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1.
J Biol Chem ; 300(4): 107121, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38417795

RESUMO

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1-/- rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions.


Assuntos
Defeitos Congênitos da Glicosilação , Transferência Ressonante de Energia de Fluorescência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Animais , Humanos , Masculino , Ratos , Envelhecimento/metabolismo , Encéfalo/metabolismo , Defeitos Congênitos da Glicosilação/diagnóstico , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência
2.
FEBS J ; 291(5): 884-896, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37997624

RESUMO

It is known that oligosaccharyltransferase (OST) has hydrolytic activity toward dolichol-linked oligosaccharides (DLO), which results in the formation of free N-glycans (FNGs), i.e. unconjugated oligosaccharides with structural features similar to N-glycans. The functional importance of this hydrolytic reaction, however, remains unknown. In this study, the hydrolytic activity of OST was characterized in yeast. It was shown that the hydrolytic activity of OST is enhanced in ubiquitin ligase mutants that are involved in endoplasmic reticulum-associated degradation. Interestingly, this enhanced hydrolysis activity is completely suppressed in asparagine-linked glycosylation (alg) mutants, bearing mutations related to the biosynthesis of DLO, indicating that the effect of ubiquitin ligase on OST-mediated hydrolysis is context-dependent. The enhanced hydrolysis activity in ubiquitin ligase mutants was also found to be canceled upon treatment of the cells with dithiothreitol, a reagent that potently induces protein unfolding in the endoplasmic reticulum (ER). Our results clearly suggest that the hydrolytic activity of OST is enhanced under conditions in which the formation of unfolded proteins is promoted in the ER in yeast. The possible role of FNGs on protein folding is discussed.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Hexosiltransferases , Proteínas de Membrana , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Hidrólise , Retículo Endoplasmático , Ubiquitina , Dolicóis , Ligases , Oligossacarídeos , Polissacarídeos
3.
Biochim Biophys Acta Gen Subj ; 1867(12): 130494, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37865174

RESUMO

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1.


Assuntos
Aminoácidos , Fatores de Transcrição , Aminoácidos/metabolismo , Fatores de Transcrição/metabolismo , Proteólise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Eletroforese em Gel de Poliacrilamida
4.
Cell Rep ; 41(8): 111679, 2022 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-36417860

RESUMO

N-glycans are processed mainly in the Golgi, and a well-organized Golgi structure is required for accurate glycosylation. However, during mitosis the Golgi undergoes severe fragmentation. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes. It is unclear how cells avoid glycosylation defects during mitosis. In this study, we report that Golgi α-1,2-mannosidase IA (MAN1A1), the first enzyme that cargo proteins encounter once arriving the Golgi, is phosphorylated at serine 12 by CDK1 in mitosis, which attenuates its activity, affects the production of glycan isomers, and reduces its interaction with the subsequent glycosyltransferase, MGAT1. Expression of wild-type MAN1A1, but not its phosphomimetic mutant, rescues the glycosylation defects in mannosidase I-deficient cells, whereas expression of its phosphorylation-deficient mutant in mitosis increases the formation of complex glycans. Our study reveals that glycosylation is regulated by cytosolic signaling during the cell cycle.


Assuntos
Complexo de Golgi , Manosidases , Fosforilação , Manosidases/metabolismo , Complexo de Golgi/metabolismo , Mitose , Polissacarídeos/metabolismo
5.
J Biochem ; 171(2): 169-176, 2022 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-34791337

