Corticosteroid binding globulin, testosterone-estradiol binding globulin, and androgen binding protein belong to protein families distinct from steroid receptors

The cDNA nucleotide sequences and the deduced amino acid sequences of human corticosteroid binding globulin (hCBG), human testosterone-estradiol binding globulin (hTeBG), and rat androgen binding protein (rABP) were determined. Studies of the steroid binding sites suggest they are toward the carboxy-terminus in hTeBG and rABP and more central in hCBG. hCBG has remarkable sequence homology with members of a superfamily whose functions have diverged; these include thyroxine-binding protein, serine protease inhibitors, egg white proteins, and angiotensinogen. hTeBG and rABP have a 68% amino acid sequence identity. Hybridization studies suggest that hTeBG is probably even more closely related, if not identical, to hABP. The carboxy-terminal sequences of hTeBG and rABP are also similar to that of protein S, a vitamin-K-dependent clotting factor. There were no nucleotide or amino acid sequence homologies between hCBG, hTeBG, or rABP and other steroid binding proteins such as steroid receptors, albumin, alpha-fetoprotein, and vitamin D binding protein. We conclude that the "extracellular steroid binding proteins" and steroid receptors do not appear to have descended from a common ancestor.     

Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.     

Physical mapping of four serpin genes: alpha 1-antitrypsin, alpha 1-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome I4q32.1.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.     

Physical and genetic mapping of the serpin gene cluster at 14q32.1: allelic association and a unique haplotype associated with alpha 1-antitrypsin deficiency.

The alpha 1-antitrypsin (PI) gene is part of a cluster of structurally related serine protease inhibitor genes localized at chromosome 14q32.1, a cluster that includes the alpha 1-antichymotrypsin (AACT), protein C inhibitor (PCI), and corticosteroid-binding globulin (CBG) genes and the alpha 1-antitrypsin-like pseudogene (PIL). The order of the genes is refined here by genetic mapping using simple tandem repeat polymorphisms (STRPs) and by physical mapping in YACs. The order of the genes is (centromere)-CBG-PIL-PI-PCI-AACT-(telomere). Analysis of DNA haplotypes comprising STRP and RFLP markers in the serpin genes reveals considerable allelic association throughout the cluster. Furthermore, the common alpha 1-antitrypsin deficiency allele, PI*Z, has a unique DNA haplotype at the CBG, PIL, and PI loci, which extends over 60 kb in 97% of cases and in 44% of cases includes the PCI and AACT loci. This unique haplotype will be of use in examining a number of other diseases, particularly those with an inflammatory component, thought to be associated with alpha 1-antitrypsin deficiency or partial deficiency.     

Molecular evolution of serpins: homologous structure of the human alpha 1-antichymotrypsin and alpha 1-antitrypsin genes

alpha 1-Antichymotrypsin belongs to a supergene family that includes alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal alpha 1-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the alpha 1-antichymotrypsin gene are identical with those of the human alpha 1-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the alpha 1-antichymotrypsin and alpha 1-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggests that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.     

An amino acid substitution in biobreeding rat corticosteroid binding globulin results in reduced steroid binding affinity

BioBreeding (BB) rats are derived from an outbred colony of Wistar rats and are used as a model of autoimmune diabetes mellitus. A corticosteroid binding globulin (CBG) variant with reduced affinity for glucocorticoids has now been found in the blood of these animals. The dissociation rate constants of BB CBG for cortisol (4.42 nM) and corticosterone (1.43 nM) are both about 50% higher than those associated with Wistar CBG, but no obvious difference in the steroid binding specificity of BB and Wistar CBGs was detected. Purified BB and Wistar CBGs exhibit the same size heterogeneity when examined by polyacrylamide gel electrophoresis under denaturing conditions, and the sizes of their respective hepatic mRNAs are identical. The genetic basis for this abnormality was therefore determined by comparing the cDNA sequences for BB and Wistar CBG, and this revealed a point mutation that results in a single amino acid substitution at residue 276 (Ile in BB CBG and Met in Wistar CBG). To confirm that this mutation is responsible for the reduced steroid binding affinity associated with BB CBG, the cDNAs for rat CBG-Ile276 and CBG-Met276 were expressed in Chinese hamster ovary cells. The steroid binding affinities of the CBGs secreted by these cells were essentially identical with those observed in the corresponding serum samples from these two rat strains. The amino acid substitution identified in BB rat CBG therefore clearly accounts for the reduction in its steroid binding affinity, and further analysis of this and other natural CBG variants may reveal important information about the CBG steroid binding site. It is also possible that this mutation may contribute to the etiology of pathological abnormalities that are characteristic of the BB rat.     

