Profiling Glycosyltransferase Activities on the Epithelial-Mesenchymal Transition in Breast Epithelial Cells using peptide arrays and mass spectrometry

 描述(由申请人提供)：使用多肽Asrray和质谱仪分析乳腺上皮细胞上皮-间充质转变中的糖基转移酶活性高通量生物技术已经能够发现识别具有不同表型的癌症亚型的遗传和蛋白质组学特征。然而，由于缺乏高效的高通量分析，基于糖基转移酶介导的翻译后修饰的类似研究工作一直滞后。这种酶谱技术是必要的，因为这些酶对细胞表型有很大的影响。例如，前列腺细胞上皮细胞中纤维连接蛋白被GalNAc-T3和GalNAc-T6糖基化是转化生长因子-β诱导的上皮-间充质转化(EMT)所必需的，这是癌细胞劫持以实现转移的一个发育过程。这个例子与乳腺癌特别相关，因为这种形式的纤维连接蛋白onfFN在乳腺癌组织中上调，而在正常乳腺组织中不存在。此外，GalNAc-T6的过表达诱导永生化乳腺上皮细胞系MCF10A出现EMT样表型。尽管有这些证据，但目前尚不清楚GalNAc-T3和GalNAc-T6是否通过与前列腺细胞类似的机制来调节乳腺组织中的EMT。因此，我的假设是GalNAc-T3和GalNAc-T6通过纤维连接蛋白的糖基化促进乳腺细胞的EMT，它们的活性可以作为EMT的新的生物标志物。该提案旨在研究GalNAc-T3/-T6在乳腺细胞中转化生长因子-βEMT中的作用，并使用一种名为SAMDI质谱仪的创新技术直接从细胞裂解物中描述它们的活性。这种方法使用多肽底物阵列来分析样品中的酶活性，并已证明对包括糖基化在内的几种酶化学研究是有效的。初步数据进一步表明，通过检测三阴性乳腺癌细胞系MDA-MB-231的内源性半乳糖转移酶活性，SAMDI图谱是可行的。第一个目标是合成一个能够区分GalNAc-T3和GalNAc-T6活性的肽库，然后用SAMDI测试GalNAc-T3和GalNAc-T6的活性是否可以用来分析EMT。在第二个目标中，我将研究GalNAc-T3和-T6在多大程度上调节乳腺上皮细胞的EMT。通过过表达和敲除研究，我将检验EMT是否依赖于GalNAc-T3/-T6的表达，以及EMT是否依赖于onfFN的存在。这项建议旨在解决两个关键需求：填补与整个糖生物学领域相关的现有技术的空白，以及提供对乳腺癌社区重要的基本信息。

 DESCRIPTION (provided by applicant): Profiling Glycosyltransferase Activity on the Epithelial-Mesenchymal Transition in Breast Epithelial Cells using Peptide Asrrays and Mass Spectrometry High-throughput biotechnology has enabled discovery of genetic and proteomic profiles that identify cancer subtypes with distinct phenotypes. However, similar efforts to profil based on the posttranslational modifications mediated by glycosyltransferases have lagged due to lack of efficient high-throughput assays. Such enzymatic profiling technology is needed because these enzymes exert dramatic effects on cell phenotype. For example, fibronectin glycosylation by GalNac-T3 and GalNac-T6 in prostate cells epithelial cells is essential for the TGF-beta induced epithelial-mesenchymal transition (EMT), a developmental process that cancer cells hijack to enable metastasis. This example is particularly relevant to breast cancer because this form of fibronectin, onfFN, is upregulated in breast cancer tissue and not present in normal mammary tissue. Moreover, overexpression of GalNac-T6 induce an EMT-like phenotype in MCF10A, an immortalized breast epithelial cell line. Despite these evidence it remains unclear if GalNAc-T3 and GalNAc-T6 regulate EMT in breast tissue by a similar mechanism as that in prostate cells. Therefore my hypothesis that GalNAc-T3 and GalNAc-T6 promote EMT in breast cells via glycosylation of fibronectin and that their activity can serve as novel biomarkers of EMT. This proposal seeks to investigate the role of GalNAc-T3/-T6 on TGF-beta EMT in breast cells and use an innovative technology to profile their activity directly from cell lysate, known as SAMDI mass spectrometry. This method uses peptide substrate arrays to profile enzyme activities in a sample and has demonstrated effectiveness for studying several enzymatic chemistries, including glycosylation. Preliminary data further demonstrate that SAMDI profiling is feasible via detection endogenous galactosyltransferase activity in the triple-negativ breast cancer cell line, MDA-MB-231. The first aim will entail synthesis of a peptide library that can distinguish activities between GalNAc-T3 and GalNAc-T6 and then test by SAMDI whether GalNAc-T3 and GalNac-T6 activities can be used to profile EMT. In the second aim, I will investigate the extent to which GalNAc-T3 and -T6 regulate EMT in breast epithelial cells. Using overexpression and knockdown studies, I will test whether EMT is dependent on the expression of GalNAc-T3/-T6 and whether EMT is dependent on the presence of onfFN. This proposal aims to address two critical needs: fill a gap in available technology relevant to the field of glycobiology as a whole as well as provide fundamental information important to the breast cancer community.