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.

Highlights

  • Colorectal cancer (CRC) is the third most diagnosed cancer worldwide, with 1.8 million cases in 2018 and 880,792 deaths according to the World HealthOrganization

  • Protein O-fucosyltransferase 1 (POFUT1) proteins carried by colorectal cancer (CRC) patients, this study provides additional information on the structure–function relationships of this glycosyltransferase

  • When structure-function studies should be performed on POFUT1, major drawbacks related to the isolation of this glycosyltransferase should be considered due to its status of endoplasmic reticulum (ER)-resident protein

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Summary

Introduction

Colorectal cancer (CRC) is the third most diagnosed cancer worldwide (second in females and third in males), with 1.8 million cases in 2018 and 880,792 deaths according to the World Health. Given the importance of the O-fucosylation in the regulation of Notch receptors-ligands interactions and its consequences on Notch signaling activation, we chose to investigate the functional significance of these seven POFUT1 variants found in CRC. For this purpose, we first produced and purified soluble forms of WT and mutated POFUT1 variants in stable CHO cell lines and a correctly folded and non-glycosylated EGF-LD, as a substrate, in the bacterial periplasm. POFUT1 proteins carried by CRC patients, this study provides additional information on the structure–function relationships of this glycosyltransferase

Frequency and Location of Missense Mutations Affecting POFUT1 in CRC
Production of Soluble Forms for Human WT and Mutated POFUT1 Variants
Production of Soluble Forms for Human
Western
Evidencing of the O-Fucosyltransferase Activity for POFUT1 Variants
15 High exposure
Quantification
Discussion
Location
Plasmid Constructs to Produce Recombinant EGF-LDs and POFUT1 Variants
Cell Culture and Transfection
Cell Lines
Protein Production and Purification
SDS-PAGE and Blotting Techniques
Glycosyltransferase Reaction
Targeted Mass Spectrometry
Alignments and Automatic Molecular Modelling
4.10. Statistical Analyses
Conclusions
Full Text
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