RESUMO

Cytosolic peptide:N-glycanase (NGLY1 in mammals), a highly conserved enzyme in eukaryotes, catalyses the deglycosylation of N-glycans that are attached to glycopeptide/glycoproteins. In 2012, an autosomal recessive disorder related to the NGLY1 gene, which was referred to as NGLY1 deficiency, was reported. Since then, more than 100 patients have been identified. Patients with this disease exhibit various symptoms, including various motor deficits and other neurological problems. Effective therapeutic treatments for this disease, however, have not been established. Most recently, it was demonstrated that the intracerebroventricular administration of an adeno-associated virus 9 vector expressing human NGLY1 during the weaning period allowed some motor functions to be recovered in Ngly1-/- rats. This observation led us to hypothesize that a therapeutic intervention for improving these motor deficits or other neurological symptoms found in the patients might be possible. To achieve this, it is critical to establish robust and facile methods for assaying NGLY1 activity in biological samples, for the early diagnosis and evaluation of the therapeutic efficacy for the treatment of NGLY1 deficiency. In this mini review, we summarize progress made in the development of various assay methods for NGLY1 activity, as well as a recent progress in the identification of NGLY1 deficiency-specific biomarkers.


Assuntos
Defeitos Congênitos da Glicosilação , Animais , Biomarcadores , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Humanos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeos , Ratos
6.
Glycobiology ; 32(2): 110-122, 2022 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-34939090

RESUMO

Cytosolic peptide: N-glycanase (PNGase; NGLY1), an enzyme responsible for de-glycosylation of N-glycans on glycoproteins, is known to play pivotal roles in a variety of biological processes. In 2012, NGLY1 deficiency, a rare genetic disorder, was reported and since then, more than 100 patients have now been identified worldwide. Patients with this disease exhibit several common symptoms that are caused by the dysfunction of NGLY1. However, correlation between the severity of patient symptoms and the extent of the reduction in NGLY1 activity in these patients remains to be clarified, mainly due to the absence of a facile quantitative assay system for this enzyme, especially in a crude extract as an enzyme source. In this study, a quantitative, non-radioisotope (RI)-based assay method for measuring recombinant NGLY1 activity was established using a BODIPY-labeled asialoglycopeptide (BODIPY-ASGP) derived from hen eggs. With this assay, the activities of 27 recombinant NGLY1 mutants that are associated with the deficiency were examined. It was found that the activities of three (R469X, R458fs and H494fs) out of the 27 recombinant mutant proteins were 30-70% of the activities of wild-type NGLY1. We further developed a method for measuring endogenous NGLY1 activity in crude extracts derived from cultured cells, patients' fibroblasts, iPS cells or peripheral blood mononuclear cells (PBMCs), using a glycosylated cyclopeptide (GCP) that exhibited resistance to the endogenous proteases in the extract. Our methods will not only provide new insights into the molecular mechanism responsible for this disease but also promises to be applicable for its diagnosis.


Assuntos
Leucócitos Mononucleares , Peptídeos Cíclicos , Animais , Galinhas , Misturas Complexas , Feminino , Glicosilação , Humanos , Leucócitos Mononucleares/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo
8.
Mol Brain ; 14(1): 91, 2021 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-34120625

RESUMO

N-glycanase 1 (NGLY1) deficiency is a rare inherited disorder characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, motor deficits, and other neurological symptoms. The underlying mechanisms of the NGLY1 phenotype are poorly understood, and no effective therapy is currently available. Similar to human patients, the rat model of NGLY1 deficiency, Ngly1-/-, shows developmental delay, movement disorder, somatosensory impairment, scoliosis, and learning disability. Here we show that single intracerebroventricular administration of AAV9 expressing human NGLY1 cDNA (AAV9-hNGLY1) to Ngly1-/- rats during the weaning period restored NGLY1 expression in the brain and spinal cord, concomitant with increased enzymatic activity of NGLY1 in the brain. hNGLY1 protein expressed by AAV9 was found predominantly in mature neurons, but not in glial cells, of Ngly1-/- rats. Strikingly, intracerebroventricular administration of AAV9-hNGLY1 normalized the motor phenotypes of Ngly1-/- rats assessed by the rota-rod test and gait analysis. The reversibility of motor deficits in Ngly1-/- rats by central nervous system (CNS)-restricted gene delivery suggests that the CNS is the primary therapeutic target organs for NGLY1 deficiency, and that the Ngly1-/- rat model may be useful for evaluating therapeutic treatments in pre-clinical studies.


