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

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. 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. Human corticosteroid-binding globulin (CBG) 1The abbreviations used are:CBGcorticosteroid-binding globulinBPCbase peak chromatogramETDelectron transfer dissociationFAformic acidGalgalactoseGlcNAcN-acetylglucosamineHILIChydrophilic interaction chromatographyLacNAcN-acetyllactosamineNeuAcN-acetylneuraminic acidXICextracted ion chromatogramRTroom temperatureLC-MS/MSliquid chromatography-tandem MSACNacetonitrileTFAtrifluoroacetic acidESIelectrospray ionizationCIDcollision-induced dissociation., also referred to as transcortin in the literature, is the major plasma transport protein that binds cortisol and progesterone with high affinity in a single steroid binding site, providing a reservoir for the circulating steroids and regulating the amount of free hormones that are available for entry into target tissues (1Dunn J.F. Nisula B.C. Rodbard D. 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PubMed Google Scholar, and of corticosteroid-binding globulin: of Biol. 53: PubMed Scopus Google Scholar). was that the N-glycans on site are not for steroid binding the protein folded to its terminal acid (NeuAc) residues of CBG N-glycans to in and of native and desialylated and human 1981; PubMed Scopus Google and that N-glycosylation is for and of CBG G.L. of human corticosteroid-binding and of individual PubMed Scopus Google Scholar, G.L. of human corticosteroid-binding globulin is for steroid Biol. PubMed Google Scholar, on the of glycosylation for human corticosteroid-binding globulin: with that for PubMed Scopus Google Scholar). the of the used human CBG human CBG produced in in human cell in to the of the CBG N-glycans. This in protein glycosylation is to be and and the structure of Rev. Mol. Biol. PubMed Scopus Google Scholar, site-specific and of J. 1987; PubMed Scopus Google Scholar, of in a of of the in Natl. Acad. Sci. U.S.A. 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Smith C.L. N.A. for corticosteroid-binding globulin in of cortisol to Clin. Endocrinol. Metab. 1990; PubMed Scopus Google Scholar). The and of the of the glycosylation that to N-glycans were from to and six to NeuAc residues were from and and that was of N-glycans. The functional was on the that MCF-7 cells cAMP as a CBG with its cell surface W. of in a cell by human corticosteroid-binding PubMed Scopus Google Scholar). the cAMP amount be used to the CBG:receptor interaction and the for W. of in a cell by human corticosteroid-binding PubMed Scopus Google Scholar). we that cAMP is produced in a (CBG) and MCF-7 cells were challenged with CBG not was also that of cortisol to CBG was in the cAMP was produced upon with CBG cortisol W. of in a cell by human corticosteroid-binding PubMed Scopus Google Scholar). 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As from of with the produced higher cAMP cells the of CBG N-glycans in the was that the individual not cAMP the not is that of CBG upon and as for other The of the human in 2008; PubMed Scopus Google Scholar, J. of and protein in binding PubMed Scopus Google the cAMP into the increased cAMP upon with the glycosylation analogs be that N-glycans are the CBG:receptor interaction and that the CBG N-glycans and the NeuAc residues are the interaction the CBG is a glycosylated protein and the with the of is to that the N-glycans are for the structure and of by performing a structural characterization of the CBG and through functional the importance of the N-glycans for CBG The of to CBG from human CBG is that and CBG glycosylation and that the of CBG glycosylation be of for are The structural characterization of the CBG N-glycans was predominantly performed to glycosylation site-specific This as the six N-glycosylation sites were the glycoforms from the individual sites were and the all were complex type the degree of was The the N-glycans on the individual sites are in the and are the site-specific differences in the of could be correlated with the of the glycosylation sites on the protein surface by of the to the individual glycosylation sites and This of the is to on the maturely folded in the The of N-glycans and in the to PubMed Scopus Google and steric on the protein surface relatively to the protein surface from the protein surface glycoforms from the relatively glycosylation site and of CBG contained a higher degree of and and a higher degree of branching and glycoforms from the glycosylation site and and and and the structure of human CBG with on which is not glycosylation site and the high of and