RESUMO
Sialic acids have a special place in vertebrate glycobiology, where they constitute the dominant terminal saccharides on many cell surface glycans. From early studies that identified sialoglycans as receptors for important pathogens and toxins to more recent discoveries of sialic acid-binding proteins essential for immune system (and other) functions in humans, sialic acids and sialoglycans have become cornerstones in understanding vertebrate glycobiology and pathology. During a remarkable 3-year period in the late 1950s, a newly minted postdoctoral fellow (Donald G. Comb) and his young mentor (Saul Roseman) made a surprising series of discoveries that put sialic acid research on sound chemical and biochemical footing. A detailed personal letter written by Dr. Roseman that describes this period of intense sialic acid discovery, complete with inserted figures, was given to one of us (Y.C.L.) several years later. The text and figures of this letter provide a look back at the enthusiasm, rigor and serendipity that led to their important findings through the eyes of one of the key figures in sialic acid research.
Assuntos
Bioquímica de Carboidratos/história , Ácido N-Acetilneuramínico do Monofosfato de Citidina/química , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , História do Século XXRESUMO
Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell's macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of "super-IR resistant" mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum "super-IR resistant" mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes.
Assuntos
Raios gama , Halobacterium salinarum/efeitos da radiação , Manganês/metabolismo , Tolerância a Radiação , Proteínas Arqueais/metabolismo , Coenzimas/metabolismo , Metabolismo Energético , Halobacterium salinarum/genética , Halobacterium salinarum/metabolismo , Homeostase , Mutação , Estresse Oxidativo , Proteoma/metabolismoRESUMO
We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20-80min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA-primaquine conjugate (TriGalNAc-PGA-PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250ng per million cells of the conjugate bound to one million rat hepatocytes at 0°C, and approximately 2µg per million cells of the conjugate was taken up over 7h incubation at 37°C. Furthermore, our results suggested that TriGalNAc-PGA-PQ was almost completely degraded over 24h, and small degradation products were secreted into cell culture medium. The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.
Assuntos
Hepatócitos/metabolismo , Ácido Poliglutâmico/metabolismo , Primaquina/química , Animais , Células Cultivadas , Feminino , Camundongos , Microscopia de Fluorescência , Ácido Poliglutâmico/química , RatosRESUMO
Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the alpha1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le(x)). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-alpha expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-alpha expression in MDDCs via, in part, Raf-1 signaling pathways.
Assuntos
Alérgenos/imunologia , Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Receptores de Superfície Celular/imunologia , Alérgenos/metabolismo , Alérgenos/farmacologia , Animais , Moléculas de Adesão Celular/metabolismo , Cynodon/imunologia , Células Dendríticas/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Monócitos/citologia , Pólen/imunologia , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Pyroglyphidae/imunologia , Receptores de Superfície Celular/metabolismo , Soroalbumina Bovina/imunologia , Soroalbumina Bovina/farmacologia , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
C-type lectins (CTLs) are proteins that contain one or more carbohydrate-recognition domains (CRDs) that require calcium for sugar binding and share high degree of sequence homology and tertiary structure. CTLs whose CRD contain EPN (Glu-Pro-Asn) tripeptide motifs have potential to bind mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc) and l-fucose (Fuc), whereas those with QPD (Glu-Pro-Asp) tripeptide motifs bind galactose (Gal) and N-acetylgalactosamine (GalNAc). We report here for the first time a direct comparison of monosaccharide (and some di- and trisaccharides)-binding characteristics of 11 EPX-containing (X = N, S or D) immune-related CTLs using a competition assay and an enzyme-linked immunosorbent assay, and neoglycoproteins as ligand. The EPX CTLs studied are DC-SIGN, L-SIGN, mSIGNR1, human and mouse mannose receptors, Langerin, BDCA-2, DCIR, dectin-2, MCL and MINCLE. We found that: (1) they all bound Man and Fuc; (2) binding of Glc and GlcNAc varied considerably among these lectins, but was always less than Man and Fuc; (3) in general, Gal and GalNAc were not bound. However, dectin-2, DCIR and MINCLE showed ability to bind Gal/GalNAc; (4) DC-SIGN, L-SIGN, mSIGNR1 and Langerin showed enhanced binding of Manα2Man over Man, whereas all others showed no enhancement; (5) DC-SIGN bound Le(x) trisaccharide structure, which has terminal Gal and Fuc residues, more avidly than Fuc, whereas L-SIGN, mSIGNR1, DCIR and MINCLE bound Le(x) less avidly than Fuc. BDCA-2, dectin-2, Langerin, MCL and mannose receptor did not bind Le(x) at all.
