RESUMO
BACKGROUND: N-Glycan branching regulates various functions of glycoproteins. N-Acetylglucosaminyltransferase V (GnT-V) is a GlcNAc transferase that acts on N-glycans and the GnT-V-producing branch is highly related to cancer progression. This indicates that specific GnT-V inhibitors may be drug candidates for cancer treatment. To design novel GnT-V inhibitors, we focused on the unique and weak recognition of the donor substrate UDP-GlcNAc by GnT-V. On the basis of the catalytic pocket structure, we hypothesized that UDP-GlcNAc analogs with increasing hydrophobicity may be GnT-V inhibitors. METHODS: We chemically synthesized 10 UDP-GlcNAc analogs in which one or two phosphate groups were replaced with hydrophobic groups. To test these compounds, we set up an HPLC-based enzyme assay system for all N-glycan-branching GlcNAc transferases in which GnT-I-V activity was measured using purified truncated enzymes. Using this system, we assessed the inhibitory effects of the synthesized compounds on GnT-V and their specificity. RESULTS: Several UDP-GlcNAc analogs inhibited GnT-V activity, although the inhibition potency was modest. Compared with other GnTs, these compounds showed a preference for GnT-V, which suggested that GnT-V was relatively tolerant of hydrophobicity in the donor substrate. Docking models of the inhibitory compounds with GnT-V suggested the mechanisms of how these compounds interacted with GnT-V and inhibited its action. CONCLUSIONS: Chemical modification of the donor substrate may be a promising strategy to develop selective inhibitors of GnT-V. GENERAL SIGNIFICANCE: Our findings provide new insights into the design of GnT inhibitors and how GnTs recognize the donor substrate.
Assuntos
Neoplasias , Polissacarídeos , Glicoproteínas , Humanos , Polissacarídeos/química , Polissacarídeos/farmacologia , Difosfato de UridinaRESUMO
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
Fucose, a terminal sugar in glycoconjugates, critically regulates various physiological and pathological phenomena, including cancer development and inflammation. However, there are currently no probes for efficient labeling and detection of this sugar. We chemically synthesized a novel series of alkynyl-fucose analogs as probe candidates and found that 7-alkynyl-fucose gave the highest labeling efficiency and low cytotoxicity. Among the fucose analogs, 7-alkynyl-fucose was the best substrate against all five fucosyltransferases examined. We confirmed its conversion to the corresponding guanosine diphosphate derivative in cells and found that cellular glycoproteins were labeled much more efficiently with 7-alkynyl-fucose than with an existing probe. 7-Alkynyl-fucose was detected in the N-glycan core by mass spectrometry, and 7-alkynyl-fucose-modified proteins mostly disappeared in core-fucose-deficient mouse embryonic fibroblasts, suggesting that this analog mainly labeled core fucose in these cells. These results indicate that 7-alkynyl-fucose is a highly sensitive and powerful tool for basic glycobiology research and clinical application for biomarker discovery.
Assuntos
Biomarcadores Tumorais/análise , Fucose/farmacologia , Sondas Moleculares/farmacologia , Polissacarídeos/análise , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fucose/análogos & derivados , Fucose/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Sondas Moleculares/síntese química , Sondas Moleculares/químicaRESUMO
We previously reported that knockout mice for α1,6-fucosyltransferase (Fut8), which catalyzes the biosynthesis of core-fucose in N-glycans, develop emphysema and that Fut8 heterozygous knockout mice are more sensitive to cigarette smoke-induced emphysema than wild-type mice. Moreover, a lower FUT8 activity was found to be associated with a faster decline in lung function among chronic obstructive pulmonary disease (COPD) patients. These results led us to hypothesize that core-fucosylation levels in a glycoprotein could be used as a biomarker for COPD. We focused on a lung-specific glycoprotein, surfactant protein D (SP-D), which plays a role in immune responses and is present in the distal airways, alveoli, and blood circulation. The results of a glycomic analysis reported herein demonstrate the presence of a core-fucose in an N-glycan on enriched SP-D from pooled human sera. We developed an antibody-lectin enzyme immunoassay (EIA) for assessing fucosylation (core-fucose and α1,3/4 fucose) in COPD patients. The results indicate that fucosylation levels in serum SP-D are significantly higher in COPD patients than in non-COPD smokers. The severity of emphysema was positively associated with fucosylation levels in serum SP-D in smokers. Our findings suggest that increased fucosylation levels in serum SP-D are associated with the development of COPD. BIOLOGICAL SIGNIFICANCE: It has been proposed that serum SP-D concentrations are predictive of COPD pathogenesis, but distinguishing between COPD patients and healthy individuals to establish a clear cut-off value is difficult because smoking status highly affects circulating SP-D levels. Herein, we focused on N-glycosylation in SP-D and examined whether or not N-glycosylation patterns in SP-D are associated with the pathogenesis of COPD. We performed an N-glycomic analysis of human serum SP-D and the results show that a core-fucose is present in its N-glycan. We also found that the N-glycosylation in serum SP-D was indeed altered in COPD, that is, fucosylation levels including core-fucosylation are significantly increased in COPD patients compared with non-COPD smokers. The severity of emphysema was positively associated with fucosylation levels in serum SP-D in smokers. Our findings shed new light on the discovery and/or development of a useful biomarker based on glycosylation changes for diagnosing COPD. This article is part of a Special Issue entitled: HUPO 2014.
Assuntos
Fucose , Doença Pulmonar Obstrutiva Crônica/sangue , Proteína D Associada a Surfactante Pulmonar/sangue , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/sangue , Feminino , Glicosilação , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-IdadeRESUMO
In order to verify the protein enriched from pooled human sera to be a lung-specific protein surfactant protein-D (SP-D), we performed peptide mass fingerprinting (PMF)-based protein identification. MASCOT search results of the obtained PMF unequivocally demonstrated that it is identical to human SP-D. Meanwhile, we performed MALDI-QIT-TOF mass spectrometry-based N-glycomic analysis of the recombinant human SP-D produced in murine myeloma cells. The obtained mass spectra of N-glycans from the recombinant SP-D demonstrated that the recombinant protein is almost exclusively modified with core-fucosylated N-glycans [1].
RESUMO
N-Acetylglucosaminyltransferase (GnT) III is a glycosyltransferase which produces bisected N-glycans by transferring GlcNAc to the 4-position of core mannose. Bisected N-glycans are involved in physiological and pathological processes through the functional regulation of their carrier proteins. An understanding of the biological functions of bisected glycans will be greatly accelerated by use of specific inhibitors of GnT-III. Thus far, however, such inhibitors have not been developed and even the substrate-binding mode of GnT-III is not fully understood. To gain insight into structural features required of the substrate, we systematically synthesized four N-glycan units, the branching parts of the bisected and non-bisected N-glycans. The series of syntheses were achieved from a common core trimannose, giving bisected tetra- and hexasaccharides as well as non-bisected tri- and pentasaccharides. A competitive GnT-III inhibition assay using the synthetic substrates revealed a vital role for the Manß(1-4)GlcNAc moiety. In keeping with previous reports, GlcNAc at the α1,3-branch is also involved in the interaction. The structural requirements of GnT-III elucidated in this study will provide a basis for rational inhibitor design.
Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Polissacarídeos/farmacologia , Configuração de Carboidratos , Sequência de Carboidratos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/síntese química , Polissacarídeos/química , Relação Estrutura-AtividadeRESUMO
O-glycans are suitable targets as novel and useful tumor markers. The structures of O-glycans in human sera from four healthy controls were precisely analyzed to obtain the reference O-glycan database. O-glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine and identified using two-dimensional mapping, enzymatic digestion and mass spectrometry (MS) together with methanolysis and the use of newly synthesized sulfated oligosaccharides as standards. O-glycans, present at more than 0.01% of the total O-glycans, were analyzed, and 18 kinds of acidic and 2 kinds of neutral glycans were identified. NeuAcα2-3Galß1-3N-acetylgalactosamine (GalNAc) (61-64%), NeuAcα2-3Galß1-3(NeuAcα2-6)GalNAc (15-26%) and Galß1-3GalNAc (6-14%) were major components while other sialylated glycans, Galß1-3(NeuAcα2-6)GalNAc, Galß1-4GlcNAcß1-6(NeuAcα2-3Galß1-3)GalNAc and NeuAcα2-3Galß1-4GlcNAcß1-6(NeuAcα2-3Galß1-3)GalNAc were relatively minor components, accounting for â¼1-2%. Very minor glycans accounting for â¼0.01-0.1% of the total include (i) the neutral glycan, Galß1-4GlcNAcß1-6(Galß1-3)GalNAc, (ii) sialylated glycans, having sialyl Tn antigen, agalacto and trisialylated structures, (iii) fucosylated glycans forming blood type H antigen, blood type A antigen, blood type B antigen, Lewis X antigen and sialyl Lewis X antigen and (iv) sulfated glycans, having 6-sulfo and 3'-sulfo structures. Two kinds of clinically applied tumor markers namely sialyl Tn antigen and sialyl Lewis X antigen in healthy controls sera were revealed to be present at â¼0.1-0.2% of the total. However, other markers such as CA19-9 and DU-PAN-2 were not found, suggesting the relative amounts of these glycans to be <0.01%. These detailed O-glycan profiles will help to find novel carbohydrate tumor markers.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/sangue , Oligossacarídeos/sangue , Polissacarídeos/sangue , Gangliosídeos , Glicosilação , Humanos , Neoplasias/patologiaRESUMO
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
In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/citologia , Ácido Glutâmico/metabolismo , Homeostase/fisiologia , Tenascina/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Astrócitos/ultraestrutura , Células Cultivadas , Córtex Cerebral/metabolismo , Sulfatos de Condroitina/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Glicoproteínas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Tenascina/genéticaRESUMO
Our previous studies on a ß1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.
Assuntos
Regulação Enzimológica da Expressão Gênica , N-Acetilglucosaminiltransferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Animais , Células COS , Carboidratos/química , Chlorocebus aethiops , Glicosilação , Glicosiltransferases/metabolismo , Células HEK293 , Humanos , Hidrólise , Camundongos , Nucleosídeos/química , Plasmídeos/metabolismo , Interferência de RNARESUMO
In demyelinating diseases such as multiple sclerosis, a critical problem is failure of remyelination, which is important for protecting axons against degeneration and restoring conduction deficits. However, the underlying mechanism of demyelination/remyelination remains unclear. N-acetylglucosaminyltransferase-IX (GnT-IX; also known as GnT-Vb) is a brain-specific glycosyltransferase that catalyzes the branched formation of O-mannosyl glycan structures. O-Mannosylation of α-dystroglycan is critical for its function as an extracellular matrix receptor, but the biological significance of its branched structures, which are exclusively found in the brain, is unclear. In this study, we found that GnT-IX formed branched O-mannosyl glycans on receptor protein tyrosine phosphatase ß (RPTPß) in vivo. Since RPTPß is thought to play a regulatory role in demyelinating diseases, GnT-IX-deficient mice were subjected to cuprizone-induced demyelination. Cuprizone feeding for 8 weeks gradually promoted demyelination in wild-type mice. In GnT-IX-deficient mice, the myelin content in the corpus callosum was reduced after 4 weeks of treatment, but markedly increased at 8 weeks, suggesting enhanced remyelination under GnT-IX deficiency. Furthermore, astrocyte activation in the corpus callosum of GnT-IX-deficient mice was significantly attenuated, and an oligodendrocyte cell lineage analysis indicated that more oligodendrocyte precursor cells differentiated into mature oligodendrocytes. Together, branched O-mannosyl glycans in the corpus callosum in the brain are a necessary component of remyelination inhibition in the cuprizone-induced demyelination model, suggesting that modulation of O-mannosyl glycans is a likely candidate for therapeutic strategies.
Assuntos
Astrócitos/metabolismo , Doenças Desmielinizantes/enzimologia , Doenças Desmielinizantes/patologia , N-Acetilglucosaminiltransferases/deficiência , Fatores Etários , Animais , Encéfalo/patologia , Antígeno CD11b/metabolismo , Células Cultivadas , Cadeias Pesadas de Clatrina/metabolismo , Corpo Caloso/patologia , Cuprizona/toxicidade , Doenças Desmielinizantes/etiologia , Modelos Animais de Doenças , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , N-Acetilglucosaminiltransferases/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Polissacarídeos/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismoRESUMO
Determining glycosyltransferase activities gives a clue for better understanding an underlying mechanism for glycomic alterations of carrier molecules. N-glycan branch formation is concertedly regulated by cooperative and competitive activities of N-acetylglucosaminyltransferases (GnTs). Here, we describe methods for large scale preparation of the oligosaccharide acceptor substrate, fluorescence-labeling of oligosaccharides by pyridylamination, quality control, and reversed phase HPLC-based measurement of GnT activities including GnT-III, IV, V, and IX.
Assuntos
Ensaios Enzimáticos/métodos , N-Acetilglucosaminiltransferases/metabolismo , Oligossacarídeos/metabolismo , Aminação , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Humanos , N-Acetilglucosaminiltransferases/isolamento & purificação , Oligossacarídeos/análiseRESUMO
The glucose transporter isoform, GLUT2, -mediated glucose sensing is essential for maintaining normal glucose-stimulated insulin secretion in pancreatic beta cells. We previously reported that GnT-IVa glycosyltransferase is required for the production of an N-glycan structure that acts as a ligand for galectins to form the glycan-galectin lattice that maintains the stable cell surface expression of GLUT2, and cellular glucose transport activity, although the functional relevance of the N-glycosylation of GLUT2 to its membrane sub-domain distribution is not fully understood. In the present study, we demonstrated that disruption of the GLUT2 N-glycan-galectin lattice by the genetic inactivation of GnT-IVa, or by treatment of pancreatic beta cells with competitive glycan mimetics, induced the re-distribution of GLUT2 into the lipid-raft microdomain. This subsequently resulted in the binding of Stomatin to GLUT2 and an attenuation of cellular glucose transport activity. Moreover, disruption of the lipid-raft microdomain by treatment with methyl-ß-cyclodextrin caused the GLUT2 to be released from lipid-rafts and reactivation of the cellular glucose transport activity in GnT-IVa deficient beta cells. These results indicate that the membrane sub-domain distribution of GLUT2 is associated with the glucose transport activity of beta cells, in which the GnT-IVa-dependent formation of the N-glycan-galectin lattice plays an important role. This provides a novel pathophysiological insight into the mechanism of beta cell failure in the pathogenesis of type 2 diabetes.
Assuntos
Transportador de Glucose Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Transporte Biológico , Proteínas Sanguíneas/metabolismo , Galectinas/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Glicosilação , Imunoprecipitação , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , beta-Ciclodextrinas/farmacologiaRESUMO
We previously reported on the accumulation of a substantial amount of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans in human pancreatic cancer cells (Yabu M, Korekane H, Takahashi H, Ohigashi H, Ishikawa O, Miyamoto Y. 2013. Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers. Glycoconj J, 30(3):247-256). In the present paper, we further extend our cancer glycomic study of human prostate cancer. Specifically, we demonstrate that, in addition to the free Neu5Ac-containing N-glycans, significant amounts of free deaminoneuraminic acid (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid)-containing N-glycans had accumulated in the prostate cancer tissues from four of five patients. Indeed, in one of the four cases, the free KDN glycans accumulated as major components in prostate cancer tissue. The structures of the KDN-containing free oligosaccharides were analyzed by a variety of methods. Specifically, we used fluorescent labeling with aminopyridine combined with two-dimensional mapping, KDNase digestion and mass spectrometry to facilitate identification. The analysis also utilized newly synthesized KDN-linked oligosaccharides as standards. The prostate-specific glycans were composed of five species having the following sequence, KDN-Gal-GlcNAc-Man-Man-GlcNAc (α2,6-KDN-linked glycans being the dominant form). The most abundant free KDN-containing N-glycan was KDNα2-6Galß1-4GlcNAcß1-2Manα1-3Manß1-4GlcNAc followed by KDNα2-6Galß1-4GlcNAcß1-2Manα1-6Manß1-4GlcNAc. This is the first study to show unequivocal chemical evidence for the occurrence of KDN glycoconjugates in human tissues together with their detailed structures. These oligosaccharides might be developed as tumor markers, especially for prostate cancer.
Assuntos
Oligossacarídeos/metabolismo , Neoplasias da Próstata/metabolismo , Ácidos Siálicos/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicosídeo Hidrolases/química , Glicoesfingolipídeos/química , Glicoesfingolipídeos/isolamento & purificação , Glicoesfingolipídeos/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Ácidos Neuramínicos/química , Ácidos Neuramínicos/isolamento & purificação , Ácidos Neuramínicos/metabolismo , Neuraminidase/química , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Ácidos Siálicos/química , Ácidos Siálicos/isolamento & purificaçãoRESUMO
We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galß1-4GlcNAcß1-2Manα1-3Manß1-4GlcNAc, followed by Neu5Acα2-6Galß1-4GlcNAcß1-2Manα1-6Manß1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.
Assuntos
Adenocarcinoma/metabolismo , Glucanos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neoplasias Pancreáticas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Carboidratos , Feminino , Glucanos/química , Glicômica , Humanos , Masculino , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/químicaRESUMO
Glycobiology has contributed tremendously to the discovery and characterization of cancer-related biomarkers containing glycans (i.e., glyco-biomarkers) and a more detailed understanding of cancer biology. It is now recognized that most chronic diseases involve some elements of chronic inflammation; these include cancer, Alzheimer's disease, and metabolic syndrome (including consequential diabetes mellitus and cardiovascular diseases). By extending the knowledge and experience of the glycobiology community regarding cancer biomarker discovery, we should be able to contribute to the discovery of diagnostic/prognostic glyco-biomarkers of other chronic diseases that involve chronic inflammation. Future integration of large-scale "omics"-type data (e.g., genomics, epigenomics, transcriptomics, proteomics, and glycomics) with computational model building, or a systems glycobiology approach, will facilitate such efforts.
Assuntos
Inflamação/metabolismo , Polissacarídeos/metabolismo , Doença de Alzheimer/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Feminino , Glicômica , Humanos , Masculino , Polissacarídeos/química , Doença Pulmonar Obstrutiva Crônica/metabolismo , Biologia de SistemasRESUMO
We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-ß1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8(+/-)) that were exposed to cigarette smoke (CS). The Fut8(+/-) mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8(+/-) mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-ß-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.
Assuntos
Fucosiltransferases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 9 da Matriz/biossíntese , Enfisema Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Fator de Crescimento Transformador beta/metabolismo , Animais , Fucosiltransferases/genética , Regulação Enzimológica da Expressão Gênica/genética , Heterozigoto , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Knockout , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologia , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad7/genética , Proteína Smad7/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Fucosylation is one of the most important types of glycosylations related to cancer. Our previous studies of the enzymatic basis and structural studies of α-fetoprotein (AFP) samples from liver cancer patients indicated that core-fucosylation by α1,6-fucosyltransferase (FUT8) resulted in the production of fucosylated AFP, and in fact fucosylated AFP allowed differential diagnosis in some types of liver cancer from liver cirrhosis. This served as a predictive biomarker for the development of liver cancer 3 to 18 months before it could be detected using imaging techniques. Fucosylated AFP is currently measured by means of a liquid-phase binding assay (LBA) or by an electrokinetic analyte transport assay (EATA). However, these methods require special instrumentation that is currently available only in major medical laboratories. To overcome this problem, we attempted to develop an enzyme immunoassay (EIA) based on the sandwich technique with specific antibody and lectin. RESULTS: Dilute solutions of highly fucosylated AFP in human sera were assayed using a microtiter plate coated with a periodate-oxidized anti-AFP antibody, a fucose-specific biotinylated Aleuria aurantia lectin (AAL), a peroxidase-conjugated streptoavidin, and a chemiluminescent detection system. The technique was able to measure highly fucosylated AFP diluted to 5 to 80ng/ml in human sera using the developed antibody-lectin EIA in combination with the enrichment of AFP. CONCLUSION: A simple method using an antibody-lectin EIA for quantifying fucosylated AFP that does not require special instrumentation was developed. GENERAL SIGNIFICANCE: The method can be generally applied to the quantitative measurement of various fucosylated glycoproteins using specific antibodies. This article is part of a Special Issue entitled Glycoproteomics.
Assuntos
Fucose/metabolismo , Técnicas Imunoenzimáticas/métodos , alfa-Fetoproteínas/análise , Anticorpos , Análise Química do Sangue/métodos , Sequência de Carboidratos , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fucose/imunologia , Fucosiltransferases/metabolismo , Glicosilação , Humanos , Lectinas , Hepatopatias/sangue , Hepatopatias/diagnóstico , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Modelos Biológicos , Valor Preditivo dos Testes , alfa-Fetoproteínas/química , alfa-Fetoproteínas/isolamento & purificação , alfa-Fetoproteínas/metabolismoRESUMO
Scavenger receptor expressed by endothelial cells (SREC-I) mediates the endocytosis of chemically modified lipoproteins such as acetylated low-density lipoprotein (Ac-LDL) and oxidized LDL and is implicated in atherogenesis. We produced recombinant SREC-I in Chinese hamster ovary-K1 cells and identified three potential glycosylation sites, Asn(289), Asn(382) and Asn(393), which were all glycosylated. To determine the function of N-glycans in SREC-I, we characterized SREC-I mutant proteins by intracellular distribution and the cellular incorporation rate of Ac-LDL. N382Q/N393Q and N289Q/N382Q/N393Q were sequestered in the endoplasmic reticulum, resulting in a severe reduction in the cellular incorporation of Ac-LDL. N382Q showed a normal cell surface residency and an enhanced affinity for Ac-LDL, resulting in an elevated Ac-LDL cellular incorporation. These results indicate that the N-glycan of Asn(393) regulates the intracellular sorting of SREC-I and that the N-glycan of Asn(382) controls ligand-binding affinity. Furthermore, we detected an enhanced trypsin sensitivity of the N289Q. Glycan structure analyses revealed that the core-fucosylated bi-antennary is the common major structure at all glycosylation sites. In addition, tri- and tetra-antennary were detected as minor constituents at Asn(289). A bisecting GlcNAc was also detected at Asn(382) and Asn(393). Structural analyses and homology modeling of SREC-I suggest that the N-glycan bearing a ß1-6GlcNAc branch at Asn(289) protects from proteinase attack and thus confers a higher stability on SREC-I. These data indicate that Asn(289)-, Asn(382)- and Asn(393)-linked N-glycans of SREC-I have distinct functions in regulating proteolytic resistance, ligand-binding affinity and subcellular localization, all of which might be involved in the development of atherogenesis.