CBG regulates the bioavailability of this anti-inflammatory steroid hormone by binding up to 90% of the entire cortisol pool with high affinity and specificity in a single binding pocket in a temperature-sensitive manner ( 1, 2). Human corticosteroid-binding globulin (CBG) 5 is the main carrier of cortisol in the blood circulatory system. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. Molecular dynamics simulations of various Asn 347 glycoforms of uncleaved CBG indicated that multiple Asn 347 glycan features are modulating the RCL digestion efficiencies by NE/PAE. In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr 345-Leu 346 and Asn 347-Leu 348, was abolished by the presence of Asn 347 glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn 347 NeuAc-type sialylation (all p < 0.05). The site-specific (Val 344-Thr 345) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn 347 glycan features ( i.e. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn 347 glycosylation as a function of digestion time. We document that RCL-localized Asn 347 glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL).
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