Deciphering the mechanisms of macrophage polarization through comprehensive analysis of cellular and cell-surface protein glycosylation.

Targeting tumor-associated macrophages holds great promise for cancer immunotherapy. Surface glycoproteins in macrophages regulate their interactions with tumor cells and represent emerging therapeutic targets. Given the importance of glycoproteins in immunity and cancer, we systematically investigate the total and surface glycoproteomes in polarized macrophages using multiplexed proteomics coupled with selective enrichment. In monocyte-derived macrophages, distinct changes of N-glycoproteins are observed between M1 and M2 phenotypes, with more pronounced alterations in pro-inflammatory M1. In M1, glycoproteins involved in chondroitin sulfate-type glycosaminoglycan (CS-GAG) biosynthesis are upregulated, validated by the elevated CS-GAG levels, and enzymes associated with nucleotide sugar biosynthesis are also upregulated, consistent with the increased hexokinase activity. Furthermore, surface glycoproteomic analysis of primary human macrophages with M1 or M2 phenotype (HM1 and HM2) reveals phenotype-specific remodeling: HM1 display enhanced innate immune signaling, whereas surface glycoproteins related to adhesion and endocytosis are upregulated in HM2. Integrative analyses of cell-surface glycoproteomic, transcriptomic, and tissue-specific datasets enable prioritization of candidate surface proteins associated with M2 phenotype, including LIPA, CD200R1, and CR1, and phenotype-specific changes of several surface glycoproteins are validated by immunofluorescence imaging. Together, this study provides insights into macrophage functional specialization and advances our understanding of cell-surface glycoproteins.