Fringe proteins are β3-N-acetylglucosaminyltransferases that modulate Notch activity by modifying O-fucose residues on epidermal growth factor-like (EGF) repeats of Notch. Mammals have three Fringes: ...Lunatic, Manic, and Radical. While Lunatic and Manic Fringe inhibit Notch1 activation from Jagged1 and enhance activation from Delta-like 1, Radical Fringe enhances signaling from both. We used a mass spectrometry approach to determine whether the variable effects of Fringes on Notch1 result from generation of unique glycosylation patterns on Notch1. We found that Lunatic and Manic Fringe modified similar sites on Notch1, while Radical Fringe modified a subset. Fringe modifications at EGF8 and EGF12 enhanced Notch1 binding to and activation from Delta-like 1, while modifications at EGF6 and EGF36 (added by Manic and Lunatic but not Radical) inhibited Notch1 activation from Jagged1. Combined, these results suggest that Fringe modifications “mark” different regions in the Notch1 extracellular domain for activation or inhibition.
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•Fringe modulates Notch activity by addition of GlcNAc to O-fucose on EGF repeats•O-fucose was found on 17 EGF repeats, but only a few are modified by any Fringe•Fringe modifications at EGF8 and 12 enhance activation of Notch1 by Delta-like 1•Fringe modifications at EGF6 and 36 inhibit Notch1 activation by Jagged1
Kakuda and Haltiwanger examine how Fringes regulate Notch1. They show that Radical Fringe modifies a subset of EGF repeats modified by Lunatic and Manic Fringe. Modifications at distinct EGF repeats “mark” the Notch1 extracellular domain, either enhancing or inhibiting activity.
•Protein O-fucosyltransferases 1 and 2 (POFUT1 and POFUT2) are ER-localized and modify EGF repeats and TSRs.•Both POFUT1 and POFUT2 are exquisitely selective for properly folded substrates.•O-Fucose ...on Notch EGF repeats directly participates in intermolecular interactions with Notch ligands.•O-Fucose glycans on both EGF repeats and TSRs interact with underlying amino acids, stabilizing the folded domains.•POFUT1 and POFUT2 participate in non-canonical ER quality control pathways for folding of EGF repeats and TSRs, respectively.
Fucose is a common terminal modification on protein and lipid glycans. Fucose can also be directly linked to protein via an O-linkage to Serine or Threonine residues located within consensus sequences contained in Epidermal Growth Factor-like (EGF) repeats and Thrombospondin Type 1 Repeats (TSRs). In this context, fucose is added exclusively to properly folded EGF repeats and TSRs by Protein O-fucosyltransferases 1 and 2, respectively. In both cases, the O-linked fucose can also be elongated with other sugars. Here, we describe the biological importance of these O-fucose glycans and molecular mechanisms by which they affect the function of the proteins they modify. O-Fucosylation of EGF repeats modulates the Notch signaling pathway, while O-fucosylation of TSRs is predicted to influence secretion of targets including several extracellular proteases. Recent data show O-fucose glycans mediate their effects by participating in both intermolecular and intramolecular interactions.
Significance of glycosylation in Notch signaling Takeuchi, Hideyuki; Haltiwanger, Robert S.
Biochemical and biophysical research communications,
10/2014, Letnik:
453, Številka:
2
Journal Article
Recenzirano
Odprti dostop
•Notch signaling is regulated by glycosylation of its extracellular domain.•Multiple O-linked carbohydrate modifications are found on the epidermal growth factor-like repeats of Notch.•Defects in ...glycosylation of Notch leads to a variety of human disorders.
Notch signaling is essential for cell-fate specification in metazoans, and dysregulation of the pathway leads to a variety of human diseases including heart and vascular defects as well as cancer. Glycosylation of the Notch extracellular domain has emerged as an elegant means for regulating Notch activity, especially since the discovery that Fringe is a glycosyltransferase that modifies O-fucose in 2000. Since then, several other O-glycans on the extracellular domain have been demonstrated to modulate Notch activity. Here we will describe recent results on the molecular mechanisms by which Fringe modulates Notch activity, summarize recent work on how O-glucose, O-GlcNAc, and O-GalNAc glycans affect Notch, and discuss several human genetic disorders resulting from defects in Notch glycosylation.
Biological functions of fucose in mammals Schneider, Michael; Al-Shareffi, Esam; Haltiwanger, Robert S
Glycobiology (Oxford),
07/2017, Letnik:
27, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Fucose is a 6-deoxy hexose in the l-configuration found in a large variety of different organisms. In mammals, fucose is incorporated into N-glycans, O-glycans and glycolipids by 13 ...fucosyltransferases, all of which utilize the nucleotide-charged form, GDP-fucose, to modify targets. Three of the fucosyltransferases, FUT8, FUT12/POFUT1 and FUT13/POFUT2, are essential for proper development in mice. Fucose modifications have also been implicated in many other biological functions including immunity and cancer. Congenital mutations of a Golgi apparatus localized GDP-fucose transporter causes leukocyte adhesion deficiency type II, which results in severe developmental and immune deficiencies, highlighting the important role fucose plays in these processes. Additionally, changes in levels of fucosylated proteins have proven as useful tools for determining cancer diagnosis and prognosis. Chemically modified fucose analogs can be used to alter many of these fucose dependent processes or as tools to better understand them. In this review, we summarize the known roles of fucose in mammalian physiology and pathophysiology. Additionally, we discuss recent therapeutic advances for cancer and other diseases that are a direct result of our improved understanding of the role that fucose plays in these systems.
