Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase–mediated proteolysis of corticosteroid-binding globulin

Anastasia Chernykh, Jodie L. Abrahams, Oliver C. Grant, Lucas Kambanis, Zeynep Sumer-Bayraktar, Julian Ugonotti, Rebeca Kawahara, Leo Corcilius, Richard J. Payne, Robert J. Woods, Morten Thaysen-Andersen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)
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Abstract

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure–function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified 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 glycosylation was experimentally observed and supported in silico by modeling of a CBG–GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (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. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

Original languageEnglish
Article number105519
Pages (from-to)1-20
Number of pages20
JournalJournal of Biological Chemistry
Volume300
Issue number1
Early online date30 Nov 2023
DOIs
Publication statusPublished - Jan 2024

Bibliographical note

Copyright the Author(s) 2023. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • corticosteroid-binding globulin
  • glycosylation
  • N-glycosylation
  • proteolysis
  • O-glycosylation
  • reactive center loop
  • neutrophil elastase
  • cortisol

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