PXD046405-1

PXD046405 is an original dataset announced via ProteomeXchange.

Title	Position-specific N- and O-glycosylation of the reactive centre loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin
Description	Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through the proteolytic cleavage of an exposed reactive centre loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localised Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterisation of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we firstly performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics (MD) simulations to study their impact on NE proteolysis. Importantly, we also identified novel O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 sites was experimentally observed and supported in silico by modelling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and -T3 abundantly expressed by liver and gallbladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG (rCBG) was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialo- (disialyl T) and asialo-glycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. MD substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on strategically-positioned and biologically-relevant CBG RCL glycans, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.
HostingRepository	PRIDE
AnnounceDate	2023-12-06
AnnouncementXML	Submission_2023-12-05_16:18:06.343.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Anastasia Chernykh
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	complex glycosylation
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2023-10-25 23:23:42	ID requested
⏵ 1	2023-12-05 16:18:06	announced

Dataset with its publication pending

submitter keyword: glycosylation, O-glycosylation,Corticosteroid-binding globulin, cortisol, N-glycosylation, proteolysis, reactive centre loop, neutrophil elastase

Morten Thaysen-Andersen
contact affiliation	1. School of Natural Sciences, Macquarie University, Sydney, NSW 2109, Australia 2. Institute for Glyco-core Research (iGCORE), Nagoya University, Nagoya, Japan
contact email	morten.andersen@mq.edu.au
lab head
Anastasia Chernykh
contact affiliation	Macquarie University
contact email	nayachernykh@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD046405
     1. Label: PRIDE project
     2. Name: Position-specific N- and O-glycosylation of the reactive centre loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin