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Download fileBiosynthesis, structure and function of protein paucimannosylation in human neutrophils
thesis
posted on 2022-03-28, 03:13 authored by Ju Wen Ian LokeAsparagine (N)-linked glycosylation, a common post-translational modification of human proteins, is widely involved in molecular and cellular functions. Inflammation and infection are commonly associated with N-glycoprotein dysregulation. Truncated N-glycans, consisting of GlcNAc2Man1-3Fuc0-1, known as paucimannosylation, have previously been considered as an invertebrate-specific glyco-signature and are perceived to be neglible or arising from degradation in humans. However, emerging evidence indicates that protein paucimannosylation may play pivotal roles in human biology. Therefore the presence and potential functions of protein paucimannosylation in the human N-glycoproteome remain controversial, warranting further investigation.
The granulated neutrophils are amongst the first responders to inflammation and path inflammation and pathogen infection during human innate immunity. Recently, protein paucimannosylation was encountered in pathogen-infected, neutrophil -rich sputum. This thesis extends from these initial findings, by performing an in-depth molecular and (sub)cellular characterisation of the biosynthesis, structure and function of these unconventional glycoproteins in human neutrophils. LC-MS/MS-based glycomics, glycoproteomics and molecular glycobiological techniques were used to study human neutrophil-like cells (HL-60) and primary neutrophils isolated from healthy and isolated from healthy and β-hexosaminidase-deficient (Sandhoff disease; deficient (Sandhoff disease: HEXB) individuals. The latter was diminished in paucimannosylated neutrophil proteins, shown to normally reside in dedicated azurophilic granules, suggesting the involvement of human hexosaminidases in the biosynthesis of paucimannosylation in human neutrophils.
Infection of HL-60 cells with Pseudomonas aeruginosa induced a virulence- and time-dependent secretion of paucimannosidic protein, indicating active degranulation of azurophilic granule proteins. Expressions of paucimannose-rich human neutrophil elastase (HNE) on activated neutrophil cell surface and their interactions with α1-antitrypsin and mannose binding lectin, demonstrated immunomodulatory roles of paucimannosylated glycoforms of HNE in neutrophil-mediated immune functions. Finally, protein paucimannosylation was found to be present in macrophages, lymphocytes, platelets, and neuronal progenitor stem cells, suggesting wider functional roles of these glycoepitopes during physiological and pathological conditions. These findings detail and facilitate a deeper understanding of the unique N-glycoproteome of human neutrophils. This knowledge contributes to a greater appreciation of the biosynthesis, structure and function of paucimannosylated glycoproteins in neutrophil biology.