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/deficiênciaRESUMO
Chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic bronchitis, is now the third cause of death worldwide, and COVID-19 infection has been reported as an exacerbation factor of them. In this study, we report that the intratracheal administration of the keratan sulfate-based disaccharide L4 mitigates the symptoms of elastase-induced emphysema in a mouse model. To know the molecular mechanisms, we performed a functional analysis of a C-type lectin receptor, langerin, a molecule that binds L4. Using mouse BMDCs (bone marrow-derived dendritic cells) as langerin-expressing cells, we observed the downregulation of IL-6 and TNFa and the upregulation of IL-10 after incubation with L4. We also identified CapG (a macrophage-capping protein) as a possible molecule that binds langerin by immunoprecipitation combined with a mass spectrometry analysis. We identified a portion of the CapG that was localized in the nucleus and binds to the promoter region of IL-6 and the TNFa gene in BMDCs, suggesting that CapG suppresses the gene expression of IL-6 and TNFa as an inhibitory transcriptional factor. To examine the effects of L4 in vivo, we also generated langerin-knockout mice by means of genome editing technology. In an emphysema mouse model, the administration of L4 did not mitigate the symptoms of emphysema as well as the inflammatory state of the lung in the langerin-knockout mice. These data suggest that the anti-inflammatory effect of L4 through the langerin-CapG axis represents a potential therapeutic target for the treatment of emphysema and COPD.
Assuntos
Dissacarídeos , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Animais , Camundongos , Dissacarídeos/farmacologia , Modelos Animais de Doenças , Interleucina-6/genética , Sulfato de Queratano/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/genética , Enfisema Pulmonar/induzido quimicamente , Lectinas Tipo C/metabolismoRESUMO
Mutations in the human peptide:N-glycanase gene (NGLY1), which encodes a cytosolic de-N-glycosylating enzyme, cause a congenital autosomal recessive disorder. In rodents, the loss of Ngly1 results in severe developmental delay or lethality, but the underlying mechanism remains unknown. In this study, we found that deletion of Fbxo6 (also known as Fbs2), which encodes a ubiquitin ligase subunit that recognizes glycoproteins, rescued the lethality-related defects in Ngly1-KO mice. In NGLY1-KO cells, FBS2 overexpression resulted in the substantial inhibition of proteasome activity, causing cytotoxicity. Nuclear factor, erythroid 2-like 1 (NFE2L1, also known as NRF1), an endoplasmic reticulum-associated transcriptional factor involved in expression of proteasome subunits, was also abnormally ubiquitinated by SCFFBS2 in NGLY1-KO cells, resulting in its retention in the cytosol. However, the cytotoxicity caused by FBS2 was restored by the overexpression of "glycan-less" NRF1 mutants, regardless of their transcriptional activity, or by the deletion of NRF1 in NGLY1-KO cells. We conclude that the proteasome dysfunction caused by the accumulation of N-glycoproteins, primarily NRF1, ubiquitinated by SCFFBS2 accounts for the pathogenesis resulting from NGLY1 deficiency.
Assuntos
Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Açúcares/metabolismo , Animais , Comportamento Animal , Morte Celular , Núcleo Celular/metabolismo , Proliferação de Células , Citosol/metabolismo , Células HCT116 , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Atividade Motora , Mutação/genética , Fator 1 Nuclear Respiratório/metabolismo , Polissacarídeos/metabolismo , Transporte Proteico , UbiquitinaçãoRESUMO
Recent studies demonstrated the occurrence of sialyl free N-glycans (FNGs) in sera from a variety of animals. Unlike the intracellular FNGs that mainly carry a single N-acetylglucosamine at their reducing termini (Gn1-type), these extracellular FNGs have an N,N'-diacetylchitobiose at their reducing termini (Gn2-type). The detailed mechanism for how they are formed, however, remains unclarified. In this study, we report on an improved method for isolating FNGs from sera and found that, not only sialyl FNGs, but also neutral FNGs are present in animal sera. Most of the neutral oligomannose-type FNGs were found to be Gn1-type. We also found that a small portion of sialyl FNGs were Gn1-type. The ratio of Gn1-type sialyl FNGs varies between species, and appears to be partially correlated with the distribution of lysosomal chitobiase activity. We also identified small sialylated glycans similar to milk oligosaccharides, such as sialyl lactose or sialyl N-acetyllactosamine in sera. Our results indicate that there are varieties of free oligosaccharides in sera and the mechanism responsible for their formation is more complicated than currently envisaged.