Assuntos
Fucose/metabolismo , Lectinas Tipo C/metabolismo , Manose/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Motivos de Aminoácidos , Animais , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Oligossacarídeos/metabolismoRESUMO
Three perglycosylated nordihydroguaiaretic acids (NDGA) were synthesized through the Huiseng 1,3-dipolar cycloaddition reaction. These sugar-NDGA conjugates containing triazole-linkages possessed good solubility in water. NDGA-(triazol-galactose)(4) (12b) and NDGA-(triazol-glucose)(4) (12c) were found to act as inhibitors against human hepatocellular carcinoma Hep3B cells in culture.
Assuntos
Antineoplásicos/química , Galactosídeos/química , Glucosídeos/química , Masoprocol/análogos & derivados , Masoprocol/química , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Galactosídeos/síntese química , Galactosídeos/uso terapêutico , Glucosídeos/síntese química , Glucosídeos/uso terapêutico , Glicosilação , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Masoprocol/síntese química , Masoprocol/uso terapêutico , Triazóis/síntese química , Triazóis/química , Triazóis/uso terapêuticoRESUMO
New multi-valent, carbohydrate ligands that contain terminal N-acetylgalactosamine (GalNAc) or lactose (Lac) were prepared using a nitrilotriacetic acid (NTA) derivative of L-lysine as scaffold. Tri-valent structures were prepared by attaching an ω-amino glycoside of GalNAc or Lac to each of the three carboxyl groups of N(ε)-protected N(α)-dicarboxymethyl-L-lysine. In addition, a hexa-valent lactoside was synthesized by attaching N(ε)-deprotected trivalent lactoside to each of the carboxyl group of N(α)-(trifluoroacetamido)hexanoyl L-aspartic acid. Tri-valent GalNAc glycosides and the hexa-valent lactoside had high affinity (dissociation constants approaching nM) for rat hepatocytes. The hexa-valent lactoside, after de-N(ε)-protection, was modified with a chelator, diethylenetriaminepentaacetic acid (DTPA), through which a fluorescent or radioactive tag, such as europium or indium, can be firmly attached. Intravenous infusion of (111)Indium-tagged hexa-valent lactoside to rats and mice resulted in nearly exclusive accumulation of radioactivity in the liver.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Glicosídeos/metabolismo , Hepatócitos/química , Acetilgalactosamina , Animais , Células Cultivadas , Glicosídeos/administração & dosagem , Glicosídeos/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Lactose , Ligantes , Fígado/metabolismo , Camundongos , Ligação Proteica , RatosRESUMO
We previously showed that the expression of (Gal alpha 1-4Gal)-bearing glycoproteins among birds is related to their phylogeny. However, precise structures of (Gal alpha 1-4Gal)-containing N-glycans were only known for pigeon egg white glycoproteins and IgG. To compare structural features of (Gal alpha 1-4Gal)-containing N-glycans from other species, we analyzed N-glycans of gull egg white (GEW)-glycoproteins, ovomucoid, and ovotransferrin, and gull egg yolk IgG by HPLC, mass spectrometry (MS), and MS/MS analyses. GEW-glycoproteins included neutral, monosialyl, and disialyl N-glycans, and some of them contained Gal alpha 1-4Gal sequences. Bi-, tri-, and tetra-antennary oligosaccharides that lacked bisecting GlcNAc were the major core structures, and incomplete alpha-galactosylation and sialylation as well as the presence of diLacNAc on the branches generated microheterogeneity of the N-glycan structures. Moreover, unlike pigeon egg white glycoproteins, the major sialylation in GEW-glycoproteins is alpha2,3-, but not alpha2,6-linked sialic acids (NeuAc). In addition to the complex-type oligosaccharide, hybrid-type oligosaccharides that lack bisecting GlcNAc were also abundant in GEW-glycoproteins. Gull egg yolk IgG also contained Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequences, but unlike pigeon IgG, no Gal alpha 1-4Gal beta 1-4Gal beta 1-4GlcNAc beta 1- sequence was detected. Bi- and tri-antennary complex-type oligosaccharides with bisecting GlcNAc and with core fucosylation as well as high-mannose-type oligosaccharides were the major structures in gull IgG. Our data indicated that some N-glycans from both GEW-glycoproteins and gull IgG contain the Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequence, but the ratio of alpha-Gal-capped residues to non-alpha-Gal-capped residues in the nonreducing termini of N-glycans is much lower than that in those of pigeon glycoproteins.
