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1.
Angew Chem Int Ed Engl ; 55(33): 9482-512, 2016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27435524

RESUMO

In metabolic glycoengineering (MGE), cells or animals are treated with unnatural derivatives of monosaccharides. After entering the cytosol, these sugar analogues are metabolized and subsequently expressed on newly synthesized glycoconjugates. The feasibility of MGE was first discovered for sialylated glycans, by using N-acyl-modified mannosamines as precursor molecules for unnatural sialic acids. Prerequisite is the promiscuity of the enzymes of the Roseman-Warren biosynthetic pathway. These enzymes were shown to tolerate specific modifications of the N-acyl side chain of mannosamine analogues, for example, elongation by one or more methylene groups (aliphatic modifications) or by insertion of reactive groups (bioorthogonal modifications). Unnatural sialic acids are incorporated into glycoconjugates of cells and organs. MGE has intriguing biological consequences for treated cells (aliphatic MGE) and offers the opportunity to visualize the topography and dynamics of sialylated glycans in vitro, ex vivo, and in vivo (bioorthogonal MGE).

2.
Chem Sci ; 7(6): 3928-3933, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30155038

RESUMO

Synthetically accessible C6-analogs of N-acetylmannosamine (ManNAc) were tested as potential inhibitors of the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK), the key enzyme of sialic acid biosynthesis. Enzymatic experiments revealed that the modification introduced at the C6 saccharide position strongly influences the inhibitory potency. A C6-ManNAc diselenide dimer showed the strongest kinase inhibition in the low µM range among all the substrates tested and successfully reduced cell surface sialylation in Jurkat cells.

3.
J Biol Chem ; 290(45): 27345-27359, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26370074

RESUMO

Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.


Assuntos
Vírus da Leucemia Murina de Moloney/patogenicidade , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/química , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Gangliosídeos/química , Gangliosídeos/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interferon-alfa/fisiologia , Leucemia Experimental/fisiopatologia , Leucemia Experimental/virologia , Linfócitos/fisiologia , Linfócitos/virologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/fisiologia , Ácido N-Acetilneuramínico/química , Receptores Virais/química , Receptores Virais/fisiologia , Infecções por Retroviridae/fisiopatologia , Infecções por Retroviridae/virologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Infecções Tumorais por Vírus/fisiopatologia , Infecções Tumorais por Vírus/virologia
4.
Neural Regen Res ; 10(6): 976-81, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26199617

RESUMO

Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-acyl side chain. N-Propionylmannosamine (ManNProp) increases neurite outgrowth and accelerates the reestablishment of functional synapses in vitro. We investigated the influence of systemic ManNProp application using a specific in vivo mouse model. Using mice expressing axonal fluorescent proteins, we quantified the extension of regenerating axons, the number of regenerating axons, the number of arborising axons and the number of branches per axon 5 days after injury. Sciatic nerves from non-expressing mice were grafted into those expressing yellow fluorescent protein. We began a twice-daily intraperitoneal application of either peracetylated ManNProp (200 mg/kg) or saline solution 5 days before injury, and continued it until nerve harvest (5 days after transection). ManNProp significantly increased the mean distance of axonal regeneration (2.49 mm vs. 1.53 mm; P < 0.005) and the number of arborizing axons (21% vs. 16%; P = 0.008) 5 days after sciatic nerve grafting. ManNProp did not affect the number of regenerating axons or the number of branches per arborizing axon. The biochemical glycoengineering of the N-acyl side chain of sialic acid might be a promising approach for improving peripheral nerve regeneration.

5.
J Neural Transm (Vienna) ; 122(9): 1211-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25850639

RESUMO

The key enzyme of sialic acid (Sia) biosynthesis is the bifunctional UDP-N-acetylglucosamine 2-epimerase/ManNAc kinase (GNE/MNK). It metabolizes the physiological precursor ManNAc and N-acyl modified analogues such as N-propionylmannosamine (ManNProp) to the respective modified sialic acid. Polysialic acid (polySia) is a crucial compound for several functions in the nervous system and is synthesized by the polysialyltransferases ST8SIA2 and ST8SIA4. PolySia can be modified in vitro and in vivo by metabolic glycoengineering of the N-acyl side chain of Sia. In vitro studies show that the application of ManNProp increases neurite outgrowth and accelerates the re-establishment of functional synapses. In this study, we investigate in vivo how ManNProp application might benefit peripheral nerve regeneration. In mice expressing axonal fluorescent proteins (thy-1-YFP), we transected the sciatic nerve and then replaced part of it with a sciatic nerve graft from non-expressing mice (wild-type mice or St8sia2(-/-) mice). Analyses conducted 5 days after grafting showed that systemic application of ManNProp (200 mg/kg, twice a day, i.p.), but not of physiological ManNAc (1 g/kg, twice a day, i.p.), significantly increased the extent of axonal elongation, the number of arborizing axons and the number of branches per regenerating axon within the grafts from wild-type mice, but not in those from St8sia2(-/-) mice. The results demonstrate that the application of ManNProp has beneficial effects on early peripheral nerve regeneration and indicate that the stimulation of axon growth depends on ST8SIA2 activity in the nerve graft.