Assuntos
Oligossacarídeos , Polissacarídeos , Acetilglucosamina , Animais , CitosolRESUMO
N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1-/- rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1-/- rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1-/- rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1-/- rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1-/- rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Transtornos dos Movimentos/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Modelos Animais de Doenças , Degradação Associada com o Retículo Endoplasmático/genética , Oftalmopatias Hereditárias , Técnicas de Inativação de Genes , Glicosilação , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Doenças do Aparelho Lacrimal , Transtornos dos Movimentos/patologia , Mutação/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/patologia , Fenótipo , Complexo de Endopeptidases do Proteassoma/genética , RatosRESUMO
N-Glycanase 1 (NGLY1) deficiency is a congenital disorder caused by mutations in the NGLY1 gene. Because systemic Ngly1-/- mice with a C57BL/6 (B6) background are embryonically lethal, studies on the mechanism of NGLY1 deficiency using mice have been problematic. In this study, B6-Ngly1-/+ mice were crossed with Japanese wild mice-originated Japanese fancy mouse 1 (JF1) mice to produce viable F2 Ngly1-/- mice from (JF1×B6)F1 Ngly1-/+ mice. Systemic Ngly1-/- mice with a JF1 mouse background were also embryonically lethal. Hybrid F1 Ngly1-/- (JF1/B6F1) mice, however, showed developmental delay and motor dysfunction, similar to that in human patients. JF1/B6F1 Ngly1-/- mice showed increased levels of plasma and urinary aspartylglycosamine, a potential biomarker for NGLY1 deficiency. JF1/B6F1 Ngly1-/- mice are a useful isogenic animal model for the preclinical testing of therapeutic options and understanding the precise pathogenic mechanisms responsible for NGLY1 deficiency.
Assuntos
Defeitos Congênitos da Glicosilação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Acetilglucosamina/análogos & derivados , Acetilglucosamina/sangue , Acetilglucosamina/genética , Animais , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/sangue , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genéticaRESUMO
BACKGROUND: Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application. METHODS: We performed binding analysis between purified long and short forms of langerin and a series of KS disaccharide components. We also chemically synthesized oligomeric derivatives of L4 to develop a new high-affinity ligand of langerin. RESULTS: We show that the binding critically requires the 6-sulfation of Gal and that the long form of langerin displays higher affinity than the short form. The synthesized trimeric (also designated as triangle or Tri) and polymeric (pendant) L4 derivatives displayed over 1000-fold higher affinity toward langerin than monomeric L4. The pendant L4, but not the L4 monomer, was found to effectively transduce langerin signaling in a model cell system. CONCLUSIONS: L4 is a specific ligand for langerin. Oligomerization of L4 unit increased the affinity toward langerin. GENERAL SIGNIFICANCE: These results suggest that oligomeric L4 derivatives will be useful for clarifying the langerin functions and for the development of new glycan-based anti-inflammatory drugs.
Assuntos
Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Dissacarídeos/metabolismo , Sulfato de Queratano/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Antígenos CD/química , Antígenos de Superfície/química , Líquido da Lavagem Broncoalveolar/química , Citocinas/metabolismo , Células Dendríticas/metabolismo , Dissacarídeos/química , Dissacarídeos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Galactose/metabolismo , Humanos , Sulfato de Queratano/química , Lectinas Tipo C/química , Ligantes , Lectinas de Ligação a Manose/química , Ligação Proteica , Isoformas de Proteínas/metabolismo , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Core fucosylation, a posttranslational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4)-deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-ß (IFN-ß) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-ß production, impaired IFN-ß production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling.
