岩藻糖基转移酶1敲除的DAMI细胞系的N-糖基化组学分析  

N-Glycoproteomic Analysis of FUT1 Knockout DAMI Cell Lines

作　　者：朱慧君[1] 陆萍[1] ZHU Huijun;LU Ping(Shanghai Blood Center,Shanghai 200051)

机构地区：[1]上海市血液中心,上海200051

出　　处：《临床输血与检验》2024年第4期440-446,共7页Journal of Clinical Transfusion and Laboratory Medicine

基　　金：国家自然科学基金青年项目(No.82100246);上海市卫生健康委员会卫生行业临床研究专项(No.202140060)资助。

摘　　要：目的糖蛋白对血小板的功能至关重要,最近发现组织血型抗原Lewis y在血小板中有表达。由于血小板无核,不能直接操纵其基因,我们在先前的研究中构建了岩藻糖基转移酶1(FUT1)敲除的人巨核细胞白血病细胞DAMI细胞系。通过对FUT1敲除的DAMI细胞系进行全范围的N-糖基化组学分析以研究FUT1对DAMI中糖蛋白表达及功能的影响,为研究其在血小板中的功能提供思路和Lewis y修饰的候选蛋白。方法FUT1敲除的DAMI细胞和野生型DAMI细胞各三个复样,提取蛋白质并消化成肽段,使用混合凝集素富集后用N-糖酰胺酶F处理切除糖蛋白上的糖链,随后进行高效液相色谱-质谱分析。对所得结果进行亚定位分析、基因功能分析和KEGG通路分析。最后,对FUT1敲除及野生型DAMI细胞进行静态和流动粘附实验,以验证前述分析中差异化糖肽富集的功能。结果在FUT1敲除和野生型DAMI细胞中共鉴定到1110个N连接的糖基化位点、792个N-糖肽,以及592个糖蛋白。相比于野生型DAMI细胞,FUT1敲除细胞中有59个N-糖肽的表达量有显著变化,其中20个显著下调(log_(2)(倍数变化)<‒1)而39个显著上调(log_(2)(倍数变化)>1)。对结果的生物信息学分析显示FUT1敲除显著影响的这些糖肽主要包含在粘附相关的通路和生物学过程。粘附实验显示FUT1敲除的DAMI细胞粘附能力显著低于野生型细胞。结论N-糖基化组学分析揭示FUT1对DAMI细胞的粘附功能有影响,为后续在血小板中功能的研究提供思路。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 1110 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(log_(2)(fold-change)<‒1)and 39 were significantly up-regulated(log_(2)(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

关 键 词：岩藻糖基转移酶 血小板功能 N-糖基化组学 细胞粘附 

分 类 号：R446[医药卫生—诊断学]