Poster Presentation 26th Annual Lorne Proteomics Symposium 2021

Unexpected glycan signatures of the RCL of human corticosteroid-binding globulin impact its susceptibility to proteolytic cleavage (#90)

Anastasia Chernykh 1 , Jodie L. Abrahams 1 2 , Zeynep Sumer-Bayraktar 1 3 , Rebeca Kawahara 1 , Morten Thaysen-Andersen 1 4
  1. Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia
  2. Glycosciences Laboratory, Department of Metabolism, Digestion and Reproduction, Imperial College London, London W12 0NN, United Kingdom
  3. Glycometabolic Biochemistry Team, Cluster of Pioneering Research, RIKEN, Wako, Saitama 351-0198, Japan
  4. Biomolecular Discovery Research Centre, Macquarie University, Sydney, NSW 2109, Australia

The liver-derived corticosteroid-binding globulin (CBG) is a heavily N-glycosylated protein that transports anti-inflammatory cortisol in human plasma. We have previously demonstrated that Asn347-glycans positioned on the exposed reactive centre loop (RCL) of human CBG, impact the release of cortisol by modulating the RCL cleavage process catalysed by neutrophil elastase (NE). However, a comprehensive structural and functional characterisation of the glycans decorating the RCL is still required to unravel the molecular mechanisms underpinning cortisol delivery to inflamed tissues. To this end, we have glycoprofiled human CBG isolated from pooled healthy donor sera (hCBG) using porous graphitised carbon-LC-MS/MS-based glycomics with parallel exoglycosidase digestion and in combination with C18-LC-MS/MS-based glycopeptide analysis employing CID-, HCD- and EThcD-MS/MS. Deep glycan and glycopeptide profiling was also performed on recombinant human CBG (rhCBG) produced in HEK293 cells. Longitudinal NE-based cleavage experiments with downstream glycopeptide detection were performed using rhCBG to study the impact of the RCL specific glycosylation on the NE-mediated cleavage process. Glycomics of the isolated RCL-glycopeptides of hCBG provided a quantitative profile of Asn347-N-glycans and their fine structural features including the assignment of core- and antenna-fucosylation, the elucidation of bi- and tri-antennary N-glycans and multiple α2,3- and α2,6-sialyl linkage isomers. Surprisingly, glycoproteomics of hCBG revealed a hitherto unknown presence of RCL O-glycosylation. The data confirmed that NeuAc1-2Gal1GalNAc1O-glycan structures occupy, albeit at low levels, four out of six possible O-glycosylation sites within the RCL region including a strategically positioned Thr345 site in close proximity to the NE cleavage site. Quantitative analysis revealed that the hCBG RCL was ~18.7% non-glycosylated, ~80.0% N-glycosylated, ~0.5% O-glycosylated, and ~0.8% both N- and O-glycosylated. In contrast, the rhCBG glycoprofiling revealed a relatively high level of Thr345 O-glycosylation (NeuAc1-2Gal1GalNAc1) (5.9%) while glycosylation of other possible O-glycosylation sites and Asn347 was negligible. Interestingly, cleavage experiments indicated that the Thr345-glycosylation of rhCBG strongly impedes NE-mediated proteolysis of the RCL. This suggests a functional significance of the RCL O-glycosylation, reported here for the first time. In conclusion, this is the first comprehensive structural and functional site-specific characterisation of the glycans decorating the RCL of native and recombinant human CBG. We present new functionally-important glycosylation features of the RCL that improve our understanding of the molecular mechanisms governing the timely and tissue-specific delivery of cortisol to inflammatory sites.