Assuntos
Charadriiformes , Clara de Ovo/química , Gema de Ovo/química , Glicoproteínas/química , Imunoglobulina G/química , Polissacarídeos/análise , Polissacarídeos/química , Animais , Glicoproteínas/isolamento & purificação , Espectrometria de Massas , Especificidade da Espécie , Fatores de TempoRESUMO
Existing HPLC methods can provide detailed structure and isomeric information, but are often slow and require large initial sample sizes. In this study, a previously established two-dimensional HPLC technique was adapted to a two-step identification method for smaller sample sizes. After cleavage from proteins, purification, and fluorescent labeling, glycans were analyzed on a 2-mm reverse phase HPLC column on a conventional HPLC and spotted onto a MALDI-TOF MS plate using an automated plate spotter to determine molecular weights. A direct correlation was found for 25 neutral oligosaccharides between the 2-mm Shim-Pack VP-ODS HPLC column (Shimadzu) and the 6-mm CLC-ODS column (Shimadzu) of the standard two- and three-dimensional methods. The increased throughput adaptations allowed a 100-fold reduction in required amounts of starting protein. The entire process can be carried out in 2-3 days for a large number of samples as compared to 1-2 weeks per sample for previous two-dimensional HPLC methods. The modified method was verified by identifying N-glycan structures, including specifying two different galactosylated positional isomers, of an IgG antibody from human sera samples. Analysis of tissue plasminogen activator (t-PA) from CHO cell cultures under varying culture conditions illustrated how the method can identify changes in oligosaccharide structure in the presence of different media environments. Raising glutamine concentrations or adding ammonia directly to the culture led to decreased galactosylation, while substituting GlutaMAX-I, a dipeptide of L-alanine and L-glutamine, resulted in structures with more galactosylation. This modified system will enable glycoprofiling of smaller glycoprotein samples in a shorter time period and allow a more rapid evaluation of the effects of culture conditions on expressed protein glycosylation.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ácidos , Animais , Células CHO , Sequência de Carboidratos , Cricetinae , Cricetulus , Meios de Cultura/farmacologia , Glicoproteínas/metabolismo , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Imunoglobulina G/análise , Indicadores e Reagentes/química , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Dados de Sequência Molecular , Polissacarídeos/química , Tamanho da Amostra , Silanos/química , Coloração e RotulagemRESUMO
PA-IL is a galactophilic lectin that is found on the outer membrane of Pseudomonas aeruginosa. Pigeon ovalbumin (POA), a phosphoprotein, contains high levels of terminal Gal alpha(1-->4)Gal units. Thus, magnetic nanoparticles with immobilized POA can be used as affinity probes for P. aeruginosa, functioning via the recognition of galactophilic PA-IL. We fabricated POA-bound nanoparticles (NPs) by immobilizing POA onto the surface of core/shell magnetic iron oxide/alumina NPs via metal-phosphate chelation. We then used the generated NPs to probe target bacteria from complex samples. The trapped bacterial cells were characterized based on their mass peak profiles obtained from MALDI MS analyses. In addition, we confirmed the determination of P. aeruginosa using a proteomic strategy: combining the resultant MALDI MS/MS spectra of its tryptic digest with protein database searching. The feasibility of using this approach to rapidly characterize P. aeruginosa from clinical samples without the need to perform culturing steps was also demonstrated.