Assuntos
Fucose/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Animais , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Emphysema is a typical component of chronic obstructive pulmonary disease (COPD), a progressive and inflammatory airway disease. However, no effective treatment currently exists. Here, we show that keratan sulfate (KS), one of the major glycosaminoglycans produced in the small airway, decreased in lungs of cigarette smoke-exposed mice. To confirm the protective effect of KS in the small airway, a disaccharide repeating unit of KS designated L4 ([SO3--6]Galß1-4[SO3--6]GlcNAc) was administered to two murine models: elastase-induced-emphysema and LPS-induced exacerbation of a cigarette smoke-induced emphysema. Histological and microcomputed tomography analyses revealed that, in the mouse elastase-induced emphysema model, administration of L4 attenuated alveolar destruction. Treatment with L4 significantly reduced neutrophil influx, as well as the levels of inflammatory cytokines, tissue-degrading enzymes (matrix metalloproteinases), and myeloperoxidase in bronchoalveolar lavage fluid, suggesting that L4 suppressed inflammation in the lung. L4 consistently blocked the chemotactic migration of neutrophils in vitro. Moreover, in the case of the exacerbation model, L4 inhibited inflammatory cell accumulation to the same extent as that of dexamethasone. Taken together, L4 represents one of the potential glycan-based drugs for the treatment of COPD through its inhibitory action against inflammation.
Assuntos
Dissacarídeos/uso terapêutico , Progressão da Doença , Sulfato de Queratano/uso terapêutico , Pneumonia/tratamento farmacológico , Pneumonia/prevenção & controle , Enfisema Pulmonar/tratamento farmacológico , Animais , Líquido da Lavagem Broncoalveolar , Dexametasona/farmacologia , Dissacarídeos/farmacologia , Modelos Animais de Doenças , Sulfato de Queratano/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Elastase Pancreática/metabolismo , Pneumonia/complicações , Pneumonia/patologia , Alvéolos Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/complicações , Enfisema Pulmonar/patologia , Células RAW 264.7 , Fumar , Sus scrofaRESUMO
Glycans play key roles in a variety of protein functions under normal and pathological conditions, but several glycosyltransferase-deficient mice exhibit no or only mild phenotypes due to redundancy or compensation of glycan functions. However, we have only a limited understanding of the underlying mechanism for these observations. Our previous studies indicated that 70% of Fut8-deficient (Fut8(-/-)) mice that lack core fucose structure die within 3 days after birth, but the remainder survive for up to several weeks although they show growth retardation as well as emphysema. In this study, we show that, in mouse embryonic fibroblasts (MEFs) from Fut8(-/-) mice, another N-glycan branching structure, bisecting GlcNAc, is specifically up-regulated by enhanced gene expression of the responsible enzyme N-acetylglucosaminyltransferase III (GnT-III). As candidate target glycoproteins for bisecting GlcNAc modification, we confirmed that level of bisecting GlcNAc on ß1-integrin and N-cadherin was increased in Fut8(-/-) MEFs. Moreover using mass spectrometry, glycan analysis of IgG1 in Fut8(-/-) mouse serum demonstrated that bisecting GlcNAc contents were also increased by Fut8 deficiency in vivo. As an underlying mechanism, we found that in Fut8(-/-) MEFs Wnt/ß-catenin signaling is up-regulated, and an inhibitor against Wnt signaling was found to abrogate GnT-III expression, indicating that Wnt/ß-catenin is involved in GnT-III up-regulation. Furthermore, various oxidative stress-related genes were also increased in Fut8(-/-) MEFs. These data suggest that Fut8(-/-) mice adapted to oxidative stress, both ex vivo and in vivo, by inducing various genes including GnT-III, which may compensate for the loss of core fucose functions.
Assuntos
Fucose/metabolismo , N-Acetilglucosaminiltransferases/genética , Polissacarídeos/metabolismo , Regulação para Cima , Proteínas Wnt/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with NGLY1 deficiency, preclinical studies have not been conducted due to the lack of animal models for epileptic seizures in NGLY1 deficiency. Here, we observed the behaviors of male and female Ngly1-/- mice by video monitoring and found that these mice exhibit spontaneous seizure-like behaviors. Gene expression analyses and enzyme immunoassay revealed significant decreases in oxytocin, a well-known neuropeptide, in the hypothalamus of Ngly1-/- mice. Seizure-like behaviors in Ngly1-/- mice were transiently suppressed by a single intranasal administration of oxytocin. These findings suggest the therapeutic potential of oxytocin for epileptic seizure in patients with NGLY1 deficiency and contribute to the clarification of the disease mechanism.
