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1.
Proc Natl Acad Sci U S A ; 112(48): E6654-62, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26627256

RESUMO

Sialylated glycans on the surface of mammalian cells act as part of a "self-associated molecular pattern," helping the immune system to recognize "self" from "altered self" or "nonself." To escape the host immune system, some bacterial pathogens have evolved biosynthetic pathways for host-like sialic acids, whereas others recruited host sialic acids for decorating their surfaces. Prions lack nucleic acids and are not conventional pathogens. Nevertheless, prions might use a similar strategy for invading and colonizing the lymphoreticular system. Here we show that the sialylation status of the infectious, disease-associated state of the prion protein (PrP(Sc)) changes with colonization of secondary lymphoid organs (SLOs). As a result, spleen-derived PrP(Sc) is more sialylated than brain-derived PrP(Sc). Enhanced sialylation of PrP(Sc) is recapitulated in vitro by incubating brain-derived PrP(Sc) with primary splenocytes or cultured macrophage RAW 264.7 cells. General inhibitors of sialyltranserases (STs), the enzymes that transfer sialic acid residues onto terminal positions of glycans, suppressed extrasialylation of PrP(Sc). A fluorescently labeled precursor of sialic acid revealed ST activity associated with RAW macrophages. This study illustrates that, upon colonization of SLOs, the sialylation status of prions changes by host STs. We propose that this mechanism is responsible for camouflaging prions in SLOs and has broad implications.


Assuntos
Tecido Linfoide/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Encéfalo/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Macrófagos/metabolismo , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPC/química , Proteínas PrPSc/química , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Scrapie/metabolismo , Ácidos Siálicos/química , Baço/citologia , Baço/metabolismo
2.
Chembiochem ; 18(13): 1216-1225, 2017 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-28374962

RESUMO

CD22 is a member of the Siglec family. Considerable attention has been drawn to the design and synthesis of new Siglec ligands to explore target biology and innovative therapies. In particular, CD22-ligand-targeted nanoparticles with therapeutic functions have proved successful in preclinical settings for blood cancers, autoimmune diseases, and tolerance induction. Here we report the design, synthesis and affinity evaluation of a new class of Siglec ligands: namely sialic acid derivatives with a triazole moiety replacing the natural glycoside oxygen atom. In addition, we describe important and surprising differences in binding to CD22 expressed at the cell surface for compounds with distinct valences. The new class of compounds might serve as a template for the design of ligands for other members of the Siglec family and next-generation CD22-ligand-based targeted therapies.


Assuntos
Linfócitos B/efeitos dos fármacos , Glicosídeos/síntese química , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Ácidos Siálicos/química , Triazóis/síntese química , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Glicosídeos/farmacologia , Humanos , Imunoglobulina M/química , Ligantes , Ligação Proteica , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/química , Triazóis/farmacologia
3.
Bioorg Med Chem ; 23(17): 5915-21, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26234906

RESUMO

Siglecs (sialic acid recognizing immunoglobulin like lectins) are a family of lectins with specificity for sialic acid containing carbohydrates. Synthetic sialic acid derivatives with high affinity proved useful to unravel the biological role of the ligand binding domain, although many of their functions in immunity remain unknown. Here we present design, synthesis, affinity evaluation and molecular modeling of novel 9-N-oxamoyl modified sialosides as Siglec-7 ligands.


Assuntos
Ácido N-Acetilneuramínico/química , Ácidos Neuramínicos/química , Lectinas/metabolismo , Ligantes , Estrutura Molecular
4.
J Biol Chem ; 287(14): 11489-97, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22334707

RESUMO

Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.


Assuntos
Biologia Computacional , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/química , Simportadores/metabolismo , Azepinas/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Indóis/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/genética , Projetos Piloto , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Relação Estrutura-Atividade , Simportadores/genética
5.
Eur J Immunol ; 42(10): 2792-802, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22777817

