title: 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 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.
authors.affiliation: Department of Biological Sciences and Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Sumer-Bayraktar
authors.forename: Daniel
authors.initials: D
authors.lastname: Kolarich
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Campbell
authors.forename: Sinan
authors.initials: S
authors.lastname: Ali
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
mesh_terms: D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D054794:Glycomics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009438:Neuraminic Acids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism; D015290:Second Messenger Systems; D014156:Transcortin / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
chemical_list: D009438:Neuraminic Acids; D011134:Polysaccharides; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; C534935:SERPINA6 protein, human; D014156:Transcortin; D000242:Cyclic AMP