N-Glycoproteomic Analysis of FUT1 Knockout DAMI Cell Lines
Objective Glycoproteins are critical in platelet physiology.In a recent study,histo-blood group Lewis y(Ley)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 Ley 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.
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