Oral Presentation 27th Annual Lorne Proteomics Symposium 2022

Unexpected O-glycosylation of human corticosteroid-binding globulin protects against neutrophil elastase and impedes cortisol release (#4)

Anastasia Chernykh 1 , Jodie L. Abrahams 1 2 , Zeynep Sumer-Bayraktar 1 3 , Oliver C. Grant 4 , Robert J. Woods 4 , Rebeca Kawahara 1 , Morten Thaysen-Andersen 1 5
  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. Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602, USA
  5. Biomolecular Discovery Research Centre, Macquarie University, Sydney, NSW 2109, Australia

Human corticosteroid-binding globulin (hCBG) is a heavily N-glycosylated protein that transports cortisol in plasma. We previously demonstrated that Asn347-glycans positioned on the exposed reactive centre loop (RCL) of hCBG impact the release of cortisol by modulating the neutrophil elastase (NE)-mediated RCL cleavage at inflammatory sites. However, a comprehensive structure-function characterisation of the RCL glycosylation is still required to unravel the molecular mechanisms underpinning cortisol delivery to inflamed tissues. To this end, we have performed a deep RCL-centric glycoprofiling of hCBG isolated from healthy sera using glycomics and glycoproteomics. Glycoprofiling and longitudinal NE-centric cleavage experiments were also performed on HEK293-derived recombinant human CBG (rhCBG) to study how the RCL glycosylation impacts the NE-mediated cleavage process and cortisol release. Surprisingly, the hCBG glycoprofiling revealed a hitherto unknown presence of RCL O-glycosylation. Two core 1-type O-glycans (NeuAc1-2Gal1GalNAc1) were found to occupy four O-glycosylation sites within the RCL, including the strategically-positioned Thr345 site proximal to the Val344-Thr345 cleavage site targeted by NE. Interestingly, some hCBG glycoforms displayed an intriguing N- and O-glycan co-occurrence involving the co-occupancy of Asn347 and either Thr338 or Thr342 suggesting a crosstalk between N- and O-glycosylation on the RCL. In contrast, rhCBG glycoprofiling revealed high levels of core 1/2-type O-glycosylation (NeuAc1-2Gal1-2GalNAc1-2) at the Thr345 site and negligible glycosylation of other potential RCL sites. Notably, longitudinal NE-centric cleavage experiments demonstrated that sialo-, asialo-, and agalacto-glycans decorating Thr345 of rhCBG strongly protect against NE proteolysis, which was supported by molecular dynamics simulations that substantiated that a Thr345-positioned O-GalNAc residue can protect against NE interactions and, in turn, block cortisol release from CBG. In conclusion, we are the first to report on strategically-positioned and functionally-relevant N- and O-glycans occupying the RCL of hCBG. These findings improve our understanding of the molecular mechanisms governing the timely and tissue-specific delivery of cortisol to inflammatory sites.