Identification of O-Glycosylation Sites and Partial Characterization of Carbohydrate Structure and Disulfide Linkages of Human Insulin-like Growth Factor Binding Protein 6†

摘要: The actions of insulin-like growth factors (IGFs) are modulated by a family high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated. Glycosylated nonglycosylated recombinant human expressed in Chinese hamster ovary cells Escherichia coli, respectively, were purified using affinity chromatography reverse-phase medium-pressure chromatography. Electrospray ionization mass spectrometry (ESMS) glycosylated IGFBP-6 revealed considerable heterogeneity carbohydrate composition. Major glycoforms contained 8-16 monosaccharides, N-acetylhexosamine, hexose, N-acetylneuraminic acid. Glycosylation sites identified as Thr126, Ser144, Thr145, Thr146, Ser152 combination ESMS Edman sequencing tryptic fragments separated high-pressure liquid One oligosaccharide chain 5-6 whereas the others 2-4 monosaccharides. exhibited greater resistance to proteolysis chymotrypsin trypsin than IGFBP-6. Native disulfide bond positions localized means observed disulfide-linked fragments, revealing that there two subdomains within each N- C-terminal regions confirming previous suggestion latter not interconnected. A model developed these distinct domains central region