Avian corticosteroid-binding globulin: biological function and regulatory mechanisms in physiological stress responses.
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ABSTRACT:
SUBMITTER: Lin HY
PROVIDER: S-EPMC8086359 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six N-glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are N-glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved N-glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one N-glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its N-glycan is removed. When expressed in N-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2-4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an N-glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the Pseudomonas aeruginosa protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, N-glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.
| S-EPMC5793714 | biostudies-literature
Project description:ObjectiveDiscrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG.Methods and subjectsMolecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects.ResultsWe demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients.ConclusionsPlasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.
| S-EPMC6686952 | biostudies-literature
Project description:Produced by the liver, corticosteroid-binding globulin (CBG) regulates the plasma distribution and actions of glucocorticoids. A sex difference in pituitary growth hormone secretion patterns established during puberty in rats results in increased hepatic CBG production and two-fold higher plasma corticosterone levels in females. Glucocorticoids control hepatic development and metabolic activities, and we have therefore examined how disrupting the SerpinA6 gene encoding CBG influences plasma corticosterone dynamics, as well as liver gene expression in male and female rats before and after puberty. Comparisons of corticosterone plasma clearance and hepatic uptake in adult rats, with or without CBG, indicated that CBG limits corticosterone clearance by reducing its hepatic uptake. Hepatic transcriptomic profiling revealed minor sex differences (207 differentially expressed genes) and minimal effect of CBG deficiency in 30-day-old rats before puberty. While liver transcriptomes in 60-day-old males lacking CBG remained essentially unchanged, 2,710 genes were differentially expressed in wild-type female versus male livers at this age. Importantly, ~10% of these genes lost their sexually dimorphic expression in adult females lacking CBG, including those related to cholesterol biosynthesis, inflammation, and lipid and amino acid catabolism. Another 203 genes were altered by the loss of CBG specifically in adult females, including those related to xenobiotic metabolism, circadian rhythm, and gluconeogenesis. Our findings reveal that CBG consolidates the sexual dimorphism of the rat liver initiated by sex differences in growth hormone secretion patterns and provide insight into how CBG deficiencies are linked to glucocorticoid-dependent diseases.
2023-11-03 | GSE245348 | GEO
Project description:Corticosteroid-binding globulin (CBG) transports glucocorticoids and progesterone in the blood and thereby modulates the tissue availability of these hormones. As a member of the serine protease inhibitor (SERPIN) family, CBG displays a reactive center loop (RCL) that is targeted by proteinases. Cleavage of the RCL is thought to trigger a SERPIN-typical stressed-to-relaxed (S-to-R) transition that leads to marked structural rearrangements and a reduced steroid-binding affinity. To characterize structure-function relationships in CBG we studied various conformational states of E. coli-produced rat and human CBG. In the 2.5 Å crystal structure of human CBG in complex with progesterone, the RCL is cleaved at a novel site that differs from the known human neutrophil elastase recognition site. Although the cleaved RCL segment is five residues longer than anticipated, it becomes an integral part of β-sheet A as a result of the S-to-R transition. The atomic interactions observed between progesterone and CBG explain the lower affinity of progesterone in comparison to corticosteroids. Surprisingly, CD measurements in combination with thermal unfolding experiments show that rat CBG fails to undergo an S-to-R transition upon proteolytic cleavage of the RCL hinting that the S-to-R transition observed in human CBG is not a prerequisite for CBG function in rat. This observation cautions against drawing general conclusions about molecular mechanisms by comparing and merging structural data from different species.
| S-EPMC3530532 | biostudies-literature
Project description:BackgroundPatients with adrenal insufficiency are treated with oral hydrocortisone (HC) to compensate for the loss of endogenous cortisol production. Intrinsic imperfections of cortisol replacement strategies in mimicking normal cortisol secretion are the underlying cause of the increased morbidity and mortality of patients suffering from secondary adrenal insufficiency (SAI). To improve oral hydrocortisone substitution therapy, a better understanding of its pharmacokinetics (PK) is necessary. The previous PK model did not include protein binding. It is known that protein binding can impact hydrocortisone pharmacokinetics. The aim of this study is to describe HC pharmacokinetics including the protein-binding state using Edsim++ (Mediware, Prague) pharmacokinetic modeling software, paving the way for an in-silico tool suitable for drug delivery design.MethodsA total of 46 patients with SAI participated in a randomized double-blind crossover study Patients randomly received a low dose of HC (0.2-0.3 mg/kg body weight/day) for 10 weeks, followed by a high dose (0.4-0.6 mg/kg body weight/day) for another 10 weeks, or vice versa. Plasma samples were obtained and analyzed for free and total hydrocortisone. Single compartment population pharmacokinetic analysis was performed using an extended Werumeus-Buning model built in Edsim++. This model includes a mathematical approach for estimating free cortisol by Nguyen et al., taking the protein binding of HC to albumin and hydrocortisone-binding globulin (CBG, transcortin) into consideration, as well as different states of CBG which affect binding kinetics to HC. The goodness of fit for observed versus predicted values was calculated.Results and conclusionsNguyen's formula for free cortisol estimation was successfully implemented in a pharmacokinetic model. The model shows high Spearman's correlation for observed versus predicted hydrocortisone concentrations. Significantly higher correlations (Spearman's r, 0.901 vs. 0.836) between total and free hydrocortisone AUC24 (area-under the curve over 24 h) are found when comparing new and old models. This new model was used to simulate the plasma concentration-time behavior of a more suitable hydrocortisone formulation.
