Analysis of endogenous NOTCH1 from POFUT1 S162L patient fibroblasts reveals the importance of the O-fucose modification on EGF12 in human development.

NOTCH1 is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of NOTCH1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-fucose on NOTCH1 in POFUT1 S162L patient fibroblasts was the cause of these effects, we immunopurified endogenous NOTCH1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. NOTCH1 EGF8 to EGF12 comprise the ligand binding domain, and O-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of NOTCH1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-fucose at EGF12. Since the loss of O-fucose on EGF12 is known to have significant effects on NOTCH1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.

Analysis of endogenous NOTCH1 from POFUT1 S162L patient fibroblasts reveals the importance of the O -fucose modification on EGF12 in human development.

NOTCH1 (N1) is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O -fucosyltransferase 1 (POFUT1) adds O -fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of N1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O -fucose on N1 in POFUT1 S162L patient fibroblasts was the cause of these effects, we immunopurified endogenous N1 from control and patient fibroblasts and analyzed O -fucosylation using mass spectral glycoproteomics methods. N1 EGF8 to EGF12 comprise the ligand binding domain, and O -fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of N1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O -fucose at EGF12. Since the loss of O -fucose on EGF12 is known to have significant effects on N1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.

1H, 15N, 13C backbone and sidechain resonance assignments and secondary structure of mouse NOTCH1 EGF27.

NOTCH1 is a transmembrane receptor in metazoans that is linked to a variety of disorders. The receptor contains an extracellular domain (ECD) with 36 tandem epidermal growth factor-like (EGF) repeats. The ECD is responsible for intercellular signaling via protein-ligand interactions with neighboring cells. Each EGF repeat consists of approximately 40 amino acids and 3 conserved disulfide bonds. The Abruptex region (EGF24-29) is critical for NOTCH1 signaling and is known for its missense mutations. Certain EGF repeats are modified with the addition of O-linked glycans and many have calcium binding sites, which give each EGF repeat a unique function. It has been shown that the loss of the O-fucose site of EGF27 alters NOTCH1 activity. To investigate the role of glycosylation in the NOTCH1 signaling pathway, nuclear magnetic resonance spectroscopy has been employed to study the structures of EGF27 and its glycoforms. Here, we report the backbone and sidechain 1H, 15N, and 13C-resonance assignments of the unmodified EGF27 protein and the predicted secondary structure derived from the assigned chemical shifts.

Diseases related to Notch glycosylation.

The Notch receptors are a family of transmembrane proteins that mediate direct cell-cell interactions and control numerous cell-fate specifications in humans. The extracellular domains of mammalian Notch proteins contain 29-36 tandem epidermal growth factor-like (EGF) repeats, most of which have O-linked glycan modifications: O-glucose added by POGLUT1, O-fucose added by POFUT1 and elongated by Fringe enzymes, and O-GlcNAc added by EOGT. The extracellular domain is also N-glycosylated. Mutations in the glycosyltransferases modifying Notch have been identified in several diseases, including Dowling-Degos Disease (haploinsufficiency of POFUT1 or POGLUT1), a form of limb-girdle muscular dystrophy (autosomal recessive mutations in POGLUT1), Spondylocostal Dysostosis 3 (autosomal recessive mutations in LFNG), Adams-Oliver syndrome (autosomal recessive mutations in EOGT), and some cancers (amplification, gain or loss-of-function of POFUT1, Fringe enzymes, POGLUT1, MGAT3). Here we review the characteristics of these diseases and potential molecular mechanisms.

Expression, purification, and glycosylation of epidermal growth factor-like repeat 27 from mouse NOTCH1.

