背景:蛋白O-岩藻糖基转移酶1(POFUT1)过表达,这在许多癌症中观察到,如结直肠癌(CRC),导致与肿瘤过程相关的NOTCH信号传导失调。在罕见的CRC病例中,没有POFUT1过表达,在人类POFUT1中发现了7个错义突变。
方法:产生并纯化人WTPOFUT1的重组分泌形式及其7种突变对应物。使用化学酶促方法,以叠氮标记的GDP-岩藻糖作为供体底物和在大肠杆菌周质中产生的NOTCH1EGF-LD26,在体外测定了它们的O-岩藻糖基转移酶活性,作为相关的受体底物。进行靶向质谱(MS)以定量所有POFUT1蛋白的O-岩藻糖基转移酶能力。
结果:MS分析显示六个POFUT1变体的O-岩藻糖基转移酶活性显着提高(R43H,Y73C,T115A,I343V,D348N,和R364W)与WTPOFUT1相比。
结论:这项研究提供了关于这7个错义突变可能参与结直肠肿瘤的见解。过度活跃的形式可能导致CRC患者POFUT1蛋白靶标如NOTCH受体的O-岩藻糖基化增加,从而导致NOTCH信号传导失调。这是这种关键糖基转移酶功能获得突变的首次证明,调节NOTCH活性,以及其他潜在的糖蛋白。
BACKGROUND: Protein O-fucosyltransferase 1 (POFUT1) overexpression, which is observed in many cancers such as colorectal cancer (CRC), leads to a NOTCH signaling dysregulation associated with the tumoral process. In rare CRC cases, with no POFUT1 overexpression, seven missense mutations were found in human POFUT1.
METHODS: Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their O-fucosyltransferase activities were assayed in vitro using a chemo-enzymatic approach with azido-labeled GDP-fucose as a donor substrate and NOTCH1 EGF-LD26, produced in E. coli periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the O-fucosyltransferase ability of all POFUT1 proteins.
RESULTS: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1.
CONCLUSIONS: This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased O-fucosylation of POFUT1 protein targets such as NOTCH receptors in CRC patients, thereby leading to a NOTCH signaling dysregulation. It is the first demonstration of gain-of-function mutations for this crucial glycosyltransferase, modulating NOTCH activity, as well as that of other potential glycoproteins.