Assuntos
Marcadores de Afinidade/química , Marcadores de Afinidade/metabolismo , Magnetismo , Nanopartículas/química , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Micro-Ondas , Dados de Sequência Molecular , Ovalbumina/química , Ovalbumina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
Lectins are carbohydrate-binding proteins with many biological functions including cellular recognition and innate immunity. In this study, a major l-fucose-binding lectin from the serum of Nile tilapia (Oreochromis niloticus L.), designated as TFBP, was isolated by l-fucose-BSA Sepharose CL6B affinity chromatography. The SDS-PAGE (10%) analysis of TFBP revealed a major band of approximately 23 kDa with an N-terminal amino acid sequence of DQTETAGQQSXPQDIHAVLREL which did not give significant similarities to the protein databases using BLASTp searches. Ruthenium red staining indicate positive calcium-binding property of TFBP. The purified TFBP agglutinated human type O erythrocytes but not the type A and B fresh erythrocytes. Live Aeromonas hydrophila and Enterococcus faecalis cells were also agglutinated by the lectin. The fucose-binding proteins were detected in the soluble protein extracts from the gills, gut, head kidneys, liver, serum and spleen using a fucose-binding protein probe (l-fucose-BSA-horseradish peroxidase). The binding of TFBP with the l-fucose-BSA probe was inhibited by l-fucose but not by alpha-methyl-d-mannose.
Assuntos
Ciclídeos/genética , Lectinas/isolamento & purificação , Lectinas/metabolismo , Testes de Aglutinação , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Cálcio/metabolismo , Ciclídeos/metabolismo , Eritrócitos/metabolismo , Fucose/metabolismo , Humanos , Lectinas/química , Dados de Sequência Molecular , Peso MolecularRESUMO
Escherichia coli and Staphylococcus saprophyticus are the most common causes of urinary tract infections, with 80% of these infections caused by uropathogenic E. coli. Because the P fimbriae of E. coli have specificity toward Gal(alpha1-4)Gal beta units, pigeon ovalbumin (POA), whose structure contains terminal Gal(alpha1-4)Gal beta moieties, was used as a probe for interaction with P fimbriated E. coli. The functional affinity probes for these bacteria by immobilizing POA--a phosphoprotein--onto the surface of magnetic iron oxide nanoparticles (NPs) coated with alumina (Fe3O4@Al2O3), using the phosphate units of POA as linking groups for the formation of phosphate-alumina complexes. The immobilization process occurred within 30 s when performing the reaction under microwave heating. The magnetic POA-Fe3O4@Al2O3 NPs generated using this facile approach exhibited specificity toward P fimbriated E. coli. The bacteria targeted by the affinity probes were characterized by matrix-assisted laser desorption/ionization mass spectrometry. The detection limit toward uropathogenic bacteria when using this approach was approximately 9.60 x 10(4) cfu/mL (0.5 mL).
Assuntos
Óxido de Alumínio/química , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Compostos Férricos/química , Magnetismo , Nanopartículas Metálicas/química , Ovalbumina/química , Animais , Columbidae , Escherichia coli/ultraestrutura , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus/isolamento & purificação , Staphylococcus/patogenicidadeRESUMO
Galalpha1-4Gal is typically found in mammalian glycolipids in small quantities, and recognized by some pathogens, such as uropathogenic Escherichia coli. In contrast, glycoproteins containing Galalpha1-4Gal were rarely found in vertebrates except in a few species of birds and amphibians until recently. However, we had previously reported that pigeon (Columba livia) egg white and serum glycoproteins are rich in N-glycans with Galalpha1-4Gal at non-reducing termini. Our investigation with egg white glycoproteins from 181 avian species also revealed that the distribution of (Galalpha1-4Gal)-containing glycoproteins was not rare among avians, and is correlated with the phylogeny of birds. The differentiated expression was most likely emerged at earlier stage of diversification of modern birds, but some birds might have lost the facility for the expression relatively recently.
Assuntos
Aves/genética , Dissacarídeos/análise , