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Formation and functions of the paucimannosidic-rich N-glycosylation of human myeloperoxidase

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posted on 28.03.2022, 09:54 authored by Julian Daniel Ugonotti
Myeloperoxidase (MPO) is a key neutrophil glycoprotein critically involved in neutrophil-mediated innate immunity. Recently, it was discovered that MPO and other important neutrophil glycoproteins are decorated with paucimannosidic glycans (Man1-3GlcNAc2Fuc0-1), an under-explored N-glycan class in human glycobiology. Our knowledge of the formation and functions of these peculiar and heavily truncated glycoproteins expressed by human neutrophils remains incomplete. Firstly, I employed enzyme activity assays and mass spectrometry-based glycomics on a collection of CRISPR-Cas9-edited neutrophil-like (HL-60) mutants displaying impaired HEXA and HEXB genes in order to provide in vitro-based evidence for a N-acetyl-β-hexosaminidase (Hex)-driven production of paucimannosidic proteins in human neutrophils. Secondly, solvent accessibility measurements, three-dimensional structural assessments, chlorination activity assays, and glycopeptide analysis of granule-separated and in vitro-generated MPO glycoforms were used to characterise the formation and functions of the N-glycans carried by MPO. These studies provided mechanistic insight into the site- and granule-specific glycosylation of monomeric and dimeric MPO, and, importantly, demonstrated that MPO exhibits glycoform-dependent chlorination activities and ceruloplasmin-mediated inhibition characteristics. The peculiar N-glycans decorating Asn355 andAsn391 were found to be critical in defining the MPO activity and inhibition potential. In conclusion, this thesis has generated novel data that advance our understanding of the fascinating complex neutrophil glycobiology -- abstract.

History

Table of Contents

Chapter 1: Introduction and aims -- Chapter 2: Materials and methods -- Chapter 3: Results -- Chapter 4: Discussion -- Chapter 5: Conclusions and future directions -- References -- Appendices.

Notes

Theoretical thesis. Bibliography: pages 51-58

Awarding Institution

Macquarie University

Degree Type

Thesis MRes

Degree

MRes, Macquarie University, Faculty of Science and Engineering, Department of Molecular Sciences

Department, Centre or School

Department of Molecular Sciences

Year of Award

2019

Principal Supervisor

Morten Thaysen-Andersen

Rights

Copyright Julian Daniel Ugonotti 2019. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (xi, 70 pages) diagrams, graphs, tables

Former Identifiers

mq:72066 http://hdl.handle.net/1959.14/1281034