Sialylation and desialylation dynamics of monocytes upon differentiation and polarization to macrophages

Sialic acids (SAs) often exist as the terminal sugars of glycan structures of cell surface glycoproteins and glycolipids. The level and linkages of cell surface SAs, which are controlled by both sialylation and desialylation processes and environment cues, can dramatically impact cell properties and represent different cellular status. In this study, we systematically examined the sialylation and desialylation profiles of THP-1 monocytes after differentiation to M0 macrophages, and polarization to M1 and M2 macrophages by the combination of LC-MS/MS, flow cytometry and confocal microscopy. Interestingly, both α2-3- and α2-6-linked SAs on the cell surface decreased after monocytes were differentiated to macrophages, which was in accordance with the increased level of free SA in the cell culture medium and the elevated activity of endogenous Neu1 sialidase. Meanwhile, the siaoglycoconjugates inside the cells increased as confirmed by confocal microscopy and the total SA inside the cells increased as determined by LC-MS/MS. Western blot analysis showed higher expression levels of sialyltransferases, including ST3Gal-I, ST3Gal-V, ST6Gal-I and ST6GalNAc-II. Further, upon polarization, the cell surface sialylation levels of M1 and M2 macrophages remained the same as M0 macrophages, while a slight decrease of cellular SAs in the M1 macrophages but increase in the M2 macrophages were confirmed by LC-MS/MS.