrolase 1 (Fragment) (Fragment)
Putative uncharacterized protein Chloride intracellular channel -1.48 DKFZp762A2415 (Fragment) protein
Glycerol-3-phosphate dehydrogenase 1-like Histone H1.5 +INF protein
cDNA FLJ10144 fis, clone HEMBA1003286, cDNA FLJ53366, highly similar to highly similar to Beta-1,4- Probable ATP-dependent RNA 2.27 galactosyltransferase 4 (EC 2.4.1.-) helicase DDX5 (EC 3.6.1.-)
cDNA FLJ77456, highly similar to Homo 2,4-dienoyl-CoA reductase, sapiens interleukin enhancer binding factor 1.61 mitochondrial 3, 90kDa (ILF3), transcript variant 2, mRNA
Phospholipid scramblase 1 Sperm surface protein Sp17 -0.73
cDNA FLJ78677, highly similar to Homo Neutrophil gelatinase-associated sapiens splicing factor 3b, subunit 3, -3.27 lipocalin 130kDa (SF3B3), mRNA
Glucosidase I P37 AUF1 0.79 RNA binding protein (Autoantigenic, Soluble calcium-activated hnRNP-associated with lethal yellow) long 2.08 nucleotidase 1 isoform variant (Fragment)
cDNA FLJ12766 fis, clone NT2RP2001520, Dopamine receptor interacting highly similar to Calcium-binding -0.92 protein 4 mitochondrial carrier protein Aralar1
cDNA, FLJ94213, highly similar to Calcium-binding mitochondrial carrier Homo sapiens pregnancy-zone -1.42 protein Aralar2 protein (PZP), mRNA
Flotillin-2 Lysozyme C 3.59
261 Synaptotagmin binding, cytoplasmic RNA Chaperonin containing TCP1, 1.47
interacting protein variant (Fragment) subunit 7 (Eta) variant (Fragment)
Heterogeneous nuclear ribonucleoprotein R Cystatin-S 11.57 N(4)-(beta-N-acetylglucosaminyl)- Testis-specific gene 10 protein (Fragment) -INF L-asparaginase
Medium-chain specific acyl-CoA 40S ribosomal protein S11 2.38 dehydrogenase, mitochondrial
cDNA FLJ46662 fis, clone TRACH3006800, highly similar to cDNA FLJ53342, highly similar to Granulins Homo sapiens mucin 16, cell -2.58 surface associated (MUC16), mRNA
Phosphoenolpyruvate carboxykinase ATP-binding cassette sub-family D 4.02 [GTP], mitochondrial member 3
cDNA, FLJ93804, highly similar to Homo cDNA FLJ55694, highly similar to sapiens gp25L2 protein (HSGP25L2G), Dipeptidyl-peptidase 1 (EC 2.07 mRNA 3.4.14.1)
Potassium-transporting ATPase Protein FAM3D 0.78 alpha chain 2
40S ribosomal protein S12 CD133 isoform H -2.56
Malic enzyme Intelectin 1 11.98
Discoidin domain receptor family, member Galectin-3-binding protein -1.29 1, isoform CRA_b
ATP-binding cassette sub-family A Ganglioside GM2 activator -2.42 member 13
Nicotinamide phosphoribosyltransferase Choline transporter-like protein 4 -0.56 cDNA FLJ75299, highly similar to Xenopus
262 laevis proteasome (prosome, macropain) Heat shock protein 105 kDa 1.31
26S subunit, ATPase 3, mRNA
IQ motif containing GTPase activating Glutathione S-transferase pi 0.47 protein 2, isoform CRA_b (Fragment)
26S protease regulatory subunit 4 Profilin-1 0.49 Glucosidase, alpha acid (Pompe Polyadenylate-binding protein disease, glycogen storage disease -1.87 type II), isoform CRA_a
Ubiquitin carboxyl-terminal hydrolase 5 Gelsolin 2.05
Actin-related protein 2/3 complex subunit 5 Dipeptidyl peptidase 1 2.14