Notch receptors have large extracellular domains containing up to 36 tandem epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding ligands on neighboring cells. Notch receptors play major roles in a variety of developmental processes by controlling cell fate decisions. Each EGF repeat consists of about 40 amino acids with 3 conserved disulfide bonds. Many of the EGF repeats are modified by O-linked fucose glycans, and more than half have calcium-binding sites, but the sequences of the EGF repeats vary giving distinct roles to each repeat. EGF repeat 27 (EGF27) from mouse NOTCH1 is modified with O-fucose and is 1 of 7 repeats that is differentially modified by specific Fringe enzymes, which are known to regulate NOTCH1 activation and ligand binding. To better understand the role of EGF27 in NOTCH1 function and regulation, the 3-dimensional structures of EGF27 and its glycoforms are being pursued. E. coli cells were used to produce EGF27 in sufficient quantities for nuclear magnetic resonance analysis. Previous attempts to express the repeat alone and refold the repeat under a steady redox environment were unsuccessful due to low yields and extensive mixed-disulfide bond cross-linking. A new strategy using a cleavable maltose binding protein fusion tag increased the solubility and yield of EGF27. With the fusion tag, EGF27 was refolded to produce the correct disulfide bond arrangement, which was verified enzymatically with the glycosyltransferases, Protein O-fucosyltransferase 1 (POFUT1) and Lunatic Fringe (LFNG).

Recombinant expression of hydroxylated human collagen in Escherichia coli.

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.

Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme.

Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology.

O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1.

Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C(1)XSXPC(2). We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C(1)XSX(A/P)C(2). We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.

Structural and mechanistic insights into lunatic fringe from a kinetic analysis of enzyme mutants.

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of beta1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-alpha-L-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.

Role of unusual O-glycans in intercellular signaling.

In the last two decades, our knowledge of the role of glycans in development and signal transduction has expanded enormously. While most work has focused on the importance of N-linked or mucin-type O-linked glycosylation, recent work has highlighted the importance of several more unusual forms of glycosylation that are the focus of this review. In particular, the ability of O-fucose glycans on the epidermal growth factor-like (EGF) repeats of Notch to modulate signaling places glycosylation alongside phosphorylation as a means to modulate protein-protein interactions and their resultant downstream signals. The recent discovery that O-glucose modification of Notch EGF repeats is also required for Notch function has further expanded the range of glycosylation events capable of modulating Notch signaling. The prominent role of Notch during development and in later cell-fate decisions underscores the importance of these modifications in human biology. The role of glycans in intercellular signaling events is only beginning to be understood and appears ready to expand into new areas with the discovery that thrombospondin type 1 repeats are also modified with O-fucose glycans. Finally, a rare form of glycosylation called C-mannosylation modifies tryptophans in some signaling competent molecules and may be a further layer of complexity in the field. We will review each of these areas focusing on the glycan structures produced, the consequence of their presence, and the enzymes responsible.

Notch signaling in normal and disease States: possible therapies related to glycosylation.

The Notch signaling pathway is involved in a wide variety of highly conserved developmental processes in mammals. Importantly, mutations of the Notch protein and components of its signaling pathway have been implicated in an array of human diseases (T-cell leukemia and other cancers, Multiple Sclerosis, CADASIL, Alagille Syndrome, Spondylocostal Dysostosis). In mammals, Notch becomes activated upon binding of its extracellular domain to ligands (Delta and Jagged/Serrate) that are present on the surface of apposed cells. The extracellular domain of Notch contains up to 36 tandem Epidermal Growth Factor-like (EGF) repeats. Many of these EGF repeats are modified at evolutionarily-conserved consensus sites by an unusual form of O-glycosylation called O-fucose. Work from several groups indicates that O-fucosylation plays an important role in ligand mediated Notch signaling. Recent evidence also suggests that the enzyme responsible for addition of O-fucose to Notch, protein O-fucosyltransferase-1 (POFUT1), may serve a quality control function in the endoplasmic reticulum. Additionally, some of the O-fucose moieties are further elongated by the action of members of the Fringe family of beta-1,3-N-acetylglucosaminyltransferases. The alteration in O-fucose saccharide structure caused by Fringe modulates the response of Notch to its ligands. Thus, glycosylation serves an important role in regulating Notch activity. This review focuses on the role of glycosylation in the normal functioning of the Notch pathway. As well, potential roles for glycosylation in Notch-related human diseases, and possible roles for therapeutic targeting of POFUT1 and Fringe in Notch-related human diseases, are discussed.

O-fucosylation is required for ADAMTS13 secretion.