branching for glycoforms on glycosylation site with its on a and the degree of not to be by the the degree of to be with the degree of the degree of to the protein surface on the of the protein surface the glycosylation on the was that glycosylation site is to the to the steroid binding site, The branching of the N-glycans on site bi- and tri-antennary be to accessibility of the steroid to the binding N-glycans from site contained which also the to the steroid binding site the N-glycans to site are involved in the of the accessibility of the steroid to its binding site, in to for the of the steroid binding site of CBG G.L. of human corticosteroid-binding and of individual PubMed Scopus Google Scholar, G.L. of human corticosteroid-binding globulin is for steroid Biol. PubMed Google Scholar, and of corticosteroid-binding globulin: of Biol. 53: PubMed Scopus Google Scholar). The of the CBG N-glycans was with that the binding of CBG to its cell surface by the amount of cAMP produced by MCF-7 cells upon with native and glycan-modified The in cAMP production upon with as with that the N-glycans are the CBG:receptor interaction and that the N-glycans are the CBG interaction with its of the CBG:receptor interaction could be by six to NeuAc residues This the NeuAc residues of the CBG N-glycans as for the CBG:receptor interaction and indicates that be not by steric but also by electrostatic This with the that the CBG also to be of human CBG with cell Mol. Biol. PubMed Scopus Google electrostatic the The mechanism of the in regulating the CBG:receptor interaction could in be both by the degree of and by the in is by the and of the involved the and the of the N-glycans the CBG:receptor interaction a higher is the binding site of human CBG to its to be the of CBG protein is that the binding site be in to of the six N-glycans a of the CBG the structure available for human CBG was produced from a of CBG protein with of the structural are to the binding site of CBG and to the of the residues of the glycosylation analogs used in the is that the residues were the was performed native CBG binding complex W. of in a cell by human corticosteroid-binding PubMed Scopus Google Scholar, R.W. of steroid Scholar, R.W. globulin with target cells and plasma Acad. Sci. PubMed Scopus Google cortisol to target cells W. The functions of corticosteroid-binding globulin and sex hormone-binding globulin: recent advances.Endocr. Rev. 1990; 11: 80-91Crossref PubMed Scopus (535) Google Scholar, W. of the binding of corticosteroid-binding globulin to cell PubMed Scopus Google and cAMP W. of in a cell by human corticosteroid-binding PubMed Scopus Google Scholar). a of functions from the of and CBG N-glycosylation could a in regulating which of are with This is the of a of CBG are N.A. W. binding of human corticosteroid-binding globulin to cell Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, of the transcortin binding to human plasma PubMed Scopus Google Scholar, for the of the transcortin in the interaction with the plasma of human PubMed Scopus Google Scholar, of the complex with plasma of human 53: Google are and as the CBG N-glycans of individual on the and of corticosteroid-binding Mol. Biol. PubMed Scopus Google Scholar, G.L. A. D. in plasma corticosteroid-binding globulin in is with increased and in PubMed Google Scholar, and serum of of human 1987; PubMed Scopus Google Scholar, corticosteroid-binding globulin: high of Metab. PubMed Scopus Google Scholar, of human serum transcortin and PubMed Scopus Google Scholar, W. of the of and human corticosteroid-binding globulin: Mol. Biol. PubMed Scopus Google Scholar). hormone could be to cells a CBG to CBG that is a to the binding the other major of CBG is to transport and release corticosteroids in the The binding of steroid is in a binding of CBG and the release is to be by a of CBG by a by G.L. Smith C.L. N.A. for corticosteroid-binding globulin in of cortisol to Clin. Endocrinol. Metab. 1990; PubMed Scopus Google Scholar, Underhill G.L. globulin, a structural for steroid transport and release.J. Biol. PubMed Scopus Google Scholar). CBG N-glycans are involved in both of by regulating the accessibility of steroid to the binding and its as as regulating the accessibility of to the the the site is in to glycosylation site N-glycans from site are the accessibility of to the site and be involved in the of steroid release from functional be to investigate the CBG in to the CBG:receptor interaction as are also involved in the corticosteroid This that CBG is through of glycosylation to the glycosylation The N-glycans on CBG were to its by the binding of CBG to its for and with