Epidermal growth factor receptor Coatomer subunit beta -1.63 kinase substrate 8-like protein 2
Heterogeneous nuclear Hexokinase 2.70 ribonucleoprotein F
Epidermal growth factor receptor Adenylate kinase 2, mitochondrial -3.15 kinase substrate 8
Neural cell adhesion molecule L1- 40S ribosomal protein S6 -2.26 like protein
Adenylosuccinate synthetase isozyme 2 Attractin -2.72 Electron transfer flavoprotein cDNA FLJ55051 2.70 subunit beta
cDNA, FLJ92620, highly similar to Homo EGF-containing fibulin-like sapiens staphylococcal nuclease domain -2.38 extracellular matrix protein 1 containing 1 (SND1),mRNA
cDNA, FLJ94599, highly similar to Homo sapiens GDP-mannose 4,6-dehydratase ADP-ribosylation factor 4 0.87 (GMDS), mRNA
263 cDNA FLJ75883, highly similar to Alpha/beta hydrolase domain-containing Homo sapiens glucosamine (N- -INF
protein 11 acetyl)-6-sulfatase (Sanfilippo disease IIID) (GNS), mRNA
NADPH:adrenodoxin 40S ribosomal protein S7 1.78 oxidoreductase, mitochondrial
Membrane alanine aminopeptidase variant Cystatin-SN 12.91 (Fragment)
cDNA FLJ53308, highly similar to 2-oxoglutarate dehydrogenase E1 Inorganic pyrophosphatase 2, mitochondrial +INF component, mitochondrial (EC 1.2.4.2)
Histidine triad nucleotide-binding Serine/threonine-protein kinase 26 +INF protein 2, mitochondrial
Glutamine--fructose-6-phosphate Proliferation-associated protein 2G4 +INF aminotransferase [isomerizing] 1
Cytochrome b-c1 complex subunit Rieske, Tubulin alpha-1A chain 0.81 mitochondrial
cDNA FLJ55705, highly similar to Threonyl- Heat shock protein HSP 90-alpha 0.88 tRNA synthetase, cytoplasmic (EC 6.1.1.3)
Adhesion G-protein coupled RPS4X protein (Fragment) -3.88 receptor F1
cDNA FLJ44920 fis, clone cDNA, FLJ94025, highly similar to Homo BRAMY3011501, highly similar to sapiens tripartite motif-containing 28 1.55 Heterogeneous nuclear (TRIM28), mRNA ribonucleoprotein U
ATP-binding cassette sub-family D member Antileukoproteinase -1.04 3
cDNA FLJ78635, highly similar to Homo sapiens ATP synthase, H+
264 transporting, mitochondrial F0 Casemin kinase II subunit alpha 2.46 complex, subunit b, isoform 1 (ATP5F1), transcript variant 1, mRNA
cDNA, FLJ94267, highly similar to phospholipase A2 inhibitor and Ly6/PLAUR Homo sapiens glutathione S- 3.26 domain-containing protein transferase omega 1 (GSTO1), mRNA
Protein disulfide-isomerase A6 MICOS complex subunit MIC60 3.03
Amyloid beta A4 protein Protein S100-A13 +INF
cDNA FLJ57602, highly similar to Catenin delta-1 Creatine kinase M-type (EC -0.38 2.7.3.2)
Succinate dehydrogenase Intraflagellar transport protein 25 homolog [ubiquinone] iron-sulfur subunit, 2.75 mitochondrial
Stabilizer of axonemal microtubules 2 Multivesicular body subunit 12A -2.51
Cathepsin Z Hexokinase 4.65
Insulin-like growth factor-binding Single-stranded DNA-binding protein -1.00 protein 7
N-acetyllactosaminide beta-1,3-N- Acyl-CoA-binding protein 0.63 acetylglucosaminyltransferase 3
von Willebrand factor A domain- Armadillo repeat-containing protein 4 -6.49 containing protein 7