Glycans linked to proteins and lipids play key roles in biology; thus, accurate replication of cellular glycans is crucial for maintaining function following cell division. The fact that glycans are ...not copied from genomic templates suggests that fidelity is provided by the catalytic templates of glycosyltransferases that accurately add sugars to specific locations on growing oligosaccharides. To form new glycosidic bonds, glycosyltransferases bind acceptor substrates and orient a specific hydroxyl group, frequently one of many, for attack of the donor sugar anomeric carbon. Several recent crystal structures of glycosyltransferases with bound acceptor substrates reveal that these enzymes have common core structures that function as scaffolds upon which variable loops are inserted to confer substrate specificity and correctly orient the nucleophilic hydroxyl group. The varied approaches for acceptor binding site assembly suggest an ongoing evolution of these loop regions provides templates for assembly of the diverse glycan structures observed in biology.
Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the ...extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor–like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes upon Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. This mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.
Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 ...and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4–NOTCH1 activation was more than twice that of DLL4–NOTCH2, whereas all other ligands activated NOTCH2 similarly or slightly more than NOTCH1. However, NOTCH2 showed less sensitivity to the Fringes. Lunatic fringe (LFNG) enhanced NOTCH2 activation by DLL1 and -4, and Manic fringe (MFNG) inhibited NOTCH2 activation by JAG1 and -2. Mass spectral analysis showed that O-fucose occurred at high stoichiometry at most consensus sequences of NOTCH2 and that the Fringe enzymes modified more O-fucose sites of NOTCH2 compared with NOTCH1. Mutagenesis studies showed that LFNG modification of O-fucose on EGF8 and -12 of NOTCH2 was responsible for enhancement of DLL1–NOTCH2 activation, similar to previous reports for NOTCH1. In contrast to NOTCH1, a single O-fucose site mutant that substantially blocked the ability of MFNG to inhibit NOTCH2 activation by JAG1 could not be identified. Interestingly, elimination of the O-fucose site on EGF12 allowed LFNG to inhibit JAG1-NOTCH2 activation, and O-fucosylation on EGF9 was important for trafficking of both NOTCH1 and NOTCH2. Together, these studies provide new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes.
Glycosylation in the endoplasmic reticulum (ER) is closely associated with protein folding and quality control. We recently described a non-canonical ER quality control mechanism for folding of ...thrombospondin type 1 repeats by protein O-fucosyltransferase 2 (POFUT2). Epidermal growth factor-like (EGF) repeats are also small cysteine-rich protein motifs that can be O-glycosylated by several ER-localized enzymes, including protein O-glucosyltransferase 1 (POGLUT1) and POFUT1. Both POGLUT1 and POFUT1 modify the Notch receptor on multiple EGF repeats and are essential for full Notch function. The fact that POGLUT1 and POFUT1 can distinguish between folded and unfolded EGF repeats raised the possibility that they participate in a quality control pathway for folding of EGF repeats in proteins such as Notch. Here, we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat and that addition of both O-glucose and O-fucose enhances stability in an additive manner. Finally, we solved the crystal structure of a single EGF repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.
The Notch receptor initiates a unique intercellular signaling pathway that is evolutionarily conserved across all metazoans and contributes to the development and maintenance of numerous tissues. ...Consequently, many diseases result from aberrant Notch signaling. Emerging roles for Notch in disease are being uncovered as studies reveal new information regarding various components of this signaling pathway. Notch activity is regulated at several levels, but O-linked glycosylation of Epidermal Growth Factor (EGF) repeats in the Notch extracellular domain has emerged as a major regulator that, depending on context, can increase or decrease Notch activity. Three types of O-linked glycosylation occur at consensus sequences found within the EGF repeats of Notch: O-fucosylation, O-glucosylation, and O-GlcNAcylation. Recent studies have investigated the site occupancy of these types of glycosylation and also defined specific roles for these glycans on Notch structure and function. Nevertheless, there are many functional aspects to each type of O-glycosylation that remain unclear. Here, we will discuss molecular mechanisms of how O-glycosylation regulates Notch signaling and describe disorders associated with defects in Notch O-glycosylation.
ROLE OF GLYCOSYLATION IN DEVELOPMENT Haltiwanger, Robert S; Lowe, John B
Annual review of biochemistry,
2004, Letnik:
73, Številka:
1
Journal Article
Recenzirano
Researchers have long predicted that complex carbohydrates on cell surfaces
would play important roles in developmental processes because of the
observation that specific carbohydrate structures ...appear in specific spatial
and temporal patterns throughout development. The astounding number and
complexity of carbohydrate structures on cell surfaces added support to the
concept that glycoconjugates would function in cellular communication during
development. Although the structural complexity inherent in glycoconjugates has
slowed advances in our understanding of their functions, the complete
sequencing of the genomes of organisms classically used in developmental
studies (e.g., mice,
Drosophila melanogaster
, and
Caenorhabditis
elegans
) has led to demonstration of essential functions for a number of
glycoconjugates in developmental processes. Here we present a review of recent
studies analyzing function of a variety of glycoconjugates (O-fucose,
O-mannose, N-glycans, mucin-type O-glycans, proteoglycans, glycosphingolipids),
focusing on lessons learned from human disease and genetic studies in mice,
D. melanogaster
, and
C. elegans
.
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