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

Abstract

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...

Functional implications of corticosteroid-binding globulin N-glycosylation

“…Human CBG produced in insect Sf9 cells that produce only oligosaccharides of the high-mannose type bound cortisol with high affinity, while unglycosylated CBG produced in Sf9 cells in the presence of tunicamycin was inactive ( Ghose-Dastidar et al . 1991 ).…”

Aberrant trafficking of human melanocortin 1 receptor variants associated with red hair and skin cancer: Steady‐state retention of mutant forms in the proximal golgi

“…This suggests that oligosaccharide processing in the Golgi is not essential for MC1R traffic and activity. Although this is not a common situation, it has been found for a few proteins of the secretory pathway, such as the human corticosteroid binding globulin (Ghose-Dastidar et al, 1991). …”

N-Glycans Modulate the Function of Human Corticosteroid-Binding Globulin

“…Interestingly, this variant also shows substantially higher binding to other membrane preparations compared with normal CBG, indicating the presence of multiple CBG receptors with different binding preferences on some cell surfaces (22,23). In another study, it was shown that CBG N-glycans are essential for the biosynthesis of CBG that has steroid-binding activity (24). Later it was specified that it is the Asn 238 N-glycans (glycosylation site 4) that are required for the production of CBG with steroid binding properties (25)(26)(27).…”