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [(3)H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-beta1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.

Identification and characterization of abeta1,3-glucosyltransferase that synthesizes the Glc-beta1,3-Fuc disaccharide on thrombospondin type 1 repeats.

Thrombospondin type 1 repeats (TSRs) are biologically important domains of extracellular proteins. They are modified with a unique Glcbeta1,3Fucalpha1-O-linked disaccharide on either serine or threonine residues. Here we identify the putative glycosyltransferase, B3GTL, as the beta1,3-glucosyltransferase involved in the biosynthesis of this disaccharide. This enzyme is conserved from Caenorhabditis elegans to man and shares 28% sequence identity with Fringe, the beta1,3-N-acetylglucosaminyltransferase that modifies O-linked fucosyl residues in proteins containing epidermal growth factor-like domains, such as Notch. beta1,3-Glucosyltransferase glucosylates properly folded TSR-fucose but not fucosylated epidermal growth factor-like domain or the non-fucosylated modules. Specifically, the glucose is added in a beta1,3-linkage to the fucose in TSR. The activity profiles of beta1,3-glucosyltransferase and protein O-fucosyltransferase 2, the enzyme that carries out the first step in TSR O-fucosylation, superimpose in endoplasmic reticulum subfractions obtained by density gradient centrifugation. Both enzymes are soluble proteins that efficiently modify properly folded TSR modules. The identification of the beta1,3-glucosyltransferase gene allows us to manipulate the formation of the rare Glcbeta1,3Fucalpha1 structure to investigate its biological function.

Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD], 20 microM) and low-affinity (KD, 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.

Lunatic fringe, manic fringe, and radical fringe recognize similar specificity determinants in O-fucosylated epidermal growth factor-like repeats.

Notch signaling is a component of a wide variety of developmental processes in many organisms. Notch activity can be modulated by O-fucosylation (mediated by protein O-fucosyltransferase-1) and Fringe, a beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose in the context of epidermal growth factor-like (EGF) repeats. Fringe was initially described in Drosophila, and three mammalian homologues have been identified, Manic fringe, Lunatic fringe, and Radical fringe. Here for the first time we have demonstrated that, similar to Manic and Lunatic, Radical fringe is also a fucose-specific beta1,3-N-acetylglucosaminyltransferase. The fact that three Fringe homologues exist in mammals raises the question of whether and how these enzymes differ. Although Notch contains numerous EGF repeats that are predicted to be modified by O-fucose, previous studies in our laboratory have demonstrated that not all O-fucosylated EGF repeats of Notch are further modified by Fringe, suggesting that the Fringe enzymes can differentiate between them. In this work, we have sought to identify specificity determinants for the recognition of an individual O-fucosylated EGF repeat by the Fringe enzymes. We have also sought to determine differences in the biochemical behavior of the Fringes with regard to their in vitro enzymatic activities. Using both in vivo and in vitro experiments, we have found two amino acids that appear to be important for the recognition of an O-fucosylated EGF repeat by all three mammalian Fringes. These amino acids provide an initial step toward defining sequences that will allow us to predict which O-fucosylated EGF repeats are modified by the Fringes.

Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila.

Phosphotransacetylase (Pta) [EC 2.3.1.8] is ubiquitous in the carbon assimilation and energy-yielding pathways in anaerobic prokaryotes where it catalyzes the reversible transfer of the acetyl group from acetyl phosphate to CoA forming acetyl CoA and inorganic phosphate. The crystal structure of Pta from the methane-producing archaeon Methanosarcina thermophila, representing the first crystal structure of any Pta, was determined by multiwavelength anomalous diffraction at 2.7 A resolution. In solution and in the crystal, the enzyme forms a homodimer. Each monomer consists of two alpha/beta domains with a cleft along the domain boundary, which presumably contains the substrate binding sites. Comparison of the four monomers present in the asymmetric unit indicates substantial variations in the relative orientation of the two domains and the structure of the putative active site cleft. A search for structural homologs revealed the NADP(+)-dependent isocitrate and isopropylmalate dehydrogenases as the only homologs with a similar two-domain architecture.