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1.
Methods Mol Biol ; 2043: 25-43, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463900

RESUMO

Metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 repeats) superfamily are extensively modified with glycan moieties. Glycosylation occurs as these enzymes are trafficked through the endoplasmic reticulum (ER) and Golgi apparatus on their way to the extracellular space and includes N-linked glycosylation, O-linked fucosylation and C-linked mannosylation. This chapter focuses on O-linked fucose, which is added to properly folded thrombospondin type 1 repeats (TSRs) in the ER by protein O-fucosyltransferase 2 (POFUT2) and elongated to a Glucoseß1-3Fucose disaccharide by ß3-glucosyltransferase (B3GLCT). Knockout of POFUT2 results in embryonic lethality in mice, and inactivating mutations in B3GLCT cause Peters plus syndrome, a congenital disorder of glycosylation in humans. Addition of the disaccharide by POFUT2 and B3GLCT stabilizes folded TSRs, enhancing folding in the ER and secretion efficiency of several ADAMTS proteins from cells. Thus, POFUT2 and B3GLCT both function as an ER quality control pathway for folding of TSRs in ADAMTS proteins. In this chapter we describe in detail the methods developed to analyze secretion defects of ADAMTS proteins upon loss of either POFUT2 or B3GLCT. The methods described include creation of CRISPR/Cas9-mediated knockout cell lines of POFUT2 and B3GLCT and use of these cell lines to analyze and quantify secretion defects of ADAMTS proteins.

2.
Nat Chem Biol ; 15(9): 853-864, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31427814

RESUMO

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.

3.
Nature ; 567(7746): 56-60, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814731

RESUMO

The cytokine interferon-γ (IFNγ) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNγ pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNγ receptor IFNγR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNγ-IFNγR1-IFNγR2 signalling complex at 3.25 Å resolution. The structure reveals the mechanism underlying deficits in IFNγ responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNγR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNγ variants to tune IFNγ receptor signalling output. Unexpectedly, we found that several partial IFNγ agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNγ for therapeutic applications.


Assuntos
Desenho de Drogas , Interferon gama/agonistas , Interferon gama/imunologia , Receptores de Interferon/química , Receptores de Interferon/metabolismo , Antígeno B7-H1/biossíntese , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Agonismo Parcial de Drogas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Interferon gama/química , Interferon gama/genética , Ligantes , Modelos Moleculares , Mutação , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Estabilidade Proteica , Receptores de Interferon/genética , Transdução de Sinais , Relação Estrutura-Atividade
4.
Curr Opin Struct Biol ; 56: 78-86, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30690220

RESUMO

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.

6.
Proc Natl Acad Sci U S A ; 115(36): E8395-E8402, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30127001

RESUMO

The Notch-signaling pathway is normally activated by Notch-ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.


Assuntos
Glucosiltransferases/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Receptor Notch3/metabolismo , Transdução de Sinais/fisiologia , Animais , Glucosiltransferases/genética , Glicosilação , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Transporte Proteico/fisiologia , Receptor Notch1/genética , Receptor Notch2/genética , Receptor Notch3/genética , Sequências Repetitivas de Aminoácidos
7.
Adv Exp Med Biol ; 1066: 59-78, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30030822

RESUMO

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.

8.
Glycobiology ; 28(5): 276-283, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29452367

RESUMO

Protein O-fucosyltransferase-1 (POFUT1) adds O-fucose monosaccharides to epidermal growth factor-like (EGF) repeats found on approximately 100 mammalian proteins, including Notch receptors. Haploinsufficiency of POFUT1 has been linked to adult-onset Dowling Degos Disease (DDD) with hyperpigmentation defects. Homozygous deletion of mouse Pofut1 results in embryonic lethality with severe Notch-like phenotypes including defects in somitogenesis, cardiogenesis, vasculogenesis and neurogenesis, but the extent to which POFUT1 is required for normal human development is not yet understood. Here we report a patient with a congenital syndrome consisting of severe global developmental delay, microcephaly, heart defects, failure to thrive and liver disease with a previously unreported homozygous NM_015352.1: c.485C>T variant (p.Ser162Leu) in POFUT1 detected by exome sequencing. Both parents are heterozygotes and neither manifests any signs of DDD. No other detected variant explained the phenotype. This variant eliminated a conserved N-glycosylation sequon at Asn160 in POFUT1 and profoundly decreased POFUT1 activity in patient fibroblasts compared to control fibroblasts. Purified p.Ser162Leu mutant protein also showed much lower POFUT1 activity with a lower affinity for EGF acceptor substrate than wild type POFUT1. Eliminating the N-glycan sequon by replacing Asn160 with Gln had little effect on POFUT1 activity, suggesting that loss of the glycan is not responsible for the defect. Furthermore, the p.Ser162Leu mutant showed weaker ability to rescue Notch activity in cell-based assays. These results suggest that this N-glycan of POFUT1 is not required for its proper enzymatic function, and that the p.Ser162Leu mutation of POFUT1 likely causes global developmental delay, microcephaly with vascular and cardiac defects.


