Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors.

Abstract

We have isolated and sequenced cDNAs for corticosteroid binding globulin (CBG) Corticosteroid binding globulin (CBG) is the major transport protein for glucocorticoids in the blood of almost all vertebrate species (1), and >90% of the cortisol in human plasma is bound by this protein (2). The remaining fraction is distributed more evenly between albumin and the pool of nonprotein-bound or "free" steroid that is generally assumed to be biologically active (2, 3). In humans, CBG is an acidic, -58-kDa glycoprotein (4-6) comprising five N-linked oligosaccharide chains (7) that collectively represent -23% of the molecule by mass (6, 7). The binding site for natural glucocorticoids appears to be a hydrophobic pocket containing one of two cysteine residues that have been identified by amino acid composition analyses (8)(9)(10)(11). Apart from this information, and the identification of eight residues at the NH2 terminus of human CBG (5, 11), there is virtually no information about its primary structure or the location of its steroid binding site.Like many other plasma transport proteins, CBG is produced and secreted by hepatocytes (12), but has also been identified in a number of glucocorticoid responsive cells (2, 13), and may even interact directly with the plasma membranes of some cells (14,15). The objectives of this study were, therefore, to predict the amino acid sequence of human CBG from a cDNA and to determine whether tissues other than the liver possess the capacity to produce this protein. § METHODS cDNA Cloning. A monospecific rabbit antiserum for human CBG (6) was initially used to screen a Xgtll human liver cDNA library that was kindly provided by S. L. C. Woo (Baylor College of Medicine, Houston). The screening method was based on the technique described by Young and Davis (16), with the exception that peroxidase-labeled protein A was used to detect antibody-antigen complexes in the presence of the chromogenic substrate 4-chloro-1-naphthol. The recombinant phage isolated in this way were used to prepare plate lysates using NZC top agar (GIBCO). The phage were harvested and purified, and the cDNA inserts were excised and inserted into the EcoRI site of pBR322 according to Maniatis et al. (17). Plasmids containing CBG cDNAs were used to transform competent Escherichia coli (strain MM 294), and transformants were propagated in Luria broth in the presence of ampicillin and chloramphenicol to amplify the plasmid (17). Plasmids were isolated by the alkaline lysis method and purified using benzoylated-naphthoylated-DEAE cellulose (Sigma) according to Gamper et al. (18). The cDNAs were routinely excised from the plasmid and purified by polyacrylamide gel electrophoresis, prior to nick-translation with 32P-labeled dCTP (17).In an attempt to isolate a full-length CBG cDNA, the radiolabeled cDNA was employed to rescreen the library. Nitrocellulose filters (Schleicher & Schuell; BA85, 0.45-,um pore size) were used to transfer DNA and were hybridized with 2 x 106 dpm of the CBG cDNA probe per ml, in the pr...

Corticosteroid-binding Globulin, a Structural Basis for Steroid Transport and Proteinase-triggered Release

“…In the case of CBG, this causes a pronounced alteration of its conformation that is marked by an increase in thermostability (17), a behavior typical of serpins that undergo the S 3 R transition. In CBG, this essentially eliminates its ability to bind steroid (16,17) and supports the hypothesis that proteinase cleavage of CBG facilitates the targeted delivery of its anti-inflammatory steroids to sites of action (18).…”

“…Moreover, if the RCL in unliganded CBG is partially inserted, it could also be protected against accidental cleavage by proteinases, and this would ensure that biologically costly and irreversible proteolysis only occurs when CBG is loaded with steroid and its RCL is fully exposed. This would add a new dimension to the concept that CBG provides a means of targeting the delivery of anti-inflammatory steroids to site of inflammation (18), the importance of which has been elegantly demonstrated in mice with a targeted deletion of the cbg gene (50).…”

Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain

“…Thyroxin is transported in blood by transthyretin (pre-albumin) and thyroxin-binding globulin. The monomers in tetrameric transthyretin consist of two four-stranded β-sheets (Blake and Oatley, 1977), while sequence analyses classify thyroxin-binding globulin (Flink et al, 1986) and corticosteroid-binding globulin (Hammond et al, 1987) in the serine proteinase inhibitor (serpin) superfamily. Thus, with the exception of proteins of the serpin superfamily, which have a significant proportion of α-helical structures (Huber and Carrell, 1989), these structurally dissimilar plasma hormone-binding 506 proteins have a predominantly β secondary structure, as opposed to the almost entirely α-helical ligand-binding domains that characterize the intracellular hormone receptors (Tanenbaum et al, 1998).…”

Cellular uptake of steroid carrier proteins—Mechanisms and implications

“…Similarly, phenotypic characterization of mice lacking CBG revealed some surprising findings. CBG is the major transport protein for glucocorticoids in plasma of mammalian species with more than 90% of circulating corticosteroid molecules being bound by this carrier (Breuner and Orchinik, 2002;Hammond et al, 1987;Rosner, 1990). CBG is a 55 kDa monomeric glycoprotein that is mainly secreted by the liver, but is also produced in lung, kidney, and testis (Hammond et al, 1987).…”