High-affinity Corticosteroid-binding Globulin Research Articles

Mass spectrometric detection of neutrophil elastase cleaved corticosteroid binding globulin and its association with Asn347 site glycosylation, in septic shock patients.

Corticosteroid-binding globulin (CBG) modulates tissue cortisol availability via modification of cortisol:CBG binding affinity in response to multiple factors, including neutrophil elastase (NE) cleavage of the reactive centre loop (RCL), converting high affinity CBG (haCBG) to low affinity CBG (laCBG). In vitro, glycosylation of the RCL at Asn347 affects NE cleavage susceptibility. To date, no direct measurement of laCBG, which would verify NE cleavage, has been reported. To measure serum laCBG in septic shock patients by mass spectrometry to confirm NE cleavage in vivo, with Asn347 site glycosylation profiling to determine its impact on NE cleavage, and determine effect of %laCBG on septic shock clinical outcome. Serum from septic shock patients from a tertiary hospital intensive care unit was analysed by mass spectrometry for CBG affinity forms and Asn347 glycosylation profile, following CBG immunoprecipitation. Data was correlated with septic shock clinical outcome. N-terminal peptide of NE cleaved RCL (AVLQLNEEGVDTAGSTGV) was consistently detected in patient serum, although at low concentrations. Mean %laCBG/total CBG was 0.23 % in septic shock (range = 0.07-0.74 %, SD = 0.12 %); in comparison healthy controls mean %laCBG was 0.04 % (range 0.02-0.08 %, SD = 0.03 %). There was a negative correlation between %laCBG and serum concentrations of CBG with triantennary Asn347 glycans; TS3 (r = -0.190, p = 0.040) and TS3F (r = -0.252, p = 0.006). NE cleaved CBG (laCBG) is present in septic shock patient serum, and %laCBG correlates with Asn347 glycosylation occupancy and composition. However, serum %laCBG did not correlate with septic shock clinical outcome.

Pyrexia and acidosis act independently of neutrophil elastase reactive center loop cleavage to effect cortisol release from corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) transports cortisol and other steroids. High-affinity CBG (haCBG) undergoes proteolysis of the reactive center loop (RCL) by neutrophil elastase (NE) altering conformation to low-affinity CBG (laCBG). Elevated temperature reduces CBG:cortisol binding affinity. Surface plasmon resonance was used to determine binding profiles of 19 steroids to haCBG and laCBG at 25, 37, and 39°C mimicking pyrexia and pH 7.4 and 7.0 mimicking acidosis, pathophysiological conditions relevant to sepsis. An expected 4-8-fold reduction in affinity for cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, 17-hydroxyprogesterone, and prednisolone occurred with NE-mediated haCBG-to-laCBG conversion. CBG:cortisol binding affinity was further reduced 3.5-fold at 39°C relative to 37°C, binding affinity was also reduced by acidosis for both haCBG and laCBG. Using a conformational antibody generated against the RCL, we confirmed RCL antibody binding was eliminated by NE cleavage, but preserved in pyrexia and acidosis. Molecular modeling studies performed at 40°C confirmed a critical role for Trp371, positioned within the steroid-binding pocket, in ligand binding. These studies demonstrated CBG binding affinity to range of steroids is ligand specific and is reduced with NE-mediated haCBG-to-laCBG transition. Reduced CBG:cortisol binding occurs with increased temperature and in acidosis. Increased flexibility of the Trp371 side chain is proposed in the thermo-coupling mechanism of cortisol release. The synergy of NE cleavage, pyrexia, and acidosis on CBG:cortisol binding may serve to enhance cortisol delivery to the interstitial space in inflammation.

OR19-05 Steroid:Corticosteroid-Binding Globulin Interactions; Effects of Neutrophil Elastase Cleavage, Pyrexia and Acidosis

Context Corticosteroid-binding globulin (CBG) transports cortisol and other steroid hormones1,2. High-affinity CBG (haCBG) undergoes proteolysis of the reactive centre loop (RCL) by neutrophil elastase (NE) at inflammatory sites, liberating immunomodulatory cortisol and altering conformation to low-affinity CBG (laCBG). Pyrexia reduces CBG:cortisol binding affinity, an interaction at the RCL is speculated3. Objective To measure the equilibrium binding constants of a panel of steroids to glycosylated haCBG and laCBG over temperature and pH ranges mimicking the pathophysiological conditions of septic shock. Design Surface plasmon resonance was used to determine the binding profiles of 19 steroid ligands to haCBG and laCBG at temperatures 25°C, 37°C and 39°C and pH 7.4 and 7.0. The RCL-recognizing 9G12 antibody was used to assess cleavage and epitope availability of the RCL across conditions. Results A 4–8 fold reduction in affinity for cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, 17-hydroxyprogesterone and prednisolone occurred with NE-mediated haCBG-to-laCBG conversion, cortisol expectedly displayed the highest binding affinity. Binding affinity consistently decreased at higher temperatures and at acidic pH for both haCBG and laCBG. 9G12 antibody RCL binding was preserved for haCBG across temperatures. Conclusions These studies reveal that steroid binding to CBG is selective and in all cases reduced upon NE-mediated haCBG-to-laCBG transition. Moreover, reduced CBG:cortisol binding affinity at elevated temperature occurs with an intact and accessible RCL epitope, suggesting a non-RCL mechanism for the delivery of anti-inflammatory cortisol in pyrexia. Synergy of NE cleavage and pyrexia/acidosis may serve for local inflammatory site cortisol delivery and increase free cortisol. 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 multiorgan-organ failure in patients with septic shock and its association with mortality4.1. Pemberton PA, Stein PE, Pepys MB, et al. Hormone binding globulins undergo serpin conformational change in inflammation. Nature. 1988;336(6196):257–258.2. Pugeat MM, Dunn JF, Nisula BC. Transport of steroid hormones: interaction of 70 drugs with testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab. 1981;53(1):69–75.3. Cameron A, Henley D, Carrell R, et al. Temperature-responsive release of cortisol from its binding globulin: a protein thermocouple. J Clin Endocrinol Metab. 2010;95(10):4689–4695.4. 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.

