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

Abstract

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.

Beyond the Sequon: Sites of N-Glycosylation

“…However, even the definition of this threshold is an analytical artefact, as it is increasingly apparent that most glycosylated Asn are only partially modified, with some portion ranging from a fraction of a percent to essentially all copies of a protein, actually glycosylated (Hülsmeier et al, 2007, Sumer-Bayraktar et al, 2011. This pattern seems to contrast with the general requirement of many proteins for Nglycosylation for correct and efficient protein folding (Helenius & Aebi, 2004).…”

“…However, as glycosylation generally results in a complex mixture of glycan structures at each glycosylation site, measurement of the abundance of the glycosylated form of a given site is not trivial. Some approaches have used detection of entire glycopeptides, although this approach generally requires more specialized and targeted LC-MS technologies (Sumer-Bayraktar et al, 2011). Other approaches have focused on improving quantification of occupancy, and have discarded information on site-specific glycan structure by endoglycosidase treatment ).…”

Corticosteroid-Binding Globulin Gene Mutations and Chronic Fatigue/Pain Syndromes: An Overview of Current Evidence

“…This has led some to speculate that CBG may act as a hormone and there may be a direct contribution of bound cortisol in glucocorticoid bioavailability via this yet unidentified CBG receptor 34 . Accumulation of cyclic AMP 35 occurs as a result of this CBG:cell receptor interaction. Recently, it has been shown that the NeuAc residues on the N-glycans restrict the binding of CBG to the cell receptor.…”

“…Recently, it has been shown that the NeuAc residues on the N-glycans restrict the binding of CBG to the cell receptor. Removing these NeuAc residues resulted in marked increase in cyclic AMP levels 35 . Dilution of CBG results in release of cortisol and thus suggests, at the very least, an indirect contribution of bound cortisol in glucocorticoid bioavailability 36 .…”

The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment

“…For example, human corticosteroid-binding globulin, a major plasma glycoprotein with six N-glycosites, has variable degrees in occupancy among its six glycosites (ranging from 70 to 99.5%) that also seem to change with pregnancy (16). Although the biological implications of partial occupancy in N-glycosylation are not well understood, to date, there are no well-defined strategies that can readily identify PO glycosites, particularly in a complex mixture.…”