Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer

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.

Findings: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1.

Interpretation: 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.

EGF-like domain; POFUT1; colorectal cancer; mutations.