title: Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6.
abstract: The actions of insulin-like growth factors (IGFs) are modulated by a family of high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated. Glycosylated and nonglycosylated recombinant human IGFBP-6, expressed in Chinese hamster ovary cells and Escherichia coli, respectively, were purified using IGF-II affinity chromatography and reverse-phase medium-pressure chromatography. Electrospray ionization mass spectrometry (ESMS) of glycosylated IGFBP-6 revealed considerable heterogeneity of carbohydrate composition. Major glycoforms contained 8-16 monosaccharides, including N-acetylhexosamine, hexose, and N-acetylneuraminic acid. Glycosylation sites of IGFBP-6 were identified as Thr126, Ser144, Thr145, Thr146, and Ser152 by using a combination of ESMS and Edman sequencing of tryptic fragments separated by reverse-phase high-pressure liquid chromatography. One oligosaccharide chain contained 5-6 monosaccharides, whereas the others contained 2-4 monosaccharides. Glycosylated IGFBP-6 exhibited greater resistance to proteolysis by chymotrypsin and trypsin than nonglycosylated IGFBP-6. Native disulfide bond positions in IGFBP-6 were localized by means of observed disulfide-linked tryptic fragments, revealing that there are two disulfide-linked subdomains within each of the N- and C-terminal regions and confirming a previous suggestion that the latter regions are not interconnected. A model of IGFBP-6 is developed in which these distinct domains are separated by a central region which is O-glycosylated.
authors.affiliation: School of Biochemistry, La Trobe University, Bundoora, Victoria 3083, Australia.
authors.forename: G M
authors.initials: GM
authors.lastname: Neumann
authors.forename: J A
authors.initials: JA
authors.lastname: Marinaro
authors.forename: L A
authors.initials: LA
authors.lastname: Bach
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping
chemical_list: D004220:Disulfides; D018976:Insulin-Like Growth Factor Binding Protein 6