Intact mass analysis reveals the novel O-linked glycosylation on the stalk region of PD-1 protein

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.

Emerging therapeutic frontiers in cancer: insights into posttranslational modifications of PD-1/PD-L1 and regulatory pathways

“…It consists of 288 amino acids and features an ectodomain with a signal peptide, an N-loop, an IgV-like domain (with four N-linked glycosylation sites: N49, N58, N74, and N116, and a polyubiquitination site at K48), a transmembrane domain, and a cytoplasmic region with the key signaling motifs ITIM and ITSM and phosphorylation sites at Y223 and Y248 [10][11][12][13][14]. Recent research has identified additional O-glycosylation sites (T153, S157, S159, and T168) in the stalk region, indicating complex posttranslational modifications [15]. PD-1 interacts with two ligands, PD-L1 and PD-L2, from the B7 family, with PD-L2 binding to PD-1 with over three times the affinity of PD-L1 [16].…”