RESUMO

CD22 is an inhibitory co-receptor of the B-cell receptor (BCR) on B cells. Since CD22 is ubiquitously expressed in the B-cell lineage and CD22 endocytosis can be triggered efficiently, antibodies and antibody-based immunotoxins against CD22 are used to target B cells both in B-cell lymphomas and leukemias, as well as in autoimmune diseases. CD22 recognizes α2,6-linked sialic acids as endogenous ligands. We have developed new synthetic sialosides as ligands for human CD22. These sialosides bind CD22 on human B cells with high affinity and can efficiently enhance IgM-triggered Ca(2+) signaling. We coupled these sialosides to Pseudomonas exotoxin A to generate a novel CD22 ligand-based immunotoxin. This sialoside-exotoxin-A construct can specifically kill CD22-positive B-cell lymphoma cells. It binds specifically to CD22-positive B-cell lymphoma cells and is dominant over endogenous cis-ligands on the B-cell surface. The sialoside-exotoxin-A construct is efficiently internalized by endocytosis into B-cell lymphoma cell lines. Thus we show the development of a new therapeutic compound for targeting CD22 on human B cells, both for B-cell lymphoma, as well as for B-cell-mediated autoimmune diseases.


Assuntos
ADP Ribose Transferases/metabolismo , Doenças Autoimunes/terapia , Linfócitos B/imunologia , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Imunotoxinas/metabolismo , Linfoma de Células B/terapia , Ácido N-Acetilneuramínico/metabolismo , Pseudomonas/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/genética , Doenças Autoimunes/imunologia , Linfócitos B/efeitos dos fármacos , Toxinas Bacterianas/genética , Sinalização do Cálcio , Cátions Bivalentes/química , Diferenciação Celular , Linhagem da Célula , Citotoxicidade Imunológica , Exotoxinas/genética , Células HL-60 , Humanos , Imunoglobulina M/imunologia , Imunotoxinas/genética , Linfoma de Células B/imunologia , Terapia de Alvo Molecular , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Receptores de Antígenos de Linfócitos B/metabolismo , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosa
6.
J Exp Med ; 195(9): 1207-13, 2002 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-11994426

RESUMO

CD22 is a B cell-specific transmembrane protein of the Siglec family. It binds specifically to alpha2,6-linked sialic acid (Sia) residues, which are also present on glycoproteins on the B cell surface. CD22 acts as a negative regulator in B cell receptor-mediated signaling by recruitment of Src homology 2 domain-containing tyrosine phosphatase (SHP)-1 to its intracellular tail. To analyze how ligand-binding of CD22 influences its intracellular signaling domain, we designed synthetic sialosides as inhibitors for the lectin domain of CD22. One of these compounds inhibited binding of human CD22-Fc to target cells over 200-fold better than Sia and was highly selective for human CD22. When Daudi cells or primary B cells were stimulated with anti-immunoglobulin (Ig)M in presence of this sialoside inhibitor, a higher Ca(2+) response was observed, similar to CD22-deficient B cells. Accordingly, a lower tyrosine-phosphorylation of CD22 and SHP-1 recruitment was demonstrated in presence of the sialoside. Thus, by interfering with ligand binding of CD22 on the B cell surface, we have shown for the first time that the lectin domain of CD22 has a direct, positive influence on its intracellular inhibitory domain. Also, we have developed a novel low molecular weight compound which can enhance the response of human B cells.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Moléculas de Adesão Celular , Lectinas , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Antígenos CD/química , Antígenos de Diferenciação de Linfócitos B/química , Sítios de Ligação , Linhagem Celular , Humanos , Fragmentos Fc das Imunoglobulinas , Cinética , Ligantes , Camundongos , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Células Tumorais Cultivadas , Domínios de Homologia de src
7.
FEBS Lett ; 594(1): 135-143, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325365

RESUMO

Researchers have observed that a sialic acid (Sia)-supplemented neonatal diet leads to improved cognition in weanling piglets. However, whether cognitive improvement appears with different physiological backgrounds and persists into adulthood is not known. Here, we have established a convenient mouse model and used an 19 F NMR approach to address these questions, test the conditionally essential nutrient hypothesis about Sia supplementation, and assess the prospect of measuring Sia metabolism directly in vivo. Indeed, the neonatal mouse brain uptakes more Sia than the adult brain, and Sia supplementation of neonatal mice improves the cognitive performance of adult mice. The non-invasive 19 F NMR approach and viable mouse model opens unique opportunities for clarifying the interplay of nutritional supplementation, metabolism, and cognitive development.