Assuntos
Defeitos Congênitos da Glicosilação , Ocitocina , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Convulsões , Animais , Feminino , Masculino , Camundongos , Administração Intranasal , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Hipotálamo/metabolismo , Hipotálamo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ocitocina/administração & dosagem , Ocitocina/farmacologia , Convulsões/tratamento farmacológico , Convulsões/etiologia , Defeitos Congênitos da Glicosilação/complicações , Defeitos Congênitos da Glicosilação/tratamento farmacológico , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiênciaRESUMO
Dendritic cells, macrophages, neutrophils, and other antigen-presenting cells express various C-type lectin receptors that function to recognize the glycans associated with pathogens. The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds various pathogens such as HIV glycoprotein 120, the Ebola glycoprotein, hemagglutinin, and the dengue virus glycoprotein in addition to the SARS-CoV-2 spike protein, and also triggers antigen-presenting cell endocytosis and immune escape from systemic infections. Many studies on the binding of SARS-CoV-2 spike protein with glycans have been published, but the underlying mechanism by which intracellular signaling occurs remains unclear. In this study, we report that the S1 spike protein of SARS-CoV-2 induces the phosphorylation of extracellular signal-regulated kinases (ERKs) in THP-1 cells, a DC-SIGN-expressing human monocytic leukemic cell line. On the other hand, the phosphorylation level of NF-κB remained unchanged under the same conditions. These data suggest that the major cell signaling pathway regulated by the S1 spike protein is the ERK pathway, which is superior to the NF-κB pathway in these DC-SIGN-expressing THP-1 cells and may contribute to immune hyperactivation in SARS-CoV-2 infections. Additionally, several glycans such as mannans, mannosylated bovine serum albumin, the serum amyloid beta protein, and intracellular adhesion molecule 3 suppressed ERK phosphorylation, suggesting that these molecules are target molecules for SARS-CoV-2 infection by suppressing immune hyperactivation that occurs in the ERK signaling pathway.
Assuntos
COVID-19 , Receptores de Superfície Celular , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , NF-kappa B/metabolismo , SARS-CoV-2/metabolismo , Sistema de Sinalização das MAP Quinases , Células THP-1 , Peptídeos beta-Amiloides , COVID-19/metabolismo , Moléculas de Adesão Celular/metabolismo , Transdução de Sinais , Lectinas Tipo C/metabolismo , Polissacarídeos/metabolismo , Células Dendríticas/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD), manifested as emphysema and chronic airway obstruction, can be exacerbated by bacterial and viral infections. Although the frequency of exacerbations increases as the disease progresses, the mechanisms underlying this phenomenon are largely unknown, and there is a need for a simple in vivo exacerbation model. In this study, we compared four groups of mice treated with PBS alone, elastase alone, LPS alone, and elastase plus LPS. A single intratracheal administration of LPS to mice with elastase-induced emphysema provoked infiltration of inflammatory cells, especially CD8(+) T cells, into alveolar spaces and increased matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and perforin production in bronchoalveolar lavage fluid at the acute inflammatory phase compared with the other groups. We also measured the percentage of low-attenuation area (LAA%) in the above mice using micro-computed X-ray tomography. The LAA% was the most sensitive parameter for quantitative assessments of emphysema among all the parameters evaluated. Using the parameter of LAA%, we found significantly more severe alveolar destruction in the group treated with elastase plus LPS compared with the other groups during long-term longitudinal observations. We built three-dimensional images of the emphysema and confirmed that the lungs of elastase plus LPS-treated mice contained larger emphysematous areas than mice treated with elastase alone. Although human exacerbation of COPD is clinically and pathologically complicated, this simple mouse model mimics human cases to some extent and will be useful for elucidating its mechanism and developing therapeutic strategies.
Assuntos
Lipopolissacarídeos/toxicidade , Doença Pulmonar Obstrutiva Crônica/etiologia , Enfisema Pulmonar/complicações , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Imageamento Tridimensional , Lipopolissacarídeos/administração & dosagem , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Elastase Pancreática/administração & dosagem , Elastase Pancreática/toxicidade , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/diagnóstico por imagem , Enfisema Pulmonar/patologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Microtomografia por Raio-XRESUMO
BACKGROUND: Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice. METHODS: Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice. RESULTS: The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver. CONCLUSIONS: AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins. GENERAL SIGNIFICANCE: AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.
Assuntos
Aldeído Redutase/fisiologia , Aldeído Redutase/genética , Animais , Ácido Ascórbico/análise , Proteínas de Ligação ao Cálcio/análise , Feminino , Glucuronatos/metabolismo , Ácido Glucurônico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Fígado/química , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCerSyn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected.
Assuntos
Perfilação da Expressão Gênica , Glicoesfingolipídeos/biossíntese , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Compartimento Celular , Linhagem Celular , Citometria de Fluxo , Imunofluorescência , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Retroviridae/genética , Frações Subcelulares/enzimologiaRESUMO
Fut8 (α1,6-Fucosyltransferase) heterozygous knock-out (Fut8(+/-)) mice had an increased influx of inflammatory cells into the lungs, and this was associated with an up-regulation of matrix metalloproteinases, MMP-2 and MMP-9, after treatment with porcine pancreatic elastase (PPE), exhibiting an emphysema-prone phenotype as compared with wild type mice (Fut8(+/+)). The present data as well as our previous data on cigarette-smoke-induced emphysema [8] led us to hypothesize that reduced Fut8 levels leads to COPD with increased inflammatory response in humans and is associated with disease progression. To test this hypothesis, symptomatic current or ex-smokers with stable COPD or at risk outpatients were recruited. We investigated the association between serum Fut8 activity and disease severity, including the extent of emphysema (percentage of low-attenuation area; LAA%), airflow limitation, and the annual rate of decline in forced expiratory volume in 1 s (FEV(1)). Association with the exacerbation of COPD was also evaluated over a 3-year period. Serum Fut8 and MMP-9 activity were measured. Fut8 activity significantly increased with age among the at risk patients. In the case of COPD patients, however, the association was not clearly observed. A faster annual decline of FEV(1) was significantly associated with lower Fut8 activity. Patients with lower Fut8 activity experienced exacerbations more frequently. These data suggest that reduced Fut8 activity is associated with the progression of COPD and serum Fut8 activity is a non-invasive predictive biomarker candidate for progression and exacerbation of COPD.
Assuntos
Fucosiltransferases/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/patologia , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fucosiltransferases/sangue , Fucosiltransferases/genética , Humanos , Masculino , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Elastase Pancreática/farmacologia , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/enzimologia , Enfisema Pulmonar/sangue , Enfisema Pulmonar/enzimologiaRESUMO
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 , TransgenesRESUMO
Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.
Assuntos
Carboidratos/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Nucleotídeos/química , Linhagem Celular Tumoral , Glicosilação , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Nucleotídeos/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Ativação Linfocitária/imunologia , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/metabolismo , Animais , Linfócitos B/citologia , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Epitopos/imunologia , Marcação de Genes , Humanos , Lectinas/metabolismo , Ligantes , Camundongos , Oxigenases de Função Mista/deficiência , Oxigenases de Função Mista/genética , Fosfotirosina/metabolismo , Polissacarídeos/metabolismo , Ratos , Receptores de Antígenos de Linfócitos B/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Sialiltransferases/metabolismo , Baço/imunologia , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Xenopus laevis is an excellent animal for analyzing early vertebrate development. Various effects of glycosaminoglycans (GAGs) on growth factor-related cellular events during embryogenesis have been demonstrated in Xenopus. To elucidate the relationship between alterations in fine structure and changes in the specificity of growth factor binding during Xenopus development, heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) chains were isolated at four different embryonic stages and their structure and growth factor-binding capacities were compared. The total amounts of both HS and CS/DS chains decreased from the pre-midblastula transition to the gastrula stage, but increased exponentially during the following developmental stages. The length of HS chains was not significantly affected by development, whereas that of CS/DS chains increased with development. The disaccharide composition of GAGs in embryos also changed during development. The degree of sulfation of the HS chains gradually decreased with development. The predominant sulfation position in the CS/DS chains shifted from C4 to C6 of GalNAc during embryogenesis. Growth factor-binding experiments using a BIAcore system demonstrated that GAGs bound growth factors including fibroblast growth factors-1 and -2, midkine, and pleiotrophin, with comparable affinities. These affinities significantly varied during development, although the correlation between the structural alterations of GAGs and the change in the ability to bind growth factors remains to be clarified. The expression of saccharide sequences, which specifically interact with a growth factor, might be regulated during development.