title: Discovery and investigation of O-xylosylation in engineered proteins containing a (GGGGS)n linker.
abstract: Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, greater safety, reduced immunogenicity, and better delivery. GGGGS [(G4S)n] linkers are commonly used when engineering a protein, because of their flexibility and resistance to proteases. However, post-translational modifications (PTMs) can occur at the Ser residue in these linkers. Here, we report, for the first time, the occurrence of O-xylosylation at the serine residue in (G4S)n>2 linkers. The O-xylosylation was discovered as a result of molecular mass determination, peptide mapping analysis, and MS/MS sequencing. Our investigation showed that (i) O-xylosylation is a common PTM for (G4S)(n>2) linkers; (ii) GSG is the motif for O-xylosylation; and (iii) the total amount of xylosylation per linker increases as the number of GSG motifs in the linker increases. Our investigation has also shown that the O-xylosylation level is clone-dependent, to a certain degree, but the xylosylation level varies considerably among the proteins examined-from <2% to >25% per linker-likely depending on the accessibility to the sites by the xylosyltransferase. Our work demonstrates that potential therapeutic proteins containing (G4S)n linkers should be closely monitored for O-xylosylation in order to ensure that drugs are homogeneous and of high quality. The strategies for elimination and reduction of O-xylosylation were also examined and are discussed.
authors.affiliation: Analytical Biochemistry, Department of Biologics Drug Discovery, Biogen Idec, 12 Cambridge Center, Cambridge, Massachusetts 02142, United States. dingyi.wen@biogenidec.com
authors.forename: Dingyi
authors.initials: D
authors.lastname: Wen
authors.forename: Susan F
authors.initials: SF
authors.lastname: Foley
authors.forename: Xiaoping L
authors.initials: XL
authors.lastname: Hronowski
authors.forename: Sheng
authors.initials: S
authors.lastname: Gu
authors.forename: Werner
authors.initials: W
authors.lastname: Meier
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D010449:Peptide Mapping; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014994:Xylose / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011506:Proteins; D012694:Serine; D014994:Xylose