Assuntos
Encéfalo/efeitos dos fármacos , Cognição , Ácido N-Acetilneuramínico/farmacologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Suplementos Nutricionais , Feminino , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/administração & dosagem
8.
J Leukoc Biol ; 84(4): 1075-81, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18664529

RESUMO

Endogenous polymorphonuclear leukocyte (PMN)-associated sialidase activity enhances PMN adhesion to and migration across the endothelium through the removal of sialylated cell-surface residues. We tested the hypothesis that PMNs also express sialyltransferase (ST) activity that restores sialyl residues to the PMN surface. We developed a highly sensitive fluorometric assay to demonstrate that intact human PMNs can mediate and accept sialyl residue transfer. This ST activity is inhibited by a ST inhibitor, CMP, which also inhibits the transendothelial migration of PMNs in response to IL-8 in vitro and in vivo. We conclude that intact PMNs express sialidase and ST activities that permit rapid modulation of their surface sialylation and their ability to adhere to and migrate across the endothelium.


Assuntos
Neutrófilos/enzimologia , Sialiltransferases/genética , Movimento Celular/efeitos dos fármacos , Eritrócitos/enzimologia , Regulação da Expressão Gênica , Humanos , Interleucina-8/farmacologia , Microscopia Confocal , Neuraminidase , Neutrófilos/ultraestrutura , Sialiltransferases/antagonistas & inibidores , Sialiltransferases/sangue , beta-D-Galactosídeo alfa 2-6-Sialiltransferase
9.
J Mol Biol ; 365(5): 1469-79, 2007 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-17137591

RESUMO

The Siglec family of receptors mediates cell-surface interactions through recognition of sialylated glycoconjugates. Previously reported structures of the N-terminal domain of the Siglec sialoadhesin (SnD1) in complex with various sialic acid analogs revealed the structural template for sialic acid binding. To characterize further the carbohydrate-binding properties, we have determined the crystal structures of SnD1 in the absence of ligand, and in complex with 2-benzyl-Neu5NPro and 2-benzyl-Neu5NAc. These structures reveal that SnD1 undergoes very few structural changes on ligand binding and detail how two novel classes of sialic acid analogs bind, one of which unexpectedly can induce Siglec dimerization. In conjunction with in silico analysis, this set of structures informs us about the design of putative ligands with enhanced binding affinities and specificities to different Siglecs, and provides data with which to test the effectiveness of different computational drug design protocols.


Assuntos
Biologia Computacional , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Ácidos Siálicos/metabolismo , Algoritmos , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Estrutura Secundária de Proteína , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Ácidos Siálicos/química
10.
Biochem J ; 397(2): 271-8, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16623661

RESUMO

Siglecs (sialic acid binding Ig-like lectins) are transmembrane receptors for sialylated glycoconjugates that modulate cellular interactions and signalling events in the haematopoietic, immune and nervous systems. Siglec-7 is a structural prototype for the recently described family of immune inhibitory CD33-related siglecs and is predominantly expressed on natural killer cells and monocytes, as well as subsets of CD8 T-cells. Siglec-specific inhibitors are desired for the detection of masked and unmasked forms of siglecs, to aid in dissection of signalling pathways and as tools to investigate siglecs as potential therapeutic targets. As a first step towards this end, we present the crystal structure of siglec-7 in complex with a sialylated ligand, the ganglioside analogue DSLc4 [alpha(2,3)/alpha(2,6) disialyl lactotetraosyl 2-(trimethylsilyl)ethyl], which allows for a detailed description of the binding site, required for structure-guided inhibitor design. Mutagenesis and binding assays were used to demonstrate a key structural role for Lys131, a residue that changes conformation upon sialic acid binding. Differences between the binding sites of siglec family members were then exploited using alpha-methyl Neu5Ac (N-acetylneuraminic acid) as a basic scaffold. A co-crystal of siglec-7 in complex with the sialoside inhibitor, oxamido-Neu5Ac [methyl alpha-9-(amino-oxalyl-amino)-9-deoxy-Neu5Ac] and inhibition data for the sialosides gives clear leads for future inhibitor design.


Assuntos
Antígenos de Diferenciação Mielomonocítica/química , Antígenos de Diferenciação Mielomonocítica/fisiologia , Lectinas/química , Lectinas/fisiologia , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cristalografia por Raios X , Desenho de Fármacos , Eritrócitos/metabolismo , Humanos , Leucócitos/metabolismo , Conformação Molecular , Ácido N-Acetilneuramínico/química , Ácidos Siálicos/química
11.
J Med Chem ; 60(3): 941-956, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28103033

RESUMO

Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar Kd values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.