| S-EPMC9231005 | biostudies-literature
Project description:Human corticosteroid-binding globulin (CBG), a heavily glycosylated protein containing six N-linked glycosylation sites, transports cortisol and other corticosteroids in blood circulation. Here, we investigate the biological importance of the N-glycans of CBG derived from human serum by performing a structural and functional characterization of CBG N-glycosylation. Liquid chromatography-tandem MS-based glycoproteomics and glycomics combined with exoglycosidase treatment revealed 26 complex type N-glycoforms, all of which were terminated with α2,3-linked neuraminic acid (NeuAc) residues. The CBG N-glycans showed predominantly bi- and tri-antennary branching, but higher branching was also observed. N-glycans from all six N-glycosylation sites were identified with high site occupancies (70.5-99.5%) and glycoforms from all sites contained a relatively low degree of core-fucosylation (0-34.9%). CBG showed site-specific glycosylation and the site-to-site differences in core-fucosylation and branching could be in silico correlated with the accessibility to the individual glycosylation sites on the maturely folded protein. Deglycosylated and desialylated CBG analogs were generated to investigate the biological importance of CBG N-glycans. As a functional assay, MCF-7 cells were challenged with native and glycan-modified CBG and the amount of cAMP, which is produced as a quantitative response upon CBG binding to its cell surface receptor, was used to evaluate the CBG:receptor interaction. The removal of both CBG N-glycans and NeuAc residues increased the production of cAMP significantly. This confirms that N-glycans are involved in the CBG:receptor interaction and indicates that the modulation is performed by steric and/or electrostatic means through the terminal NeuAc residues.
| S-EPMC3149095 | biostudies-literature
Project description:Corticosteroid-binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG steroid-binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only ?40% identity, and their steroid-binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG steroid-binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its steroid-binding affinity, but we present evidence that it may participate in protein folding and steroid-binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct steroid-binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts steroid binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex steroids in these species.
| S-EPMC4900275 | biostudies-literature
Project description:Predation is a key organizing force in ecosystems. The threat of predation may act to programme the endocrine hypothalamic-pituitary-adrenal axis during development to prepare offspring for the environment they are likely to encounter. Such effects are typically investigated through the measurement of corticosteroids (Cort). Corticosteroid-binding globulin (CBG) plays a key role in regulating the bioavailability of Cort, with only free unbound Cort being biologically active. We investigated the effects of prenatal predator odour exposure (POE) in mice on offspring CBG and its impact on Cort dynamics before, during and after restraint stress in adulthood. POE males, but not females, had significantly higher serum CBG at baseline and during restraint and lower circulating levels of Free Cort. Restraint stress was associated with reduced liver transcript abundance of SerpinA6 (CBG-encoding gene) only in control males. POE did not affect SerpinA6 promoter DNA methylation. Our results indicate that prenatal exposure to a natural stressor led to increased CBG levels, decreased per cent of Free Cort relative to total and inhibited restraint stress-induced downregulation of CBG transcription. These changes suggest an adaptive response to a high predator risk environment in males but not females that could buffer male offspring from chronic Cort exposure.
| S-EPMC8634628 | biostudies-literature
Project description:Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.
| S-EPMC6347282 | biostudies-literature
Project description:Recent studies of corticosteroid-binding globulin (CBG) indicate that it does not merely transport cortisol passively but also actively regulates its release in the circulation. We show how CBG binding affinity can vary to give changes in free cortisol concentration in a physiologically relevant range.The objective was to determine how the binding affinity of plasma CBG is affected by glycosylation, changes in body temperature, and the conformational change induced by proteases at sites of inflammation.Binding assays were performed over a range of temperatures with plasma and recombinant CBG to determine the contribution of glycosylation. The role of conformational change was assessed by measuring binding affinities of plasma CBG before and after reactive loop cleavage by neutrophil elastase.Determination of binding constants allows calculation of clinically relevant changes in CBG saturation and free cortisol concentrations.On reactive loop cleavage at inflammation sites, CBG can continue to act as a buffered source of cortisol, although with a much reduced affinity, to give a potential quadrupling of free cortisol. Predicted increases in systemic free cortisol resulting from elevated body temperatures, previously reported based on affinity measurements using nonglycosylated recombinant CBG, were shown here to be considerably increased using glycosylated plasma CBG, with a doubling for every 2°C rise in body temperature.The ability of CBG to modulate free cortisol levels in blood must be considered in the understanding and management of disease processes, as illustrated here with predictable changes in inflammation and fever.
| S-EPMC3813945 | biostudies-literature