Assuntos
Defeitos Congênitos da Glicosilação/fisiopatologia , Atividade Motora/fisiologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Acetilglucosamina/análogos & derivados , Animais , Defeitos Congênitos da Glicosilação/enzimologia , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/administração & dosagem , Gliose/complicações , Gliose/patologia , Humanos , Inflamação/patologia , Injeções Intraventriculares , Neurônios/patologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Ratos , Ratos Transgênicos , Transgenes
9.
Elife ; 92020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32720893

RESUMO

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Glicoproteínas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Translocação Genética/fisiologia , Animais , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Glicoproteínas/genética , Glicosilação
10.
Biochim Biophys Acta Mol Basis Dis ; 1866(3): 165588, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733337

RESUMO

The cytoplasmic peptide:N-glycanase (Ngly1) is a de-N-glycosylating enzyme that cleaves N-glycans from misfolded glycoproteins and is involved in endoplasmic reticulum-associated degradation. The recent discovery of NGLY1-deficiency, which causes severe systemic symptoms, drew attention to the physiological function of Ngly1 in mammals. While several studies have been carried out to reveal the physiological necessity of Ngly1, the semi-lethal nature of Ngly1-deficient animals made it difficult to analyze its function in adults. In this study, we focus on the physiological function of Ngly1 in liver (hepatocyte)-specific Ngly1-deficient mice generated using the cre-loxP system. We found that hepatocyte-specific Ngly1-deficient mice showed abnormal hepatocyte nuclear size/morphology with aging but did not show other notable defects in unstressed conditions. This nuclear phenotype did not appear to be related to the function of the only gene currently reported to rescue Ngly1-deficient murine lethality so far, endo-ß-N-acetylglucosaminidase. We also found that under a high fructose diet induced stress, the hepatocyte-specific Ngly1-deletion resulted in liver transaminases elevation and increased lipid droplet accumulation. We showed that the processing and localization of the transcription factor, nuclear factor erythroid 2-like 1 (Nfe2l1), was impaired in the Ngly1-deficient hepatocytes. Therefore, Nfe2l1, at least partially, contributes to the phenotypes observed in hepatocyte-specific Ngly1-deficient mice. Our results indicate that Ngly1 plays important roles in the adult liver impacting nuclear morphology and lipid metabolism. Hepatocyte-specific Ngly1-deficient mice could thus serve as a valuable animal model for assessing in vivo efficacy of drugs and/or treatment for NGLY1-deficiency.


Assuntos
Defeitos Congênitos da Glicosilação/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Estresse Fisiológico/fisiologia , Animais , Linhagem Celular , Citoplasma/metabolismo , Dieta , Modelos Animais de Doenças , Degradação Associada com o Retículo Endoplasmático/fisiologia , Feminino , Frutose/metabolismo , Glicosilação , Hepatócitos/metabolismo , Masculino , Camundongos , Fenótipo
11.
J Biol Chem ; 294(44): 15900-15911, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31311856

RESUMO

In eukaryotic cells, unconjugated oligosaccharides that are structurally related to N-glycans (i.e. free N-glycans) are generated either from misfolded N-glycoproteins destined for the endoplasmic reticulum-associated degradation or from lipid-linked oligosaccharides, donor substrates for N-glycosylation of proteins. The mechanism responsible for the generation of free N-glycans is now well-understood, but the issue of whether other types of free glycans are present remains unclear. Here, we report on the accumulation of free, O-mannosylated glycans in budding yeast that were cultured in medium containing mannose as the carbon source. A structural analysis of these glycans revealed that their structures are identical to those of O-mannosyl glycans that are attached to glycoproteins. Deletion of the cyc8 gene, which encodes for a general transcription repressor, resulted in the accumulation of excessive amounts of free O-glycans, concomitant with a severe growth defect, a reduction in the level of an O-mannosylated protein, and compromised cell wall integrity. Our findings provide evidence in support of a regulated pathway for the degradation of O-glycoproteins in yeast and offer critical insights into the catabolic mechanisms that control the fate of O-glycosylated proteins.


