Title : Micro- and macroheterogeneity of N-glycosylation yields size and charge
isoforms of human
sex hormone binding globulin circulating in serum
Abstract :
- Human sex hormone binding globulin ( hSHBG ) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid-sensitive tissues
- Several molecular mechanisms of action of hSHBG , including the function of its attached glycans remain unknown
- Here, we perform a detailed site-specific characterization of the N- and O-linked glycosylation of serum-derived hSHBG
- MS-driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom-up and top-down manner to determine glycosylation sites , site-specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG
- It was found that serum-derived hSHBG is N-glycosylated at Asn(351) and Asn(367) with average molar occupancies of 85.1 and 95.3%, respectively
- Both sites are occupied by the same six sialylated and partly core fucosylated bi- and triantennary N-Glycoforms with lactosamine-type antennas of the form (±NeuAcα6)Galβ4GlcNAc.
- N-Glycoforms of Asn(367) were slightly more branched and core fucosylated than Asn(351) N-glycoforms due probably to a more surface-exposed glycosylation site
- The N-terminal Thr(7) was fully occupied by the two O-linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio
- Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum
- Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn(351) glycan occupancies and N-glycan sialylation that may modulate the hSHBG activity
- To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans