RESUMO
Infection with Brachyspira hyodysenteriae results in mucoid hemorrhagic diarrhea. This pathogen is associated with the colonic mucus layer, mainly composed of mucins. Infection regulates mucin O-glycosylation in the colon and increases mucin secretion as well as B. hyodysenteriae binding sites on mucins. Here, we analyzed potential mucin epitopes for B. hyodysenteriae adhesion in the colon, as well as the effect of colonic mucins on bacterial growth. Associations between B. hyodysenteriae binding to pig colonic mucins and mucin glycan data showed that B. hyodysenteriae binding was associated with the presence of N-glycolylneuraminic acid (NeuGc) on mucins. The role of sialic acid in B. hyodysenteriae adhesion was analyzed after the removal of sialic acid residues on the mucins by enzymatic treatment with sialidase A, which decreased bacterial binding to the mucins. The effect of pig colonic mucins on B. hyodysenteriae growth was determined in carbohydrate-free medium. B. hyodysenteriae growth increased in the presence of mucins from two out of five infected pigs, suggesting utilization of mucins as a carbon source for growth. Additionally, bacterial growth was enhanced by free sialic acid and N-acetylglucosamine. The results highlight a role of sialic acid as an adhesion epitope for B. hyodysenteriae interaction with colonic mucins. Furthermore, the mucin response and glycosylation changes exerted in the colon during B. hyodysenteriae infection result in a potentially favorable environment for pathogen growth in the intestinal mucus layer.
Assuntos
Aderência Bacteriana/fisiologia , Brachyspira hyodysenteriae/fisiologia , Mucinas/fisiologia , Ácido N-Acetilneuramínico/fisiologia , Animais , Brachyspira hyodysenteriae/crescimento & desenvolvimento , Colo/metabolismo , SuínosRESUMO
Human milk not only has nutritional value, but also provides a wide range of biologically active molecules, which are adapted to meet the needs of newborns and infants. Mother's milk is a source of sialylated oligosaccharides and glycans that are attached to proteins and lipids, whose concentrations and composition are unique. Sialylated human milk glycoconjugates and oligosaccharides enrich the newborn immature immune system and are crucial for their proper development and well-being. Some of the milk sialylated oligosaccharide structures can locally exert biologically active effects in the newborn's and infant's gut. Sialylated molecules of human milk can be recognized and bound by sialic acid-dependent pathogens and inhibit their adhesion to the epithelial cells of newborns and infants. A small amount of intact sialylated oligosaccharides can be absorbed from the intestine and remain in the newborn's circulation in concentrations high enough to modulate the immunological system at the cellular level and facilitate proper brain development during infancy. Conclusion: The review summarizes the current state of knowledge on sialylated human milk oligosaccharides and glycoconjugates, discusses the significance of sialylated structures of human milk in newborn protection and development, and presents the advantages of human milk over infant formula.
Assuntos
Desenvolvimento Infantil , Glicoconjugados , Imunidade Materno-Adquirida , Leite Humano , Ácido N-Acetilneuramínico , Oligossacarídeos , Aleitamento Materno , Glicoconjugados/química , Glicoconjugados/imunologia , Glicoconjugados/fisiologia , Humanos , Recém-Nascido , Leite Humano/química , Leite Humano/imunologia , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/imunologia , Ácido N-Acetilneuramínico/fisiologia , Oligossacarídeos/química , Oligossacarídeos/imunologia , Oligossacarídeos/fisiologiaRESUMO
Streptococcus gordonii is an early colonizer of the oral cavity. Although a variety of S. gordonii adherence mechanisms have been described, current dogma is that the major receptor for S. gordonii is sialic acid. However, as many bacterial species in the oral cavity produce neuraminidase that can cleave terminal sialic acid, it is unclear whether S. gordonii relies on sialic acid for adherence to oral surfaces or if this species has developed alternative binding strategies. Previous studies have examined adherence to immobilized glycoconjugates and identified binding to additional glycans, but no prior studies have defined the contribution of these different glycan structures in adherence to oral epithelial cells. We determined that the majority of S. gordonii strains tested did not rely on sialic acid for efficient adherence. In fact, adherence of some strains was significantly increased following neuraminidase treatment. Further investigation of representative strains that do not rely on sialic acid for adherence revealed binding not only to sialic acid via the serine-rich repeat protein GspB but also to ß-1,4-linked galactose. Adherence to this carbohydrate occurs via an unknown adhesin distinct from those utilized by Streptococcus oralis and Streptococcus pneumoniae Demonstrating the potential biological relevance of binding to this cryptic receptor, we established that S. oralis increases S. gordonii adherence in a neuraminidase-dependent manner. These data suggest that S. gordonii has evolved to simultaneously utilize both terminal and cryptic receptors in response to the production of neuraminidase by other species in the oral environment.
Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana , Proteínas de Transporte/fisiologia , Ácido N-Acetilneuramínico/fisiologia , Neuraminidase/biossíntese , Streptococcus gordonii/fisiologia , Galactose/metabolismo , Hemaglutininas Virais , Humanos , Mucosa Bucal/microbiologia , Streptococcus oralis/fisiologiaRESUMO
Influenza A virus (IAV) recognizes terminal sialic acid of sialoglyco-conjugates on host cells through the viral envelope glycoprotein hemagglutinin (HA), followed by initiation of entry into the cells. Molecular species of sialic acid are largely divided into two moieties: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). A receptor for IAV infection generally means Neu5Ac. Almost all equine IAVs and some human, swine, and duck IAVs bind not only to Neu5Ac but also to Neu5Gc. In nonhuman animals, Neu5Gc has been detected in swine and equine tracheas and the duck colon, which are the main replication sites of mammalian and avian IAVs. Therefore, Neu5Gc in these sites has been suggested to be a functional receptor for IAV infection. Humans cannot synthesize Neu5Gc due to a genetic defect of the Neu5Gc-synthesizing enzyme. We evaluated the receptor function of Neu5Gc in IAV infection in human cells. Our results indicated that Neu5Gc expression on the surface of human cells is not a functional receptor for IAV infection and that it has a negative effect on infectivity of IAV possessing Neu5Gc binding ability. IAV also binds to non-sialo 3-O-sulfated galactosylceramide (sulfatide). Sulfatide has been suggested to be a functional receptor for IAV infection. However, we have shown that sulfatide is not a functional receptor for IAV infection and that the binding of HA with sulfatide enhances progeny virus production. It is expected that functions of these glyco-molecules can be used in prevention and development of new drugs against IAV.
Assuntos
Vírus da Influenza A/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , Receptores Virais/fisiologia , Animais , Antivirais , Descoberta de Drogas , Galactosilceramidas/metabolismo , Glicoconjugados/metabolismo , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Terapia de Alvo Molecular , Ácido N-Acetilneuramínico/fisiologia , Ácidos Neuramínicos/metabolismo , Sulfoglicoesfingolipídeos/metabolismoRESUMO
Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse ß2-integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express ß2-integrin or not. Clearly, removal of sialic acid groups significantly reduces a ß2-integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express ß2-integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both ß2-integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to ß2-integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Morte Celular/fisiologia , Eritrócitos/efeitos dos fármacos , Exotoxinas/fisiologia , Ácido N-Acetilneuramínico/fisiologia , Análise de Variância , Animais , Antígenos CD18/fisiologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Eritrócitos/metabolismo , Exotoxinas/toxicidade , Humanos , Camundongos , Ácido N-Acetilneuramínico/química , Coelhos , OvinosRESUMO
As obligate intracellular parasites, viruses must bind to, and enter, permissive host cells in order to gain access to the cellular machinery that is required for their replication. The very large number of mammalian viruses identified to date is reflected in the fact that almost every human and animal cell type is a target for infection by one, or commonly more than one, species of virus. As viruses have adapted to target certain cell types for their propagation, there is exquisite specificity in cellular tropism. This specificity is frequently, but not always, mediated by the first step in the viral replication cycle: attachment of viral surface proteins to receptors expressed on susceptible cells. Viral receptors may be protein, carbohydrate, and/or lipid. Many viruses can use more than one attachment receptor, and indeed may sequentially engage multiple receptors to infect a cell. Thus, it is useful to differentiate between attachment receptors, that simply allow viruses a foothold at the limiting membrane of a cell, and entry receptors that mediate delivery the viral genome into the cytoplasm. For some viruses the attachment factors that promote binding to permissive cells are very well defined, but the sequence of events that triggers viral entry is only now beginning to be understood. For other viruses, despite many efforts, the receptors remain elusive. In this chapter we will confine our review to viruses that infect mammals, with particular focus on human pathogens. We do not intend that this will be an exhaustive overview of viral attachment receptors; instead we will take a number of examples of well-characterized virus-receptor interactions, discuss supporting evidence, and highlight any controversies and uncertainties in the field. We will then conclude with a reflection on general principles of viral attachment, consider some exceptions to these principles, and make some suggestion for future research.
Assuntos
Receptores Virais/fisiologia , Ligação Viral , Animais , Antígenos CD4 , Moléculas de Adesão Celular/fisiologia , Humanos , Integrinas/fisiologia , Lectinas Tipo C/fisiologia , Ácido N-Acetilneuramínico/fisiologia , Receptores de Superfície Celular/fisiologia , Internalização do VírusRESUMO
Mammalian orthoreoviruses (reoviruses) are members of the Reoviridae. Reoviruses contain 10 double-stranded (ds) RNA gene segments enclosed in two concentric protein shells, called outer capsid and core. These viruses serve as a versatile experimental system for studies of viral replication events at the virus-cell interface, including engagement of cell-surface receptors, internalization and disassembly, and activation of the innate immune response, including NF-κB-dependent cellular signaling pathways. Reoviruses also provide a model system for studies of virus-induced apoptosis and organ-specific disease in vivo.Reoviruses attach to host cells via the filamentous attachment protein, σ1. The σ1 protein of all reovirus serotypes engages junctional adhesion molecule-A (JAM-A), an integral component of intercellular tight junctions. The σ1 protein also binds to cell-surface carbohydrate, with the type of carbohydrate bound varying by serotype. Following attachment to JAM-A and carbohydrate, reovirus internalization is mediated by ß1 integrins, most likely via clathrin-dependent endocytosis. In the endocytic compartment, reovirus outer-capsid protein σ3 is removed by acid-dependent cysteine proteases in most cell types. Removal of σ3 results in the exposure of a hydrophobic conformer of the viral membrane-penetration protein, µ1, which pierces the endosomal membrane and delivers transcriptionally active reovirus core particles into the cytoplasm.Reoviruses induce apoptosis in both cultured cells and infected mice. Perturbation of reovirus disassembly using inhibitors of endosomal acidification or protease activity abrogates apoptosis. The µ1-encoding M2 gene is genetically linked to strain-specific differences in apoptosis-inducing capacity, suggesting a function for µ1 in induction of death signaling. Reovirus disassembly leads to activation of transcription factor NF-κB, which modulates apoptotic signaling in numerous types of cells. Inhibition of NF-κB nuclear translocation using either pharmacologic agents or expression of transdominant forms of IκB blocks reovirus-induced apoptosis, suggesting an essential role for NF-κB activation in the death response. Multiple effector pathway s downstream of NF-κB-directed gene expression execute reovirus-induced cell death. This chapter will focus on the mechanisms by which reovirus attachment and disassembly activate NF-κB and stimulate the cellular proapoptotic machinery.
Assuntos
Apoptose , Receptores Virais/fisiologia , Reoviridae/fisiologia , Transdução de Sinais/fisiologia , Internalização do Vírus , Animais , Endocitose , Humanos , Ácido N-Acetilneuramínico/fisiologia , NF-kappa B/fisiologia , Infecções por Reoviridae/etiologia , TranscriptomaRESUMO
The early stages of neurodevelopment in infants are crucial for establishing neural structures and synaptic connections that influence brain biochemistry well into adulthood. This postnatal period of rapid neural growth is of critical importance for cell migration, neurite outgrowth, synaptic plasticity, and axon fasciculation. These processes thus place an unusually high demand on the intracellular pool of nutrients and biochemical precursors. Sialic acid (Sia), a family of 9-carbon sugar acids, occurs in large amounts in human milk oligosaccharides and is an essential component of brain gangliosides and sialylated glycoproteins, particularly as precursors for the synthesis of the polysialic acid (polySia) glycan that post-translationally modify the cell membrane-associated neural cell adhesion molecules (NCAM). Human milk is noteworthy in containing exceptionally high levels of Sia-glycoconjugates. The predominate form of Sia in human milk is N-acetylneuraminic acid (Neu5Ac). Infant formula, however, contains low levels of Sia consisting of both Neu5Ac and N-glycolyneuraminic acid (Neu5Gc). Current studies implicate Neu5Gc in several human inflammatory diseases. Polysialylated NCAM and neural gangliosides both play critical roles in mediating cell-to-cell interactions important for neuronal outgrowth, synaptic connectivity, and memory formation. A diet rich in Sia also increases the level of Sia in the brains of postnatal piglets, the expression level of 2 learning-related genes, and enhances learning and memory.
Assuntos
Encéfalo/fisiologia , Cognição/fisiologia , Ácido N-Acetilneuramínico/fisiologia , Animais , Encéfalo/crescimento & desenvolvimento , Comunicação Celular , Gangliosídeos/análise , Gangliosídeos/metabolismo , Glicoconjugados/análise , Glicoconjugados/metabolismo , Humanos , Lactente , Fórmulas Infantis/química , Aprendizagem/fisiologia , Memória/fisiologia , Leite Humano/química , Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/fisiologiaRESUMO
Twenty-five years ago the author proposed new ideas of glycoprotein (GPs) and glycosphingolipid (GSLs) functions at the cell membrane. The GPs, apart from their glycan carrying capacity, were assumed to have specific, protein associated, functions. In contrast, GSLs such as those of globo and neolacto/lacto series, were considered to be energetically cheap membrane packing substances, filling in membrane spaces not covered with functional GPs. The terminal carbohydrate structures of the neolacto/lacto GSLs, i.e., sialic acid residues and ABH glycotopes, were postulated to have either regulatory or protective functions, respectively. A special active role was ascribed to terminal ß-galactosyl residues of GSLs and GPs. Gangliosides were considered to be functional GSLs. In the present review the author discusses these old ideas in context of the contemporary knowledge and comes to the conclusion that they have not aged.
Assuntos
Sistema ABO de Grupos Sanguíneos/fisiologia , Membrana Celular/metabolismo , Glicoesfingolipídeos/fisiologia , Animais , Antígenos CD/fisiologia , Diferenciação Celular/fisiologia , Glicoproteínas/fisiologia , Humanos , Lactosilceramidas/fisiologia , Lectinas/fisiologia , Modelos Biológicos , Ácido N-Acetilneuramínico/fisiologia , Neuraminidase/fisiologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.
Assuntos
Modelos Animais de Doenças , Nefropatias/genética , Animais , Biomarcadores/metabolismo , Carboidratos Epimerases/genética , Proteínas de Transporte/genética , Suplementos Nutricionais , Avaliação Pré-Clínica de Medicamentos/métodos , Hexosaminas/uso terapêutico , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/embriologia , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Mutação , Ácido N-Acetilneuramínico/fisiologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sialoglicoproteínas/metabolismoRESUMO
Although humans are genetically very similar to the evolutionarily related nonhuman hominids (chimpanzees, bonobos, gorillas, and orangutans), comparative studies suggest a surprising number of uniquely human differences in the incidence and/or severity of biomedical conditions. Some differences are due to anatomical changes that occurred during human evolution. However, many cannot be explained either by these changes or by known environmental factors. Because chimpanzees were long considered models for human disease, it is important to be aware of these differences, which appear to have been deemphasized relative to similarities. We focus on the pathophysiology and pathobiology of biomedical conditions that appear unique to humans, including several speculative possibilities that require further study. We pay particular attention to the possible contributions of uniquely human changes in the biology of cell-surface sialic acids and the proteins that recognize them. We also discuss the metabolic incorporation of a diet-derived nonhuman sialic acid, which generates a novel xeno-autoantigen reaction, and chronic inflammation known as xenosialitis.
Assuntos
Doenças dos Animais/fisiopatologia , Modelos Animais de Doenças , Hominidae/anatomia & histologia , Hominidae/fisiologia , Ácido N-Acetilneuramínico/fisiologia , Animais , Evolução Biológica , Humanos , Especificidade da EspécieAssuntos
Linfócitos B/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Acetilação , Animais , Humanos , Ligantes , Camundongos , Ácido N-Acetilneuramínico/fisiologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcr/fisiologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Several Trypanosoma cruzi molecules that stimulate macrophages activity were described as Toll-like receptor 2 (TLR2) ligands. Besides, the models of dendritic cells (DC) are poorly characterised. We have previously demonstrated that live-trypomastigotes (Tp) plus lipopolysaccharide (LPS) induce DC with tolerogenic properties that produce high levels of interleukin (IL)-10 and an impaired capacity to induce lymphoproliferation. Here, we show that the regulatory phenotype was observed with heat-killed trypomastigotes (Tphk) stimulation, ruling out DC infection. T. cruzi induced a particular DC activation state increasing LPS-activation of extracellular regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. Inhibition of ERK down-regulated IL-10 production and restored DC stimulatory capacity, showing the importance of this pathway in the DC modulation. A recent work shows that signalling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam(3)Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we showed that high levels of IL-10 were independent of TLR2 but associated with TLR4 and NF-kappaB signallization. Although sialic acid has been described as a molecule responsible of DC inhibition, we determine that it is not associated with T. cruzi-IL-10 modulatory response. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association with NF-kappaB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite-DC interactions not described yet.
Assuntos
Células Dendríticas/imunologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Interleucina-10/biossíntese , Receptor 4 Toll-Like/fisiologia , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos , Ácido N-Acetilneuramínico/fisiologia , NF-kappa B/fisiologia , Fosforilação , Fator de Transcrição STAT3/metabolismo , Receptor 2 Toll-Like/fisiologiaRESUMO
IgG antibodies have long been recognized as proinflammatory mediators of the humoral immune response. Antibodies bind and neutralize antigens to promote antibody-dependent cytotoxicity, opsonization of antigens, and the initiation of phagocytosis. Whereas the antigen specificity of antibodies is determined by the antigen-binding Fab portion, the effector functions initiated by antibodies are triggered by the Fc (crystallizable) domain. These effector functions are heavily dependent on the single N-linked, biantennary glycan of the heavy chain, which resides just below the hinge region. This glycan is believed to maintain the two heavy chains of the Fc in an open confirmation required for interactions with activating Fcgamma receptors (FcgammaRs). However, the presence of specific sugar moieties on the glycan has profound implications on Fc effector functions. The addition of terminal sialic acid to the glycan reduces FcgammaR binding and converts IgG antibodies to anti-inflammatory mediators through the acquisition of novel binding activities. Studies from our laboratory demonstrated that these sialylated IgG Fcs are important for the in vivo activity of intravenous immunoglobulin. Instead of binding with FcgammaRs, sialylated Fcs initiate an anti-inflammatory cascade through the lectin receptor SIGN-R1 or DC-SIGN. This leads to upregulated surface expression of the inhibitory FcR, FcgammaRIIb, on inflammatory cells, thereby attenuating autoantibody-initiated inflammation.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Inflamação/imunologia , Ácido N-Acetilneuramínico/fisiologia , Receptores de IgG/imunologia , Animais , Reações Antígeno-Anticorpo , Autoanticorpos/química , Autoanticorpos/imunologia , Doenças Autoimunes/terapia , Moléculas de Adesão Celular/imunologia , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Cadeias Pesadas de Imunoglobulinas/química , Imunoglobulinas Intravenosas/uso terapêutico , Inflamação/terapia , Lectinas Tipo C/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Receptores de Superfície Celular/imunologia , Receptores de IgG/química , Relação Estrutura-AtividadeRESUMO
Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that alpha2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on alpha2,6-sialic acid. We also found that the absence of alpha2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.
Assuntos
Apoptose , Ácido N-Acetilneuramínico/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Células Endoteliais , Endotélio Vascular/citologia , Glicosilação , Humanos , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Fosforilação , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
von Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by alpha2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (+/- 14%) by ADAMTS13, compared with 11% (+/- 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O > or = B > A > or = AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance.
