Rat corticosteroid-binding globulin (CBG) biosynthesis by fetal hepatocytes in culture

The ability of fetal liver to synthesize and secrete corticosteroid-binding globulin (CBG) was investigated using primary cultures of hepatocytes transplanted from 15- and 18-day-old rat fetuses. Analysis of culture medium, after labelling with [14C]leucine, by crossed immunoelectrophoresis, clearly demonstrated CBG, albumin and alpha-fetoprotein (AFP) secretion. The relative rate of CBG secretion was more important at the younger stage. Indirect immunofluorescence permitted localization of CBG in fetal hepatocytes. The results suggest that the high level of CBG found in fetal serum is mainly produced by the liver of the fetus itself.     

皮质醇结合球蛋白的生理功能及其编码基因突变

皮质醇结合球蛋白（CBG）对皮质醇具有高亲和力,在调节血液中的皮质醇水平,转运孕酮,参与炎症反应等过程发挥作用。目前已发现的影响CBG的基因突变或单核苷酸多态性有9种,往往影响CBG水平或是CBG与皮质醇的结合能力,CBG缺陷先证者多表现为慢性疲乏,往往伴随着低血压、高血压或者是肥胖,但并不是所有CBG缺陷患者均有上述症状,由于病例报道数量较少,突变与症状的相关性很难确定,仍需更多针对CBG的遗传学研究。     

Corticosteroid-binding globulin (CBG) in fetal development

In fetal sheep the prepartum increase in plasma cortisol concentration is associated with an increase in high affinity corticosteroid binding activity in plasma. This appears to reflect an increase in corticosteroid-binding globulin (CBG) biosynthesis from the fetal liver, and evidence is presented that hepatic CBG gene expression is increased by exposure to glucocorticoids in the fetus. Immunoreactive CBG is found in other fetal tissues, and CBG mRNA is present in fetal pituitary. CBG reduces the ability of cortisol to exert negative feedback on basal or CRH-stimulated ACTH output by fetal sheep pituitary cells in culture. We suggest that CBG interacts with cortisol in a manner that maintains a low negative feedback on the pituitary, and perhaps hypothalamus. This constitutes a component of the cascade of events that is associated with hypothalamic-pituitary-adrenal activation in the late gestation fetus, and with the onset of parturition.     

Progesterone measurement using "stripped" corticosteroid binding globulin (CBG)

Modifications in an existing competitive protein binding assay for progesterone have been made which provide a readily available and rich source of binding sites on the corticosteroid binding globulin (CBG). The primary and most important modification is the rapid removal of endogenous steroids from plasma by gel filtration at an elevated temperature. The ‘stripped’ protein retains full CBG activity, but is cleared of 95% of its endogenous steroids. This stripping procedure provides not only increased number of binding sites, but in conjunction with the other modifications also eliminates some of the variability in the assay.     

Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques

Temporal relationships between concentrations of sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), total and free estradiol, total and free testosterone, cortisol, and progesterone were studied in plasma obtained at 1- to 3-day intervals throughout gestation in six rhesus macaques. Concentrations of SBP and CBG were measured by diethylaminoethyl cellulose filter assays. Total and free steroids were estimated by radioimmunoassay and ultrafiltration dialysis, respectively. We found that SBP was elevated between days 30 and 50 and CBG between days 60 and 140; both then declined until term (167 days). Estradiol increased gradually throughout gestation. Testosterone was elevated between days 10 and 40, then declined, and rose slightly in late gestation until approximately 15 days before delivery, when it increased markedly. Free estradiol and testosterone increased dramatically before parturition. Progesterone was elevated between days 25 and 45 and declined to relatively constant levels thereafter. Cortisol was essentially unchanged throughout gestation. Our data show that in the pregnant rhesus, levels of SBP and CBG vary independently of one another, but both decline before term; concentrations of both total and free estradiol and testosterone increase markedly before parturition; in late gestation, elevated estrogen is not associated with increased levels of SBP or CBG (as it is in human females).     

An enzyme-linked immunosorbent assay (ELISA) for human corticosteroid-binding globulin

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for human corticosteroid-binding globulin was developed. A polyclonal rabbit anti-CBG antibody is immobilised to a microtitre plate. Following incubation of standards and samples a second monospecific rabbit anti-CBG antibody, labelled with alkaline phosphatase, is added. After colour development the microtitre plate is read at 405 nm wavelength. The assay shows good agreement to CBG binding capacity assay and commercially available RIA.     

Plasma variation of corticosteroid-binding globulin and sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) circulate in plasma and bind their cognate ligands with high affinity, offering a steroid delivery system to target tissues by a variety of mechanisms. Analysis of these steroid-binding proteins is gaining importance in the clinical setting, although more information is warranted on their diurnal and biological variation. This study shows that plasma SHBG (in normal subjects) exhibits little diurnal or biological variation over the 30 day period studied, in contrast to CBG, where plasma levels peak in the early afternoon. This leads to attenuation of the diurnal free cortisol level rhythm compared to total cortisol. We also show that plasma CBG is significantly lower in male subjects with the metabolic syndrome compared to age-matched lean counterparts, and may therefore act as a surrogate marker of insulin resistance. The consequence of lower levels of CBG in these obese male subjects is reflected by higher levels of circulating free cortisol, potentially offering a more favourable environment for adipogenesis.     

Preparative high-performance liquid partition chromatography of proteins.

Methods for preparative high-performance liquid chromatography (hplc) of proteins are described. Both normal and reverse-phase chromatography were studied and adapted to the fractionation of proteins in quantities of up to 50 mg. Lichrosorb Diol was used as a “normal phase” for chromatography of hydrophobic proteins. Lichrosorb RP-8 was used for reversephase chromatography of proteins.     

