Decoding the glycobiology of the paucimannose-rich neutrophil N-glycoproteome

Protein N-glycosylation is a post-translational modification where complex carbohydrates (or glycans) are covalently attached to polypeptides at sequon-specific asparagine residues. N-glycosylation mediates many cellular processes including cell-cell communication and cell signalling and has been implicated in several (patho)physiologies.

Neutrophils are an essential cell type of the innate immune system that mediate critical immune processes to fight invading pathogens and maintain homeostasis. Maturing in the bone marrow through a process known as granulopoiesis, neutrophils synthesise and package key antimicrobial glycoproteins, including myeloperoxidase, into cytosolic granules. These granules are mobilised sequentially upon neutrophil activation to perform their effector functions of extravasation into the tissue, phagocytosis, degranulation and the formation of neutrophil extracellular traps. While our knowledge of neutrophil biology and the involvement of neutrophils in (patho)physiology continues to improve, our understanding of the glycobiology of neutrophils, particularly the neutrophil N-glycoproteome, remains comparatively immature. Thus, the focus of this thesis was to improve our glycobiological understanding of the neutrophil N-glycoproteome with particular attention to paucimannosylation, a type of protein glycosylation that has been largely overlooked in the human glycobiological literature.

To pave the way, key biological and technical concepts related to this thesis are concisely introduced in Chapter 1. This introductory chapter also includes a detailed protocol on the glycomics-assisted glycoproteomics method (Publication 1) that is frequently utilised throughout this thesis to explore the neutrophil N-glycoproteome. Further, Chapter 1 includes a comprehensive review on the structural and functional diversity of neutrophil glycosylation in health and disease (Publication 2), which allowed me to articulate the aims of this thesis. The introductory chapter is followed by four experimental chapters and a concluding chapter.

Firstly, glycomics-assisted glycoproteomics was employed to survey the granule N-glycoproteome of resting neutrophils and the glycoproteome remodelling occurring during granulopoiesis in Chapter 2 (Publication 3). Neutrophils were found to exhibit site-, protein- and granule-specific glycophenotypes, including highly truncated paucimannosidic and chitobiose core-type N-glycans in the azurophilic granules. Further, this study revealed that profound glycoproteome remodelling underpins early granulopoiesis in the bone marrow. Collectively, these findings provided new molecular-level insights into the fascinatingly complex neutrophil N-glycoproteome and the mechanisms underpinning its formation during neutrophil maturation.

Next, after identifying that neutrophils abundantly express paucimannosidic proteins in the previous chapter, the biosynthesis of this unusual class of N-glycans was investigated in Chapter 3 (Publication 4). In short, this chapter demonstrates that N-acetyl-β-D-hexosaminidase isoenzymes mediate the biosynthesis of paucimannosidic proteins in human neutrophils via a noncanonical truncation pathway that is commonly utilised in paucimannose-rich lower organisms, however, for the first time here, it was revealed to be active in human neutrophils. Ultimately, the knowledge gained of how paucimannosidic proteins are formed in neutrophils will assist in deciphering their spatiotemporal expression, function and importance in the human immune system.

In Chapter 4 (Publication 5), the structure-biosynthesis-function relationship of the N-glycosylation of neutrophil-derived myeloperoxidase was characterised. Facilitated by multiomics tools, this chapter shows that myeloperoxidase is decorated with highly truncated paucimannosidic and chitobiose core-type N-glycans at discrete sites and that several protein factors influence the processing of myeloperoxidase. Excitingly, the N-glycans of myeloperoxidase were shown to modulate the enzyme activity and inhibition potential by the endogenous inhibitor ceruloplasmin. These efforts have generated new fundamental insight into the glycobiology of myeloperoxidase, a glycoprotein which is central to several neutrophilmediated innate immune processes in health and in neutrophil-related disorders.

Lastly, to decipher the biological and cellular functions of paucimannosylation, I began developing tools to identify potential binders and receptors of this overlooked class of N-glycans in Chapter 5. The first approach, which utilised a chemically synthesised Man₃GlcNAc₂Fuc₁ (M3F)-biotin paucimannose glycan coupled with streptavidin magnetic beads, identified that annexin A1 and annexin A2 are putative paucimannosidic glycan-binding proteins in peripheral blood mononuclear cells. The second approach, while not fully completed, aims to apply a novel forward genetic screening approach to a CRISPR-edited library comprising a complex mixture of cells with knockouts in a wide range of glycan-binding proteins in a human acute monocytic leukemia (THP-1) cell line. These preliminary findings and efforts open exciting avenues to explore the functional consequence of paucimannose recognition by putative binders and receptors, particularly in certain paucimannose-rich physiologies and diseases such as innate immunity and cancer.

Chapter 6 concludes by discussing how glycomics-assisted glycoproteomics and several other key molecular and cell biology tools have facilitated this exploration of the paucimannose-rich N-glycoproteome of neutrophils and reflects on the significance of these findings and how they advance our understanding of neutrophil glycobiology. This thesis lays a critical foundation for future explorations into the functions of paucimannosylation and neutrophil glycobiology in health and disease.