Galactose up-regulates the level of Siglec-9 glycan ligand in endothelial cells to inhibit macrophage activity

Abstract

Objective To investigate the effect and mechanism of galactose on the expression of sialic-acid-binding immunoglobulin-like lectin 9 (Siglec-9) glycan ligand on human primarily cultured pulmonary artery endothelial cells. Methods The expression of Siglec-9 glycan ligand on the endothelial cell surface and the type of glycosidic bonds at the end of this ligand were identified by flow cytometry. Endothelial cells were treated with L-glucose, glucose, N-acetylglucose, mannose, N-acetylmannose, galactose, sialic acid and sucrose for 48 h, and the expression of Siglec-9 glycan ligand in endothelial cells was detected by flow cytometry. The endothelial cells were divided into the control group and the galactose group (n=3). Western blotting, flow cytometry, and immunofluorescence assay were employed to investigate the impact of galactose on the Siglec-9 glycan ligand in endothelial cells. After α2-3, 6, 8 sialidase treatment for endothelial cells, Western blotting was used to detected the effect of galactose on the recovery time of Siglec-9 glycan ligand in endothelial cells. Endothelial cells were treated with galactose, Western blotting was used to detect and analyze the expression levels of intracellular sialic acid synthetases (GNE, NANS, NANP, CMAS, NPL and ST3Gals), and the mRNA expression levels of the relevant proteins were verified by RT-qPCR. Western blotting was used to detect the changes of Siglec-9 glycan ligand level in endothelial cells after siRNA knockdown in NANP, CMAS and ST3Gal-Ⅲ. The effect of the increase of Siglec-9 ligand on apoptosis and phagocytosis of macrophages was analyzed by macrophage co-culture experiments. Results Endothelial cells showed the expression of Siglec-9 ligand and the ligand was a sialic acid glycoprotein linked to α 2-3 sialic acid at the end. Compared with the control group, the expression level of this ligand on endothelial cells in the galactose group was increased significantly (P < 0.01), but the addition of galactose had no effect on the self-recovery time of the ligand. Compared with the control group, the expression levels of NANP, CMAS and ST3Gal-Ⅲ in endothelial cells treated with galactose were increased (P < 0.05, P < 0.01), respectively. The results of RT-qPCR verification were consistent with those results. After the expression of NANP, CMAS or ST3Gal-Ⅲ was inhibited, the level of Siglec-9 glycan ligand in endothelial cells was decreased (P < 0.01). In the co-culture experiment, compared with the untreated group, galactose-treated endothelial cells promoted the apoptosis of macrophages (P < 0.01) and reduced their phagocytosis (P < 0.05). Conclusion Galactose up-regulates the level of Siglec-9 glycan ligand on endothelial cells through the NANP-CMAS-ST3Gal-Ⅲ pathway, thereby inhibiting the activity of macrophages.

Keywords

• galactose
• endothelial cells
• siglec-9 glycan ligand
• sialic acid synthase
• macrophage