A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2

Valero‐González, Jessika; Leonhard-Melief, Christina; Lira‐Navarrete, Erandi; Jiménez‐Osés, Gonzalo; Hernández-Ruiz, Cristina; Pallarés, María Carmen; Yruela, Inmaculada; Vasudevan, Deepika; Lostao, Anabel; Corzana, Francisco; Takeuchi, Hideyuki; Haltiwanger, Robert S.; Hurtado‐Guerrero, R.

doi:10.1038/nchembio.2019

Cited by 55 publications

(97 citation statements)

References 46 publications

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“…Pulse-chase experiments have shown that C-mannosylation occurs early, potentially before or during folding (13) and likely precedes O-fucosylation, which takes place on folded proteins (37,38). This hypothesis is consistent with our observation that we never see fucose on nonmannosylated peptides.…”

Section: Restitution Of Dpy19l1 -L3 and -L4 In The Triple Knockout supporting

confidence: 82%

Distinct C- mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

Shcherbakova

Tiemann

Buettner

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Thrombospondin type 1 repeats (TSRs) occur in diverse proteins involved in adhesion and signaling. The two extracellular TSRs of the netrin receptor UNC5A contain WxxWxxWxxC motifs that can be C-mannosylated on all tryptophans. A single C-mannosyltransferase , modifying the first two tryptophans, occurs in Caenorhabditis elegans, but four putative enzymes (DPY-19-like 1-4, DPY19L1-4) exist in mammals. Single and triple CRISPR-Cas9 knockouts of the three homologs that are expressed in Chinese hamster ovary cells (DPY19L1, DPY19L3, and DPY19L4) and complementation experiments with mouse homologs showed that DPY19L1 preferentially mannosylates the first two tryptophans and DPY19L3 prefers the third, whereas DPY19L4 has no function in TSR glycosylation. Mannosylation by DPY19L1 but not DPY19L3 is required for transport of UNC5A from the endoplasmic reticulum to the cell surface. In vertebrates, a new C-mannosyltransferase has apparently evolved to increase glycosylation of TSRs, potentially to increase the stability of the structurally essential tryptophan ladder or to provide additional adhesion functions.glycosylation | thrombospondin type 1 repeat | C-mannosylation

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Section: Restitution Of Dpy19l1 -L3 and -L4 In The Triple Knockout supporting

confidence: 82%

Distinct C- mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

Shcherbakova

Tiemann

Buettner

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…We confirm that at least three sites in MIC2 (S 285 , S 485 and T 546 , Figure 3) are modified by GluFuc in addition to seven sites of C-glycosylation. These O-glycosylation sites lie within the previous proposed POFUT2 consensus sequon CXX(S/T)C (11,30,31) and suggest that TgPOFUT2 has a similar substrate preference to metazoan POFUT2 enzymes. Our data demonstrates that these sites are modified to a high occupancy with few peptide species lacking the O-glycans observed in parental MIC2 preparations.…”

Section: Discussionsupporting

confidence: 77%

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

Khurana

Coffey

John

et al. 2018

Preprint

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Toxoplasma gondii is a ubiquitous obligate intracellular eukaryotic parasite that causes congenital birth defects, disease of the immunocompromised and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune response of many eukaryotic parasites and is also of great relevance to vaccine design. Here, we demonstrate that MIC2, the motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat domains (TSR). At least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at O-glycosylation sites. We demonstrate that the addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homologue (TgPOFUT2) encoded by TGGT1_273550. While POFUT2 homologues are important for stabilizing motility associated adhesins and host infection in other apicomplexan parasites, in T. gondii loss of TgPOFUT2 has only a modest impact on MIC2 levels and the wider proteome. Consistent with this, both plaque formation and tachyzoite infectivity are broadly similar in the presence or absence of TgPOFUT2. These findings demonstrate that TgPOFUT2 Oglycosylates MIC2 and that this glycan is dispensable in T. gondii tachyzoites.

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“…This may suggest that GalNAc-T11 is unique among the GalNAc-T isoforms in recognizing folded domains as substrates. Other glycosyltransferases initiating O-Fuc, O-Glc, and O-GlcNAc recognize small folded domains such as epidermal growth factor-like (EGF) and thrombospondin type 1 repeats (TSR) (26,36), and these domains share a design with three conserved disulphide bridges with the LA-modules (37)(38). However, in all cases the acceptor sites are located in the folded domain, while the substrate site for GalNAc-T11 is located in the short linker between the folded domains.…”

Section: Discussionmentioning

confidence: 99%

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

Wang

Mao

Narimatsu

et al. 2018

Journal of Biological Chemistry

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The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved Oglycan-sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptors localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of geneedited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell surface expression and stability of these receptors, but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by approximately 5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.

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A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2

Cited by 55 publications

References 46 publications

Distinct C- mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

Distinct C- mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

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