Coupling Aptamer‐based Protein Tagging with Metabolic Glycan Labeling for In Situ Visualization and Biological Function Study of Exosomal Protein‐Specific Glycosylation

Abstract

Exosomal glycoproteins play important roles in many physiological and pathological functions. Herein, we developed a dual labeling strategy based on a protein‐specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes. The glycosylation of exosomal PD‐L1 (exoPD‐L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD‐L1 aptamers bound on exoPD‐L1 and fluorescent tags on glycans introduced via metabolic glycan labeling. This method enables in situ visualization and biological function study of exosomal protein glycosylation. Exosomal PD‐L1 glycosylation was confirmed to be required in interaction with PD‐1 and participated in inhibiting of CD8+ T cell proliferation. This is an efficient and non‐destructive method to study the presence and function of exosomal protein‐specific glycosylation in situ, which provides a powerful tool for exosomal glycoproteomics research.

Research progress of PD‐L1 non‐glycosylation in cancer immunotherapy

“…This exosomes Ap‐MGL strategy has been used to compare the quality of interactions between glycosylated exoPD‐L1/PD‐1 and its deglycosylated form. These data show, for the first time, that exosomes PD‐L1 glycosylation is the critical structural basis for exoPD‐L1/PD‐1 interaction and inhibition of CD8+ T cell proliferation 39 . Exosomes PD‐L1 glycosylation may be a promising new target for immunotherapy.…”

“…Exosomes PD‐L1 is highly N‐glycosylated and plays an important role in regulating immune escape. Zhu et al 39 developed a novel exosomes Ap‐Metabolic glycan labelling (MGL) strategy based on protein‐specific aptamer tagging and metabolic polysaccharide labelling for the identification and functional study of glycosylation of extracellular proteins, which is efficient and non‐destructive. This exosomes Ap‐MGL strategy has been used to compare the quality of interactions between glycosylated exoPD‐L1/PD‐1 and its deglycosylated form.…”

Isolation of circulating exosomes and identification of exosomal PD-L1 for predicting immunotherapy response

“…PD-L1 is heavily glycosylated; 40 to investigate the binding properties of exosomes with the PD-L1 antibody or PD-L1 aptamer, the experiments were divided into a deglycosylation group and glycosylation group, and deglycosylation was conducted as described in the literature. 41 Briefly, B16F10 cells seeded in the confocal dish were fixed with 4% paraformaldehyde and the fixed cells were incubated with glycoprotein denaturing buffer and treated with PNGase F at 37 °C. For glycosylation, the reagents were replaced with PBS.…”

“…2E), and the size and morphology were consistent with previous reports. 41,46 Next, the proportion of exosomes with sizes between 40 and 160 nm was assessed using NTA. As can be seen from Fig.…”

“…Because PD-L1 was heavily glycosylated and the PD-L1 aptamer was smaller than the antibody, the aptamer was more likely to get through the PD-L1 glycosylation. 23,40,41 To validate this hypothesis, B16F10 cells were treated with PNGase F to remove glycosylation before subjecting them to PD-L1 aptamer or antibody staining. As observed in Fig.…”