Oral Presentation ESA-SRB-AOTA 2019

Binding affinities of high and low affinity corticosteroid-binding globulin; effect of fever and acidosis. (#126)

Emily J Meyer 1 2 , David J Torpy 1 2 , Anastasia Chernykh 3 , Morten Thaysen-Andersen 3 , John Lewis 4 , Wayne Rankin 1 5 , Steven Polyak 6
  1. Endocrine and Metabolic Unit, Royal Adelaide Hospital, Adelaide, SA, Australia
  2. Discipline of Medicine, University of Adelaide, Adelaide, South Australia, Australia
  3. Department of Molecular Sciences, Macquarie University, Sydney, New South Wales, Australia
  4. Steroid & Immunobiochemistry Laboratory, Canterbury Health Laboratories, Christchurch, New Zealand
  5. Chemical Pathology Directorate, SA Pathology, Adelaide, South Australia, Australia
  6. School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia

Corticosteroid-binding globulin (CBG) is the principal transport protein for cortisol in the circulation1. Secreted high-affinity (ha) CBG undergoes an irreversible conformational change to low-affinity (la) CBG following proteolytic digestion of an exposed reactive centre loop (RCL) by neutrophil elastase (NE). Cortisol is liberated from haCBG upon NE proteolysis at inflammatory sites or elevated body temperatures2. CBG also binds other hormones including progesterone, 17-hydroxyprogesterone and cortisone3-5.

A homogenous ligand binding assay using Surface plasmon resonance (SPR) technology measured binding affinities of haCBG and laCBG to a panel of 19 steroid ligands at temperatures 25oC, 37oC and 39oC, and neutral (7.4) and acidic (7.0) pH to mimic the pathophysiological conditions of septic shock. 9G12, an in-house monoclonal antibody recognising the RCL epitope encompassing the NE cleavage site, was used to access the RCL integrity across temperature ranges.

Our newly developed SPR binding assay demonstrated a 4-8-fold decrease in relative binding affinity between haCBG and laCBG for cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, 17-hydroxyprogesterone and prednisolone, the former expectedly displaying the highest binding affinity. Hormone binding affinity also decreased 7-12-fold at higher temperatures and acidic pH for both haCBG and laCBG. This is the first study to determine the binding affinity of haCBG and laCBG to a large panel of steroid ligands and importantly demonstrate intact thermo-coupling even after NE cleavage.  The 9G12 antibody recognition was preserved for haCBG across the temperature range confirming that the RCL remains intact and available for NE proteolysis, providing insight into the mechanism at which haCBG loses ligand affinity during pyrexia due to temperature-dependent flexibility of the hormone binding pocket.

These findings demonstrate the modifiable hormone binding characteristics of CBG in (patho-)physiological conditions, supporting its significance in cortisol delivery in obviating systemic inflammation and multi-organ failure in patients with septic shock and its association with mortality6.

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  6. Meyer EJ, Nenke MA, Rankin W, et al. Total and high-affinity corticosteroid-binding globulin depletion in septic shock is associated with mortality. Clin Endocrinol (Oxf). 2019;90(1):232-240.