Moreover, upon NE-based RCL cleavage, the cortisol affinity of glycosylated CBG was reduced more than non-glycosylated forms, indicating that CBG glycosylation may yield a more rapid surge of free cortisol at target tissues to thereby facilitate a quicker resolution of inflammation (29). CBG liver, endometrium, placenta, and prostate) (20, 21); 2) direct internalization of an intact cortisol-bound CBG complex in specific tissues (placental syncytiotrophoblasts) (20); and, the most documented, 3) extracellular elastase-based proteolytic cleavage of the exposed reactive center loop (RCL) of CBG, resulting in cortisol release and cellular uptake of cortisol upon a stressed-to-relaxed conformational change of CBG at the site of inflammation (22,C27). Readers are directed Mouse monoclonal antibody to UCHL1 / PGP9.5. The protein encoded by this gene belongs to the peptidase C12 family. This enzyme is a thiolprotease that hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin. This gene isspecifically expressed in the neurons and in cells of the diffuse neuroendocrine system.Mutations in this gene may be associated with Parkinson disease to a recent review as a source of more in depth coverage of the CBG biology (28). The highly flexible RCL spanning the Glu333CIle354 region of CBG is the target of human neutrophil elastase (NE), which cleaves Val344-Thr345 to form two complementary fragments: the large (50C55 kDa) N-terminal fragment (CBG-Nt) and the small (5C10 kDa) C-terminal fragment (CBG-Ct) (22). The reduced cortisol affinity of cleaved CBG increases the local concentration of free cortisol, which is beneficial toward resolving inflammation in affected tissues by the anti-inflammatory effects of cortisol (17, 29). It was recently suggested that in basal, low inflammatory conditions, proteases other than NE may be causing systemic CBG cleavage (30). Both endogenous and exogenous proteases were documented to cleave the RCL and reduce the cortisol affinity of CBG. It was also shown that chymotrypsin cleaves the RCL of CBG at Leu346-Asn347 and Leu348-Thr349, but the biological relevance of this pancreatic protease in the context of the host immune response in inflamed tissues remains unknown (31). In addition, the Thr349-Ser350 and Ser350-Lys351 sites of elastase (PAE), the major virulence factor of this opportunistic Gram-negative pathogen and a zinc metalloprotease that is structurally unrelated to the serine proteases NE and chymotrypsin, was shown to cleave the RCL of CBG primarily between Asn347 and Leu348, leading to reduced cortisol affinity and release of the hormone from CBG (33). It was also reported that PAE cleaves several RCL sites (Thr345-Leu346, Leu346-Asn347, Asn347-Leu348 (main cleavage site), and Leu348-Thr349) (33). Interestingly, PAE and NE are known to co-exist in the inflamed and bacteria-infected respiratory tract of individuals with cystic fibrosis and chronic obstructive pulmonary disease (34). Although growing evidence supports an elastase-driven release of cortisol from CBG Zaldaride maleate as a mechanism for cortisol delivery to inflamed tissues, the molecular basis for the regulation of such a delivery mechanism by endogenous and exogenous elastases remains poorly understood. The six occupied glycosylated CBG was found to bind cortisol with significantly higher affinity and was more temperature-sensitive relative to non-glycosylated CBG) (29). Moreover, upon NE-based RCL cleavage, the cortisol affinity of glycosylated CBG was reduced more than non-glycosylated forms, indicating that CBG glycosylation may yield a more rapid surge of free cortisol at target tissues to thereby facilitate a quicker resolution of inflammation (29). Intriguingly, the C-terminally located Asn347, which is 84.7% occupied by primarily triantennary sialoglycans, is located on the RCL in close proximity to the reported cleavage sites (36). Similarly, other highly occupied glycosylation sites of CBG displaying mainly bi- and triantennary sialoglycans appear to be in relatively close spatial vicinity of the RCL when evaluated on the three-dimensional structure of human CBG (32, 37). However, the site-specific effects of CBG CBG-Ct (bands 1C3; 12, 11, and 5 kDa, respectively) and CBG-Nt (50C55 kDa) fragments) Zaldaride maleate (Fig. 1CBG-Ct (band 1, 12 kDa (BS2, BS2f (biantennary (and Zaldaride maleate indicate the identified cleavage sites of NE and trypsin, respectively. Asn347 Glycan Occupancy, Core Fucosylation, and Branching Reduce the NE-based RCL Cleavage Efficiency The influence of the Asn347 glycan moiety on the NE-based RCL cleavage was investigated by monitoring three volume-enhancing features (glycan site occupancy, core fucosylation, and outer antennae GlcNAc branching) of the Asn347 glycan as a function of NE digestion time. The Asn347 glycan occupancy significantly affected the efficiency of the NE-induced RCL cleavage, as evidenced by a significant increase of the site occupancy over the short (5C25 s) (20.0 2.7%) to medium (30C150 s) (54.6 11.7%, 0.05) incubation period (Fig. 2for an extended NE-based incubation of CBG (10 min and.