title: Corticosteroid-binding globulin (CBG) reactive centre loop antibodies and surface plasmon resonance interrogate the proposed heat dependent "flip-flop" mechanism of human CBG.
abstract: Corticosteroid-binding globulin (CBG) is the predominant carrier of cortisol in circulation and is a non-inhibitory member of the serpin superfamily of serine protease inhibitors. In the stressed or "S" conformation, CBG possesses an intact exposed reactive centre loop (RCL) that can be irreversibly cleaved by elastase released from activated human neutrophils whereupon it adopts a relaxed or "R" conformation. The latter conformation has decreased affinity for cortisol, allowing the release of the majority of cortisol at sites of inflammation. Recently there has been speculation that mild increments in heat such as found in pyrexia (39-40°C) may also induce a reversible "flip-flop" of the RCL into the body of the protein structure, without cleavage, facilitating a reversible temperature-dependent release of cortisol. Here we raised a new monoclonal antibody to the RCL of human CBG and used this in concert with an existing RCL antibody and show by surface plasma resonance that, at temperatures up to 40°C, the RCL of purified CBG and the RCL of CBG in intact plasma is accessible to these two antibodies. Together, the epitopes of these antibodies span 11 consecutive amino acids (STGVTLNLTSK) of the 18 residues of the RCL. This adequate antibody cover of the RCL sequence leads to the conclusion that the proposed temperature-dependent "flip-flop" of the RCL of CBG is doubtful.
authors.affiliation: Steroid & Immunobiochemistry Laboratory, Canterbury Health Laboratories, P.O. Box 151, Christchurch 8001, New Zealand. Electronic address: john.lewis@cdhb.health.nz.
authors.forename: John G
authors.initials: JG
authors.lastname: Lewis
authors.affiliation: Biomolecular Interaction Centre, University of Canterbury, Private bag 4800, Christchurch 8140, New Zealand.
authors.forename: Rayleen
authors.initials: R
authors.lastname: Fredericks
authors.affiliation: Biomolecular Interaction Centre, University of Canterbury, Private bag 4800, Christchurch 8140, New Zealand.
authors.forename: Conan J
authors.initials: CJ
authors.lastname: Fee
authors.affiliation: Steroid & Immunobiochemistry Laboratory, Canterbury Health Laboratories, P.O. Box 151, Christchurch 8001, New Zealand.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Elder
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping; D006358:Hot Temperature; D006801:Humans; D055767:Immobilized Proteins / Q000276:immunology / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013954:Thyroglobulin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014156:Transcortin / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D055767:Immobilized Proteins; D010455:Peptides; C534935:SERPINA6 protein, human; D013954:Thyroglobulin; D014156:Transcortin; D010196:Pancreatic Elastase
keywords: CBG; Corticosteroid-binding globulin; Cortisol; Elastase; Reactive centre loop; Serpins; Temperature