POFUT1-mediated O-fucosylation of glycoproteins expressed in the baculovirus Sf9 insect cell expression system.

The baculovirus-insect cell expression system allows addition of O-fucose to EGF-like domains of glycoproteins, following the action of the protein O-fucosyltransferase 1 named POFUT1. In this study, recombinant Spodoptera frugiperda POFUT1 from baculovirus-infected Sf9 cells was compared to recombinant Mus musculus POFUT1 produced by CHO cells. Contrary to recombinant murine POFUT1 carrying two hybrid and/or complex type N-glycans, Spodoptera frugiperda POFUT1 exhibited paucimannose N-glycans, at least on its highly evolutionary conserved across Metazoa NRT site. The abilities of both recombinant enzymes to add in vitro O -fucose to EGF-like domains of three different recombinant mammalian glycoproteins were then explored. In vitro POFUT1-mediated O-fucosylation experiments, followed by click chemistry and blot analyses, showed that Spodoptera frugiperda POFUT1 was able to add O-fucose to mouse NOTCH1 EGF-like 26 and WIF1 EGF-like 3 domains, similarly to the murine counterpart. As proved by mass spectrometry, full-length human WNT Inhibitor Factor 1 expressed by Sf9 cells was also modified with O-fucose. However, Spodoptera frugiperda POFUT1 was unable to modify the single EGF-like domain of mouse PAMR1 with O-fucose, contrary to murine POFUT1. Absence of orthologous proteins such as PAMR1 in insects may explain the enzyme's difficulty in adding O-fucose to a domain that it never encounters naturally.

Cancer-associated Notch receptor variants lead to O-fucosylation defects that deregulate Notch signaling.

NOTCH1 is a transmembrane receptor that initiates a signaling pathway involved in embryonic development of adult tissue homeostasis. The extracellular domain of NOTCH1 is composed largely of epidermal growth factor-like repeats (EGFs), many of which can be O-fucosylated at a specific consensus sequence by protein O-fucosyltransferase 1 (POFUT1). O-fucosylation of NOTCH1 is necessary for its function. The Notch pathway is deregulated in many cancers, and alteration of POFUT1 has been reported in several cancers, but further investigation is needed to assess whether there is deregulation of the Notch pathway associated with mutations that affect O-fucosylation in cancers. Using Biomuta and COSMIC databases, we selected nine NOTCH1 variants that could cause a change in O-fucosylation of key EGFs. Mass spectral glycoproteomic site mapping was used to identify alterations in O-fucosylation of EGFs containing the mutations. Cell-based NOTCH-1 signaling assays, ligand-binding assays, and cellsurface analysis were used to determine the effect of each mutation on Notch activation. Two variants led to a gain of function (GOF), six to a loss of function (LOF), and one had minimal effects. Most GOF and LOF were associated with a change in O-fucosylation. Finally, by comparing our results with known NOTCH1 alterations in cancers from which our mutations originated, we were able to establish a correlation between our results and the known GOF or LOF of NOTCH1 in these cancers. This study shows that point mutations in N1 can lead to alterations in O-fucosylation that deregulate the Notch pathway and be associated with cancer processes.

Fringe GlcNAc-transferases differentially extend O-fucose on endogenous NOTCH1 in mouse activated T cells.

NOTCH1 is a transmembrane receptor that initiates a cell-cell signaling pathway controlling various cell fate specifications in metazoans. The addition of O-fucose by protein O-fucosyltransferase 1 (POFUT1) to epidermal growth factor-like (EGF) repeats in the NOTCH1 extracellular domain is essential for NOTCH1 function, and modification of O-fucose with GlcNAc by the Fringe family of glycosyltransferases modulates Notch activity. Prior cell-based studies showed that POFUT1 modifies EGF repeats containing the appropriate consensus sequence at high stoichiometry, while Fringe GlcNAc-transferases (LFNG, MFNG, and RFNG) modify O-fucose on only a subset of NOTCH1 EGF repeats. Previous in vivo studies showed that each FNG affects naïve T cell development. To examine Fringe modifications of NOTCH1 at a physiological level, we used mass spectral glycoproteomic methods to analyze O-fucose glycans of endogenous NOTCH1 from activated T cells obtained from mice lacking all Fringe enzymes or expressing only a single FNG. While most O-fucose sites were modified at high stoichiometry, only EGF6, EGF16, EGF26, and EGF27 were extended in WT T cells. Additionally, cell-based assays of NOTCH1 lacking fucose at each of those O-fucose sites revealed small but significant effects of LFNG on Notch-Delta binding in the EGF16 and EGF27 mutants. Finally, in activated T cells expressing only LFNG, MFNG, or RFNG alone, the extension of O-fucose with GlcNAc in the same EGF repeats was diminished, consistent with cooperative interactions when all three Fringes were present. The combined data open the door for the analysis of O-glycans on endogenous NOTCH1 derived from different cell types.

