{"doi":"10.1016/j.jbc.2023.105519","title":"Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase–mediated proteolysis of corticosteroid-binding globulin","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.","journal":"Journal of Biological Chemistry","year":2023,"id":343660,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":14,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9539,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1081679,"name":"Jodie L. Abrahams","orcid":"0000-0002-2988-7843","position":1,"is_corresponding":false},{"id":227810,"name":"Oliver C. Grant","orcid":"0000-0003-4835-5161","position":2,"is_corresponding":false},{"id":1081680,"name":"Lucas Kambanis","orcid":"0000-0001-8278-5363","position":3,"is_corresponding":false},{"id":696943,"name":"Zeynep Sumer‐Bayraktar","orcid":"0000-0001-7064-2728","position":4,"is_corresponding":false},{"id":696939,"name":"Julian Ugonotti","orcid":"0000-0002-3455-6766","position":5,"is_corresponding":false},{"id":557105,"name":"Rebeca Kawahara","orcid":"0000-0002-0292-2637","position":6,"is_corresponding":false},{"id":1081681,"name":"Leo Corcilius","orcid":"0009-0003-0155-3619","position":7,"is_corresponding":false},{"id":1081682,"name":"Richard J. Payne","orcid":"0000-0002-3618-9226","position":8,"is_corresponding":false},{"id":39814,"name":"Robert J. Woods","orcid":"0000-0002-2400-6293","position":9,"is_corresponding":false},{"id":557107,"name":"Morten Thaysen‐Andersen","orcid":"0000-0001-8327-6843","position":10,"is_corresponding":false},{"id":628459,"name":"Anastasia Chernykh","orcid":"0000-0002-1493-6964","position":0,"is_corresponding":true}],"reference_count":80,"raw_metadata":null,"created_at":"2026-07-19T01:11:26.556051Z","pmid":"38042488","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}