Protein kinase C and casemin kinase Carbonyl reductase [NADPH] 1 1.42 substrate in neurons 2, isoform CRA_a
cDNA, FLJ92896, highly similar to Full-length cDNA 5-PRIME end of clone Homo sapiens proteasome CS0DF013YM24 of Fetal brain of Homo (prosome, macropain) 26S 2.43
265 sapiens (Human) variant (Fragment) subunit, non-ATPase, 1 (PSMD1), mRNA
Nicastrin RuvB-like 2 1.58
Coronin 78 kDa glucose-regulated protein 0.96
Tetratricopeptide repeat protein 21A Tubulin alpha-4A chain 0.88 cDNA, FLJ95513, highly similar to Homo ATP synthase subunit beta, 1.78 sapiens cyclin fold protein 1 (CFP1), mRNA mitochondrial
cDNA FLJ51907, highly similar to RAB13 protein (Fragment) 3.53 Stress-70 protein, mitochondrial
Calpastatin Elongation factor Tu, mitochondrial 2.33
Calcium-activated chloride Periplakin +INF channel regulator 1
Beta-glucuronidase Aminopeptidase B 2.10
Very long-chain acyl-CoA synthetase Tubulin alpha-1B chain 0.83
Heterogeneous nuclear ribonucleoprotein L Elafin -2.01
Epidermal growth factor receptor NADH-cytochrome b5 reductase 1 -0.77 kinase substrate 8-like protein 1
Acidic leucine-rich nuclear phosphoprotein Nucleobindin 2, isoform CRA_b 1.10 32 family member A
cDNA FLJ76886, highly similar to Homo sapiens loss of heterozygosity, 11, MUC5AC (Fragment) 11.05 chromosomal region 2, gene A (LOH11CR2A), transcript variant 1, mRNA
ATP synthase subunit alpha, Inositol monophosphatase 1 1.82 mitochondrial
Dynein heavy chain 11, axonemal Ferritin heavy chain 4.26
266 Succinate dehydrogenase [ubiquinone] Transmembrane channel-like -4.51
iron-sulfur subunit, mitochondrial protein
Pyruvate dehydrogenase E1 Alpha/beta hydrolase domain-containing component subunit alpha, somatic +INF protein 14B form, mitochondrial
Serine/threonine-protein phosphatase 2A 60S ribosomal protein L7a 3.39 catalytic subunit alpha isoform
Quinone oxidoreductase PIG3 Uncharacterized protein -1.88
Copine-1 Outer dense fiber protein 3B 1.02
60S ribosomal protein L7a Transketolase 0.65
Serine/arginine-rich splicing factor 40S ribosomal protein S2 (Fragment) 1.31 3
N(G),N(G)-dimethylarginine Alpha/beta hydrolase domain- 2.01 dimethylaminohydrolase 1 containing protein 14B
cDNA FLJ60299, highly similar to Persulfide dioxygenase ETHE1, Rab GDP dissociation inhibitor -0.95 mitochondrial beta
Glycerol-3-phosphate dehydrogenase, Na(+)/H(+) exchange regulatory -1.54 mitochondrial cofactor NHE-RF1
Catechol O-methyltransferase Keratin, type I cytoskeletal 17 1.36
Lamin-B1 Arylsulfatase A, isoform CRA_a -0.92
cDNA FLJ56357, highly similar to Homo sapiens apolipoprotein A-I binding protein HCG40889, isoform CRA_b -3.59 (APOA1BP), mRNA
Cadherin-related family member 4 Trefoil factor 3 +INF
Perilipin-3 Syntenin-2 0.75 Delta(3,5)-Delta(2,4)-dienoyl-CoA 267 Monoglyceride lipase (Fragment) 2.78 isomerase, mitochondrial
Protein kinase C substrate 80K-H, isoform Tetraspanin-1 -1.02 CRA_a
Insulin-like growth factor binding Deoxyribonuclease II (Fragment) -3.33 protein 3
ATP synthase-coupling factor 6, Synaptosomal-associated protein -1.70 mitochondrial 23
cDNA FLJ53573, highly similar to Myosin Ic Superoxide dismutase 1.21 Pleckstrin homology domain-containing Glutamate--cysteine ligase +INF family S member 1 regulatory subunit
Cytochrome b-c1 complex subunit NADPH--cytochrome P450 reductase 4.04 2, mitochondrial
cDNA FLJ75066, highly similar to Homo cDNA FLJ54170, highly similar to sapiens complement component 1, r 0.97 Cytosolic nonspecific dipeptidase subcomponent (C1R), mRNA
Ribosomal protein L7, isoform CRA_a Myeloid leukemia factor 1 2.42 GTP:AMP phosphotransferase Acetyl-CoA acetyltransferase, mitochondrial +INF AK3, mitochondrial
Voltage-dependent anion-selective ATP synthase subunit g, mitochondrial 2.40 channel protein 1
Cartilage intermediate layer protein 1 Cathepsin S -1.90 HLA class I histocompatibility antigen, A-68 RuvB-like 1 1.23 alpha chain
Spliceosome RNA helicase DDX39B Isochorismatase domain-containing protein 268 2, mitochondrial
Soluble calcium-activated nucleotidase 1
V-type proton ATPase subunit B, brain
isoform
Oxygen-regulated protein 1
5'(3')-deoxyribonucleotidase, cytosolic type
60S ribosomal protein L6 Vacuolar protein sorting-associated protein
28 homolog
60S ribosomal protein L27a cDNA FLJ75549, highly similar to Homo sapiens ribosomal protein, large, P0 (RPLP0), transcript variant 1, mRNA
Peptidyl-prolyl cis-trans isomerase
Programmed cell death protein 6
Isocitrate dehydrogenase [NAD] subunit
alpha, mitochondrial
Calsequestrin-2
COL21A1 protein
Sepiapterin reductase
60S ribosomal protein L32 (Fragment) cDNA, FLJ96923, highly similar to Homo
sapiens ribophorin II (RPN2), mRNA
Lysosome-associated membrane
glycoprotein 2
269 Centrin-2
Endoplasmic reticulum resident protein 29
cDNA FLJ38699 fis, clone KIDNE2002168, highly similar to Short chain 3-hydroxyacyl-
CoA dehydrogenase, mitochondrial (EC 1.1.1.35)
cDNA, FLJ95650, highly similar to Homo sapiens karyopherin (importin) beta 1 (KPNB1), mRNA
Specifically androgen-regulated gene
protein
cDNA FLJ78528, highly similar to Homo sapiens vacuolar protein sorting 4B (yeast) (VPS4B), mRNA
Glyoxylate reductase/hydroxypyruvate
reductase
Microsomal glutathione S-transferase 1
Glycogen phosphorylase, brain form
Ras-related protein Rab-2A
Acyl-protein thioesterase 1 (Fragment)
Neutral alpha-glucosidase AB
Interleukin enhancer-binding factor 2 Acetyltransferase component of pyruvate
dehydrogenase complex
Alanine--tRNA ligase, cytoplasmic
270 60S ribosomal protein L14
Electron transfer flavoprotein subunit beta
Capping protein (Actin filament) muscle Z-
line, alpha 2 variant (Fragment)
cDNA FLJ54854, highly similar to Junctional
adhesion molecule A
Heparin-binding protein HBp15
ATP-dependent RNA helicase DDX1 Intraflagellar transport 88 homolog
(Chlamydomonas), isoform CRA_d
Thymidine phosphorylase (Fragment)
Catalase
cDNA, FLJ79405, highly similar to Homo sapiens solute carrier family 25, member 24, transcript variant 1, mRNA
Aldo-keto reductase family 1 member B10
