Application of StrucGP in medical immunology: site-specific N-glycoproteomic analysis of macrophages

Abstract

The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals. Meanwhile, the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions. In this work, we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis. The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry (MS)-based glycoproteomic approaches, followed by the large-scale mapping of site-specific glycan structures via StrucGP. Results revealed that bisected GlcNAc, core fucosylated, and sialylated glycans (e.g., HexNAc₄Hex₅Fuc₁Neu5Ac₁, N₄H₅F₁S₁) were increased in M1 and M2 macrophages, especially in the latter. The findings indicated that these structures may be closely related to macrophage polarization. In addition, a high level of glycosylated PD-L1 was observed in M1 macrophages, and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1, and Asn-200 contained Lewis epitopes. The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.

Keywords

macrophage / glycoproteome / glycopeptides / N-glycan structures / PD-L1

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2019YFA0905200) and the National Natural Science Foundation of China (Nos. 91853123, 81773180, and 21705127).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11684-022-0964-8 and is accessible for authorized users.

Compliance with ethics guidelines

Pengfei Li, Zexuan Chen, Shanshan You, Yintai Xu, Zhifang Hao, Didi Liu, Jiechen Shen, Bojing Zhu, Wei Dan, and Shisheng Sun declare that they have no conflict of interest. All authors read and approved the final manuscript and, therefore, had full access to all the data in the study and take responsibility for the integrity and security of data. This article does not contain any studies involving human or animal subjects.

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