Protein and glycosylation asymmetry in human IgG antibodies Open Access
Azzam, Tala (Fall 2025)
Abstract
IgG antibodies regulate the immune system through interactions with Fc γ receptors that promote downstream signaling. These interactions are mediated by glycoforms on the homodimeric Fc region of antibodies. However, most studies that investigate the structure and function of IgG Fcs assume symmetry at the protein and glycan levels. There have been efforts to design IgG heterodimers. However, preventing homodimerization is still a challenge. Glycoengineering approaches mostly result in symmetrical glycoforms, and current glycoprofiling studies require cleavage of the glycan from the Fcs. As a result, an important feature of IgG glycosylation has gone uncharacterized: asymmetrical glycosylation (i.e., Fcs that have different glycans on each of the otherwise identical chains). In this project, we proposed the hypothesis that investigating the asymmetry of IgG Fc will improve its therapeutic potential and that asymmetrically glycosylated IgGs modulate the immune response by exhibiting antibody effector functions that are distinct from symmetrically glycosylated IgG. Thus, we developed a protein engineering strategy to design novel heterodimeric IgGs as a tool to make each Fc protomer uniquely identifiable. We also developed a novel intact LC/MS-based workflow that enables the glycoprofiling of IgG Fc glycans as pairs rather than individual glycans. Finally, we showed that asymmetric glycosylation – specifically monofucosylation – drives severe dengue disease in humans. We engineered the first monoclonal monofucosylated IgG and demonstrated that it binds to FcγRIIIA in a high-affinity orientation, mimicking the antibody effector functions of fully afucosylated IgGs in vivo and in vitro. The results of these studies have expanded our understanding of how Fc glycans mediate effector functions and will improve antibody-based therapeutic designs.
Table of Contents
Chapter 1: Introduction ...................................................................................................................... 1
Structure and folding of IgG Fc ............................................................................................................... 1
Type I Fcγ receptors ................................................................................................................................ 4
Engineering IgGs ....................................................................................................................................... 5
IgG-specific enzymes ............................................................................................................................... 6
IdeS is an IgG-specific endopeptidase that targets the hinge ................................................................. 6
EndoS and EndoS2 are IgG-specific ENGases that target the N297 glycan ............................................ 7
CU43 is an IgG-specific ENGase that targets the N297 glycan .............................................................. 14
IgG Heterodimers ..................................................................................................................................... 15
Overview ................................................................................................................................................... 16
References ................................................................................................................................................. 18
Chapter 2: Combinatorially-Restricted Computational Design of Protein-Protein Interfaces to Produce IgG Heterodimers .................................................................................................................. 22
Abstract ..................................................................................................................................................... 23
Introduction ............................................................................................................................................... 24
Results ....................................................................................................................................................... 27
Serine and tyrosine mutations at numerous positions promote Fc heterodimer formation .................... 27
Mutations identified by combYSelect result in maximal heterodimerization ......................................... 29
Heterodimer mutations do not impact IgG stability or FcRn binding ..................................................... 31
Conformationally variant residues exist in the heterodimer interface .................................................... 32
All combYSelect mutations are required for optimal heterodimerization .............................................. 33
IgG1 heterodimers predicted by combYSelect have therapeutic potential ............................................. 34
Discussion ................................................................................................................................................. 37
Materials and Methods ............................................................................................................................. 43
Acknowledgments .................................................................................................................................... 50
References ................................................................................................................................................. 52
Main Figures ............................................................................................................................................ 57
Supplementary Figures and Tables .......................................................................................................... 65
Chapter 3: Asymmetrically glycosylated IgG1 antibodies are universal and prevalent in humans .................................................................................................................................................... 74
Abstract ..................................................................................................................................................... 75
Introduction ............................................................................................................................................... 76
Results ....................................................................................................................................................... 78
WIgGWAM enables intact LC/MS glycoprofiling of polyclonal human IgG1 antibodies while preserving the spatial pairing of the Asn297-linked glycans .................................................... 78
WIgGWAM identifies both symmetric and asymmetric IgG glycoforms ................................................ 80
Asymmetric glycosylation is prevalent in the periphery of humans and IgG1 humanized mice .............. 82
Asymmetric glycosylation is present in every individual human sample and is regulated by the B-cell differentiation pathway .................................................................................................................... 84
Discussion ................................................................................................................................................. 86
Materials and Methods ............................................................................................................................. 88
Acknowledgments .................................................................................................................................... 95
References ................................................................................................................................................. 96
Main Figures ............................................................................................................................................ 98
Supplementary Figures and Tables .......................................................................................................... 103
Chapter 4: Asymmetrically glycosylated IgG1 antibodies drive human disease .............................. 122
Abstract ..................................................................................................................................................... 123
Introduction ............................................................................................................................................... 124
Results ....................................................................................................................................................... 127
Monofucosylation is a prevalent feature of peripheral hIgG1 antibodies and is associated with secondary and severe dengue disease ......................................................................................... 127
Monofucosylated IgG1 binds Fc receptors and induces effector functions similarly to fully afucosylated IgG1 ................................................................................................................................... 128
Monofucosylated IgG1 binds to FcγRIIIA in a high-affinity orientation .................................................. 131
Discussion ................................................................................................................................................. 134
Materials and Methods ............................................................................................................................. 138
Acknowledgments .................................................................................................................................... 145
References ................................................................................................................................................. 146
Main Figures ............................................................................................................................................ 150
Supplementary Figures and Tables .......................................................................................................... 153
Chapter 5: Discussion .......................................................................................................................... 164
Conclusion ................................................................................................................................................ 164
IgG glycosylation as a disease biomarker and diagnostic ........................................................................ 165
IgGs as a therapeutic target ...................................................................................................................... 166
Limitations and Future Directions ............................................................................................................ 169
References ................................................................................................................................................. 171
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