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 ␣2,3-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.

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid-sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site-specific characterization of the N- and O-linked glycosylation of serum-derived hSHBG. MS-driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom-up and top-down manner to determine glycosylation sites, site-specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum-derived hSHBG is N-glycosylated at Asn(351) and Asn(367) with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi- and triantennary N-Glycoforms with lactosamine-type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N-Glycoforms of Asn(367) were slightly more branched and core fucosylated than Asn(351) N-glycoforms due probably to a more surface-exposed glycosylation site. The N-terminal Thr(7) was fully occupied by the two O-linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn(351) glycan occupancies and N-glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.

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Previous studies demonstrated that gonadal steroids secreted during perinatal life permanently 'organize' the mechanisms governing hypothalamo-pituitary-adrenal (HPA) function, leading to sexually differentiated patterns of pituitary-adrenal activity under basal and stress conditions. In this paper, we show that gonadal steroids can also exert 'activational' effects upon the HPA system. Examination of the ability of different doses of dexamethasone to suppress the nocturnal increase in corticosterone secretion and to attenuate the gene expression of CRH in the hypothalamic paraventricular nucleus of intact and gonadectomized male and female rats revealed that ovarian steroids make an important contribution to the higher sensitivity of the pituitary-adrenal axis in females to glucocorticoid suppression, whereas testicular steroids may be causal to the male's moderate responsiveness to glucocorticoid feedback. These findings may be implicated in a number of psychiatric and neurological disease states commonly associated with impaired HPA regulation, but which may be primarily rooted in altered gonadal steroid secretion.

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