Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer

Abstract

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.

Significant Roles of Notch O-Glycosylation in Cancer

“…In CRC, POFUT1 silencing inhibited cell proliferation, decreased cell invasion and migration, arrested cell cycle progression, and stimulated CRC cell apoptosis in vitro and suppressed CRC tumor growth and transplantation in vivo [ 130 ]. Furthermore, structure and function studies on seven missense mutations in human POFUT1 in rare CRC cases showed that six of the missense mutations lead to an increase in the protein O -fucosyltransferase activity in vitro [ 131 ]. High levels of POFUT1 were found in glioblastoma (GBM) tissue, and GBM patients with high POFUT1 expression had a shorter survival rate.…”

Mouse WIF1 Is Only Modified with O-Fucose in Its EGF-like Domain III Despite Two Evolutionarily Conserved Consensus Sites

“…Thus, we first determined the propensity of recombinant mouse WIF1 EGF-LDs III and V purified from E.coli BL21 strain to be specifically in vitro modified with O -fucose by recombinant POFUT1 (recPOFUT1). As previously reported [ 11 , 37 , 56 ], O -fucosylation assays were followed either by click chemistry (CuAAC) and blotting technique or by trypsin digestion and mass spectrometry to specifically reveal and/or quantify EGF-LDs O -fucosylation ( Figure 4 ).…”

Hyperdense Pancreatic Ductal Adenocarcinoma: Clinical Characteristics and Proteomic Landscape

“…This may correlate with changes in the tumor microenvironment, such as inevitably diminished drug delivery caused by a reduction in tumor blood vessels. The differentially expressed proteins POFUT1 (24) and PKP2 (25) correlate with angiogenesis and fibrosis in other cancer species. Antiangiogenic therapy may be more effective for hyperdense PDAC patients than for hypodense PDAC patients.…”