Corticosteroid-binding globulin (CBG) reactive centre loop antibodies and surface plasmon resonance interrogate the proposed heat dependent "flip-flop" mechanism of human CBG. Issue 158 (April 2016)

• Record Type: Journal Article
• Authors: Lewis, John G.; Fredericks, Rayleen; Fee, Conan J.; Elder, Peter A.
• Is Part Of: Journal of steroid biochemistry and molecular biology. Issue 158(2016)
• Page Start: 38
• Page End: 45
• Publication Date: 2016-04
• Subjects: Corticosteroid-binding globulin -- CBG -- Serpins -- Elastase -- Reactive centre loop -- Cortisol -- Temperature; Steroid hormones -- Periodicals; Biochemistry -- Periodicals; Hormones -- Periodicals; Molecular Biology -- Periodicals; Hormones stéroïdes -- Périodiques; Steroid hormones; Periodicals; 572.579
• DOI: 10.1016/j.jsbmb.2016.01.013
• Languages: English
• ISSNs: 0960-0760

Abstract

Highlights: We interrogate the proposed heat dependent "flip-flop" of the reactive centre loop (RCL) of human CBG using antibodies and surface plasmon resonance. These antibodies span 11 of the 18 residues of the RCL. Surface plasmon resonance shows that up to 40 °C the RCL remains accessible to RCL antibodies. The release of cortisol from CBG by heat may be independent of the RCL 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.