Application of StrucGP in medical immunology: site-specific <i>N</i>-glycoproteomic analysis of macrophages

doi:10.1007/s11684-022-0964-8

Front. Med.

2023, Vol. 17

Issue (2) : 304-316 https://doi.org/10.1007/s11684-022-0964-8

RESEARCH ARTICLE

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

Pengfei Li, Zexuan Chen, Shanshan You, Yintai Xu, Zhifang Hao, Didi Liu, Jiechen Shen, Bojing Zhu, Wei Dan, Shisheng Sun(

)

College of Life Sciences, Northwest University, Xi’an 710069, China

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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

Corresponding Author(s): Shisheng Sun

Just Accepted Date: 15 November 2022 Online First Date: 27 December 2022 Issue Date: 26 May 2023

Cite this article:

Pengfei Li,Zexuan Chen,Shanshan You, et al. Application of StrucGP in medical immunology: site-specific N-glycoproteomic analysis of macrophages[J]. Front. Med., 2023, 17(2): 304-316.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-022-0964-8
https://academic.hep.com.cn/fmd/EN/Y2023/V17/I2/304

Fig.1 N-glycoproteomic analysis of THP-1-derived M0, M1, and M2 macrophages. (A) Workflow for the glycoproteomic analyses of three THP-1-derived macrophage subtypes. (B) Heat map of all identified intact glycopeptides in three THP-1-derived macrophage subtypes. The PSMs of intact glycopeptides, which comprises different glycan structures (upper) and glycosites (left), were shown in the heat map. The numbers of glycosites modified by each glycan and glycan numbers at each glycosite were summarized at the bottom (blue lines) and right (orange lines) parts of the figure, respectively. More details can be found in Table S1. (C) Venn diagram of all intact glycopeptides, glycosites, and glycoproteins identified in M0, M1, and M2 macrophages.

Fig.2 Glycan isoforms of THP-1-derived M0, M1, and M2 macrophages. N-glycan isoforms identified in M0, M1, and M2 macrophages. The location of each branch structure cannot actually be identified by StrucGP.

Fig.3 N-glycan structure interpretation of three human THP-1 macrophage subtypes. (A, B) Distributions of different glycan subtypes in various macrophage subtypes. (C, D) Distributions of four core structures and the top 10 branch structures (including the null Ø) identified in the different macrophage subtypes. The percentages in A?D were calculated on the basis of the numbers of unique glycopeptides. F: Fucose and S: Sialic acid.

Fig.4 Analysis of bisected GlcNAc-modified glycoproteins in three macrophage subtypes. (A) Heat map of the bisected GlcNAc-modified glycoproteins in the different macrophage subtypes with Z-scored of PSMs. Each column corresponds to a protein gene ID. Z-score was measured on the basis of the standard deviations from the mean, and a high (low) Z-score represented a high (low) number of PSMs. The heat map was used only with the aim of ordering the PSMs of glycopeptides according to the column obtained Z-score (z = (x − mean)/s.d.). (B) Biological function analysis of all bisected GlcNAc-modified glycoproteins in THP-1-derived macrophages. Enrichment P value < 0.05. The counts indicate how many proteins are annotated with a particular term. Digital labels of HSA (human sapiens) and GO (gene ontology) indicate the entries of annotation terms. Green represents the reactome pathways, yellow represents the Kyoto Encyclopedia of Genes and Genomes, blue represents the cellular component, orange represents the molecular function, and pink represents the biological process.

Fig.5 Site-specific N-glycan structure analysis of different macrophage subtypes. (A) Top 10 N-glycans detected on the glycoproteins of different macrophage subtypes based on the numbers of glycosites and the numbers of PSMs. (B) Analysis of the N₄H₅F₁S₁-modified glycopeptides (total PSM ≥ 2) in three macrophage subtypes. Cluster analysis of N₄H₅F₁S₁-modified glycopeptides in different macrophage subtypes with Z-scored of PSMs (left). Z-score was measured on the basis of the standard deviations from the mean, and a high (low) Z-score represented a high (low) number of PSMs. The heat map was used only with the aim of ordering the PSMs of glycopeptides according to the row obtained Z-score (z = (x − mean)/s.d.). Red means upregulation, and blue represents the downregulation of expression. The biological function analysis of N₄H₅F₁S₁-modified glycoproteins in THP-1-derived macrophages (right). The counts indicate how many proteins are annotated with a particular term. Digital labels of HSA (human sapiens) and GO (gene ontology) indicate the entries of annotation terms. Enrichment P value < 0.05. The location of each branch structure (e.g., α2,3- and α2,6-linked sialic acids) cannot actually be identified by StrucGP.

Fig.6 Differentially expressed site-specific N-glycans and glycoproteins in M1 and M2 macrophages. (A) Top five unique N-glycan structures in THP-1-derived M1 and M2 macrophages based on the numbers of PSMs. (B) Biological function analysis of unique glycoproteins in THP-1-derived M1 and M2 macrophages. The counts indicate how many proteins are annotated with a particular term. Enrichment P value < 0.05. (C) Protein–protein interaction (PPI) networks of highly interactive glycoproteins in M1 (left) and M2 (right) macrophages. The line colors varying from light to dark represent the combined score of proteins from 0.4 to 1. The location of each branch structure (e.g., α2,3- and α2,6-linked sialic acids) cannot actually be identified by StrucGP.

Fig.7 N-glycan structures of PD-L1 protein in THP-1-derived M1 macrophages. (A) N-glycan structures on glycosites Asn-192 and Asn-200 of PD-L1. (B) Representative MS/MS spectra for the identification of an intact glycopeptide from PD-L1. Upper: peptide sequence identification using a MS/MS spectrum of high HCD energy (HCD = 33%). Lower: glycan structure determination using MS/MS spectra of low HCD energy (HCD = 20%). The location of each branch structure (such as α2,3- and α2,6-linked sialic acids) cannot actually be identified by StrucGP.

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