N-glycosylation of neutrophil granules in infection and inflammation
N-glycosylation, an important type of post-translational modification of proteins, plays critical roles in innate immunity. Paucimannose is a less studied class of protein N-glycans in human glycobiology. This class of truncated glycans was only just recently found to be a significant glyco-feature of neutrophils, an abundant innate immune cell type and a first responder to pathogenic infection and inflammation. Neutrophils are granulocytic cells hosting several distinct types of cytosolic compartments (granules) comprising distinct biomolecular signatures including many glycosylated microbicidal enzymes e.g. myeloperoxidase (MPO) utilised to combat invading pathogens. Despite being recognised as a modulator of immune functions, the glycobiology of the neutrophil granules and the role of paucimannose decorating the granular glycoproteins remain relatively unexplored. To this end, this PhD thesis aimed to use advanced mass spectrometry including glycomics and glycoproteomics to explore the structure and function of the granule protein N-glycosylation of resting (non-activated) and activated neutrophils in normal physiology and in relevant pathophysiological conditions including in patients with pathogenic blood-stream infection (bacteremia). Following, firstly, a short thesis introduction that will briefly introduce the biology and methods used in the rest of the thesis (Chapter 1), and, secondly, a comprehensive literature survey of protein paucimannosylation across the eukaryotic domain synthesising new knowledge of the roles of paucimannose in human glycobiology (Chapter 2, Paper I), this thesis comprises three experimental result chapters including i) a deep characterisation of the structure-biosynthesis-activity relationship of the highly N-glycosylated neutrophil MPO (Chapter 3, Paper II), ii) glycomics profiling of the granule N-glycome of resting and Staphylococcus aureus activated neutrophils (Chapter 4-5, Paper III-IV, respectively), and iii) in vivo exploration of the serum N-glycoproteome during neutrophil activation in bacteremic patients suffering from bloodstream infections by S. aureus, Pseudomonas aeruginosa and Escherichia coli (Chapter 6, Paper V). This thesis has used innovative 'omics tools and multi-faceted strategies to produce novel data that has generated new insight into the structure and function of neutrophil granule N-glycosylation (and left many enticing research questions unanswered) of value to glycobiologists and glycoimmunologists studying the fundamentals of the human innate immune system.