Incomplete clearance of apoptotic cells by core 1-derived O-glycan-deficient resident peritoneal macrophages

The core 1 β1,3-galactosyltransferase-specific molecular chaperon (Cosmc) is essential for the synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of core 1-derived O-glycans in macrophages, we exploited the LysM-Cre transgene to generate a conditional Cosmc mutant allele (conditional Cosmc knockout; cKO) in myeloid cells. cKO mice developed normally with no gross phenotypic abnormalities or abnormal peripheral blood counts. Resident peritoneal macrophages (rpMacs) of cKO mice exhibited impaired engulfment of apoptotic cells but showed normal macrophage differentiation and counts. T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim4) is a phosphatidylserine (PS) receptor expressed on rpMacs and possesses a heavily O-glycosylated domain. Tim4 tethers apoptotic cells through PS binding. Expression of the Tim4 transcript was unchanged in cKO rpMacs, whereas flow cytometric, Western and dot blot analyses revealed that Tim4 protein expression in cKO rpMacs was significantly lower than that in wild-type (WT) rpMacs. Moreover, the expression levels of other efferocytosis-related molecules, Mertk, Itgav and Itgb3, were normal in rpMacs. In addition, hypoglycosylated Tim4-FLAG fusion protein sufficiently recognized PS. These results demonstrated that core 1-derived O-glycan is required for Tim4-dependent normal efferocytosis and may contribute to the stable expression of the Tim4 glycoprotein.