岩藻糖基转移酶1敲除的DAMI细胞系的N-糖基化组学分析*

doi:10.3969/j.issn.1671-2587.2024.04.002

临床输血与检验 ›› 2024, Vol. 26 ›› Issue (4): 440-445.DOI: 10.3969/j.issn.1671-2587.2024.04.002

岩藻糖基转移酶1敲除的DAMI细胞系的N-糖基化组学分析^*

朱慧君, 陆萍

上海市血液中心,上海 200051

收稿日期:2024-06-21 出版日期:2024-08-20 发布日期:2024-09-23
通讯作者: 陆萍,主要从事血小板免疫血液学研究,（E-mail）luping@sbc.org.cn。
作者简介:朱慧君,主要从事血小板免疫血液学研究,（E-mail）zhuhuijun@sbc.org.cn。
基金资助:
*本课题受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

N-Glycoproteomic Analysis of FUT1 Knockout DAMI Cell Lines

ZHU Huijun, LU Ping

Shanghai Blood Center, Shanghai 200051

Received:2024-06-21 Online:2024-08-20 Published:2024-09-23

摘要/Abstract

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1 110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（log₂（倍数变化）<-1）而39个显著上调（log₂（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。

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

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

中图分类号:

R446

朱慧君, 陆萍. 岩藻糖基转移酶1敲除的DAMI细胞系的N-糖基化组学分析^*[J]. 临床输血与检验, 2024, 26(4): 440-445.

ZHU Huijun, LU Ping. N-Glycoproteomic Analysis of FUT1 Knockout DAMI Cell Lines[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2024, 26(4): 440-445.

参考文献

[1] LI R H,HOFFMEISTER K M,FALET H.Glycans and the platelet life cycle[J]. Platelets,2016,27(6):505-511.
[2] FALET H,RIVADENEYRA L,HOFFMEISTER K M.Clinical impact of glycans in platelet and megakaryocyte biology[J]. Blood,2022,139(22):3255-3263.
[3] WANG Y C,JOBE S M,DING X K,et al.Platelet biogenesis and functions require correct protein O-glycosylation[J]. Proc Natl Acad Sci USA,2012, 109(40):16143-16148.
[4] VLACHADIS N,TSAMADIAS V,VRACHNIS N,et al.Associations of combined polymorphisms of the platelet membrane glycoproteins Ia and IIIa and the platelet-endothelial cell adhesion molecule-1 and P-Selectin genes with IVF implantation failures[J]. J Obstet Gynaecol,2017,37(3):363-369.
[5] NURDEN A T.Platelet membrane glycoproteins:a historical review[J]. Semin Thromb Hemost,2014, 40(5):577-584.
[6] KUNICKI T J,CHELI Y,MOROI M,et al.The influence of N-linked glycosylation on the function of platelet glycoprotein VI[J]. Blood,2005,106(8):2744-2749.
[7] PETERSON R,CHEAH W Y,GRINYER J,et al.Glycoconjugates in human milk:protecting infants from disease[J]. Glycobiology,2013,23(12):1425-1438.
[8] DETTKE M,PÁLFI G,LOIBNER H. Activation-dependent expression of the blood group-related lewis Y antigen on peripheral blood granulocytes[J]. J Leukoc Biol,2000,68(4):511-514.
[9] ZHU H J,MA Q Q,LI R S,et al.Blood group-related antigen Le^y on human platelets and its involvement in platelet aggregation via a possible interaction with CD61[J]. Transfusion,2020,60(9):2130-2138.
[10] BLANAS A,SAHASRABUDHE N M,RODRÍGUEZ E,et al. Fucosylated antigens in cancer:an alliance toward tumor progression,metastasis,and resistance to chemotherapy[J]. Front Oncol,2018,8:39.
[11] LEV P R,GOETTE N P,GLEMBOTSKY A C,et al.Production of functional platelet-like particles by the megakaryoblastic DAMI cell line provides a model for platelet biogenesis[J]. Platelets,2011,22(1):28-38.
[12] 朱慧君,马勤勤,赵俸涌,等. 组织血型抗原Le^y低表达的人巨核细胞系的建立和鉴定[J]. 中国输血杂志, 2022, 35(9):891-895.
[13] WIŚNIEWSKI J R,ZOUGMAN A,NAGARAJ N,et al. Universal sample preparation method for proteome analysis[J]. Nat Methods,2009,6(5):359-362.
[14] SCHULZ C,VON BRÜHL M L,BAROCKE V,et al. EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall[J]. J Thromb Haemost, 2011,9(5):1007-1019.
[15] HANDIN R I.DAMI cell line[J]. Blood,1997,89(11):4238.
[16] GUIDETTI G F,CANOBBIO I,TORTI M.PI3K/Akt in platelet integrin signaling and implications in thrombosis[J]. Adv Biol Regul,2015,59:36-52.
[17] CHEN Z S,LI T L,KAREEM K,et al.The role of PI3K/Akt signaling pathway in non-physiological shear stress-induced platelet activation[J]. Artif Organs,2019,43(9):897-908.
[18] WEBER K S,ALON R,KLICKSTEIN L B.Sialylation of ICAM-2 on platelets impairs adhesion of leukocytes via LFA-1 and DC-SIGN[J]. Inflammation,2004,28(4):177-188.
[19] GARCÍA-VALLEJO J J,VAN LIEMPT E,DA COSTA MARTINS P,et al. DC-SIGN mediates adhesion and rolling of dendritic cells on primary human umbilical vein endothelial cells through LewisY antigen expressed on ICAM-2[J]. Mol Immunol,2008,45(8):2359-2369.
[20] JIN X,BU Q P,ZOU Y Y,et al.Lewis-antigen-containing ICAM-2/3 on Jurkat leukemia cells interact with DC-SIGN to regulate DC functions[J]. Glycoconj J, 2018,35(3):287-297.
[21] KUO C H,CHEN P K,CHANG B I,et al.The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen[J]. Blood,2012,119(5):1302-1313.
[22] TERRANEO L,AVAGLIANO L,CARETTI A,et al.Expression of carbohydrate-antigen sialyl-Lewis a on colon cancer cells promotes xenograft growth and angiogenesis in nude mice[J]. Int J Biochem Cell Biol, 2013,45(12):2796-2800.
[23] HOU R,JIANG L,LIU D W,et al.Lewis(y) antigen promotes the progression of epithelial ovarian cancer by stimulating MUC1 expression[J]. Int J Mol Med, 2017,40(2):293-302.
[24] LIN W L,CHEN C C,SHI G Y,et al.Monocytic thrombomodulin promotes cell adhesion through interacting with its ligand,Lewis^y[J]. Immunol Cell Biol, 2017,95(4):372-379.

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

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics

本文评价

关于本刊

作者中心

在线期刊

访问统计

联系我们

[1]	王笑欢, 郭凯, 刘倩, 牛子健, 王孟键, 郭婧, 马曙轩. 凝血和血小板功能分析仪在检测川崎病患儿不同时期凝血功能中的应用[J]. 临床输血与检验, 2023, 25(5): 607-612.
[2]	张笑盼, 宋继军, 马曙轩. 血栓弹力图在儿童免疫性血小板减少症治疗中的应用[J]. 临床输血与检验, 2017, 19(3): 240-242.