人乳腺癌细胞多肽-N-乙酰半乳糖胺转移酶4（ppGalNAc-T4）新底物的发现与功能研究

Polypeptide N-acetylgalacosaminyltransferases (ppGalNAc-Ts,Ts) catalyze the O-linked of N-acetylgalacosamin (GalNAc) to serine and threonine residues of glycoprotiens and initiate the first step of O-GalNAc glycosylation of protein. So far, 20 isoenzymes belong to ppGalNAc-Ts family but isoform specific substrate structure, O-GalNAc glycosylation sites and function of ppGalNAc-Ts are still to be elucidated. Our evidences from Human breast cancer cells suggested that the transcription factor FOXA1 may function as the substrate of Polypeptide N-acetylgalacosaminyltransferase 4, ppGalNAc-T4. The main objects of this proposal are: 1) to identify and characterize a unique or novel and potential ppGalNAc-T4 specific substrate peptide structures and O-GalNAc glycosylation sites in Human breast cancer cells by the so-called “SimpleCell (SC)” comparative O-glycoproteome strategy suggested by Dr. Henrik Clausen with some modifications. The COSMC and/or GANLT4 gene will be targeting deleted in Human breast cancer cells respectively. O-GalNAc glycopeptides yield from proteinase digestion are harvested and enriched by Lectin Affinity Charomatography column. By LC/MS analysis, candidates of ppGalNAc-T4 specific substrate peptide and potential O-GalNAc glycosylation sites will be characterized. 2) To conform these candidates, the synthetic ppGalNAc-T4 specific substrate peptide and potential O-GalNAc glycosylation sites are glycosylated in vitro by the recombinated ppGalNAc-T4 enzyme protein. 3) To determine the total and ppGalNAc-T4 specific O-GalNAc glycosylation sites of FOXA1 by LC/MS analysis of FOXA1 protein expressed in Human breast cancer cells with or without ppGalNAc-T4 expression. 4) To validate the function of ppGalNAc-T4 and O-GalNAc glycosylation sites of FOXA1 in Human breast cancer cells. This study will contribute to the understanding the role of O-GalNAc glycosylation.

糖蛋白O-GalNAc糖基化的起始由多肽-N-乙酰半乳糖胺转移酶（ppGalNAc-Ts，简称Ts）催化N-乙酰半乳糖胺（GalNAc）与多肽链上丝氨酸或苏氨酸残基O-相连，而延伸依赖分子伴侣COSMC。Ts家族至少有20种同工酶,其中ppGalNAc-T4（简称T4）特异的底物糖肽结构、糖基化位点及功能不明。申请人在人乳腺癌组织和细胞中观察到T4表达且转录因子FOXA1可能是T4底物之一。本研究拟1）在人乳腺癌细胞中，采用COSMC或/和GANLT4基因敲除的SimplyCell比较O-糖蛋白质组学策略，寻找T4特异的底物肽/糖肽和糖基化位点；2）确定FOXA1中T4特异的糖基化位点；3）用纯化的T4酶、FOXA1蛋白与合成肽，在体外验证T4特异的底物糖基化及其位点；3）考查T4和FOXA1等底物T4特异的O-糖基化对人乳腺癌细胞生物学行为的影响与分子机制，旨在发现T4的新底物和新功能。

蛋白质O-GalNAc糖基化由多肽-N-乙酰半乳糖胺转移酶（ppGalNAc-Ts）起始催化N-乙酰半乳糖胺（GalNAc）与多肽链上丝氨酸或苏氨酸残基O-相连。其中ppGalNAc-T4（T4）特异的底物糖蛋白底物和功能不明。本研究在人乳腺癌细胞通过Cas9技术敲除T4和分子伴侣COSMC，利用Simple Cell策略比较分析获得了多种T4酶的特异性底物候选蛋白；进一步利用蛋白质谱分析获得了底物蛋白FOXA1和TGFR2的O-GalNAc糖基化位点分别为S355和S31；通过考查O-GalNAc对人乳腺癌细胞生物学行为的影响与分子机制，揭示了T4催化的O-GalNAc糖基化抑制了TGFR1和R2的二聚化，通过抑制TGFβ信号通路的激活最终阻止乳腺癌细胞的EMT过程和肿瘤细胞转移。本研究的相关结果为乳腺癌诊断、治疗提供新的理论依据。