N-glycans modulate the function of human corticosteroid-binding globulin

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.     

Concentration decrease of corticosteroid binding globulin (CBG) in plasma of the mare throughout pregnancy

A significant decrease of CBG binding capacity in plasma of the mare throughout pregnancy was demonstrated using equilibrium dialysis and gel equilibration methods. As indicated with immunoelectrophoresis experiments, the pregnancy related fall of CBG binding capacity was linked to an actual decrease in blood CBG concentration. This result contrasts sharply with data on most other mammalian species, with the exception of the gestating rhesus monkey.     

Regulation of plasma corticosteroid-binding globulin in adult cynomolgus monkey (Macaca fascicularis) during different reproductive states

The plasma concentration of the corticosteroid-binding globulin (mCBG) has been measured in Macaca fascicularis, during different stages of reproduction and under hormonal treatments. The mCBG level was determined by a specific electroimmunoassay. There was no difference between females in the follicular phase and intact males; mCBG concentrations were respectively (mean +/- SEM) 469 +/- 53 and 443 +/- 25.6 nmol/l. The mCBG levels levels were similar during both the luteal (469 +/- 33.5 nmol/l) and the follicular phase (469 +/- 53 nmol/l). Compared to intact males, the mCBG levels were higher (P less than 0.05) in castrated males (527 +/- 6.6 nmol/l). During gestation, no systematic variations were found and the mCBG levels were not statistically different from the values found during the follicular phase. When estradiol benzoate was administered to castrated animals, the mCBG concentrations increased rapidly. In contrast, the values were reduced slightly by testosterone treatment. The sex-steroid action on the mCBG levels was discussed and compared with the mSBP levels. We question also, the mechanisms involved in the regulation of the mCBG levels during pregnancy.     

Preparation of milligrams of pure enantiomers using analytical-scale high-performance liquid chromatography

Pure enantiomers of an agrochemical process intermediate, (RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)-pentan-3-one ( 1 ), have been prepared on the milligram scale under overload chromatographic conditions on an analytical chiral column (250 × 4.6 mm i.d.). The effects of variation of temperature and mobile phase composition on retention factor, separation factor, and peak resolution have been investigated. Effects of flow rate, enantiomer ratio, sample concentration, and column load on productivity are also studied. Seven milligrams of the less retained (+)-enantiomer and 5 mg of the (?)-enantiomer were obtained from a single injection of 21 mg of (RS)- 1 . © 1994 Wiley-Liss, Inc.     

A new role for corticosteroid binding globulin (CBG), member of SERPIN superfamily]

Recent advances in molecular endocrinology have shed a new light on the role and mode of action of CBG. It is now not only demonstrated that this plasma glycoprotein, a steroid carrier, can be internalized by glucocorticoid target tissues, but it is also certain that CBG mRNA is synthesized by extra-hepatic tissues. Moreover, some authors have reported a modulation of CBG properties by free fatty acids. The existence of CBG receptors (or high affinity membrane-binding sites), and even a positive effect of CBG on adenylate cyclase activity, have also been reported. To progress in the understanding of these diverse results, one must first integrate them in a general scheme where it is considered that CBG is a member of the SERPIN (SERine Protease INhibitors) superfamily. In the case of CBG, that means a protein which functions as a substrate for elastase at the surface of neutrophils, for instance at sites of inflammation. CBG is specifically cleaved by this protease at a precise site close to its carboxy-terminus. This induces a conformation change and disrupts the binding between glucocorticoids and CBG, and promotes a significant and local release of glucocorticoids (over 90% of them are bound to CBG in human plasma). In this context, CBG directs glucocorticoids to sites of inflammation, and plays in consequence a crucial role in efficient glucocorticoid action in physiology. The elucidation of the CBG sequence, the knowledge of its gene structure, and the discovery of its chromosomal localization near two other SERPIN genes, are three sets of data in concordance to demonstrate that CBG is a SERPIN; and this has allowed the understanding of a new role for CBG, possibly with important consequences in pathology. Moreover, it could be more appropriate to say that CBG is a member of the SERine Protease INhibitors and Substrates superfamily (SERPINS).     

Purification of monoclonal antibodies using high performance liquid chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.     

Structure and function of corticosteroid-binding globulin: Role of carbohydrates

To study the site-specificity of human corticosteroid-binding globulin (CBG) glycosylation and the functional significance of individual carbohydrate chains in its molecule, a panel of recombinant CBG mutants containing each of the six potential glycosylation sites alone and in various combinations has been expressed in Chinese hamster ovary (CHO) cells. Analyses of these mutant glycoproteins showed that three of the glycosylation sites are only partially utilized, and this may contribute to the production of glycoforms with distinct physiological functions. Processing of individual carbohydrate chains (branching and fucosylation) is site-specific and may, thus, account for the formation of structural determinants essential for the recognition of CBG by cell membranes. Glycosylation at the only phylogenetically conserved consensus site, Asn²³⁸-Gly²³⁹-Thr²⁴⁰, is essential for the biosynthesis of CBG with steroid-binding activity. Evidence has been obtained to support the hypothesis that transient carbohydrate-polypeptide interactions between Trp²⁶⁶ and the maturing carbohydrate chain at Asn²³⁸ occur during early stages of the CBG biosynthesis which affect protein folding and formation of the steroid-binding site. Another tryptophan residue, Trp³⁷¹, has been found to be critical for CBG-steroid interactions and is likely located in the steroid-binding site.