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Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes

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 invo...

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Autores principales: Song, Yuanli, Qian, Yueming, Huang, Zhe, Khattak, Sarwat F., Li, Zheng Jian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Research Network of Computational and Structural Biotechnology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734232/
https://www.ncbi.nlm.nih.gov/pubmed/33335689
http://dx.doi.org/10.1016/j.csbj.2020.11.037
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author Song, Yuanli
Qian, Yueming
Huang, Zhe
Khattak, Sarwat F.
Li, Zheng Jian
author_facet Song, Yuanli
Qian, Yueming
Huang, Zhe
Khattak, Sarwat F.
Li, Zheng Jian
author_sort Song, Yuanli
collection PubMed
description 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.
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spelling pubmed-77342322020-12-16 Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes Song, Yuanli Qian, Yueming Huang, Zhe Khattak, Sarwat F. Li, Zheng Jian Comput Struct Biotechnol J Research Article 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. Research Network of Computational and Structural Biotechnology 2020-12-01 /pmc/articles/PMC7734232/ /pubmed/33335689 http://dx.doi.org/10.1016/j.csbj.2020.11.037 Text en © 2020 Bristol Myers Squibb Co. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Article
Song, Yuanli
Qian, Yueming
Huang, Zhe
Khattak, Sarwat F.
Li, Zheng Jian
Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes
title Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes
title_full Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes
title_fullStr Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes
title_full_unstemmed Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes
title_short Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes
title_sort computational insights into o-glycosylation in a ctla4 fc-fusion protein linker and its impact on protein quality attributes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734232/
https://www.ncbi.nlm.nih.gov/pubmed/33335689
http://dx.doi.org/10.1016/j.csbj.2020.11.037
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