Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:300 |
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| Enthalten in: |
The Journal of biological chemistry - 300(2024), 1 vom: 11. Jan., Seite 105519 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Chernykh, Anastasia [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 07.02.2024 Date Revised 10.02.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jbc.2023.105519 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM365334758 |
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| 100 | 1 | |a Chernykh, Anastasia |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin |
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| 520 | |a Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a N-glycosylation | |
| 650 | 4 | |a O-glycosylation | |
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| 650 | 4 | |a cortisol | |
| 650 | 4 | |a glycosylation | |
| 650 | 4 | |a neutrophil elastase | |
| 650 | 4 | |a proteolysis | |
| 650 | 4 | |a reactive center loop | |
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| 700 | 1 | |a Abrahams, Jodie L |e verfasserin |4 aut | |
| 700 | 1 | |a Grant, Oliver C |e verfasserin |4 aut | |
| 700 | 1 | |a Kambanis, Lucas |e verfasserin |4 aut | |
| 700 | 1 | |a Sumer-Bayraktar, Zeynep |e verfasserin |4 aut | |
| 700 | 1 | |a Ugonotti, Julian |e verfasserin |4 aut | |
| 700 | 1 | |a Kawahara, Rebeca |e verfasserin |4 aut | |
| 700 | 1 | |a Corcilius, Leo |e verfasserin |4 aut | |
| 700 | 1 | |a Payne, Richard J |e verfasserin |4 aut | |
| 700 | 1 | |a Woods, Robert J |e verfasserin |4 aut | |
| 700 | 1 | |a Thaysen-Andersen, Morten |e verfasserin |4 aut | |
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