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Intact mass analysis reveals the novel O-linked glycosylation on the stalk region of PD-1 protein
Phanthakarn Tit-Oon1, Arisa Wonglangka2, Klaichan Boonkanta2
1Translational Research Unit, Chulabhorn Research Institute, Bangkok, 10210, Thailand.
Abstract:
Programmed cell death protein 1 (PD-1) is a key receptor in the immune checkpoint pathway and has emerged to be a promising target for cancer therapy. PD-1 consists of an intracellular domain followed by a transmembrane domain that is connected to the extracellular domain by the stalk region. Although the PD-1 structure has been studied for more than two decades, the posttranslational modification of this protein has been incompletely characterized. In this study, we identified the previously undescribed modification sites of O-linked glycan on the stalk region of PD-1 protein using O-protease digestion coupling with intact mass analysis. The result indicates that T153, S157, S159, and T168 are modified by sialylated mucin-type O-glycan with core 1- and core 2-based structures. This study provides both information on potential novel modification sites on the PD-1 protein and an attractive method for identifying O-linked glycosylation using a specific enzyme and intact mass analysis.
Insights
Researchers identified new O-linked glycan modifications on the Programmed cell death protein 1 (PD-1) stalk region. This discovery offers novel insights into PD-1 posttranslational modifications and cancer therapy targets.
Area of Science:
- Immunology
- Biochemistry
- Glycobiology
Background:
- Programmed cell death protein 1 (PD-1) is a crucial immune checkpoint receptor.
- PD-1 is a promising target for cancer immunotherapy.
- The posttranslational modifications of PD-1 remain incompletely understood.
Purpose of the Study:
- To identify previously undescribed O-linked glycosylation sites on the PD-1 protein.
- To characterize the nature of these O-linked glycans.
- To develop a method for identifying O-linked glycosylation on PD-1.
Main Methods:
- O-protease digestion of PD-1 protein.
- Intact mass analysis.
- Characterization of glycan structures.
Main Results:
- Identified novel O-linked glycan modification sites at T153, S157, S159, and T168 on the PD-1 stalk region.
- These sites are modified by sialylated mucin-type O-glycans with core 1- and core 2-based structures.
- A robust method combining O-protease digestion and intact mass analysis was established.
Conclusions:
- Novel O-linked glycosylation sites on PD-1 have been identified and characterized.
- These findings contribute to a deeper understanding of PD-1 biology and its regulation.
- The developed method offers a valuable tool for future studies on PD-1 glycosylation and other glycoproteins.
