Discovery and synthesis of immunogenic glycan antigens from Schistosoma mansoni Open Access

Abstract

Glycans are pervasive at the host-pathogen interface, both as stimulators of innate immunity and targets of adaptive immunity for a multitude of pathogens including parasitic helminthes. In the case of schistosomiasis, a disease caused by infection with trematodes of the Schistosoma species, glycans play a prominent role the modulation of the host immune system and humoral responses. Glycan structures on schistosome glycoconjugates have gained increasing attention as alternative vaccine and serodiagnostic targets, and a deeper understanding of worm glycans could potentially ignite innovative new strategies for lessening the mortality and morbidity caused by these parasites. A major limitation in the study of the immunogenicity and biological functions of schistosome glycans has been the lack of adequate quantities of the parasite glycans as well as the lack of specific tools needed for identification and study of immune relevant glycans. We sought to generate high affinity and specific IgG monoclonal antibodies to schistosome glycans to facilitate the purification and tracking of immunogenic parasite glycans, and developed a natural N-glycan microarray from S. mansoni egg glycoproteins to identify specific antigenic epitopes within the egg glycome which are the targets of adaptive immunity in infected hosts. We then began to "decipher the schistosome glycogenome" by utilizing genomics technologies to develop a semi-synthetic approach for heterologous expression of schistosome glycosyltransferases to generate significant glycan structures. These studies demonstrated the importance of the unique linkages and high levels of fucosylation found in complex schistosome glycans, the success of shotgun glycomics to elucidate host immune targets, and the extremely complex and still enigmatic specificity and functionality of the glycosyltransferase families responsible for the generation of the worm glycome. This work should encourage and support future studies on Schistosoma's uniquely fucosylated glycans to elucidate their role in worm biology, explore their feasibility as immune targets, and facilitate the development of novel diagnostic approaches and glycoconjugate vaccine platforms.

Table of Contents

Distribution Agreement -i

Abstract -iv

Acknowledgements -vi

Table of Contents -viii

List of Figures - xiii

List of Tables - xv

Abbreviation List - xvi

Chapter 1.Introduction -1

1.1 Global state of schistosomiasis -1

1.1.1 Burden of helminth disease -1

1.1.2 Schistosoma life cycle -2

1.1.3 Clinical manifestations -3

1.1.4 Diagnostics -4

1.1.5 Current treatment and control measures -5

1.2 Importance of schistosome glycoconjugates -6

1.2.1 Structural features of helminthic glycans -7

1.2.2 Parasitic glycan gimmickry -12

1.2.3 Immune modulation -12

1.2.4 Innate immune responses to schistosome glycans -13

1.2.5 Adaptive immune responses to schistosome glycans -18

1.2.6 Glycans interactions with intermediate hosts -25

1.3 Glycotechnologies and approaches -27

1.3.1 Glycan synthesis and glycoconjugate production -27

1.3.2 Glycan microarrays -28

1.3.3 Limitations in glycan research and historical approaches -30

1.3.4 A genomic approach -31

1.4 Schistosome glycan biosynthesis pathways -35

1.4.1 N-glycans -37

1.4.2 O-glycans -38

1.4.3 Glycolipids -39

1.4.4 GPI-anchored glycoproteins -40

1.4.5 Glycosaminoglycans and proteoglycans -41

1.4.6 Terminal motifs -43

1.5 Project aims -49

Chapter 2. IgG monoclonal antibody F2D2 binds the difucosylated FLDNF glycan epitope -52

2.1 Introduction -52

2.2 Materials and Methods -55

2.2.1 Materials -55

2.2.2 Parasites -56

2.2.3 Production of Hybridoma -56

2.2.4 Propagation of Hybridoma Cells in Serum Free Media -57

2.2.5 Purification of Secreted mAb F2D2 by MEP HyperCel -58

2.2.6 Preparation of Neoglycoproteins -58

2.2.7 Preparation of soluble egg extracts and other detergent extracts -59

2.2.8 Preparation of Cross-Reactive Proteins -60

2.2.9 Preparation of Snail Hemolymph -60

2.2.10 ELISA of Proteins and Neoglycoconjugates -61

2.2.11 ELISA of Periodate/Borohydride Treated Antigens -62

2.2.12 ELISA in the Presence of Free Haptenic Sugars -62

2.2.13 Glycosidase Treatments -63

2.2.14 SDS-PAGE and Western blot analysis -64

2.2.15 Periodate/Borohydride Treatment and Western Blot Analysis -64

2.2.16 Beta-Elimination and Western Blotting -65

2.2.17 Enzymatic Release and Labeling of N-glycans from KLH -65

2.2.18 Immobilization of monoclonal antibody F2D2 to Ultralink hydrazine biosupport -67

2.2.19 Affinity Purification of PNGase F Released AEAB-labeled KLH Glycans on F2D2-Ultralink Column -67

2.2.20 Mass Spectrometry Analysis of KLH Glycans -68

2.3 Results -68

2.3.1 F2D2 is an IgG monoclonal antibody that recognizes a unique glycan epitope in SEA -68

2.3.2 Purification and Characterization of mAb F2D -2- 71

2.3.3 F2D2 binds to glycan epitopes from keyhole limpet hemocyanin (KLH) and Biomphalaria glabrata hemolymph (BGH) -72

2.3.4 Fucose is a major determinant of the mAb F2D2-binding epitope -74

2.3.5 The glycan epitope bound by mAb F2D2 is present on N-glycans of glycoproteins from SEA, KLH AND BGH -77

2.3.6 Beta-elimination of SEA, KLH and BGH -79

2.3.7 Identification of the structure of the glycan epitope bound by mAb F2D2 using AEAB-labeled KLH derived glycans -81

2.4 Discussion -84

2.5 Acknowledgments -87

Chapter 3. Identification of antigenic glycans from Schistosoma mansoni using a shotgun egg glycan microarray -88