Modulation of the NOTCH1 Pathway by LUNATIC FRINGE Is Dominant over That of MANIC or RADICAL FRINGE.

Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG), and RADICAL FRINGE (RFNG). Fringe modification of O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of O-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of O-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG.

The single EGF-like domain of mouse PAMR1 is modified by O-Glucose, O-Fucose and O-GlcNAc.

Epidermal growth factor-like domains (EGF-LDs) of membrane and secreted proteins can be modified by N-glycans and/or potentially elongated O-linked monosaccharides such as O-glucose (O-Glc) found at two positions (O-Glc 1 and O-Glc2), O-fucose (O-Fuc) and O-N-acetylglucosamine (O-GlcNAc). The presence of three O-linked sugars within the same EGF-LD, such as in EGF-LD 20 of NOTCH1, has rarely been evidenced. We searched in KEGG GENES database to list mouse and human proteins with an EGF-LD sequence including one, two, three or four potential O-glycosylation consensus sites. Among the 129 murine retrieved proteins, most had predicted O-fucosylation and/or O-GlcNAcylation sites. Around 68% of EGF-LDs were subjected to only one O-linked sugar modification and near 5% to three modifications. Among these latter, we focused on the peptidase domain-containing protein associated with muscle regeneration 1 (PAMR1), having only one EGF-LD. To test the ability of this domain to be glycosylated, a correctly folded EGF-LD was produced in Escherichia coli periplasm, purified and subjected to in vitro incubations with the recombinant O-glycosyltransferases POGLUT1, POFUT1 and EOGT, adding O-Glc1, O-Fuc and O-GlcNAc, respectively. Using click chemistry and mass spectrometry, isolated PAMR1 EGF-LD was demonstrated to be modified by the three O-linked sugars. Their presence was individually confirmed on EGF-LD of full-length mouse recombinant PAMR1, with at least some molecules modified by both O-Glc1 and O-Fuc. Overall, these results are consistent with the presence of a triple O-glycosylated EGF-LD in mouse PAMR1.

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

The Wnt Inhibitory Factor 1 (Wif1), known to inhibit Wnt signaling pathways, is composed of a WIF domain and five EGF-like domains (EGF-LDs) involved in protein interactions. Despite the presence of a potential O-fucosylation site in its EGF-LDs III and V, the O-fucose sites occupancy has never been demonstrated for WIF1. In this study, a phylogenetic analysis on the distribution, conservation and evolution of Wif1 proteins was performed, as well as biochemical approaches focusing on O-fucosylation sites occupancy of recombinant mouse WIF1. In the monophyletic group of gnathostomes, we showed that the consensus sequence for O-fucose modification by Pofut1 is highly conserved in Wif1 EGF-LD III while it was more divergent in EGF-LD V. Using click chemistry and mass spectrometry, we demonstrated that mouse WIF1 was only modified with a non-extended O-fucose on its EGF-LD III. In addition, a decreased amount of mouse WIF1 in the secretome of CHO cells was observed when the O-fucosylation site in EGF-LD III was mutated. Based on sequence comparison and automated protein modeling, we suggest that the absence of O-fucose on EGF-LD V of WIF1 in mouse and probably in most gnathostomes, could be related to EGF-LD V inability to interact with POFUT1.

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.

In vitro acellular method to reveal O-fucosylation on EGF-like domains.

A hundred of human proteins have one or more EGF-like domains (EGF-LD) bearing the O-fucosylation consensus motif C2X4(S/T)C3 but to date, only a few of them have been shown to be O-fucosylated. The protein O-fucosyltransferase (POFUT1) specifically recognizes correctly folded EGF-LD of the human EGF (hEGF) type and transfers fucose on serine or threonine residue within the O-fucosylation motif. Here, we propose a strategy for a rapid screening for ability of any EGF-LD to be O-fucosylated, using copper-catalyzed azide-alkyne cycloaddition (CuAAC). By an oligonucleotide hybridization approach, double-stranded fragments encoding any EGF-LD can be first rapidly cloned into the prokaryotic vector pET-25b to promote its targeting to periplasm and formation of the three conserved disulfide bonds. After protein production and purification, an in vitro POFUT1-mediated O-fucosylation can be performed with azido GDP-fucose. Successful transfer of O-fucose is finally revealed by blotting technique after CuAAC. In this study, we specially focused on mouse NOTCH1 EGF12 and EGF26, which are both known to be O-fucosylated although having different binding affinities towards POFUT1. Indeed, we clearly showed here that addition of O-fucose by POFUT1 was much more efficient for EGF26 than for EGF12. This experimental approach is rapid and sufficiently sensitive to reveal propensity of any EGF-LD to be O-fucosylated; it is thus useful prior to perform structure-function studies on target proteins containing one or several EGF-LD.

Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice.

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice.