Discovery and Investigation of O-Xylosylation in Engineered Proteins Containing a (GGGGS)n Linker

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• 作者：Dingyi Wen ; Susan F. Foley ; Xiaoping L. Hronowski ; Sheng Gu ; Werner Meier
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：May 7, 2013
• 年：2013
• 卷：85
• 期：9
• 页码：4805-4812
• 全文大小：354K
• 年卷期：v.85,no.9(May 7, 2013)
• ISSN：1520-6882

文摘

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鈥昮rom <2% to >25% per linker鈥昹ikely 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.