Human Milk Glycan Interactions with Glycan-Binding Proteins of the Gastrointestinal Tract Público

Abstract

Human milk glycans (HMGs) represent one of the major biomolecules found in human milk. These non-nutritional carbohydrates are strongly associated with health benefits towards newborns and infants, including regulation of the gut microbiome and reducing the incidence of infection. More recently, HMGs have been shown to regulate immune responses and intestinal epithelial cell gene expression, but the underlying mechanisms are unclear. The human gastrointestinal (GI) tract, specifically epithelial cells and dendritic cells, express a number of glycan-binding proteins (GBPs) that may become exposed to HMGs during breast-feeding. GBPs, including the C-type lectin, Siglec, and galectin families of GBPs, are known to bind endogenous and exogenous glycan structures and subsequently initiate signaling pathways leading to changes in gene expression and immune responses. This thesis work was guided by the hypothesis that HMGs bind to some GBPs expressed by epithelial cells and dendritic cells (DCs) in the GI tract. To test this hypothesis, a number of galectins, C-type lectins, and Siglecs expressed by GI tract epithelial cells and DCs were screened for HMG binding. This study took advantage of glycan microarray technology to test binding to over 240 natural HMGs purified from human milk as well as chemically defined HMG and non-HMG glycans in a high-throughput format. The binding of GBPs to free HMGs in solution was also used to confirm GBP-HMG interactions in a more natural setting. The results of this study show that many galectins bind specific HMG structures, with each galectin exhibiting a unique binding specificity. Additionally, the DC-expressed C-type lectin DC-SIGN specifically binds numerous fucosylated HMGs. The HMG microarray technology also uncovered hitherto undefined glycan binding structures and determinants for galectins and DC-SIGN. The affinity of these GBPs for specific HMG structures occurs at or below HMG concentrations found in human milk, suggesting that these interactions may be physiologically relevant. The results of this study suggest that multiple GBPs in the GI tract may serve as HMG receptors. Future directions are aimed at understanding if these GBP-HMG interactions occur in vivo and if these interactions underlie the mechanism of regulation of gene expression and immune responses by HMGs.

Table of Contents

Chapter 1: Introduction

I. Human Milk Glycans....................................................................................................1

II. Galectins................................................................................................................... 38

III. Glycan-Binding Proteins of Dendritic Cells .................................................................67

IV. HMG Interactions with Human Glycan-Binding Proteins ..............................................92

V. Goals of Study.............................................................................................................96

VI. Tables and Figures ....................................................................................................100

VII. References ............................................................................................................. 106

Chapter 2: Galectins are Human Milk Glycan Receptors

Title Page......................................................................................................................145

Abstract ....................................................................................................................... 146 Introduction..................................................................................................................147

Results......................................................................................................................... 149

Discussion .................................................................................................................... 158

Materials and Methods....................................................................................................164 Acknowledgements......................................................................................................... 171

Abbreviations ................................................................................................................ 172

Tables .......................................................................................................................... 173

Legends to figures.......................................................................................................... 177 Figures.......................................................................................................................... 179

Supplementary Material List............................................................................................184

Supplementary Information ............................................................................................186 References..................................................................................................................... 191

Chapter 3: Human DC-SIGN Binds Specific Human Milk Glycans

Title Page...................................................................................................................... 198

Abstract ....................................................................................................................... 199

Summary Statement ...................................................................................................... 199

Short Title .................................................................................................................... 200

Key Words .................................................................................................................... 200

Abbreviations list .......................................................................................................... 200 Introduction.................................................................................................................. 202

Materials and methods .................................................................................................. 204

Results......................................................................................................................... 207

Discussion ................................................................................................................... 217

Acknowledgements........................................................................................................ 222

Declarations of Interest ................................................................................................. 222

Funding Information ..................................................................................................... 222

Author Contribution Statement.......................................................................................222 Figures......................................................................................................................... 225

Supplementary Information ..........................................................................................234

References.................................................................................................................... 237

Chapter 4: Discussion

I. Implications of GBP-HMG Interactions.........................................................................243

II. Proposed Model......................................................................................................... 249

III. Future Directions ..................................................................................................... 254

IV. Figures .................................................................................................................... 259

V. References.................................................................................................................261

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Health Sciences, Immunology
Palavra-chave	DC-SIGN Glycobiology Glycan Microarrays Human Milk Glycans Galectins Glycan-Binding Proteins Lectins
Committee Chair / Thesis Advisor	Cummings, Richard, Emory University
Committee Members	Shafer, William M, Emory University Weiss, David S, Emory University Goldberg, Joanna B., Emory University Steinhauer, David, Emory University Conn, Graeme L, Emory University

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