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Biosynthesis, structure and function of protein paucimannosylation in human neutrophils

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posted on 28.03.2022, 03:13 authored by Ju Wen Ian Loke
Asparagine (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.

History

Table of Contents

Chapter 1. General introduction, thesis overview and research aims -- Chapter 2. Initial observation of paucimannosylated human neutrophil proteins in bacterial colonised cystic fibrosis sputum -- Chapter 3. The role of hexosaminidases in the biosynthesis of paucimannosidic N-glycoproteins in human neutrophils -- Chapter 4. Site-specific structural characterisation of the N-glycosylation of human neutrophil cathepsing using complementary LC-MS/MS strategies -- Chapter 5. Paucimannosylation modulates the innate immune functions of human neutrophil elastase across the distinct granules of human neutrophils -- Chapter 6. N-glycomics indicates that protein paucimannosylation is a multi-cellular N-glycan feature in inflammation, stemness and cancer -- Chapter 7. Summary and conclusion -- References -- Appendices.

Notes

Theoretical thesis. Bibliography: pages 252-298

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

PhD, Macquarie University, Faculty of Science and Engineering, Department of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award

2017

Principal Supervisor

Morten Thaysen Andersen

Additional Supervisor 1

Nicki Packer

Rights

Copyright Loke Ju we Ian 2017. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (xx, 306 pages) diagrams, graphs, tables

Former Identifiers

mq:71734 http://hdl.handle.net/1959.14/1277545