N-linked glycosylation of PD-L1/PD-1: an emerging target for cancer diagnosis and treatment

Abstract

During tumorigenesis and progression, the immune checkpoint programmed death-1 (PD-1) and its ligand programmed death ligand-1 (PD-L1) play critical roles in suppressing T cell-mediated anticancer immune responses, leading to T-cell exhaustion and subsequent tumor evasion. Therefore, anti-PD-L1/PD-1 therapy has been an attractive strategy for treating cancer over the past decade. However, the overall efficacy of this approach remains suboptimal, revealing an urgent need for novel insights. Interestingly, increasing evidence indicates that both PD-L1 on tumor cells and PD-1 on tumor-specific T cells undergo extensive N-linked glycosylation, which is essential for the stability and interaction of these proteins, and this modification promotes tumor evasion. In various preclinical models, targeting the N-linked glycosylation of PD-L1/PD-1 was shown to significantly increase the efficacy of PD-L1/PD-1 blockade therapy. Furthermore, deglycosylation of PD-L1 strengthens the signal intensity in PD-L1 immunohistochemistry (IHC) assays, improving the diagnostic and therapeutic relevance of this protein. In this review, we provide an overview of the regulatory mechanisms underlying the N-linked glycosylation of PD-L1/PD-1 as well as the crucial role of N-linked glycosylation in PD-L1/PD-1-mediated immune evasion. In addition, we highlight the promising implications of targeting the N-linked glycosylation of PD-L1/PD-1 in the clinical diagnosis and treatment of cancer. Our review identifies knowledge gaps and sheds new light on the cancer research field.

Keywords: Clinical diagnosis; Immune evasion; Immunotherapy; N-linked glycosylation; PD-1; PD-L1.

Fig. 1

Structures of PD-L1/PD-1 and N-linked glycosylated PD-L1/PD-1. PD-L1, which is a membrane protein that is highly expressed on tumor cells, possesses four asparagine residues that are undergo N-linked glycosylation (N35, N192, N200, and N219) and are distributed across the IgV-like and IgC-like domains of PD-L1. PD-1, which is a membrane protein that is expressed mainly on T cells, possesses four asparagine residues (N49, N58, N74, and N116) that undergo N-linked glycosylation and span the IgV-like domain of PD-1. There are also several potential O-linked glycosylation sites in the stalk domain of PD-1. The numbers represent amino acid residues. TM, transmembrane; IgV, immunoglobulin variable; IgC, immunoglobulin constant

Fig. 2

A diagram of the regulatory mechanisms of PD-L1/PD-1 N-linked glycosylation. In tumor cells, GSK3β binds to nonglycosylated PD-L1 and phosphorylates ngPD-L1 at T180/S184, which results in the polyubiquitination of ngPD-L1 via β-TcRP and its 26 S proteasomal degradation. Activated AMPK directly phosphorylates PD-L1 at S195, inducing the abnormal glycosylation of PD-L1 and ERAD. Several signaling pathways are involved in promoting PD-L1 N-glycosylation during tumorigenesis: (1) IL-6/IL-6R signaling activation leads to PD-L1 phosphorylation at Y112 via JAK1 in the ER lumen, which recruits STT3 to N-glycosylate PD-L1; (2) EGF/EGFR signaling inhibits PD-L1 degradation via GSK3β inactivation, while the EGF/EGFR axis induces PD-L1 N-glycosylation by upregulating the glycosyltransferase B3GNT3; and (3) EMT transcriptionally induces STT3 through β-catenin/TCF4, which is recruited to PD-L1 and catalyzes PD-L1 N-glycosylation. In T cells, the N-linked glycosylation of PD-1 via B3GNT2 and FUT8 inhibits PD-1 proteasomal degradation and promotes PD-1 cell-surface localization and PD-1/PD-L1 interaction; FKBP51 and sigma1 function as PD-L1 molecular chaperones, facilitating PD-L1 folding and N-linked glycosylation

Fig. 3

Comparison of heavily N-linked glycosylated PD-L1 and deglycosylated PD-L1 via an IHC assay. N-linked glycans on PD-L1 hinder its recognition by currently common anti-PD-L1 antibodies, leading to suboptimal precision of IHC assays. To address this issue, fixed FFPE tissue slides can be pretreated with the recombinant glycosidase PNGase F to remove N-glycans from PD-L1. This process makes epitopes more accessible for antibody binding, thereby increasing PD-L1 signal intensity and improving PD-L1 detection and therapeutic relevance