Assuntos
Antígenos de Diferenciação Mielomonocítica/metabolismo , Evasão da Resposta Imune , Lectinas/metabolismo , Neoplasias/imunologia , Desenho de Fármacos , Humanos , Células Matadoras Naturais/imunologia , Ligantes
12.
Structure ; 11(5): 557-67, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12737821

RESUMO

The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC(50) values (relative to methyl-alpha-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-alpha-9-N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Me-alpha-9-N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-alpha-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.


Assuntos
Lectinas/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Proteínas Nucleares/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico
13.
ACS Chem Biol ; 9(7): 1444-50, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24807582

RESUMO

Sialic acids are abundant in higher domains of life and lectins recognizing sialosaccharides are heavily involved in the regulation of the human immune system. Modified sialosides are useful tools to explore the functions of those lectins, especially members of the Siglec (sialic acid binding immunoglobulin like lectin) family. Here we report design, synthesis, and affinity evaluation of novel sialoside classes with combined modification at positions 2, 4, and 9 or 2, 3, 4, and 9 of the sialic acid scaffold as human CD22 (human Siglec-2) ligands. They display up to 7.5 × 10(5)-fold increased affinity over αMe Neu5Ac (the minimal Siglec ligand). CD22 is a negative regulating coreceptor of the B-cell receptor (BCR). In vitro experiments with a human B-lymphocyte cell line showed functional blocking of CD22 upon B-cell receptor (BCR) stimulation in the presence of nanomolar concentrations of the novel ligands. The observed increased Ca(2+) response corresponds to enhanced cell activation, providing an opportunity to therapeutically modulate B-lymphocyte responses, e.g., in immune deficiencies and infections.


Assuntos
Linfócitos B/efeitos dos fármacos , Fatores Imunológicos/química , Ácido N-Acetilneuramínico/análogos & derivados , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Linfócitos B/imunologia , Linhagem Celular , Descoberta de Drogas , Humanos , Fatores Imunológicos/farmacologia , Ligantes , Ácido N-Acetilneuramínico/farmacologia , Receptores de Antígenos de Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores
14.
Immunol Lett ; 131(1): 89-96, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20206207

RESUMO

Human monocyte-derived dendritic cells (mo-DCs) express highly sialylated structures, with recognized but poorly understood function in maturation, immunogenicity and endocytosis capacity. We have previously shown that mo-DCs surface sialylation is changeable upon different stimuli, which led us to hypothesise the existence of cell surface (non-intracellular) sialyltransferases, rapidly restoring or altering mo-DC surface sialylation, thus modulating specific functions. Here, we demonstrate that, in the presence of exogenous CMP-Neu5Ac, mo-DCs incorporate considerable amounts of sialic acids into cell surface, predominantly when mo-DCs were previously desialylated or matured. This is a genuine sialyltransferase activity, confirmed by specific inhibition assays, which is not influenced by secreted enzymes. Functionally, the ecto-sialyltransferase activity causes a significant down-regulation of mo-DCs endocytic capacity, without affecting the maturation state. These findings suggest that ecto-sialyltransferases participate in a dynamic control of mo-DC sialylation, with functional repercussions. This activity is possibly related with specific physiological and pathological conditions, as inflammation and infection, contributing to protection and homeostasis regulation.


Assuntos
Membrana Celular/enzimologia , Células Dendríticas/enzimologia , Sialiltransferases/metabolismo , Células Dendríticas/citologia , Células Dendríticas/ultraestrutura , Endocitose , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Microscopia Confocal , Monócitos/citologia , Ácidos Siálicos/metabolismo , Sialiltransferases/genética
15.
J Neurosci Res ; 85(14): 3053-63, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17722062

RESUMO

The novel myelin-associated glycoprotein (MAG) inhibitor BENZ binds to the N-acetylneuraminic acid (Neu5Ac) portion of the N-terminal Ig-like domain of MAG. Treatment of rat dorsal root ganglion (DRG) cell cultures with BENZ-induced outgrowth of neurofilament 200-positive neurites improved survival of neurons and increased the number of GFAP-positive cells, as determined by fluorescence and confocal laser microscopy and by Western immunoblotting. Furthermore, treatment of DRG cell cultures with BENZ repressed gene and protein expression of the small GTPase RhoA but induced expression of Rho GTP-activating proteins 5 and 24, likely to counteract protein kinase A activity. Specifically, expression of inhibitors of neurite outgrowth, for example, Rock2 and PAK4, was repressed, but cofilin 1, a promoter of axonal growth, was induced. We propose that the MAG inhibitor BENZ abrogates the RhoA-ROCK-cofilin pathway to promote neurite outgrowth. Our findings require confirmation by in vivo animal studies.


