Abstract: Objectives Glycoproteins are critical in platelet physiology. In a recent study, histo-blood group Lewis y (Le^y) antigen was found to be expressed on platelets. Since platelets are anucleated and we cannot directly manipulate their genes, we constructed an fucosyltransferase 1-knockout human megakaryocytic leukemia cell line (DAMI) in a previous study. A comprehensive N-glycoproteomic analysis was performed on the FUT1 knockout DAMI cell line, to reveal the roles of FUT1 on DAMI cell function and candidate proteins for Le^y modification. Methods Proteins from FUT1-knockout DAMI cells and wild-type DAMI cells (each sample in triplicate) were extracted and digested into peptides with trypsin. Glycoproteins were enriched by mixed lectins and treated with N-glycoamidase (PNGase) F to remove glycans from glycoprotein, and then detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Subcellular localization, gene function and KEGG pathway were performed in the identified N-glycopeptides. FUT1-knockout and wild-type DAMI cells were evaluated by static adhesion and flow adhesion experiments to verify the functions enriched in glycopeptides with differential expression. Results 1 110 N-linked glycosylation sites, 792 N-glycopeptides, and 592 glycoproteins were identified in FUT1 knockout and wild-type DAMI cells. Compared with wild-type DAMI cells, the expression levels of 59 N-glycopeptides in FUT1 knockout cells were significantly changed, of which 20 were significantly down-regulated (log₂ (fold-change)<-1) and 39 were significantly up-regulated (log₂ (fold-change)>1). The bioinformatics analysis showed that the knockout of FUT1 significantly changed the expression of glycopeptides involved in adhesion-related pathways and biological processes. Cell adhesion experiments showed that the adhesion ability of FUT1 knockout DAMI cells was significantly lower than that wild-type cells. Conclusion N-glycosylation analysis revealed that FUT1 had an effect on DAMI cell adhesion, which further provides ideas for subsequent research on its function in platelets.

Key words: Fucosyltransferase, Platelet function, N-glycoproteomics, Cell adhesion