3.1 Introduction -88

3.2 Materials and Methods -92

3.2.1 Materials -92

3.2.2 Isolation of Schistosoma mansoni Eggs and Life Stages -93

3.2.3 Infected Sera -94

3.2.4 Schistosome Egg Glycan Isolation and Labeling -94

3.2.5 Separation of AEAB-labeled Glycans (GAEABs) by HPLC and Mass Spectrometry Analysis -96

3.2.6 Array printing, Binding Assays, and Scanning -97

3.2.7 Parasite Lysates, SDS-Page, and Western blots -98

3.2.8 Immunofluorescence Imaging -99

3.2.9 Schistosomula Killing Assay -99

3.3 Results -100

3.3.1 Preparation of N-glycans from Schistosoma mansoni Eggs -100

3.3.2 Identification of Antigenic Egg N-glycan Fractions for Further Purification -105

3.3.3 Preparation and Characterization of the 2-Dimensional Schistosome Egg Shotgun N-glycan Microarray (2D-SSGM) -107

3.3.4 Anti-FLDNF Antibody Shares Common Binding Pattern with Sera from S. mansoni-infected Animals and Humans on 2D-SSGM -111

3.3.5 FLNDF Epitope is Expressed on S. mansoni Intramammalian Life Stages -113

3.3.6 F2D2 Kills Schistosomula in vitro -115

3.3.7 Proposed Structures for Antisera Glycan Targets using Metadata-Assisted Glycan Sequencing (MAGS) -115

3.4 Discussion -121

3.5 Acknowledgements -130

Chapter 4. Schistosoma mansoni alpha 1,3-Fucosyltransferase-F Generates the Lewis X Antigen -132

4.1 Introduction -132

4.2 Materials and Methods -134

4.2.1 Materials -134

4.2.2 Cloning of S. mansoni FuT-F -135

4.2.3 Expression of smFuT-F-pGen2 in HEK Freestyle Cells -136

4.2.4 SDS-Page and Western Blot of smFuT-F-GFP Expression -136

4.2.5 Purification of Recombinant smFuT-F-GFP -137

4.2.6 Glycan AEAB Labeling and Purification -137

4.2.7 Fucosyltransferase Assays -138

4.2.8 Mass Spectrometry of FuT Products -139

4.2.9 Glycobead Assay -139

4.2.10 Schistosoma Life Stages -140

4.2.11 Quantitative RT-PCR of FuT Transcription Levels -141

4.3 Results -143

4.3.1 Identification and cloning of a possible Lewis type fucosyltransferase -143

4.3.2 Expression of smFuT-F-pGen2 generates a recombinant GFP fusion protein -147

4.3.3 smFuT-F-GFP adds fucose to type II glycan chains -149

4.3.4 smFuT-F generates the Lewis X motif -153

4.3.5 Acceptor specificity of smFuT-F-GFP -154

4.3.6 Temperature and cation requirement smFuT-F-GFP -160

4.3.7 Expression of smFuT-F mRNA in various intra-mammalian life stages -161

4.4 Discussion -163 4.5 Acknowledgements -167

Chapter 5. Schistosoma mansoni β1,4-GalNAcTransferase generates LacdiNAc glycans -1685.

1 Introduction - 168

5.2 Materials and Methods -170

5.2.1 Materials -170

5.2.2 Cloning of S. mansoni βGalNAcT -171

5.2.3 Expression of smβGalNAcT-pGen2 in HEK Freestyle Cells -171

5.2.4 Purification of Recombinant smβGalNAcT-GFP -172

5.2.5 SDS-Page and Western Blot of smβGalNAcT-GFP -172

5.2.6 Glycan Digestion, AEAB Labeling, and Purification -173

5.2.7 GalNAcT Enzymatic Assay -173 5.2.8 Mass Spectrometry of Glycans -174

5.3 Results -175

5.3.1 Selection of smβ1-4GalNAcT -175

5.3.2 Expression of recombinant smβGalNAcT-GFP -178

5.3.3 smβGalNAcT adds GalNAc to terminal GlcNAc in a β-linkage -179

5.4 Discussion -182

5.5 Acknowledgments -186

Chapter 6. Discussion -187

6.1 Aim I: Identification of immunogenic glycan antigens and reagent development - 187

6.2 Aim II: Development of semi-synthetic approach for heterologous expression of glycosyltransferases -190

6.3 Future directions -192

6.4 Conclusions - 199

References - 200

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Parasitology Biology, Molecular
Keyword	Fucose Schistosoma mansoni Glycan Fucosyltransferase Glycan microarray
Committee Chair / Thesis Advisor	Cummings, Richard, Emory University
Committee Members	Lamb, Tracey, Emory University Cooper, Max Dale, Emory University Ortlund, Eric, Emory University Levin, Bruce, Emory University

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