Electron spin resonance study of human transcortin thiol groups and binding site topography

Defaye, G.; Basset, M.; Monnier, Nicole; Chambaz, E.M.

doi:10.1016/0005-2795(80)90256-1

Cited by 28 publications

(16 citation statements)

References 26 publications

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“…We also observed only two cysteine residues in the sequence of the mature form of CBG, and this agrees with previous estimates (9)(10)(11). These cysteines do not appear to be linked (9), and one of them is located within a hydrophobic pocket that contains the steroid binding site (8,9).…”

Section: Discussionsupporting

confidence: 79%

“…These cysteines do not appear to be linked (9), and one of them is located within a hydrophobic pocket that contains the steroid binding site (8,9). Analyses of hydropathy plots (36) and secondary structure predictions (37) indicate that both cysteines are likely to be located as the third residue in tetrapeptide P-turns that are both surrounded by relatively hydrophobic regions of the molecule.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Hammond¹,

Smith²,

Goping³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

225

122

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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...

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Hammond¹,

Smith²,

Goping³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

225

122

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“…Experiments using the technique of electron spin resonance were used in the determination of several salient characteristics of the binding site of CBG (9). Affinity labeling experiments, with 6b-bromoprogesterone, established the importance of one of the two cysteinyl residues in the binding site (10).…”

Section: Introductionmentioning

confidence: 99%

Modeling of Human Corticosteroid Binding Globulin. Use of Structure–Activity Relations in Soft Steroid Binding to Refine the Structure

Little

Rodríguez

2005

Pharm Res

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“…The interaction of hCBG with cortisol protects one of its two cysteine residues from chemical modification (11) and also quenches the fluorescence of one or more of its four tryptophans (12). Notably, Cys-228 and the four tryptophans are conserved between human, rat, and rabbit CBG sequences (3,13,14), suggesting that they have functional significance; there have been no reports, however, of site-directed mutants in which steroid binding is altered.…”

Section: Introductionmentioning

confidence: 99%

Expression of biologically active human corticosteroid binding globulin by insect cells: acquisition of function requires glycosylation and transport.

Ghose-Dastidar

Ross

Green

1991

Proc. Natl. Acad. Sci. U.S.A.

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Human corticosteroid binding globulin (hCBG) is a 50. to 55-kDa serum glycoprotein that binds cortisol and progesterone with high affinity. To map the steroid-binding domain and to investigate the folding pathways of hCBG, we have established an expression system based on infection of insect cells with a recombinant baculovirus encoding hCBG. Infected Spodoptera frugiperda (Sf9) cells secrete iminunoreactive hCBG at high levels (16-24 pmol per 106 cells per 40 h), and the recombinant protein binds cortisol with an affinity and specificity equivalent to that of human serumderived hCBG. Thus, this system has the potential to provide large amounts of wild-type and mutant hCBGs for physicalchemical analysis. Cotranslational asparagine-linked glycosylation is essential for acquisition ofsteroid-binding capability, as shown by the lack of cortisol-binding activity of unglycosylated hCBG secreted in the presence of tunicamycin. Golgiassociated oligosaccharide processing, however, is not required for activity, as demonstrated by the endoglycosidase H susceptibility of the fully active, secreted glycoprotein. Comparison of the steroid-binding properties of intracellular and secreted hCBG with that synthesized in vitro in the rabbit reticulocyte lysate system suggests that this protein undergoes a maturation process during transport through the secretory pathway. This system will be useful for identifying the molecular determinants of biological function in hCBG.The steroid-binding globulins are excellent model systems for studying how protein structure and conformational dynamics determine function. The diverse biological activities proposed for these serum proteins suggest that they contain multiple functional domains. Their high-affinity ligand binding provides a sensitive indicator of proper tertiary structure, facilitating the analysis of protein folding in vivo and in vitro. Furthermore, the postsynthetic transport of these glycoproteins through endoplasmic reticulum (ER) and Golgi compartments raises the possibility of identifying intracellular folding intermediates. To incorporate biophysical and cell biological approaches, we have established a system for high-level expression of human corticosteroid binding globulin (hCBG), and we report here our first correlations of structure and function in this polypeptide.In humans, 80-90% of the cortisol present in serum is bound with high affinity and specificity to hCBG (1, 2). hCBG is an acidic glycoprotein synthesized primarily in liver and kidney, and it is secreted into the circulation as a 50-to 55-kDa monomer containing a single steroid-binding site (2). Its cDNA sequence predicts a 405-amino acid protein, which includes a 22-residue amino-terminal signal peptide and six consensus sites for asparagine-linked glycosylation; overall, hCBG shares significant homology with members of the serpin (serine protease inhibitor) family of protease inhibitors (3).A generally accepted function of hCBG is the maintenance of an appropriate balance of free and protein-boun...

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Electron spin resonance study of human transcortin thiol groups and binding site topography

Cited by 28 publications

References 26 publications

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

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

Modeling of Human Corticosteroid Binding Globulin. Use of Structure–Activity Relations in Soft Steroid Binding to Refine the Structure

Expression of biologically active human corticosteroid binding globulin by insect cells: acquisition of function requires glycosylation and transport.

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