糖鏈對(duì)腫瘤細(xì)胞生物學(xué)行為的影響及機(jī)制研究年度報(bào)告

張嘉寧 張延 燕秋 賈莉 汪淑晶

摘 要：該研究其主要目的是利用糖基轉(zhuǎn)移酶酶學(xué)活性檢測(cè)法，篩選替代糖基轉(zhuǎn)移酶天然糖苷供體底物的糖探針，建立體外糖基化修飾蛋白、標(biāo)記糖鏈的方法，高通量篩選發(fā)現(xiàn)腫瘤相關(guān)糖基轉(zhuǎn)移酶對(duì)應(yīng)的靶蛋白和糖鏈結(jié)構(gòu)，并發(fā)現(xiàn)腫瘤特征性糖基轉(zhuǎn)移酶抑制劑。并且，觀察了一系列糖基轉(zhuǎn)移酶及相關(guān)特征糖鏈在腫瘤中的表達(dá)及結(jié)構(gòu)特點(diǎn)。得到如下結(jié)論：關(guān)于糖基轉(zhuǎn)移酶抑制劑發(fā)現(xiàn)的相關(guān)工作已達(dá)該領(lǐng)域領(lǐng)先水平。發(fā)現(xiàn)的高通量篩選方法可以推動(dòng)糖基轉(zhuǎn)移酶抑制劑的發(fā)現(xiàn)工作。同時(shí)，已發(fā)現(xiàn)的ppGlaNAc-T抑制劑可以作為分子探針工具研究此類酶的生物學(xué)功能，并為腫瘤等疾病提供基于糖靶標(biāo)的治療藥物先導(dǎo)物。此外，該研究還揭示了唾液酰基轉(zhuǎn)移酶ST6Gal-I的表達(dá)調(diào)控規(guī)律。發(fā)現(xiàn)了α5-integrin的α2，6唾液酸化修飾與肝癌細(xì)胞粘附呈正相關(guān)。提示，Cav-1上調(diào)ST6Gal-I表達(dá)及α2，6連接唾液酸水平是促進(jìn)肝癌細(xì)胞轉(zhuǎn)移的早期事件，而Cav-1、ST6Gal-I及ECM黏附的順序調(diào)控有可能為轉(zhuǎn)移性肝癌的早期治療提供新的靶點(diǎn)和思路。提出了糖蛋白N-糖鏈作為預(yù)測(cè)白血病耐藥標(biāo)志物的可能性。即N-糖鏈、糖基因的差異表達(dá)的確與白血病耐藥具有相關(guān)性，是潛在的白血病耐藥相關(guān)標(biāo)志物。

關(guān)鍵詞：糖基轉(zhuǎn)移酶 探針 腫瘤 耐藥

Annual Report-Effect and Mechanism of Sugar Chains on the Biological Behavior

of Tumor Cells

Zhang Jianing1 Zhang Yan2 Yan Qiu1 Jia Li1 Wang Shujing1

（1.Dalian Medical University；2.Shanghai Jiao Tong University）

Abstract：This study suggests that the new Y subfamily of ppGalNAc-Ts plays an important role in protein glycosylation； characterizing their functions will provide new insight into the role of ppGalNAc-Ts. Disulfide- and terminal alkyne-modified magnetic silica particles （DA-MSPs） were synthesized and used to covalently capture and reductively release azido glycopeptides via click chemistry and dithiothreitol treatment. Using DA-MSPs， an efficient and specific enrichment method for separating azido glycopeptides has been developed. The alterations of integrin glycosylation play a crucial role in tumor metastasis. Our previous studies indicated that caveolin-1 promoted the expression of the key a2，6-sialytransferase ST6Gal-I and fibronectin-mediated adhesion of mouse hepatocarcinoma cell. Herein， we investigated the role of a2，6-sialylated a5-integrin in the adhesion of mouse hepatocarcinoma H22 cell. We demonstrated that caveolin-1 up-regulated cell surface a2，6-linked sialic acid via stimulating ST6Gal-I transcription. Cell surface a2，6-sialylation was required for integrin a5b1-dependent cell adhesion to fibronectin， and an increase in a2，6-linked sialic acid on a5-subunit facilitated fibronectin-mediated focal adhesion kinase phosphorylations， suggesting that a2，6-sialylated a5-subunit promoted integrin a5b1-dependent cell adhesion. B4GALT family consists of seven members， which encode corresponding enzymes known as type II membrane-bound glycoproteins. These enzymes catalyze the biosynthesis of different glycoconjugates and saccharide structures， and have been recognized to be involved in various diseases. In this study， we sought to determine the expressional profiles of B4GALT family in four pairs of parental and chemoresistant human leukemia cell lines and in bone marrow mononuclear cells （BMMC） of leukemia patients with multidrug resistance （MDR）. The results revealed that B4GALT1 and B4GALT5 were highly expressed in four MDR cells and patients， altered levels of B4GALT1 and B4GALT5 were responsible for changed drug-resistant phenotype of HL60 and HL60/adriamycin-resistant cells. Thus， we propose that B4GALT1 and B4GALT5， two members of B4GALT gene family， are involved in the development of MDR of human leukemia cells， probably by regulating the activity of Hh signaling and the expression of P-gp and MRP1.

Key Words：Glycotransferases； Probe； Tumor； Resistance

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