Assuntos
Doenças Cardiovasculares/genética , Deficiências do Desenvolvimento/genética , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Variação Genética/genética , Microcefalia/genética , Células Cultivadas , Células HEK293 , Humanos , Mutação
9.
Nat Chem Biol ; 14(1): 65-71, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29176671

RESUMO

Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.


Assuntos
Família de Proteínas EGF/metabolismo , Fucose/análogos & derivados , Fucose/farmacologia , Fucosiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Fucose/química , Fucose/genética , Fucosiltransferases/genética , Células HEK293 , Humanos , Ligantes , Ligação Proteica
10.
PLoS One ; 12(9): e0184903, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28926587

RESUMO

Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, short stature, brachydactyly, characteristic facial features, developmental delay and other highly variable systemic defects. Classic PPS is caused by loss-of-function mutations in the B3GLCT gene encoding for a ß3-glucosyltransferase that catalyzes the attachment of glucose via a ß1-3 glycosidic linkage to O-linked fucose on thrombospondin type 1 repeats (TSRs). B3GLCT was shown to participate in a non-canonical ER quality control mechanism; however, the exact molecular processes affected in PPS are not well understood. Here we report the identification and characterization of two zebrafish orthologs of the human B3GLCT gene, b3glcta and b3glctb. The b3glcta and b3glctb genes encode for 496-aa and 493-aa proteins with 65% and 57% identity to human B3GLCT, respectively. Expression studies demonstrate that both orthologs are widely expressed with strong presence in embryonic tissues affected in PPS. In vitro glucosylation assays demonstrated that extracts from wildtype embryos contain active b3glct enzyme capable of transferring glucose from UDP-glucose to an O-fucosylated TSR, indicating functional conservation with human B3GLCT. To determine the developmental role of the zebrafish genes, single and double b3glct knockouts were generated using TALEN-induced genome editing. Extracts from double homozygous b3glct-/- embryos demonstrated complete loss of in vitro b3glct activity. Surprisingly, b3glct-/- homozygous fish developed normally. Transcriptome analyses of head and trunk tissues of b3glct-/- 24-hpf embryos identified 483 shared differentially regulated transcripts that may be involved in compensation for b3glct function in these embryos. The presented data show that both sequence and function of B3GLCT/b3glct genes is conserved in vertebrates. At the same time, complete b3glct deficiency in zebrafish appears to be inconsequential and possibly compensated for by a yet unknown mechanism.


Assuntos
Glucosiltransferases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Fenda Labial/genética , Fenda Labial/patologia , Córnea/anormalidades , Córnea/patologia , Embrião não Mamífero/metabolismo , Edição de Genes , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Glucosiltransferases/deficiência , Glucosiltransferases/genética , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Humanos , Hibridização In Situ , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
11.
J Biol Chem ; 292(38): 15964-15973, 2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28729422

RESUMO

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.


Assuntos
Fator de Crescimento Epidérmico/química , Oxigênio/metabolismo , Receptores Notch/química , Receptores Notch/metabolismo , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Animais , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Glucose/metabolismo , Glucosiltransferases/deficiência , Glucosiltransferases/genética , Glicosilação , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Receptor Notch1/química , Receptor Notch1/metabolismo
12.
Glycobiology ; 27(7): 601-618, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28430973

RESUMO

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.


Assuntos
Fucose/metabolismo , Fucosiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Fucosiltransferases/química , Fucosiltransferases/genética , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/metabolismo , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Mutação , Neoplasias/genética , Neoplasias/metabolismo
13.
Science ; 355(6331): 1320-1324, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28254785

RESUMO

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.


Assuntos
Proteína Jagged-1/química , Receptor Notch1/química , Animais , Cristalografia por Raios X , Fucose/química , Engenharia Genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína Jagged-1/genética , Proteína Jagged-1/ultraestrutura , Ligantes , Proteínas de Membrana/química , Ligação Proteica , Domínios Proteicos , Ratos , Receptor Notch1/genética , Receptor Notch1/ultraestrutura , Saccharomyces cerevisiae , Transdução de Sinais
14.
Glycobiology ; 27(3): 206-212, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28177478

RESUMO

Determining the correct enzymatic activity of putative glycosyltransferases (GTs) can be challenging as these enzymes can utilize multiple donor and acceptor substrates. Upon initial determination of the donor-sugar nucleotide(s), a GT utilizes various acceptor molecules that can then be tested. Here, we describe a quick method to screen sugar-nucleotide donor specificities of GTs utilizing a sensitive, nonradioactive, commercially available bioluminescent uridine diphosphate detection kit. This in vitro method allowed us to validate the sugar-nucleotide donor-substrate specificities of recombinantly expressed human, bovine, bacterial and protozoan GTs. Our approach, which is less time consuming than many traditional assays that utilize radiolabeled sugars and chromatographic separations, should facilitate discovery of novel GTs that participate in diverse biological processes.


