RESUMO
Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.
Assuntos
Anticorpos Monoclonais , Fucose , Imunoglobulina G , Pneumopatias , Polissacarídeos , Humanos , Células A549 , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Linfócitos B/imunologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Fucose/sangue , Fucose/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Técnicas de Inativação de Genes , Imunoensaio/normas , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pneumopatias/diagnóstico , Pneumopatias/imunologia , Polissacarídeos/metabolismo , Animais , Camundongos , Células CHO , Células HEK293 , CricetulusRESUMO
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
This paper reports an entirely unexplored concept of simultaneously recognizing two receptors using high- and low-affinity ligands through ligating them in situ on the target cell surface. This de novo approach is inspired by the pretargeting strategy frequently applied in molecular imaging, and has now evolved as the basis of a new paradigm for visualizing target cells with a high imaging contrast. A distinct advantage of using a labeled low-affinity ligand such as glycan is that the excess labeled ligand can be washed away from the cells, whereas the ligand bound to the cell, even at the milli molar affinity level, can be anchored by a bioorthogonal reaction with a pretargeted high-affinity ligand on the surface. Consequently, nonspecific background is minimized, leading to improved imaging contrast. Importantly, despite previously unexplored for molecular imaging, a notoriously weak glycan/lectin interaction can now be utilized as a highly selective ligand to the targets.
RESUMO
Extracellular superoxide dismutase (EC-SOD, SOD3) protects tissues against oxidative damage by detoxifying superoxide anions, particularly in the lungs and cardiovascular system. EC-SOD undergoes several posttranslational modifications including N-glycosylation and proteolytic cleavage. While the roles of proteolytic cleavage have been well studied, the structure and function of EC-SOD N-glycans are poorly understood. Here we analyzed glycan structures on native EC-SOD purified from human sera, and identified sialylated biantennary structures. Using glycan maturation-defective CHO mutant cells, we further revealed that the presence of terminal sialic acids in the N-glycans of EC-SOD enhanced both the secretion and furin-mediated C-terminal cleavage of EC-SOD. These results provide new insights into how the posttranslational modifications of EC-SOD control its functions.
Assuntos
Furina/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteólise , Superóxido Dismutase/metabolismo , Animais , Células CHO , Cricetulus , Furina/genética , Glicosilação , Humanos , Ácido N-Acetilneuramínico/genéticaRESUMO
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
Extracellular superoxide dismutase (EC-SOD or SOD3) protects against various oxidative stress-related diseases by scavenging reactive superoxides in the extracellular space. It is the only SOD isozyme that is secreted and glycosylated (at asparagine 89). However, the physiological roles of its glycosylation are poorly understood. In this study, we found that the glycosylation site on EC-SOD is well conserved and that a glycosylation-deficient EC-SOD mutant retains its enzymatic activity, but is not secreted. This impairment in secretion may, in part, be due to the ability of the mutants to form unusual higher order oligomers. Our findings reveal that the glycan modification is a key regulator of EC-SOD secretion and contributes to the understanding of the roles of glycans in EC-SOD-related diseases.
Assuntos
Processamento de Proteína Pós-Traducional , Superóxido Dismutase/metabolismo , Glicosilação , Células HEK293 , Humanos , Transporte Proteico , Via Secretória , Superóxido Dismutase/químicaRESUMO
Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteínas de Homeodomínio/biossíntese , Esclerose Múltipla/genética , N-Acilesfingosina Galactosiltransferase/genética , Proteínas do Tecido Nervoso/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Galactosilceramidas/biossíntese , Galactosilceramidas/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Células HeLa , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Humanos , Esclerose Múltipla/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , N-Acilesfingosina Galactosiltransferase/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/enzimologia , Oligodendroglia/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Proteínas de Peixe-ZebraRESUMO
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
Bacterial or viral infection of the airway plays a critical role in the pathogenesis and exacerbation of chronic obstructive pulmonary disease (COPD) which is expected to be the 3rd leading cause of death by 2020. The induction of inflammatory responses in immune cells as well as airway epithelial cells is observed in the disease process. There is thus a pressing need for the development of new therapeutics. Keratan sulfate (KS) is the major glycosaminoglycans (GAGs) of airway secretions, and is synthesized by epithelial cells on the airway surface. Here we report that a KS disaccharide, [SO3(-)-6]Galß1-4[SO3(-)-6]GlcNAc, designated as L4, suppressed the production of Interleukin-8 (IL-8) stimulated by flagellin, a Toll-like receptor (TLR) 5 agonist, in normal human bronchial epithelial (NHBE) cells. Such suppressions were not observed by other L4 analogues, N-acetyllactosamine or chondroitin-6-sulfate disaccharide. Moreover, treatment of NHBE cells with L4 inhibited the flagellin-stimulated phosphorylation of epidermal growth factor receptor (EGFR), the down stream signaling pathway of TLRs in NHBE cells. These results suggest that L4 specifically blocks the interaction of flagellin with TLR5 and subsequently suppresses IL-8 production in NHBE cells. Taken together, L4 represents a potential molecule for prevention and treatment of airway inflammatory responses to bacteria infections, which play a critical role in exacerbation of COPD.
Assuntos
Brônquios/metabolismo , Receptores ErbB/metabolismo , Flagelina/farmacologia , Sulfato de Queratano/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Receptor 5 Toll-Like/metabolismo , Infecções Bacterianas/complicações , Infecções Bacterianas/metabolismo , Brônquios/citologia , Brônquios/efeitos dos fármacos , Células Cultivadas , Receptores ErbB/antagonistas & inibidores , Flagelina/antagonistas & inibidores , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Fosforilação/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Receptor 5 Toll-Like/agonistasRESUMO
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
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
The alpha2,6-sialylated blood group type 2H (ST2H) antigen (Fucalpha1-2(NeuAcalpha2-6)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) is a fucoganglioside found in human colon cancer tissues. To elucidate an enzyme responsible for the ST2H antigen formation, we screened some partially purified candidate enzymes, alpha2,6-sialyltransferases, ST6Gal I and ST6Gal II, and alpha1,2-fucosyltransferases, FUT1 and FUT2 for their activities towards pyridylaminated type 2H (Fucalpha1-2Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) or LS-tetrasaccharide c (LST-c: NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) as acceptor substrates. Here we show the ST6Gal I transfers NeuAc from the donor CMP-NeuAc to the terminal Gal of PA-type 2H, which formed the ST2H antigen, but the others could not synthesize it. Using a recombinant ST6Gal I, enzymatic reactions with two types of acceptors, PA-type 2H and PA-lacto-N-neotetraose (LNnT), were kinetically analysed. On the basis of catalytic efficiency (V(max)/K(m)), the specificity of ST6Gal I towards the PA-type 2H was estimated to be 42 times lower than that for PA-LNnT. The overexpression of ST6Gal I in human colon cancer DLD-1 cells effectively resulted in the ST2H antigen formation, as judged by LC-ESI-IT-MS. Many lines of evidence suggest the up-regulation of ST6Gal I in human colon cancer specimens. Collectively, these findings indicate that ST6Gal I is responsible for ST2H antigen biosynthesis in human colon cancer cells.