Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

SUBMITTER: Anastasia Chernykh

LAB HEAD: Morten Thaysen-Andersen

PROVIDER: PXD046405 | Pride | 2023-12-05

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	211104_AC_serumCBG_1.raw	Raw
	220414_AC_serumCBG_2.raw	Raw
	310_mammalian_with_M3F_glycan_database.txt	Txt
	Asialo_rCBG_NE_120s_1.raw	Raw
	Asialo_rCBG_NE_120s_2.raw	Raw

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

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