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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 i...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352195/ https://www.ncbi.nlm.nih.gov/pubmed/32486426 http://dx.doi.org/10.3390/cancers12061430 |
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author | Deschuyter, Marlène Pennarubia, Florian Pinault, Emilie Legardinier, Sébastien Maftah, Abderrahman |
author_facet | Deschuyter, Marlène Pennarubia, Florian Pinault, Emilie Legardinier, Sébastien Maftah, Abderrahman |
author_sort | Deschuyter, Marlène |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7352195 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-73521952020-07-15 Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer Deschuyter, Marlène Pennarubia, Florian Pinault, Emilie Legardinier, Sébastien Maftah, Abderrahman Cancers (Basel) Article 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. MDPI 2020-05-31 /pmc/articles/PMC7352195/ /pubmed/32486426 http://dx.doi.org/10.3390/cancers12061430 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Deschuyter, Marlène Pennarubia, Florian Pinault, Emilie Legardinier, Sébastien Maftah, Abderrahman Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer |
title | Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer |
title_full | Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer |
title_fullStr | Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer |
title_full_unstemmed | Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer |
title_short | Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer |
title_sort | functional characterization of pofut1 variants associated with colorectal cancer |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352195/ https://www.ncbi.nlm.nih.gov/pubmed/32486426 http://dx.doi.org/10.3390/cancers12061430 |
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