Assuntos
Glicoproteínas/metabolismo , Manose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Parede Celular/metabolismo , Glicoproteínas/química , Homeostase , Proteólise , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química
12.
PLoS One ; 11(3): e0151891, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27010459

RESUMO

Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc3Man9GlcNAc2) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.


Assuntos
Polissacarídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa-Glucosidases/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Dados de Sequência Molecular , Mutação , Polissacarídeos/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , alfa-Glucosidases/genética
13.
Biosci Biotechnol Biochem ; 80(1): 152-7, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26264652

RESUMO

In the cytosol of Saccharomyces cerevisiae, most of the free N-glycans (FNGs) are generated from misfolded glycoproteins by the action of the cytoplasmic peptide: N-glycanase (Png1). A cytosol/vacuole α-mannosidase, Ams1, then trims the FNGs to eventually form a trisaccharide composed of Manß1,4GlcNAc ß1,4GlcNAc (Man1GlcNAc2). Whether or not the resulting Man1GlcNAc2 is enzymatically degraded further, however, is currently unknown. The objective of this study was to unveil the fate of Man1GlcNAc2 in S. cerevisiae. Quantitative analyses of the FNGs revealed a steady increase in the amount of Man1GlcNAc2 produced in the post-diauxic and stationary phases, suggesting that this trisaccharide is not catabolized during this period. Inoculation of the stationary phase cells into fresh medium resulted in a reduction in the levels of Man1GlcNAc2. However, this reduction was caused by its dilution due to cell division in the fresh medium. Our results thus indicate that Man1GlcNAc2 is not enzymatically catabolized in S. cerevisiae.


Assuntos
Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Trissacarídeos/metabolismo , Sequência de Carboidratos , Citosol/metabolismo , Glicosilação , Metabolismo , Dados de Sequência Molecular , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Manosidase/genética , alfa-Manosidase/metabolismo
14.
Biomolecules ; 5(3): 1499-514, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26193330

RESUMO

It is well known that the "free" form of glycans that are structurally related to asparagine (N)-linked glycans ("free N-glycans") are found in a wide variety of organisms. The mechanisms responsible for the formation/degradation of high mannose-type free N-glycans have been extensively studied in mammalian cells. Recent evidence, however, also suggests that sialylated, complex-type free N-glycans are also present in the cytosol of various mammalian-derived cultured cells/tissues. We report herein on an investigation of the mechanism responsible for the degradation of such sialyl free N-glycans. The findings show that the amount of glycans is dramatically reduced upon the co-expression of cytosolic sialidase NEU2 with cytosolic ß-glycosidase GBA3 in human stomach cancer-derived MKN45 cells. The physical interaction between NEU2 and GBA3 was confirmed by co-precipitation analyses as well as gel filtration assays. The NEU2 protein was found to be stabilized in the presence of GBA3 both in cellulo and in vitro. Our results thus indicate that cytosolic GBA3 is likely involved in the catabolism of cytosolic sialyl free N-glycans, possibly by stabilizing the activity of the NEU2 protein.


Assuntos
Citosol/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , Polissacarídeos/metabolismo , Neoplasias Gástricas/patologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Neuraminidase/química , Polissacarídeos/química , Ligação Proteica , Estabilidade Proteica , Transfecção
15.
Cell Mol Life Sci ; 72(13): 2509-33, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25772500

RESUMO

Asparagine (N)-linked protein glycosylation, which takes place in the eukaryotic endoplasmic reticulum (ER), is important for protein folding, quality control and the intracellular trafficking of secretory and membrane proteins. It is known that, during N-glycosylation, considerable amounts of lipid-linked oligosaccharides (LLOs), the glycan donor substrates for N-glycosylation, are hydrolyzed to form free N-glycans (FNGs) by unidentified mechanisms. FNGs are also generated in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins during ER-associated degradation. FNGs derived from LLOs and misfolded glycoproteins are eventually merged into one pool in the cytosol and the various glycan structures are processed to a near homogenous glycoform. This article summarizes the current state of our knowledge concerning the formation and catabolism of FNGs.


