期刊名称：Computational and Structural Biotechnology Journal
印刷版ISSN：2001-0370
出版年度：2020
卷号：18
页码：3925-3935
DOI：10.1016/j.csbj.2020.11.037
出版社：Computational and Structural Biotechnology Journal
摘要：The hinge region of immunoglobulin G1 (IgG1) is used as a common linker for Fc-fusion therapeutic proteins. With the advances of high-resolution mass spectrometry and sample treatment strategies, unexpected O-linked glycosylation has been observed in the linker. However, the molecular mechanism involved in this unusual posttranslational modification is unknown. In this study, we applied site-direct mutagenesis, mass spectrometry, analytical chromatography, and computational modeling to investigate O-glycosylation processes in a clinically used CTLA4 Fc-fusion protein and its impacts on protein quality attributes. Surprisingly, O-glycans could be formed at new sites when an initial O-glycosylation site was eliminated, and continued to occur until all potential O-glycosylation sites were nulled. Site-preference of O-glycosylation initiation was attributed to the complex formation between the linker peptide and glycan transferase whereas the O-glycosylating efficiency and the linker flexibility were correlated using molecular modeling and simulations. As predicted, O-glycan-free CTLA4 Fc-fusion proteins were more homogenous for sialylation, and interestingly less prone to protein aggregation. Attenuating protein aggregation was a desirable effect, and could be related to the reduced presence of linker O-glycans that hindered inter-chain disulfide bond reformation. Findings from this study shed light on new therapeutic protein design and development.
关键词：O-glycosylation ; Protein linker ; Fc-fusion protein ; Sialic acid ; Protein aggregation