Assuntos
Glicosiltransferases/isolamento & purificação , Nucleotídeos/química , Açúcares/química , Animais , Bactérias/enzimologia , Bovinos , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Humanos , Especificidade por Substrato
15.
Sci Rep ; 7: 41871, 2017 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-28176809

RESUMO

Secreted metalloproteases have diverse roles in the formation, remodeling, and the destruction of extracellular matrix. Recessive mutations in the secreted metalloprotease ADAMTS17 cause ectopia lentis and short stature in humans with Weill-Marchesani-like syndrome and primary open angle glaucoma and ectopia lentis in dogs. Little is known about this protease or its connection to fibrillin microfibrils, whose major component, fibrillin-1, is genetically associated with ectopia lentis and alterations in height. Fibrillin microfibrils form the ocular zonule and are present in the drainage apparatus of the eye. We show that recombinant ADAMTS17 has unique characteristics and an unusual life cycle. It undergoes rapid autocatalytic processing in trans after its secretion from cells. Secretion of ADAMTS17 requires O-fucosylation and its autocatalytic activity does not depend on propeptide processing by furin. ADAMTS17 binds recombinant fibrillin-2 but not fibrillin-1 and does not cleave either. It colocalizes to fibrillin-1 containing microfibrils in cultured fibroblasts and suppresses fibrillin-2 (FBN2) incorporation in microfibrils, in part by transcriptional downregulation of Fbn2 mRNA expression. RNA in situ hybridization detected Adamts17 expression in specific structures in the eye, skeleton and other organs, where it may regulate the fibrillin isoform composition of microfibrils.


Assuntos
Proteínas ADAMTS/metabolismo , Microfibrilas/metabolismo , Processamento de Proteína Pós-Traducional , Síndrome de Weill-Marchesani/genética , Proteínas ADAMTS/química , Proteínas ADAMTS/genética , Animais , Células Cultivadas , Fibrilina-1/metabolismo , Fibrilina-2/metabolismo , Fucose/metabolismo , Furina/metabolismo , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Ratos
16.
Dev Cell ; 40(2): 193-201, 2017 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-28089369

RESUMO

Fringe proteins are ß3-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.


Assuntos
Glicosiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1/metabolismo , Proteínas/metabolismo , Receptor Notch1/metabolismo , Sequência de Aminoácidos , Animais , Fator de Crescimento Epidérmico/metabolismo , Fucose/metabolismo , Células HEK293 , Humanos , Ligantes , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Receptor Notch1/química , Sequências Repetitivas de Aminoácidos
17.
EMBO Mol Med ; 8(11): 1289-1309, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27807076

RESUMO

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb-girdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O-glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O-glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle-specific α-dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch-dependent loss of satellite cells.


Assuntos
Glucosiltransferases/genética , Distrofias Musculares/genética , Distrofias Musculares/patologia , Mutação , Receptores Notch/metabolismo , Células Satélites de Músculo Esquelético/patologia , Transdução de Sinais , Biópsia , Glicosilação , Glicosiltransferases/metabolismo , Humanos , Músculos/patologia , Análise de Sequência de DNA , Espanha
18.
Sci Rep ; 6: 33974, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27687499

RESUMO

Peters Plus syndrome (PPS), a congenital disorder of glycosylation, results from recessive mutations affecting the glucosyltransferase B3GLCT, leading to congenital corneal opacity and diverse extra-ocular manifestations. Together with the fucosyltransferase POFUT2, B3GLCT adds Glucoseß1-3Fucose disaccharide to a consensus sequence in thrombospondin type 1 repeats (TSRs) of several proteins. Which of these target proteins is functionally compromised in PPS is unknown. We report here that haploinsufficiency of murine Adamts9, encoding a secreted metalloproteinase with 15 TSRs, leads to congenital corneal opacity and Peters anomaly (persistent lens-cornea adhesion), which is a hallmark of PPS. Mass spectrometry of recombinant ADAMTS9 showed that 9 of 12 TSRs with the O-fucosylation consensus sequence carried the Glucoseß1-3Fucose disaccharide and B3GLCT knockdown reduced ADAMTS9 secretion in HEK293F cells. Together, the genetic and biochemical findings imply a dosage-dependent role for ADAMTS9 in ocular morphogenesis. Reduced secretion of ADAMTS9 in the absence of B3GLCT is proposed as a mechanism of Peters anomaly in PPS. The functional link between ADAMTS9 and B3GLCT established here also provides credence to their recently reported association with age-related macular degeneration.

19.
Nat Chem Biol ; 12(9): 735-40, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27428513

RESUMO

Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development, including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi's critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary and ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and a conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling-Degos disease are clustered around the enzyme active site and adversely affect its activity. Our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Glucosiltransferases/química , Glucosiltransferases/genética , Animais , Biocatálise , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glucosiltransferases/metabolismo , Humanos , Camundongos , Mutação , Conformação Proteica
20.
J Biol Chem ; 291(31): 16348-60, 2016 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-27268051

RESUMO

Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the ß3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.


Assuntos
Proteínas de Drosophila , N-Acetilglucosaminiltransferases , Mapeamento de Peptídeos , Peptídeos , Receptores Notch , Animais , Linhagem Celular , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Fucose/química , Fucose/genética , Fucose/metabolismo , Glicosilação , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Receptores Notch/química , Receptores Notch/genética , Receptores Notch/metabolismo
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