Assuntos
Proteínas ADAM/metabolismo , Ácido N-Acetilneuramínico/fisiologia , Fator de von Willebrand/química , Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/metabolismo , Proteína ADAMTS13 , Biopolímeros , Configuração de Carboidratos , Colágeno/metabolismo , Cisteína Proteases/metabolismo , Galactose/química , Glicosídeo Hidrolases/farmacologia , Humanos , Ácido N-Acetilneuramínico/química , Neuraminidase/farmacologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/farmacologia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Serina Proteases/metabolismo , Especificidade por Substrato , alfa-N-Acetilgalactosaminidase/farmacologia , Fator de von Willebrand/efeitos dos fármacos , Fator de von Willebrand/metabolismoRESUMO
Although current H5N1 highly pathogenic avian influenza viruses (HPAIV) are inefficiently transmitted to humans, infected individuals can suffer from severe disease, often progressing rapidly to acute respiratory distress syndrome and multiorgan failure. This is in contrast with the situation with human influenza viruses, which in immunocompetent individuals usually cause only a respiratory disease which is less aggressive than that observed with avian H5N1 viruses. While the biological basis of inefficient transmission is well documented, the mechanisms by which the H5N1 viruses cause fatal disease remain unclear. In the present study, we demonstrate that human pulmonary microvascular endothelial cells (hPMEC) had a clearly higher susceptibility to infection by H5N1 HPAIV than to infection by human influenza viruses. This was measurable by de novo intracellular nucleoprotein production and virus replication. It was also related to a relatively higher binding capacity to cellular receptors. After infection of hPMEC, cell activation markers E-selectin and P-selectin were upregulated, and the proinflammatory cytokines interleukin-6 and beta interferon were secreted. H5N1 virus infection was also associated with an elevated rate of cell death. Reverse genetics analyses demonstrated a major role for the viral hemagglutinin in this cell tropism. Overall, avian H5N1 viruses have a particular receptor specificity targeting endothelial cells that is different from human influenza viruses, and this H5N1 receptor specificity could contribute to disease pathogenesis.
Assuntos
Células Endoteliais/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Virus da Influenza A Subtipo H5N1/fisiologia , Tropismo Viral , Animais , Apoptose , Células Cultivadas , Cães , Selectina E/biossíntese , Humanos , Interferon Tipo I/metabolismo , Interleucina-6/metabolismo , Ácido N-Acetilneuramínico/fisiologia , Selectina-P/biossínteseRESUMO
Red blood cells (RBC) have emerged as a novel regulatory cell type endowed with bioactivities toward activated human T cells. Herein we show that the RBC bioactivities act on intracellular pathways initiated by T cell receptor (TCR)-dependent and -independent stimuli,including IL-2, IL-15, and the mixture of phorbol dibutyrate and ionomycin. The RBC bioactivities preserve the antioxidant status and are capable of rescuing activated T cells from cell death induced by serum deprivation. They are not mediated by glycosylphosphatidylinositol-linked receptors or sialic acids, and kinetic studies revealed that they hasten the entrance into the cell cycle. By using cyclosporine A (CsA) and rapamycin (Rapa) we show that the RBC bioactivities are calcineurin-dependent. Thus, treatment of T cells with CsA, but not Rapa, impaired RBC bioactivities, and preincubation of RBC with CsA completely abolished their bioactivities. We have demonstrated that RBC carry out bioactivities that are sensitive to CsA.
Assuntos
Ciclosporina/farmacologia , Eritrócitos/fisiologia , Imunossupressores/farmacologia , Linfócitos T/fisiologia , Apoptose , Calcineurina/metabolismo , Calcineurina/fisiologia , Divisão Celular/fisiologia , Crescimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Glicosilfosfatidilinositóis/metabolismo , Humanos , Ativação Linfocitária , Morfolinas/farmacologia , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Sirolimo/farmacologia , Compostos de Sulfidrila/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
This report provides a perspective on metabolic glycoengineering methodology developed over the past two decades that allows natural sialic acids to be replaced with chemical variants in living cells and animals. Examples are given demonstrating how this technology provides the glycoscientist with chemical tools that are beginning to reproduce Mother Nature's control over complex biological systems - such as the human brain - through subtle modifications in sialic acid chemistry. Several metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) can be used to feed flux into the sialic acid biosynthetic pathway resulting in numerous - and sometime quite unexpected - biological repercussions upon nonnatural sialoside display in cellular glycans. Once on the cell surface, ketone-, azide-, thiol-, or alkyne-modified glycans can be transformed with numerous ligands via bioorthogonal chemoselective ligation reactions, greatly increasing the versatility and potential application of this technology. Recently, sialic acid glycoengineering methodology has been extended to other pathways with analog incorporation now possible in surface-displayed GalNAc and fucose residues as well as nucleocytoplasmic O-GlcNAc-modified proteins. Finally, recent efforts to increase the "druggability" of sugar analogs used in metabolic glycoengineering, which have resulted in unanticipated "scaffold-dependent" activities, are summarized.