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Deciphering the mechanisms of macrophage polarization through comprehensive analysis of protein glycosylation in cells and on the surface

ABSTRACT: In this work, we conducted an in-depth characterization of protein glycosylation including total glycoproteins and surface ones across different macrophage phenotypes using mass spectrometry (MS)-based proteomics. The global analysis of glycoproteins was performed using the boronic acid-conjugated dendrimer beads-based enrichment method. The N-glycoproteome was found to undergo more pronounced changes in M1 macrophages compared to M2 macrophages. To examine the alterations of surface glycoproteins, we employed enzymatic and chemical reactions to label surface glycoproteins. The remodeling of the glycoproteome in macrophage polarization is primarily driven by changes in protein expression and sugar donors rather than changes in the protein glycosylation machinery. Comparative analysis of cell-surface glycoproteins between M1 and M2 macrophages reveals phenotype-specific N-glycosylation patterns. Moreover, we identified potential targets for suppressing M2 macrophages, including CD36, FLT1, Siglec11, THSD7A, and SLC12A2. Finally, we characterized the site-specific changes of cell-surface glycoproteins between M1 and M2 macrophages related to their local protein structures and adjacent residues. The dramatic alterations in N-glycosylation sites were located within crucial protein domains, including the immunoglobulin (Ig)-like domain, fibronectin type III domain, and growth factor domain. Global and site-specific analysis of protein glycosylation and surface glycoproteins advance our understanding of different types of macrophages and provide valuable and unprecedented information for future functional and mechanistic investigations of human macrophages, leading to a better understanding of cancer immunity and the development of immunotherapy.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Monocyte, Cell Culture, Macrophage

SUBMITTER: Xing Xu

LAB HEAD: Xing Xu, Kejun Yin, and Ronghu Wu

PROVIDER: PXD053647 | Pride | 2024-08-08

REPOSITORIES: Pride

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			Action	DRS
	612995_E_XX_3_95_WG_1.mzid.gz	Mzid
	613124_E_XX_3_95_WG_7.mzid.gz	Mzid
	613125_E_XX_3_95_WG_6.mzid.gz	Mzid
	613126_E_XX_3_95_WG_5.mzid.gz	Mzid
	613127_E_XX_3_95_WG_4.mzid.gz	Mzid

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Project description:Glycosylation of proteins is one of the most common post-translational modification (PTM) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of the tear film might promote development of chronic eye diseases indicating glycoproteins as valuable source for biomarker discovery or drug target identification. The present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and the characterization of their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we accumulated first native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA and UEA I, termed as 4L) was applied for glycopeptide enrichment from human tear sample digests followed by MS-based detection and localization of their specific N-glycosylation sites. As main result, the present study identified in total 26 N glycosylation sites of 11 N-glycoproteins in tear sample pools of healthy individuals (n=3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP) as well as extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, which were already associated with the pathogenesis of various chronic eye diseases such as the dry eye syndrome (DES). In conclusion, the results of the present study will serve as important tear film N-glycoprotein catalogue for future studies focusing the human tear film and ocular surface-related inflammatory diseases.

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Deciphering the mechanisms of macrophage polarization through comprehensive analysis of protein glycosylation in cells and on the surface

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