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Structure-function of N- and O-glycosylated human corticosteroid-binding globulin

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thesis
posted on 28.03.2022, 10:52 by Anastasia Chernykh
Corticosteroid-binding globulin (CBG), a heavily glycosylated liver-derived protein, is a principal carrier of anti-inflammatory cortisol in plasma. CBG impacts the cortisol bioavailability and facilitates tissue-specific and timely hormone delivery, and is, consequently, a recognised immunomodulatory glycoprotein with an untapped therapeutic potential. The intriguingly complex glycosylation covering the CBG surface is known to modulate its function, but the underpinning glycobiology remains incompletely understood. To this end, this thesis has employed advanced mass spectrometry-based glycan and glycopeptide profiling and functional assays to characterise the structure-function of two biologically-relevant forms of HEK293-produced recombinant human CBG (rhCBG) with a focus on documenting the glycosylation covering the functionally-important reactive centre loop (RCL). rhCBG exhibited strong site-specific N-glycosylation and occupancy differences across the six N-glycosylation sites. Key glyco-features for future therapeutic applications of rhCBG including mannose-6-phosphorylation, were identified. The RCL-localised Asn347 displayed very low N-glycan occupancy relative to the native counterpart. Excitingly, the proximal Thr345 was instead occupied by core 1/2-type O-glycosylation, a previously unknown feature of human CBG. Importantly, the novel Thr345-glycosylation was shown to strongly inhibit neutrophil elastase-mediated RCL proteolysis, thereby implying its involvement in the cortisol delivery mechanisms. This thesis has generated novel data that advances our knowledge of the fascinating CBG glycobiology -- abstract.

History

Table of Contents

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

Notes

Bibliography: pages 51-61 Theoretical thesis.

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 Anastasia Chernykh 2019. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (ix, 73 pages) diagrams, graphs, tables

Former Identifiers

mq:72074 http://hdl.handle.net/1959.14/1281117