RESUMO
Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system.
Assuntos
Membrana Celular/metabolismo , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Ácidos Siálicos/metabolismo , Animais , Células CHO , Membrana Celular/genética , Cricetinae , Cricetulus , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Bicamadas Lipídicas , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Substrato Quinase C Rico em Alanina Miristoilada , Proteínas do Tecido Nervoso/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Ácidos Siálicos/genéticaRESUMO
Polysialic acid (PSA) is a large and highly negatively charged glycan that plays crucial roles in nervous system development and function in the adult. It has been suggested to facilitate cell migration, neurite outgrowth, and synaptic plasticity because its hydration volume could enhance flexibility of cell interactions. Evidence for receptors of PSA has so far been elusive. We now identified histone H1 as binding partner of PSA via a single-chain variable fragment antibody using an anti-idiotypic approach. Histone H1 directly binds to PSA as shown by ELISA. Surface biotinylation of cultured cerebellar neurons indicated an extracellular localization of histone H1. Immunostaining of live cerebellar neurons and Schwann cells confirmed that an extracellular pool of histone H1 colocalizes with PSA at the cell surface. Histone H1 was also detected in detergent-insoluble synaptosomal membrane subfractions and postsynaptic densities. When applied in vitro, histone H1 stimulated neuritogenesis, process formation and proliferation of Schwann cells, and migration of neural precursor cells via a PSA-dependent mechanism, further indicating that histone H1 is active extracellularly. These in vitro observations suggested an important functional role for the interaction between histone H1 and PSA not only for nervous system development but also for regeneration in the adult. Indeed, histone H1 improved functional recovery, axon regrowth, and precision of reinnervation of the motor branch in adult mice with femoral nerve injury. Our findings encourage investigations on the therapeutic potential of histone H1 in humans.
Assuntos
Diferenciação Celular/fisiologia , Espaço Extracelular/fisiologia , Histonas/fisiologia , Regeneração Nervosa/fisiologia , Ácidos Siálicos/fisiologia , Animais , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Nervo Femoral/metabolismo , Nervo Femoral/patologia , Nervo Femoral/fisiologia , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/fisiologia , Neuritos/fisiologia , Ligação Proteica/fisiologia , Células de Schwann/citologia , Células de Schwann/metabolismo , Ácidos Siálicos/metabolismoRESUMO
The role of neutrophils in the immune response has long been regarded as mainly phagocytic, but recent publications have indicated the production of several cytokines by polymorphonuclear leucocytes (PMN). The results of the individual reports, however, vary considerably. In this study, we established a cytokine profile of pure human neutrophils and demonstrated that minor contamination of peripheral blood mononuclear cells (PBMCs) in PMN preparations can lead to false-positive results. In our hands, peripheral blood PMN fail to produce the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha). Instead, they secrete large amounts of the chemokine IL-8 and the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Additionally, PMN preparations of a high purity show production of the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and growth-related oncogene-alpha (GRO-alpha), as well as macrophage colony-stimulating factor (M-CSF). The neutrophil therefore represents a novelty by producing the antagonist of IL-1beta (i.e. IL-1ra) in the absence of IL-1beta itself. To support our results, we differentiated stem cells from human cord blood into PMN and monocytes, respectively. These in vitro-differentiated PMN showed the same cytokine profile as peripheral blood PMN lacking IL-1beta, while differentiated monocytes produced the expected IL-1beta in addition to IL-1ra. The clear anti-inflammatory nature of their cytokine profile enables PMN to antagonize pro-inflammatory signals in experimental conditions. It is therefore possible that PMN play a key role in immune regulation by counteracting a dysregulation of the inflammatory process. Clinical studies, in which administration of recombinant G-CSF had a favourable effect on the outcome of severe infections and even sepsis without worsening inflammation, could thus be explained by our results.
Assuntos
Quimiocinas/biossíntese , Mediadores da Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/biossíntese , Neutrófilos/imunologia , Antígenos CD34/análise , Western Blotting , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Leucócitos Mononucleares/imunologiaRESUMO
With a short lifespan and containing only few ribosomes and endoplasmic reticulum structures, neutrophils are thought to have a limited capacity for protein synthesis. We here show that peripheral blood polymorphonuclear neutrophils (PMN) are able react to stimulants with differential production of two interleukin (IL)-1 receptor antagonist (IL-1ra) isoforms, secreted IL-1ra (sIL-1ra) and the 16kDa intracellular form of IL-1ra (icIL-1ra3), as well as IL-8. Neutrophils of a high purity and with a low degree of preactivation upregulate mRNA and de novo synthesize protein of both IL-1ra variants and IL-8 in response to granulocyte-macrophage colony-stimulating factor and lipopolysaccharide. The cytokines are differentially regulated and distributed in two intracellular compartments. In comparison with peripheral blood mononuclear cells (PBMC), PMN produce distinctly more sIL-1ra but significantly less IL-8. This may indicate an anti-inflammatory role, enabling PMN to antagonize proinflammatory signals. It is therefore possible that PMN play an important role in immune regulation by counteracting a dysregulation of the inflammatory process.
Assuntos
Interleucina-8/biossíntese , Neutrófilos/química , Neutrófilos/imunologia , Receptores de Interleucina-1/imunologia , Sialoglicoproteínas/biossíntese , Variação Genética , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-8/análise , Reação em Cadeia da Polimerase , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Sialoglicoproteínas/genética , Frações Subcelulares/químicaRESUMO
Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.
Assuntos
Encéfalo/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Animais , Química Encefálica , Antígenos CD57/química , Bovinos , Cromatografia Líquida de Alta Pressão , Epitopos/análise , Glicopeptídeos/química , Glicosilação , Camundongos , Modelos Moleculares , Peso Molecular , Molécula L1 de Adesão de Célula Nervosa/química , Molécula L1 de Adesão de Célula Nervosa/isolamento & purificação , Polissacarídeos/química , Ácidos Siálicos/química , Ácidos Siálicos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Recognition molecules that carry carbohydrate structures regulate cell interactions during development and play important roles in synaptic plasticity and regeneration in the adult. Glycans appear to be involved in these interactions. We have searched for binding proteins for oligomannosidic structures using the L3 antibody directed against high mannose-type glycans in an anti-idiotypic approach. A selected monoclonal anti-idiotype antibody was used for affinity chromatography and identified basigin as a binding protein from mouse brain detergent lysates. Basigin was found to bind to high mannose-carrying cell recognition molecules, such as myelin-associated glycoprotein, L1, the beta2-subunit of Na+/K+-ATPase and an oligomannosidic neoglycolipid. Furthermore, basigin was involved in outgrowth of astrocytic processes in vitro. A striking homology between the first immunoglobulin (Ig)-like domain of basigin and the fourth Ig-like domain of NCAM, previously shown to bind to oligomannosidic glycans, and the lectin domain of the mannose receptor confirms that basigin is an oligomannose binding lectin. To our knowledge this is the first report that anti-idiotypic antibodies can be used to identify binding partners for carbohydrates.
Assuntos
Anticorpos Anti-Idiotípicos/metabolismo , Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície , Proteínas Aviárias , Proteínas Sanguíneas , Proteínas de Transporte/metabolismo , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Astrócitos/citologia , Astrócitos/metabolismo , Basigina , Química Encefálica , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Células Cultivadas , Cromatografia de Afinidade , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , Glicoproteína Associada a Mielina/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Oligossacarídeos/imunologia , Polissacarídeos/química , Polissacarídeos/metabolismo , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.
Assuntos
Deleção Cromossômica , Síndrome de DiGeorge/genética , Fatores de Crescimento Endotelial/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfocinas/genética , Animais , Vasos Sanguíneos/anormalidades , Anormalidades Congênitas/genética , Face/anormalidades , Camundongos , Camundongos Knockout , Neuropilina-1/genética , Isoformas de Proteínas/genética , Crânio/anormalidades , Proteínas com Domínio T/genética , Timo/anormalidades , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Peixe-ZebraRESUMO
The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual alpha 2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N-treated PSA-NCAM was separated by SDS-PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)(2)(Man)(5) up to (GlcNAc)(2)(Man)(9). In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions.