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Comprehensive Glycomic and Glycoproteomic Analyses of Human Programmed Cell Death Protein 1 Extracellular Domain
Qiushi Chen1,2, Zhiwu Tan3, Yang Tang4
1Laboratory for Synthetic Chemistry and Chemical Biology Limited, Units 1503-1511, 15/F., Building 17W, Hong Kong Science Park, Shatin, Hong Kong SAR 999077, P. R. China.
Abstract:
Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 N-glycosylation sites and 25 potential O-glycosylation sites, which implicates the importance of glycosylation. The structure of hPD-1 has been intensively studied, but the glycosylation of this protein, especially the glycan on each glycosylation site, has not been comprehensively illustrated. In this study, hPD-1 ECD expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells was analyzed; not only N- and O-glycosylation sites but also the glycans on these sites were comprehensively analyzed using mass spectrometry. In addition, hPD-1 ECD binding to different anti-hPD-1 antibodies was tested, and N-glycans were found functioned differently. All of this glycan information will be beneficial for future PD-1 studies.
Insights
This study comprehensively analyzes human programmed cell death protein 1 (hPD-1) glycosylation. N-glycans on hPD-1 extracellular domain show varied functions, offering insights into cancer therapy responses.
Area of Science:
- Biochemistry
- Immunology
- Glycobiology
Background:
- Human programmed cell death protein 1 (hPD-1) is a key immune checkpoint receptor.
- PD-1 antibody treatments are vital in cancer therapy, but patient response varies.
- The glycosylation of hPD-1, particularly its extracellular domain (ECD), is crucial for its function but not fully understood.
Purpose of the Study:
- To comprehensively analyze N- and O-glycosylation sites and their attached glycans on the hPD-1 ECD.
- To investigate the functional differences of N-glycans in hPD-1 ECD binding to anti-hPD-1 antibodies.
- To provide detailed glycan information for future research on hPD-1.
Main Methods:
- Mass spectrometry was employed to analyze hPD-1 ECD expressed in HEK 293 and CHO cells.
- Identification and characterization of both N- and O-glycosylation sites and their associated glycans.
- Binding assays were performed to assess the interaction of hPD-1 ECD with different anti-hPD-1 antibodies.
Main Results:
- Detailed mapping of N- and O-glycosylation sites on the hPD-1 ECD.
- Comprehensive characterization of the glycans present at these sites.
- Demonstrated differential functions of N-glycans in the binding of hPD-1 ECD to anti-hPD-1 antibodies.
Conclusions:
- This study provides the first comprehensive illustration of hPD-1 glycosylation.
- The identified glycan structures and their functional variations offer critical insights into hPD-1 antibody efficacy.
- This detailed glycomic information is valuable for advancing cancer immunotherapy research and drug development.
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