Homology model of thyroxine binding globulin and elucidation of the thyroid hormone binding site.

The tertiary structure of thyroxine binding globulin (TBG) has been modelled on the basis of its close homology to alpha 1-antitrypsin, the archetype of the serine protease inhibitor (serpin) superfamily. Energy minimization was applied to the model to refine the structure further. The putative thyroid hormone binding region suggested in previous labelling studies was found to exist within a beta-barrel structure of complementary dimensions to the thyroid hormones. The model also revealed that the binding cleft provides the hydrophobic environment and specific ionic interaction sites deemed important for thyroid hormone binding. The model is in good agreement with evidence derived from previously reported T3 and T4 binding, stability and isoelectric focussing studies of TBG and TBG variants. Finally, T4 analogue and drug binding studies have enabled us to postulate the orientation and manner of hormone binding to TBG. This may prove to be of assistance in the development of potent and specific, non-thyroidal ligands and also aid in the understanding of physiological thyroid hormone binding interactions.     

Corticosteroid-binding globulin, a structural basis for steroid transport and proteinase-triggered release

Corticosteroid-binding globulin (CBG) is a serine proteinase inhibitor (serpin) family member that transports glucocorticoids in blood and regulates their access to target cells. The 1.9A crystal structure of rat CBG shows that its steroid-binding site resembles the thyroxin-binding site in the related serpin, thyroxin-binding globulin, and mutagenesis studies have confirmed the contributions of key residues that constitute the steroid-binding pocket. Unlike thyroxin-bound thyroxin-binding globulin, the cortisol-bound CBG displays an "active" serpin conformation with the proteinase-sensitive, reactive center loop (RCL) fully expelled from the regulatory beta-sheet A. Moreover, the CBG structure allows us to predict that complete insertion of the proteolytically cleaved RCL into the serpin fold occurs in concert with a displacement and unwinding of helix D that would disrupt the steroid-binding site. This allosteric coupling between RCL positioning and occupancy of the CBG steroid-binding site, which resembles the ligand (glycosamino-glycan)-dependent activation of the thrombin inhibitory serpins heparin cofactor II and anti-thrombin RCLs, ensures both optimal recognition of CBG by target proteinases and efficient release of steroid to sites of action.     

Characteristics of aldosterone binding in rat and human serum

Binding of cortisol and corticosterone by serum proteins is well established, but discrepancies exist regarding aldosterone. We have observed that approximately 1% of 3H-aldosterone incubated with rat serum was bound in a time-dependent process, although it was not competed by a large excess of non-radioactive aldosterone, assessed by Florisil separation or gel filtration on Sephadex G-50 columns. After electrophoresis on cellulose acetate of rat serum incubated with 3H-aldosterone, specific or non-specific binding to protein fractions was not obtained. Further, a 10 000-fold molar excess of aldosterone (10 microM) displaced only 34% of the bound 3H-aldosterone to rat serum, preventing the calculation of the IC50 value. Increasing concentrations of aldosterone (3-83 nM) did not displace 3H-corticosterone bound in rat serum to presumably corticosterone binding globulin (CBG). In contrast, inhibition of this binding by 3-83 nM corticosterone was concentration dependent, showing an IC50 value of 10(-8) M. In normal human serum, binding of 3H-aldosterone demonstrated competition by a 100 and 1 000-fold excess of aldosterone. Displacement curves of 3H corticosterone bound to human serum by 1.7-75 nM corticosterone or 0.05-8.8 microM aldosterone yielded IC50 values in the range of 10(-8) M for corticosterone and 10(-6) M for aldosterone. With horse serum, aldosterone's binding affinity was three orders of magnitude lower than that of corticosterone. These studies suggest that in the rat aldosterone was loosely and weakly bound to a high capacity binder, possibly albumin. In agreement with the work of others, in humans aldosterone may be bound to both CBG and albumin. The current data do not substantiate for the presence of specific aldosterone binding proteins in serum.     

Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase

AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.     

A Leu----His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol.

A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An approximately 50% discordancy between the two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu----His substitution at residue 93 and another which encodes a Ser----Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation (Kd = 2.24 +/- 1.75 nM) reduced the cortisol binding affinity of CBG (mean +/- SD) significantly (P less than 0.024) when compared to normal CBG (Kd = 0.64 +/- 0.31 nM), while the Ala224 mutation (Kd = 0.63 +/- 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.     

Functional implication of an Arg307Gly substitution in corticosteroid-binding globulin, a candidate gene for a quantitative trait locus associated with cortisol variability and obesity in pig

We previously reported that corticosteroid-binding globulin gene (Cbg) may be the causal gene of a quantitative trait locus associated with cortisol levels, fat deposition, and muscle content in a pig intercross. Sequence analysis of parental animals allowed us to identify four amino-acid substitutions. Here we have examined if any of these single amino acid substitutions could be responsible for the difference in CBG binding and affinity for cortisol between the parental breeds, using in vitro assays of Cbg variants after transfection of mammalian cells. Additionally, the Cbg coding region was analyzed in samples from a synthetic pig line to study association between polymorphism and CBG biochemical properties, carcass composition, and meat quality. Both in vitro transfection assays and the association studies suggest a role of the Arg307Gly mutation in increasing CBG capacity (by >70%) and decreasing CBG affinity for cortisol (by 30%). The Ile265Val substitution may also have an effect on decreasing CBG affinity for cortisol by 25%. The mutations Ser15Ile and Thr257Met do not seem to have an effect on CBG parameters. The Arg307Gly substitution was the only mutation associated with a parameter of meat quality and no mutation was linked to carcass composition.     

Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin.

The structure of cleaved thyroxine-binding globulin (TBG) has been modelled on the crystal structure of cleaved alpha 1-antitrypsin (a member of the serine proteinase inhibitor, serpin, superfamily) based on the high sequence homology exhibited by the two proteins. Particular attention was paid to the identification and modelled characteristics of the thyroxine binding site. The primary aim of the study was to compare the site qualitatively with the crystallographically determined binding site of transthyretin, the other major transporter of thyroxine, in an attempt to explain the higher binding affinity of the site compared with the known thyroxine binding site in transthyretin (10(10) versus 10(8) M-1). The proposed binding site shares some similar characteristics with the transthyretin binding site but also includes a cluster of aromatic residues which are entirely absent in transthyretin. It is proposed that this might account for the substantial difference in binding affinities.     

Effects of neurotrophic stress on maternal metabolism and binding of plasma cortisol in late pregnant guinea-pigs and their fetuses

The maternal metabolic clearance rate (MCR) and the binding of cortisol in the plasma of fetus and mother were estimated 8 days before term in conscious pregnant guinea-pigs, control or subjected to a neurotrophic stress (they were immobilized for 3 h in a dark room in front of an intermittent luminous flash). The maternal MCR of cortisol dropped during pregnancy related to an increase in corticosteroid binding globulin (CBG) and the stress mimicked these changes with a decrease in MCR and a rise in CBG. The increase in cortisol concentrations occurring in the fetal plasma after maternal stress could be related to the increase in free cortisol in the stressed mother. Nevertheless, although the fetal CBG did not rise after maternal stress, the increase in free cortisol in the fetal plasma remained moderate, due to the numerous free sites of CBG and albumin for cortisol.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