Assuntos
Gânglios Espinais/citologia , Glicoproteína Associada a Mielina/antagonistas & inibidores , Ácido N-Acetilneuramínico/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/ultraestrutura , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Análise em Microsséries/métodos , Modelos Biológicos , Glicoproteína Associada a Mielina/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Fatores de Tempo
16.
Glycoconj J ; 22(3): 135-44, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16133834

RESUMO

The sialylated carbohydrate antigens, sialyl-Lewisx and sialyl-Lewisa, are expressed in pancreatic tumour cells and are related to their metastatic potential. While the action of the fucosyltransferases involved in the synthesis of these antigens has already been investigated, no studies have been carried out on the activity and expression of the alpha 2,3-sialyltransferases in pancreatic tumour cells. We describe the sialyltransferase (ST) activity, mRNA expression, and analysis of the cell carbohydrate structures in four human pancreatic adenocarcinoma cell lines of a wide range of neoplastic differentiation stages and in normal human pancreatic tissues. Total ST activity measured on asialofetuin, employing a CMP fluorescent sialic acid, varied among the pancreatic cell lines and could be correlated to the expression of their cell surface antigens. However, in some of the pancreatic cell lines, no relationship could be established with their ST3Gal III and IV mRNA expression. Human pancreatic tissues also showed ST expression and activity. However, it presented a much higher expression of neutral fucosylated structures than sialylated structures. In conclusion, ST activity levels in pancreatic cells could be correlated to their expression of sialylated epitopes, which indicates their involvement in the formation of the sialyl-Lewis antigens, in addition to fucosyltransferase activities.


Assuntos
Adenocarcinoma/enzimologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Antígenos do Grupo Sanguíneo de Lewis/biossíntese , Sialiltransferases/metabolismo , Ensaio de Imunoadsorção Enzimática , Fucosiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Pâncreas/enzimologia , Neoplasias Pancreáticas/enzimologia , RNA Mensageiro/metabolismo , Sialiltransferases/fisiologia , Células Tumorais Cultivadas , beta-Galactosídeo alfa-2,3-Sialiltransferase
17.
J Biol Chem ; 277(8): 6688-95, 2002 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-11751912

RESUMO

Sialic acids are critical components of many glycoconjugates involved in biologically important ligand-receptor interactions. Quantitative and structural variations of sialic acid residues can profoundly affect specific cell-cell, pathogen-cell, or drug-cell interactions, but manipulation of sialic acids in mammalian cells has been technically limited. We describe the finding of a previously unrecognized and efficient uptake and incorporation of sialic acid analogues in mammalian cells. We added 16 synthetic sialic acid analogues carrying distinct C-1, C-5, or C-9 substitutions individually to cell cultures of which 10 were readily taken up and incorporated. Uptake of C-5- and C-9-substituted sialic acids resulted in the structural modification of up to 95% of sialic acids on the cell surface. Functionally, binding of murine sialic acid-binding immunoglobulin-like lectin-2 (Siglec-2, CD22) to cells increased after N-glycolylneuraminic acid treatment, whereas 9-iodo-N-acetylneuraminic acid abolished binding. Furthermore, susceptibility to infection by the B-lymphotropic papovavirus via a sialylated receptor was markedly enhanced following pretreatment of host cells with selected sialic acid analogues including 9-iodo-N-acetylneuraminic acid. This novel experimental strategy allows for an efficient biosynthetic engineering of surface sialylation in living cells. It is versatile, extending the repertoire of modification sites at least to C-9 and enables detailed structure-function studies of sialic acid-dependent ligand-receptor interactions in their native context.


Assuntos
Moléculas de Adesão Celular , Lectinas , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/biossíntese , Animais , Antígenos CD/biossíntese , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfoma de Burkitt , Células Clonais , Meios de Cultura Livres de Soro , Células HL-60 , Humanos , Camundongos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Sialiltransferases , Especificidade por Substrato , Células Tumorais Cultivadas
18.
Glycobiology ; 14(10): 859-70, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15190007

RESUMO

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.


Assuntos
Leucócitos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Sialoglicoproteínas/química , Sialoglicoproteínas/isolamento & purificação , Adolescente , Criança , Pré-Escolar , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Valor Preditivo dos Testes , Prognóstico , Ácidos Siálicos/síntese química , Ácidos Siálicos/farmacologia , Sialoglicoproteínas/antagonistas & inibidores
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