Cruz\Topete D, Cidlowski JA

Cruz\Topete D, Cidlowski JA. 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 heat 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 Astragaloside A 39C mimicking pyrexia and pH 7.4 and 7.0 mimicking acidosis, pathophysiological conditions relevant to sepsis. An expected 4C8\fold reduction Astragaloside A 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 39C relative to 37C, 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 40C confirmed a critical role for Trp371, positioned within the steroid\binding pocket, in ligand binding. These studies exhibited CBG binding affinity to range of steroids is usually ligand specific and is reduced with NE\mediated haCBG\to\laCBG transition. Reduced CBG:cortisol binding occurs with increased heat and in acidosis. Increased flexibility of the Trp371 side chain is usually 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. 570C2,000 with a zoom scan resolution of 0.25 at full width at half maximum (FWHM) and a source voltage of +2.7 kV. Detection was performed in unfavorable ion polarity mode with data\dependent MS/MS acquisition. The automatic gain control (AGC) for the MS1 scans was set to 5 ?104 with a maximum accumulation time of 50?ms. For the MS/MS events, the resolution was set to 0.35 FWHM, the AGC was 2 ?104 and the maximum accumulation time was 300?ms. The top\five most abundant precursors in each MS1 scan were selected for MS/MS using resonance\activation (ion trap) based collision\induced dissociation (CID) at a fixed 35% normalized collision energy (NCE). Dynamic exclusion was disabled. The natural LCCMS/MS data was browsed and interrogated using Xcalibur v2.2 (Thermo Fisher Scientific, CA). The glycan structures were characterized using relative and absolute PGC\LC retention time, monoisotopic mass, and CID\MS/MS fragmentation pattern as previously described. 21 3.4. ?0.05 was considered statistically significant. 4.?RESULTS 4.1. ligand binding studies. Open in a separate windows Physique 2 Comparative glycomic analysis of native and recombinant human CBG. Native human CBG isolated from donor blood (Native CBG, upper panel) and biotinylated recombinant human CBG expressed in HEK293 cells (Biotin\CBG, lower panel) are glycosylated with broadly comparable values as supported by CID\MS/MS data are shown above using the conventional symbol nomenclature for glycans. 33 * denotes an already depicted .0001). Glucocorticoids and progestagens were the only hormones that displayed measurable binding to laCBG (i.e., All data points are represented as the mean values and their variance indicated in brackets as SEM as decided from at least three technical replicates. Abbreviation: N/D, not determined. The effects of temperature and pH on hydrocortisone (cortisol) binding to CBG were subsequently resolved (Table ?(Table2).2). Increasing heat from 37 to 39C, as observed in inflammation, caused a significant decrease in hydrocortisone binding to haCBG (37C =?.0042). Likewise, acidification from the physiological pH 7.4 to pH 7.0 at 39C caused further loss in binding activity (pH 7.4 =?.0308). TABLE 2 Hydrocortisone (cortisol) equilibrium binding constants (All data points are represented as mean values and their variance indicated in brackets as SEM. Two\way ANOVA overall conversation = .0002, row factor = .0001, column factor .0001. Abbreviation: = .0042. b Comparison of haCBG:cortisol at 37C pH 7.4 versus 39C pH 7.0 .0001. c Comparison of haCBG:cortisol at 39C pH 7.4 versus 39C pH 7.0 = .0308. d Comparison of laCBG:cortisol at 37C pH 7.4 versus 39C pH 7.4 = .0010. e Comparison of laCBG:cortisol at 37C pH 7.4 versus 39C pH 7.0 .0001. There was an additive IL10 effect of NE treatment, heat elevation and acidification on CBG:hydrocortisone binding affinity. Hydrocortisone showed 3.5\fold reduced binding from haCBG 37C, pH 7.4 (=? .0001). NE\treatment further reduced binding Astragaloside A with seven\fold reduced affinity observed between haCBG at 37C, pH 7.4 (=? .0001). Similarly, a significant reduction in cortisol binding to laCBG was observed with increased heat (Table ?(Table2).2). The data clearly demonstrate that heat, pH, and NE\mediated proteolysis all contribute to the release of CBG bound hormone under the.