A critical region in the mineralocorticoid receptor for aldosterone binding and activation by cortisol: evidence for a common mechanism governing ligand binding specificity in steroid hormone receptors

The amino acids that confer aldosterone binding specificity to the mineralocorticoid receptor (MR) remain to be determined. We had previously analyzed a panel of chimeras created between the MR and the glucocorticoid receptor and determined that amino acids 804-874 of the MR ligand binding domain are critical for aldosterone binding. In the present study a further series of chimeras was created within this region. The chimeras were analyzed by a transactivation assay and [(3)H]aldosterone binding, and the critical region was narrowed down to amino acids 820-844. Site-directed mutagenesis was used to create single and multiple amino acid substitutions in this region. These studies identified 12 of the 16 amino acids that differ in the MR and the glucocorticoid receptor in this region as being critical to conferring aldosterone responsivity. The amino acids that differ in the region 820-844 lie on the surface of the molecule and, therefore, it appears that MR ligand binding selectivity is conferred by residues that do not form part of the ligand binding pocket. Other studies have found that the corresponding regions of the androgen and glucocorticoid receptors are critical for the binding of natural and synthetic ligands, suggesting a common mechanism governing ligand binding specificity. The new chimeras also displayed, as previously reported, a dissociation between cortisol binding and transactivation and, intriguingly, only those that bound aldosterone with high affinity were activated by cortisol, suggesting a common mechanism that underlies specificity of aldosterone binding and the ability of cortisol to activate the MR.     

The development of corticosteroid binding globulin-like activity in fetal sheep blood.

Parturition in sheep is initiated by the fetus and is preceded by a rise in fetal cortisol and corticosteroid-binding globulin (CBG) late in gestation. In this study plasma cortisol and CBG concentrations were measured in fetal and maternal circulation from 40 days gestation to early post-partum. The fetal cortisol profile was shown to be triphasic in nature; being high in both the first and last trimester but low in the middle period of gestation. In the last trimester, total cortisol increased steadily, reaching it's highest level just prior to parturition (145 days gestation), before falling to maternal levels over the first 10 days post-partum. The changes seen in CBG concentrations throughout gestation and post-partum mirrored the triphasic nature seen in cortisol levels. CBG was significantly higher at 40, 56 and 140 days gestation than at mid-gestation (77 and 90 days). However, at 145 days gestation there was a significant fall in CBG levels. CBG levels were higher at 1 day post-partum when compared to 145 days gestation, the former rapidly falling to maternal levels over the subsequent 9 days. The maximum binding capacity at 40, 56, 70 and 90 days gestation exceeds the total serum cortisol concentration. However at 140 and 145 days gestation and 1 day post-partum the total serum cortisol exceeds the Bmax. The highest cortisol:Bmax ratio is seen at 145 days gestation due to the fall of CBG binding capacity at this time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticosteroid binding globulin: a new target for cortisol-driven obesity

We present data suggesting that corticosteroid-binding globulin (CBG) may be the causal gene of a previously identified quantitative trait locus (QTL) associated with cortisol levels, fat, and muscle content in a pig intercross. Because Cbg in human and mouse maps in the region orthologous to the pig region containing this QTL, we considered Cbg as an interesting positional candidate gene because CBG plays a major role in cortisol bioavailability. Firstly, we cloned pig Cbg from a bacterial artificial chromosome library and showed by fluorescent in situ hybridization and radiation hybrid mapping that it maps on 7q26 at the peak of the QTL interval. Secondly, we detected in a subset of the pig intercross progeny a highly significant genetic linkage between CBG plasma binding capacity values and the chromosome 7 markers flanking the cortisol-associated QTL. In this population, CBG capacity is correlated positively to fat and negatively to muscle content. Thirdly, CBG capacity was three times higher in Meishan compared with Large White parental breeds and a 7-fold difference was found in Cbg mRNA expression between the two breeds. Overall, the data accumulated in this study point to Cbg gene as a key regulator of cortisol levels and obesity susceptibility.