Corticosteroid-binding globulin reactive centre loop antibodies recognise only the intact natured protein: Elastase cleaved and uncleaved CBG may coexist in circulation

The half-lives of intact and elastase cleaved human corticosteroid-binding globulin (CBG) are identical in the rabbit

“…Briefly, following "blocking" in assay buffer, phosphate buffered saline (PBS) containing 0.1% Tween 20 (v/v) and 0.1% gelatin (w/v), 150 mL/well), the wells were emptied by inversion and either 100 mL of standard (1:1000 in assay buffer) or 100 mL of dilutions of elastase and non-elastase treated CBG (1:8000 to 1:32000), appropriate controls or dilutions of timed rabbit serum samples (1:50) added for 30 min incubation at 20 C. The plates were washed and CBG monoclonal antibody supernatant from clone 12G2 (1:20 in assay buffer, 100 mL/well) was added to one plate, which measures total CBG and to the other the supernatant from the RCL specific CBG monoclonal antibody 9G12, which measures intact CBG [7], (1:20 in assay buffer, 100 mL/well) for a 30 min incubation at 20 C. The plates were again washed and either antimouse IgG1-peroxidase (1:2000 in assay buffer, 100 mL/ well) was added to the plate for total CBG determination or antimouse IgG2a-peroxidase (1:1000 in assay buffer, 100 mL/well) was added to the plate for intact CBG determination. Both plates were incubated a further 15 min at 20 C. The plates were finally washed and tetramethybenzidine substrate was added (100 mL/ well).…”

“…Therefore, the CBG ELISAs performed in parallel provide measures of total and intact or uncleaved CBG [7].…”

“…The RCL of human CBG can also be cleaved by chymotrypsin [5] and the virulence factor protease LasB from Pseudomonas aeruginosa [6] which also disrupt cortisol binding to CBG. We recently raised a monoclonal antibody to the intact RCL of human CBG [7] and the epitope STGVTLNL spans the elastase cleavage site between Val344 and Thr345 as well as the 2 chymotrypsin sites at Leu346 and Leu348. We used this in concert with another monoclonal antibody to CBG to develop an ELISA which shows both intact and elastase cleaved CBG can coexist in circulation [8].…”

Towards Engineering Hormone-Binding Globulins as Drug Delivery Agents

“…We mutated a segment of the wild type CBG's reactive centre loop from STGVTLNL to SALLSSDI and SSYYSGNL by site-directed mutagenesis, altering the presumed elastase cleavage site [43], which is located at residues P6-P5 in the canonical numbering for inhibitory serpins, to potential PSA cleavage sites on CBG. Unlike the CBG-T349R mutant, for which the protease cleavage site of the reactive centre loop was placed at the canonical P1 residue (in line with the crystal structure of the similar CBG-antitrypsin chimera that was previously solved by our group [19]) we had no a priori knowledge of the effect of inserting a PSA-specific sequence on the structure and biochemical behavior of CBG.…”

Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase

“…liver, endometrium, placenta, and prostate) (20,21); 2) direct internalization of an intact cortisolbound CBG complex in specific tissues (e.g. 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)(23)(24)(25)(26)(27). Readers are directed to a recent review as a source of more in depth coverage of the CBG biology (28 to form two complementary fragments: the large (50 -55 kDa) N-terminal fragment (CBG-Nt) and the small (5-10 kDa) C-terminal fragment (CBG-Ct) (22).…”