S-formylglutathione hydrolase (Fragment)
MICOS complex subunit MIC60
N-sulphoglucosamine sulphohydrolase
Mitochondrial carrier homolog 2
X-ray repair cross-complementing protein 5
cDNA, FLJ92896, highly similar to Homo sapiens proteasome (prosome, macropain)
26S subunit, non-ATPase, 1 (PSMD1), mRNA
271 Obg-like ATPase 1
Ribosomal protein L4 variant (Fragment)
Ectonucleotide pyrophosphatase/phosphodiesterase family member 3
cDNA FLJ46506 fis, clone THYMU3030752, highly similar to BTB/POZ domain- containing protein KCTD12
Tetratricopeptide repeat protein 30B
Tetratricopeptide repeat protein 30A
Adapter molecule crk
Cytochrome c oxidase subunit 7A2,
mitochondrial
Mitochondria-eating protein
Cell adhesion molecule 4
Dolichyl-diphosphooligosaccharide--protein
glycosyltransferase subunit 1
EF-hand domain-containing protein 1
WD repeat-containing protein 60 Medium-chain specific acyl-CoA
dehydrogenase, mitochondrial
Cytochrome b-c1 complex subunit 2,
mitochondrial
Glucose-6-phosphate 1-dehydrogenase
T-complex protein 1 subunit alpha
272 40S ribosomal protein SA
40S ribosomal protein S5 (Fragment)
Cytochrome c oxidase subunit 6B1
EH domain-containing protein 1
Ubiquitin-fold modifier-conjugating enzyme
1
Actin-related protein 2/3 complex subunit 3
Twinfilin-1
Proteasome subunit beta type-8 cDNA FLJ53366, highly similar to Probable ATP-dependent RNA helicase DDX5 (EC 3.6.1.-)
cDNA FLJ56074, highly similar to 150 kDa
oxygen-regulated protein (Orp150)
Cytochrome b5
T-complex protein 1 subunit delta cDNA FLJ54530, weakly similar to
Cadherin-related tumor suppressor homolog
60S acidic ribosomal protein P2
cDNA, FLJ95068, highly similar to Homo sapiens eukaryotic translation elongation factor 1 delta (guanine nucleotide exchange protein) (EEF1D), transcript variant 1, mRNA
DnaJ
273 Aflatoxin B1 aldehyde reductase member 2
Heterogeneous nuclear ribonucleoprotein M
X-ray repair cross-complementing protein 6
Phosphoglucomutase-2
Putative uncharacterized protein (Fragment)
2,4-dienoyl-CoA reductase, mitochondrial Cytochrome c oxidase subunit 5B,
mitochondrial
Intraflagellar transport protein 81 homolog
Serine/arginine-rich splicing factor 3 cDNA, FLJ94213, highly similar to Homo sapiens pregnancy-zone protein (PZP), mRNA
Platelet-activating factor acetylhydrolase IB
subunit beta
D-3-phosphoglycerate dehydrogenase
Stomatin-like protein 2, mitochondrial
Vesicle-associated membrane protein 8
Cytochrome c oxidase subunit 5A,
mitochondrial
S-phase kinase-associated protein 1
Cellular retinoic acid-binding protein 2
Nucleolin cDNA, FLJ93802, highly similar to Homo sapiens succinate-CoA ligase, GDP-
274 forming, alpha subunit (SUCLG1), mRNA
Aminopeptidase B
Guanine nucleotide-binding protein subunit
beta-2-like 1
cDNA FLJ76817, highly similar to Homo sapiens non-POU domain containing, octamer-binding (NONO), mRNA
Nascent polypeptide-associated complex
subunit alpha, muscle-specific form
Alpha-1-antitrypsin
Heterogeneous nuclear ribonucleoprotein
A3
Heat shock 70 kDa protein 4
Fibulin-1