Assuntos
Axônios/efeitos dos fármacos , Hexosaminas/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nervo Isquiático/lesões , Sialiltransferases/metabolismo , Animais , Axônios/patologia , Axônios/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Tamanho Celular , Modelos Animais de Doenças , Feminino , Estimativa de Kaplan-Meier , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regeneração Nervosa/fisiologia , Nervo Isquiático/fisiopatologia , Nervo Isquiático/cirurgia , Nervo Isquiático/transplante , Sialiltransferases/genética , Transplantes , Resultado do Tratamento
7.
PLoS One ; 9(11): e111917, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25369234

RESUMO

The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.


Assuntos
Proteínas Aviárias/metabolismo , Orelha Interna/metabolismo , Proteínas de Peixes/metabolismo , Salmão/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Galinhas , Proteínas de Peixes/química , Cobaias , Proteínas de Membrana/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Coelhos , Homologia de Sequência de Aminoácidos
8.
J Biol Chem ; 289(46): 32056-32063, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25278018

RESUMO

Due to its position at the outermost of glycans, sialic acid is involved in a myriad of physiological and pathophysiological cell functions such as host-pathogen interactions, immune regulation, and tumor evasion. Inhibitors of cell surface sialylation could be a useful tool in cancer, immune, antibiotic, or antiviral therapy. In this work, four different C-3 modified N-acetylmannosamine analogs were tested as potential inhibitors of cell surface sialylation. Peracetylated 2-acetylamino-2-deoxy-3-O-methyl-D-mannose decreases cell surface sialylation in Jurkat cells in a dose-dependent manner up to 80%, quantified by flow cytometry and enzyme-linked lectin assays. High-performance liquid chromatography experiments revealed that not only the concentration of membrane bound but also of cytosolic sialic acid is reduced in treated cells. We have strong evidence that the observed reduction of sialic acid expression in cells is caused by the inhibition of the bifunctional enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase. 2-Acetylamino-2-deoxy-3-O-methyl-D-mannose inhibits the human ManNAc kinase domain of the UDP-GlcNAc-2-epimerase/ManNAc kinase. Binding kinetics of the inhibitor and human N-acetylmannosamine kinase were evaluated using surface plasmon resonance. Specificity studies with human N-acetylglucosamine kinase and hexokinase IV indicated a high specificity of 2-acetylamino-2-deoxy-3-O-methyl-D-mannose for MNK. This substance represents a novel class of inhibitors of sialic acid expression in cells, targeting the key enzyme of sialic acid de novo biosynthesis.


Assuntos
Carboidratos Epimerases/química , Proteínas de Transporte/química , Hexosaminas/química , Ácido N-Acetilneuramínico/química , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Citosol/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Células Jurkat , Cinética , Lectinas , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Espalhamento de Radiação , Especificidade por Substrato , Ressonância de Plasmônio de Superfície
9.
Neuropharmacology ; 77: 68-80, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24055495

RESUMO

Basic and clinical research has demonstrated that dementia of sporadic Alzheimer's disease (sAD) type is associated with dysfunction of the insulin-receptor (IR) system followed by decreased glucose transport via glucose transporter GLUT4 and decreased glucose metabolism in brain cells. An alternative source of energy is d-galactose (the C-4-epimer of d-glucose) which is transported into the brain by insulin-independent GLUT3 transporter where it might be metabolized to glucose via the Leloir pathway. Exclusively parenteral daily injections of galactose induce memory deterioration in rodents and are used to generate animal aging model, but the effects of oral galactose treatment on cognitive functions have never been tested. We have investigated the effects of continuous daily oral galactose (200 mg/kg/day) treatment on cognitive deficits in streptozotocin-induced (STZ-icv) rat model of sAD, tested by Morris Water Maze and Passive Avoidance test, respectively. One month of oral galactose treatment initiated immediately after the STZ-icv administration, successfully prevented development of the STZ-icv-induced cognitive deficits. Beneficial effect of oral galactose was independent of the rat age and of the galactose dose ranging from 100 to 300 mg/kg/day. Additionally, oral galactose administration led to the appearance of galactose in the blood. The increase of galactose concentration in the cerebrospinal fluid was several times lower after oral than after parenteral administration of the same galactose dose. Oral galactose exposure might have beneficial effects on learning and memory ability and could be worth investigating for improvement of cognitive deficits associated with glucose hypometabolism in AD.


Assuntos
Aprendizagem da Esquiva/efeitos dos fármacos , Transtornos Cognitivos/prevenção & controle , Galactose/uso terapêutico , Aprendizagem em Labirinto/efeitos dos fármacos , Administração Oral , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Transtornos Cognitivos/induzido quimicamente , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/metabolismo , Galactose/administração & dosagem , Galactose/metabolismo , Glucose/metabolismo , Masculino , Memória/efeitos dos fármacos , Ratos , Ratos Wistar , Estreptozocina
10.
Anal Chem ; 85(17): 8112-20, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23909495

RESUMO

The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide "tube gel" followed by in-gel digestion of "tube gel" pieces significantly improved detection by electrospray ionization-liquid chromatography-tandem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Membrana Celular/química , Neoplasias Hepáticas Experimentais/diagnóstico , Proteoma/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida , Ratos , Ratos Endogâmicos BUF
11.
Glycoconj J ; 30(8): 813-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23813293

RESUMO

N-Propanoylmannosamine is an unnatural precursor of sialic acid, which is taken up by a variety of animal cells and metabolized to N-propanoylneuraminic acid. In several studies it has been demonstrated that application of unnatural precursors of sialic acids such as N-propanoylmannosamine (ManNProp) and homologues interfere with cell differentiation and proliferation of neuronal cells or embryonic stem cells. Since the function of the immune system is known to rely on the presence of sialic acid, we applied ManNProp to human peripheral blood mononuclear cells (PBMC). When culturing those lymphocytes with ManNProp 10 % of the natural sialic acid N-acetylneuraminic acid could be replaced by the newly formed N-propanoylneuraminic acid. This procedure resulted (a) in a marked stimulation in the rate of proliferation of PBMC, (b) a 10-fold increase of IL-2 production coupled with an up-regulation of its receptor CD25 on the cell surface and (c) a concomitant expression and regulation of the transferrin receptor with cell growth. The stimulation of PBMC by ManNProp might therefore introduce a new approach of immunomodulation.


Assuntos
Hexosaminas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Células Cultivadas , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo
12.
Glycobiology ; 23(8): 1004-12, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23708401

RESUMO

There is an increasing interest in the modification of cell surface glycosylation to improve the properties of therapeutic cells. For example, glycosylation affects the biodistribution of mesenchymal stromal cells (MSCs). Metabolic glycoengineering is an efficient way to modify the cell surface. The mammalian biosynthetic machinery tolerates the unnatural sialic acid precursor, N-propanoylmannosamine (ManNProp), and incorporates it into cell surface glycoconjugates. We show here by mass spectrometric analysis of cell surface N-glycans that about half of N-acetylneuraminic acid was replaced by N-propanoylneuraminic acid in the N-glycans of human umbilical cord blood-derived MSCs supplemented with ManNProp. In addition, the N-glycan profile was altered. ManNProp-supplemented cells had more multiply fucosylated N-glycan species than control cells. The fucosylated epitopes were shown in tandem mass spectrometric analysis to be Lewis x or blood group H epitopes, but not sialyl Lewis x (sLex). The amounts of tri- and tetra-antennary and polylactosamine-containing N-glycans also increased in ManNProp supplementation. In accordance with previous studies of other cell types, increased expression of the sLex epitope in ManNProp-supplemented MSCs was demonstrated by flow cytometry. In light of the N-glycan analysis, the sLex epitope in these cells is likely to be carried by O-glycans or glycolipids. sLex has been shown to target MSCs to bone marrow, which may be desirable in therapeutic applications. The present results represent the first structural analysis of an N-glycome of ManNProp-supplemented cells and demonstrate the feasibility of modifying cell surface glycosylation of therapeutic cells by this type of metabolic glycoengineering.