Total and high-affinity corticosteroid-binding globulin depletion in septic shock is associated with mortality.

Corticosteroid-binding globulin (CBG) and albumin transport circulating cortisol. Cleavage of high-affinity CBG (haCBG) by neutrophil elastase at inflammatory sites causes cortisol release into tissues, facilitating immunomodulatory effects. To determine whether depletion of haCBG is related to mortality in septic shock. A single-center prospective observational cohort study of patients recruited with critical illness or septic shock, using serum samples collected at 0, 8, 24, 48 and 72hours. Serum total and haCBG, and total and free cortisol were assayed directly. Glucocorticoid treatment was an exclusion criterion. Mortality was assessed at 28days from Intensive Care Unit admission. Thirty septic shock (SS) and 42 nonseptic critical illness (CI) patients provided 195 serum samples. SS/CI patients had lower total CBG, haCBG and low-affinity CBG (laCBG) than controls. Total CBG and haCBG were significantly lower in septic shock patients who died than in those that survived (P<0.009, P=0.021, respectively). Total and free cortisol were higher in septic than nonseptic individuals. Free/total cortisol fractions were higher in those with low haCBG as observed in septic shock. However, cortisol levels were not associated with mortality. Albumin levels fell in sepsis but were not related to mortality. Low circulating haCBG concentrations are associated with mortality in septic shock. These results are consistent with an important physiological role for haCBG in cortisol tissue delivery in septic shock.

Differential Effects of Estrogen on Corticosteroid-Binding Globulin Forms Suggests Reduced Cleavage in Pregnancy.

Corticosteroid-binding globulin (CBG) is secreted as high-affinity CBG (haCBG), which may be cleaved by tissue proteases to low-affinity CBG (laCBG), releasing free cortisol. Pregnancy and the estrogen-based combined oral contraceptive pill (COCP) increase CBG concentrations twofold to threefold. The relative effects of these two hyperestrogenic states on the CBG affinity forms are unknown. We performed an observational study in 30 pregnant women, 27 COCP takers and 23 controls. We analyzed circulating total CBG, haCBG, laCBG, and free and total cortisol concentrations. In pregnancy, total CBG and haCBG were increased compared to controls (both P < 0.0001); however, laCBG concentrations were similar. In COCP takers, total CBG and haCBG were increased [802 ± 41 vs compared to controls (both P < 0.0001)], but laCBG was also increased (P = 0.03). Pregnancy and use of COCP were associated with a comparable rise in haCBG, but laCBG was lower in pregnancy (P < 0.0001). These results were consistent with an estrogen-mediated increase in CBG synthesis in both hyperestrogenemic states but with reduced CBG cleavage in pregnancy relative to the COCP, perhaps due to pregnancy-induced CBG glycosylation. Speculatively, increased circulating haCBG concentrations in pregnancy may provide an increased reservoir of CBG-bound cortisol to prepare for the risk of puerperal infection or allow for cortisol binding in the face of competition from increased circulating progesterone concentrations.

Corticosteroid-binding globulin cleavage is paradoxically reduced in alpha-1 antitrypsin deficiency: Implications for cortisol homeostasis

High-affinity corticosteroid-binding globulin (haCBG) is cleaved by neutrophil elastase (NE) resulting in permanent transition to the low cortisol-binding affinity form (laCBG), thereby increasing cortisol availability at inflammatory sites. Alpha-1 antitrypsin (AAT) is the major inhibitor of NE. AAT deficiency (AATD) predisposes patients to early-onset emphysema due to increased proteolytic destruction from the inherent proteinase–antiproteinase imbalance. We hypothesized that AATD may result in increased CBG cleavage in vivo. We collected demographic data and blood samples from 10 patients with AATD and 28 healthy controls measuring total CBG and haCBG levels by parallel in-house ELISAs, as well as AAT, total and free cortisol levels. haCBG was higher (median [range]); 329 [210–551] vs. 250 [175–365] nmol/L; P<0.005, and laCBG lower; 174 [68–229] vs. 220 [119–348] nmol/L; P=0.016 in the AATD group, compared with controls. The ratio of haCBG:total CBG was also higher in AATD; 72 [53–83] vs. 54 [41–72] %; P=0.0001). There was a negative correlation between haCBG:total CBG and AAT levels (P<0.05, R=−0.64). Paradoxically, proteolytic cleavage of CBG was reduced in AATD, despite the recognized increase in NE activity. This implies that NE activity is not the mechanism for systemic CBG cleavage in basal, low inflammatory conditions. Relatively low levels of laCBG may have implications for cortisol action in AATD.