Heterogeneous nuclear ribonucleoprotein F cDNA, FLJ78886, highly similar to Natural
resistance-associated macrophage protein 2
Myosin regulatory light chain 12A
Catenin alpha-1
SFPQ protein (Fragment)
Ras-related protein Ral-A
Glutathione S-transferase kappa 1
60S ribosomal protein L18 (Fragment)
3-hydroxyisobutyrate dehydrogenase,
mitochondrial
275 Chaperonin containing TCP1, subunit 7
(Eta) variant (Fragment)
Flavin reductase (NADPH)
Aldehyde dehydrogenase, mitochondrial
Proteasome subunit alpha type-7
Coronin-1B
Small nuclear ribonucleoprotein Sm D1
40S ribosomal protein S3
Annexin (Fragment)
Epoxide hydrolase 1 Polypeptide N-
acetylgalactosaminyltransferase
C-X-C motif chemokine 6
Asparagine--tRNA ligase, cytoplasmic
Melanoma-derived growth regulatory protein
Keratin 23 (Histone deacetylase inducible)
Glutaredoxin-1 cDNA FLJ78473, highly similar to Homo sapiens EF-hand domain (C-terminal) containing 2 (EFHC2), mRNA
Heterogeneous nuclear ribonucleoprotein
H2
ATP synthase subunit O, mitochondrial
Pyridoxal kinase
276 cDNA, FLJ94440, highly similar to Homo sapiens chaperonin containing TCP1, subunit 6A (zeta 1)(CCT6A), mRNA
Spondin-2
Calnexin
Enoyl-CoA hydratase, mitochondrial Calcium-activated chloride channel
regulator 4
Sarcoplasmic/endoplasmic reticulum
calcium ATPase 2
SH3 domain binding glutamic acid-rich
protein like 3, isoform CRA_a (Fragment)
Adenosylhomocysteinase
Mucin-20
Biotinidase
Intraflagellar transport protein 46 homolog
(Fragment)
NADPH:adrenodoxin oxidoreductase,
mitochondrial
Arrestin domain-containing protein 1
ATP synthase subunit d, mitochondrial
High mobility group protein B1
Programmed cell death protein 10
Glutathione peroxidase
277 cDNA FLJ44920 fis, clone BRAMY3011501, highly similar to Heterogeneous nuclear ribonucleoprotein U
Kunitz-type protease inhibitor 1
Prohibitin-2
Elongation factor 1-gamma cDNA FLJ75883, highly similar to Homo sapiens glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) (GNS), mRNA
Guanine deaminase
Vimentin
Toll interacting protein variant (Fragment)
Zymogen granule protein 16 homolog B
CD82 antigen
Sialate O-acetylesterase
Sulfide:quinone oxidoreductase,
mitochondrial
HCG2020860, isoform CRA_b
Uncharacterized protein Deoxynucleoside triphosphate
triphosphohydrolase SAMHD1
Plasma protease C1 inhibitor
Follistatin-related protein 1 N(4)-(beta-N-acetylglucosaminyl)-L-
asparaginase
278 Sialic acid synthase
Prohibitin
Plectin
Complement factor D preproprotein
Trifunctional enzyme subunit alpha,
mitochondrial
Peptidyl-glycine alpha-amidating
monooxygenase
Cystatin-M
ADP/ATP translocase 1
Glutamine synthetase
Lactotransferrin
Aquaporin-5
ADP/ATP translocase 3
Voltage-dependent anion-selective channel
protein 2
Anterior gradient protein 3 homolog
Alpha-1-antichymotrypsin
Complement C4-B
AGR2
Protein CutA
CEACAM1 protein (Fragment)
Growth-regulated alpha protein
279 cDNA, FLJ92390, highly similar to Homo
sapiens microSeminoprotein, beta- (MSMB), transcript variant PSP94, mRNA
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