N-glycosylation of the reactive centre loop of corticosteroid-binding globulin regulate neutrophil elastase-based cleavage and cortisol release

Human corticosteroid-binding globulin (CBG) is a heavily N-glycosylated protein which binds and transports corticosteroids i.e. cortisol in blood circulation. We recently mapped the sitespecific N-glycosylation of human blood-derived CBG and showed that the high degree of CBG sialylation is regulating the CBG receptor interaction on cell surfaces. Herein, we describe a site-specific function of CBG N-glycosylation located at Asn347 on the reactive centre loop (RCL) of CBG. It is known that the RCL undergoes human neutrophil elastase (NE)-based proteolytical cleavage at the proximal Val344-Thr345, which induces a conformational change of CBG that, in turn, favours the release of corticosteroids. NE-based digestion assays of blood-derived CBG isolated from healthy individuals and gel electrophoresis showed that NE preferentially cleaves non-occupied Asn347 glycoforms in a concentration- and time-dependent manner. LC-MS/MS based glycopeptide profiling of the Asn347 N-glycans indicates a regulatory role of volume-enhancing glycan features including α1,6-core fucosylation, β1,4-branching and α2,3-sialylation in NE digestion efficiency. The macro- (85% N-glycan occupancy) and micro-heterogeneity (>19 N-glycoforms) of Asn347 of bloodderived CBG may be a mechanism to regulate the amount/rate of NE-induced RCL cleavage and, thus, indirectly regulate the release and availability of anti-inflammatory corticosteroids. In conclusion, we propose that the heterogeneous N-glycosylation of human CBG has multiple regulatory functions in hormone signalling.1 page(s