Protein O-Fucosyltransferase 1 Undergoes Interdomain Flexibility in Solution

Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle.ARAID: MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P and PID2019-105451GBI00 to RH-G, CTQ2017-85658-R and CTQ2014-56370-R to AO)Gobierno de Aragón (E35_R20 and LMP58_18)FEDER (2014-2020) funds for ‘Building Europe from Aragón’Juan de la Cierva fellowship IJCI-2017-3287

Structural Insights into the Mechanism of Protein O-Fucosylation

Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel SN1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future

Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4

17 pags, 4 figs, 2 tabsMucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT GAGAGAGT TPGPG (containing two α-GalNAc glycosylated Thr (T ), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.We thank synchrotron radiation sources DLS (Oxford) and in particular beamline I03 (experiment number MX10121-15). We thank ARAID, MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P, CTQ2015-67727-R, CTQ2015-70524-R, and CTQ2017-85496-P), AGAUR (SGR2017-1189), the National Institutes of Health (R01-GM113534, and instrument Grant GM113534-01S to T. A. Gerken), the Danish National Research Foundation (DNRF107), the FCT-Portugal [UID/Multi/04378/2013 cofinanced by the FEDER (POCI- 01-0145-FEDER-007728)], and the DGA (E34_R17) for financial support. I. Compañón thanks Universidad de La Rioja for the FPI grant. F. Marcelo thanks FCT-Portugal for IF Investigator grant (IF/00780/2015) and PTNMR supported by Project 022161. E. Lira-Navarrete acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 cofunded by the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Curie Actions. H. Coelho and J. Jiménez-Barbero thank EU for the TOLLerant project. The research leading to these results has also received funding from the FP7 (2007−2013) under BioStruct-X (Grant agreement 283570 and BIOSTRUCTX_5186). We would also like to acknowledge the assistance of Juwan Lee in obtaining the GalNAc-T4 random peptide motif

Sars-cov-2 seroprevalence in household domestic ferrets (Mustela putorius furo)

Animal infections with SARS-CoV-2 have been reported in different countries and several animal species have been proven to be susceptible to infection with SARS-CoV-2 both naturally and by experimental infection. Moreover, infections under natural conditions in more than 20 mink farms have been reported where humans could have been the source of infection for minks. However, little information is available about the susceptibility of pet animals under natural conditions and currently there is no SARS-CoV-2 epidemiological assessment occurrence in household ferrets. In this study, the presence of SARS-CoV-2 antibodies was evaluated in serum samples obtained from 127 household ferrets (Mustela putorius furo) in the Province of Valencia (Spain). Two ferrets tested positive to SARS-CoV-2 (1.57%) by in-house enzyme-linked immunosorbent assay based on receptor binding domain (RBD) of Spike antigen. Furthermore, anti- RBD SARS-CoV-2 antibodies persisted at detectable levels in a seropositive SARS-CoV-2 domestic ferret beyond 129 days since the first time antibodies were detected. This study reports for the first time the evidence of household pet ferrets exposure to SARS-CoV-2 in Spain to date

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

15 pags, 8 figs, 1 tab. -- This article contains supplementary material (Table S1, Figs. S1–S4, and Data Sets S1–S4.1)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 O-glycan 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 receptor 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 gene-edited 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 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.This work was supported by the Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, the Kirsten og Freddy Johansen Fonden, the Lundbeck Foundation, the A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal, the Mizutani Foundation, the Novo Nordisk Foundation, the Danish Research Council Sapere Aude Research Talent Grant (to K. T. S.), and the Danish National Research Foundation (DNRF107). The authors declare that they have no conflicts of interest with the contents of this articl

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

11 pags, 3 figs, 2 tabsThe polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.We thank synchrotron radiation sources DLS (Oxford) and in particular beamline I03 (experiment number MX10121-7). We thank ARAID, MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P, CTQ2015-67727-R, CTQ2015-70524-R, and RYC-2013-14706), the National Institutes of Health (GM113534, and instrument grant GM113534-01S), the Danish National Research Foundation (DNRF107), the FCT-Portugal (UID/Multi/04378/2013 and PTNMR Project No 022161), and the DGA (B89) for the financial support. I.C. thanks Universidad de La Rioja for the FPI grant. F.M. thanks FCT-Portugal for IF Investigator. E.L.-N. acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 co-funded by the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Curie Actions. H.C. and J.J.-B. thank EU for the TOLLerant project. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement No. 283570 and BIOSTRUCTX_5186). We also thank BIFI (Memento cluster) and CESGA for computer support.Peer reviewe

Identification of global inhibitors of cellular glycosylation

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2

A perspective on structural and mechanistic aspects of protein O-fucosylation

Protein O-fucosylation is an important post-translational modification (PTM) found in cysteine-rich repeats in proteins. Protein O-fucosyltransferases 1 and 2 (PoFUT1 and PoFUT2) are the enzymes responsible for this PTM and selectively glycosylate specific residues in epidermal growth factor-like (EGF) repeats and thrombospondin type I repeats (TSRs), respectively.Within the past six years, crystal structures of both enzymes have been reported, revealing important information on how they recognize protein substrates and achieve catalysis. Here, the structural information available today is summarized and how PoFUT1 and PoFUT2 employ different catalytic mechanisms is discussed.Peer Reviewe

Polypeptide GalNAc-Ts:from redundancy to specificity

Mucin-type O-glycosylation is a post-translational modification (PTM) that is predicted to occur in more than the 80% of the proteins that pass through the Golgi apparatus. This PTM is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) that modify Ser and Thr residues of proteins through the addition of a GalNAc moiety. These enzymes are type II membrane proteins that consist of a Golgi luminal catalytic domain connected by a flexible linker to a ricin type lectin domain. Together, both domains account for the different glycosylation preferences observed among isoenzymes. Although it is well accepted that most of the family members share some degree of redundancy toward their protein and glycoprotein substrates, it has been recently found that several GalNAc-Ts also possess activity toward specific targets. Despite the high similarity between isoenzymes, structural differences have recently been reported that are key to understanding the molecular basis of both their redundancy and specificity. The present review focuses on the molecular aspects of the protein substrate recognition and the different glycosylation preferences of these enzymes, which in turn will serve as a roadmap to the rational design of specific modulators of mucin-type O-glycosylation

Asparagine Tautomerization in Glycosyltransferase Catalysis. The Molecular Mechanism of Protein O-Fucosyltransferase 1

[Image: see text] O-glycosylation is a post-translational protein modification essential to life. One of the enzymes involved in this process is protein O-fucosyltransferase 1 (POFUT1), which fucosylates threonine or serine residues within a specific sequence context of epidermal growth factor-like domains (EGF-LD). Unlike most inverting glycosyltransferases, POFUT1 lacks a basic residue in the active site that could act as a catalytic base to deprotonate the Thr/Ser residue of the EGF-LD acceptor during the chemical reaction. Using quantum mechanics/molecular mechanics (QM/MM) methods on recent crystal structures, as well as mutagenesis experiments, we uncover the enzyme catalytic mechanism, revealing that it involves proton shuttling through an active site asparagine, conserved among species, which undergoes tautomerization. This mechanism is consistent with experimental kinetic analysis of Caenorhabditis elegans POFUT1 Asn43 mutants, which ablate enzyme activity even if mutated to Asp, the canonical catalytic base in inverting glycosyltransferases. These results will aid inhibitor development for Notch-associated O-glycosylation disorders