Deschuyter Marlène, Pennarubia Florian, Pinault Emilie, Legardinier Sébastien, Maftah Abderrahman
PEIRENE, EA 7500, Glycosylation and Cell Differentiation, Faculty of Sciences and Technology, University of Limoges, F-87060 Limoges, France.
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
Cancers (Basel). 2020 May 31;12(6):1430. doi: 10.3390/cancers12061430.
Protein -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 overexpression, seven missense mutations were found in human POFUT1.
Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their -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 periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the -fucosyltransferase ability of all POFUT1 proteins.
MS analyses showed a significantly higher -fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1.
This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased -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.
蛋白质岩藻糖基转移酶1（POFUT1）过表达在许多癌症中都有观察到，如结直肠癌（CRC），它会导致与肿瘤发生过程相关的NOTCH信号失调。在罕见的无POFUT1过表达的结直肠癌病例中，在人类POFUT1中发现了七个错义突变。
制备并纯化了人野生型POFUT1及其七个突变体的重组分泌形式。使用化学酶法，以叠氮标记的GDP-岩藻糖作为供体底物，以外周质中产生的NOTCH1 EGF-LD26作为相关受体底物，在体外测定它们的岩藻糖基转移酶活性。采用靶向质谱（MS）对所有POFUT1蛋白的岩藻糖基转移酶能力进行定量。
质谱分析表明，与野生型POFUT1相比，六个POFUT1变体（R43H、Y73C、T115A、I343V、D348N和R364W）的岩藻糖基转移酶活性显著更高。
本研究为这七个错义突变在结直肠肿瘤中的可能作用提供了见解。活性增强的形式可能导致结直肠癌患者中POFUT1蛋白靶点（如NOTCH受体）的岩藻糖基化增加，从而导致NOTCH信号失调。这是首次证明这种关键糖基转移酶的功能获得性突变可调节NOTCH活性以及其他潜在糖蛋白的活性。