Assuntos
Asparagina/metabolismo , Vias Biossintéticas/fisiologia , Retículo Endoplasmático/metabolismo , Modelos Biológicos , Polissacarídeos/biossíntese , Polissacarídeos/metabolismo , Glicosilação , Hidrólise , Lipopolissacarídeos/metabolismo , Dobramento de Proteína
16.
Semin Cell Dev Biol ; 41: 110-20, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25475175

RESUMO

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that acts on N-glycoproteins. A growing evidence exists to indicate that the cytosolic form of PNGase, which is ubiquitously distributed throughout eukaryotes, is not only implicated in the efficient degradation of misfolded glycoproteins destined for the proteasomal degradation but also in the generation of free oligosaccharides as the initial step in the non-lysosomal catabolism of N-glycans. This article summarizes the current state of our knowledge of the physiological and molecular functions of the cytosolic PNGase in a model organism, Saccharomyces cerevisiae, and also discusses the functional/structural diversities of this molecule within eukaryotes.


Assuntos
Citosol/enzimologia , Glicoproteínas/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Glicoproteínas/química , Glicosilação , Humanos , Oligossacarídeos/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
17.
Biochem Biophys Res Commun ; 449(2): 256-61, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24835952

RESUMO

N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-ß-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process.


Assuntos
Acetilglucosaminidase/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas Fúngicas/metabolismo , Trichoderma/metabolismo , Acetilglucosaminidase/genética , Sequência de Aminoácidos , Citosol/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Glicosilação , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Trichoderma/genética
18.
J Biol Chem ; 288(45): 32673-32684, 2013 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-24062310

RESUMO

Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.


Assuntos
Hexosiltransferases/metabolismo , Leishmania major/enzimologia , Proteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Glicosilação , Hexosiltransferases/genética , Leishmania major/genética , Proteínas de Membrana/genética , Oligossacarídeos/genética , Oligossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Glycobiology ; 21(10): 1341-8, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21622726

RESUMO

In eukaryotic cells, it is known that N-glycans play a pivotal role in quality control of carrier proteins. Although "free" forms of oligosaccharides (fOSs) are known to be accumulated in the cytosol, the precise mechanism of their formation, degradation and biological relevance remains poorly understood. It has been shown that, in budding yeast, almost all fOSs are formed from misfolded glycoproteins. Precise structural analysis of fOSs revealed that several yeast fOSs bear a yeast-specific modification by Golgi-resident α-1,6-mannosyltransferase, Och1. In this study, structural diversity of fOSs in och1Δ cells was analyzed. To our surprise, several fOSs in och1Δ cells have unusual α-1,3-linked mannose residues at their non-reducing termini. These mannose residues were not observed in wild-type cells, suggesting that the addition of these unique mannoses occurred as a compensation of Och1 defect. A significant increase in the amount of fOSs modified by Golgi-localized mannosyltransferases was also observed in och1Δ cells. Moreover, the amount of processed fOSs and intracellular α-mannosidase (Ams1) both increased in this mutant. Up-regulation of Ams1 activity was also apparent for cells treated with cell wall perturbation reagent. These results provide an insight into a possible link between catabolism of fOSs and cell wall stress.


Assuntos
Manosiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Complexo de Golgi/metabolismo , Manose/química , Manose/metabolismo , Manosiltransferases/genética , Glicoproteínas de Membrana/genética , Mutação , Oligossacarídeos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Regulação para Cima , alfa-Manosidase/genética , alfa-Manosidase/metabolismo
20.
J Biol Chem ; 285(32): 24324-34, 2010 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-20511219

RESUMO

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related alpha-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.


Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas/química , Manosidases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa-Manosidase/química , alfa-Manosidase/metabolismo , Sequência de Bases , Bioquímica/métodos , Cicloeximida/química , Glicosilação , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Conformação Proteica , Canais de Translocação SEC , Ubiquitina/química
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