title: O-glycosylation of glycine-serine linkers in recombinant Fc-fusion proteins: attachment of glycosaminoglycans and other intermediates with phosphorylation at the xylose sugar subunit.
abstract: A xylose-based glycosaminoglycan (GAG) core was recently identified at a Ser residue in the linker sequence of a recombinant Fc fusion protein. The linker sequence, G-S-G-G-G-G, and an upstream acidic residue were serving as a substrate for O-xylosyltransferase, resulting in a major glycan composed of Xyl-Gal-Gal-GlcA and other minor intermediates. In this paper, a portion of an unrelated protein was fused to the C-terminus of an IgG Fc domain using the common (G4S) 4 linker repeat. This linker resulted in a heterogenous population of xylose-based glycans all containing at least a core Xyl. Commonly observed glycan structures include GAG-related di-, tri-, tetra-, and penta-saccharides (e.g., Xyl-Gal, Xyl-Gal-Gal, Xyl-Gal-Gal-GlcA, and Xyl-Gal-Gal-GlcA-HexNAc), as well as Xyl-Gal-Neu5Ac. Following alkaline phosphatase or sialidase treatment combined with CID fragmentation, low-level glycans with a mass addition of 79.9 Da were confirmed to be a result of phosphorylated xylose. A minute quantity of phosphorylated GAG pentasaccharides may also be sulfated (also 79.9 Da), possibly at the HexNAc moiety due to non-reactivity to alkaline phosphatase. The xylose moiety may be randomly incorporated in one of the three G-S-G sequence motifs; and the linker peptide shows evidence for multiple additions of xylose at very low levels.
authors.affiliation: Biologics Optimization; Therapeutic Discovery; Amgen Inc.; Thousand Oaks, CA USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Spahr
authors.affiliation: Biologics Optimization; Therapeutic Discovery; Amgen Inc.; Thousand Oaks, CA USA.
authors.forename: Stone D-H
authors.initials: SD
authors.lastname: Shi
authors.affiliation: Biologics Optimization; Therapeutic Discovery; Amgen Inc.; Thousand Oaks, CA USA.
authors.forename: Hsieng S
authors.initials: HS
authors.lastname: Lu
mesh_terms: D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D006224:Cricetinae; D003412:Cricetulus; D006025:Glycosaminoglycans / Q000096:biosynthesis / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000096:biosynthesis / Q000737:chemistry*; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D014994:Xylose / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006025:Glycosaminoglycans; D007127:Immunoglobulin Constant Regions; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D014994:Xylose
keywords: Fc-fusion protein; glycosaminoglycans; linkers; phosphorylation; serine; sulfation; xylose