Assuntos
Glicômica , Hexosaminas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Glicosilação , Humanos , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/metabolismo , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Antígeno Sialil Lewis X
14.
J Biol Chem ; 287(17): 13656-65, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22343627

RESUMO

Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate · ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid.


Assuntos
Hexosaminas/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ácido Aspártico/química , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Dimerização , Inibidores Enzimáticos/química , Escherichia coli/metabolismo , Glicoconjugados/química , Glicoproteínas/química , Humanos , Ácido N-Acetilneuramínico/química , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Zinco/química
15.
Eur J Cell Biol ; 91(4): 265-73, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21856036

RESUMO

Dipeptidyl peptidase IV (DPPIV or CD26) is a multifunctional membrane glycoprotein. As an exopeptidase it regulates the activity of a series of biologically important peptides. Through its interaction with specific proteins and peptides, DPPIV is also involved in a wide range of biologically relevant processes such as cell adhesion, T cell activation and apoptosis. In this paper, we review our recent studies on the interactions of DPPIV with adenosine deaminase (ADA) and the transcription transactivator of the human immunodeficiency virus type-1 (HIV-1 Tat) as revealed by three-dimensional structure reconstructed by single particle analysis of cryo-electron microscopy (EM) and crystal structures of the human DPPIV-bovine ADA complex as well as the crystal structures of DPPIV in complex with HIV-1 Tat-derived nonapeptides. These results contribute importantly to the clarification of the molecular mechanisms of this multifunctional protein. The biological relevance of these interactions is discussed.


Assuntos
Adenosina Desaminase/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , HIV-1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Adenosina Desaminase/química , Adenosina Desaminase/genética , Animais , Bovinos , Dipeptidil Peptidase 4/genética , HIV-1/química , HIV-1/genética , Humanos , Estrutura Terciária de Proteína/genética , Relação Estrutura-Atividade , Ativação Transcricional/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
16.
Cell Mol Life Sci ; 69(7): 1179-91, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22068610

RESUMO

During development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targeting with formation of synapses on inappropriate neurons may result in neurodevelopmental sequelae, leading to psychiatric disorders. Here we show that altering the expression level of the polysialic acid moiety, which is a developmentally regulated, posttranslational modification of the neural cell adhesion molecule NCAM, critically affects correct circuit formation. Using a chemically modified sialic acid precursor (N-propyl-D: -mannosamine), we inhibited the polysialyltransferase ST8SiaII, the principal enzyme involved in polysialylation during development, at selected developmental time-points. This treatment altered NCAM polysialylation while NCAM expression was not affected. Altered polysialylation resulted in an aberrant mossy fiber projection that formed glutamatergic terminals on pyramidal neurons of the CA1 region in organotypic slice cultures and in vivo. Electrophysiological recordings revealed that the ectopic terminals on CA1 pyramids were functional and displayed characteristics of mossy fiber synapses. Moreover, ultrastructural examination indicated a "mossy fiber synapse"-like morphology. We thus conclude that homeostatic regulation of the amount of synthesized polysialic acid at specific developmental stages is essential for correct synaptic targeting and circuit formation during hippocampal development.


Assuntos
Homeostase , Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Sinapses/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL
17.
FEBS Lett ; 585(20): 3322-7, 2011 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-21945320

RESUMO

The sialic acid of complex N-glycans can be biochemically engineered by substituting the physiological precursor N-acetylmannosamine with non-natural N-acylmannosamines. The Kv3.1 glycoprotein, a neuronal voltage-gated potassium channel, contains sialic acid. Western blots of the Kv3.1 glycoprotein isolated from transfected B35 neuroblastoma cells incubated with N-acylmannosamines verified sialylated N-glycans attached to the Kv3.1 glycoprotein. Outward ionic currents of Kv3.1 transfected B35 cells treated with N-pentanoylmannosamine or N-propanoylmannosamine had slower activation and inactivation rates than those of untreated cells. Therefore, the N-acyl side chain of sialic acid is intimately connected with the activation and inactivation rates of this glycosylated potassium channel.


Assuntos
Hexosaminas/farmacologia , Engenharia Metabólica , Ácido N-Acetilneuramínico/metabolismo , Canais de Potássio Shaw/metabolismo , Linhagem Celular Tumoral , Glicosilação , Hexosaminas/metabolismo , Humanos , Transporte de Íons/genética , Ácido N-Acetilneuramínico/genética , Canais de Potássio Shaw/genética
18.
Glycobiology ; 21(10): 1277-89, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21551457

RESUMO

The Thomsen-Friedenreich antigen (TF; CD176, Galß1-3GalNAcα-) is a tumor-specific carbohydrate antigen and a promising therapeutic target. Antibodies that react with this antigen are frequently found in the sera of healthy adults and are assumed to play a role in cancer immunosurveillance. In this study, we examined the occurrence of α-anomeric TF (TFα) on a large variety of gastrointestinal bacteria using a novel panel of well-characterized monoclonal antibodies. Reactivity with at least one anti-TF antibody was found in 13% (16 of 122) of strains analyzed. A more in-depth analysis, using monoclonal antibodies specific for α- and ß-anomeric TF in combination with periodate oxidation, revealed that only two novel Bacteroides ovatus strains (D-6 and F-1), isolated from the faeces of healthy persons by TF-immunoaffinity enrichment, possessed structures that are immunochemically identical to the true TFα antigen. The TF-positive capsular polysaccharide structure of strain D-6 was characterized by mass spectrometry, monosaccharide composition analysis, glycosidase treatments and immunoblot staining with TFα- and TFß-specific antibodies. The active antigen was identified as Galß1-3GalNAc-, which was α-anomerically linked as a branching structure within a heptasaccharide repeating unit. We conclude that structures immunochemically identical to TFα are extremely rare on the surface of human intestinal bacteria and may only be identifiable by binding of both antibodies, NM-TF1 and NM-TF2, which recognize a complete immunomolecular imprint of the TFα structure. The two novel B. ovatus strains isolated in this study may provide a basis for the development of TF-based anti-tumor vaccines.


Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Trato Gastrointestinal/microbiologia , Anticorpos/imunologia , Antígenos Glicosídicos Associados a Tumores/imunologia , Bacteroidetes/imunologia , Bacteroidetes/isolamento & purificação , Fezes/microbiologia , Humanos
19.
Apoptosis ; 16(6): 636-51, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21437721

RESUMO

Apoptosis is modulated by extrinsic and intrinsic signaling pathways through the formation of the death receptor-mediated death-inducing signaling complex (DISC) and the mitochondrial-derived apoptosome, respectively. Ino-C2-PAF, a novel synthetic phospholipid shows impressive antiproliferative and apoptosis-inducing activity. Little is known about the signaling pathway through which it stimulates apoptosis. Here, we show that this drug induces apoptosis through proteins of the death receptor pathway, which leads to an activation of the intrinsic apoptotic pathway. Apoptosis induced by Ino-C2-PAF and its glucosidated derivate, Glc-PAF, was dependent on the DISC components FADD and caspase-8. This can be inhibited in FADD--/-- and caspase-8--/-- cells, in which the breakdown of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-9, -8 and -3 do not occur. In addition, the overexpression of crmA, c-Flip or dominant negative FADD as well as treatment with the caspase-8 inhibitor z-IETD-fmk protected against Ino-C2-PAF-induced apoptosis. Apoptosis proceeds in the absence of CD95/Fas-ligand expression and is independent of blockade of a putative death-ligand/receptor interaction. Furthermore, apoptosis cannot be inhibited in CD95/Fas--/-- Jurkat cells. Expression of Bcl-2 in either the mitochondria or the endoplasmic reticulum (ER) strongly inhibited Ino-C2-PAF- and Glc-PAF-induced apoptosis. In conclusion, Ino-C2-PAF and Glc-PAF trigger a CD95/Fas ligand- and receptor-independent atypical DISC that relies on the intrinsic apoptotic pathway via the ER and the mitochondria.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Mitocôndrias/metabolismo , Neoplasias/fisiopatologia , Fosfolipídeos/farmacologia , Transdução de Sinais , Caspase 8/genética , Ativação Enzimática/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/genética , Glicosilação , Humanos , Células Jurkat , Mitocôndrias/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosfolipídeos/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
20.
Biochem Pharmacol ; 81(8): 985-95, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21320472

RESUMO

New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line. Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell-matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines.


Assuntos
Antineoplásicos/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Integrinas/metabolismo , Queratinócitos/efeitos dos fármacos , Éteres Fosfolipídicos/farmacologia , Quinases da Família src/metabolismo , Western Blotting , Adesão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citometria de Fluxo , Humanos , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Fosforilação , Transdução de Sinais
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