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

Abstract

Background: 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.

Objective: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Keywords: CBG; Corticosteroid binding globulin; Cortisol; N-glycosylation; Neutrophil elastase; Septic shock; low affinity CBG.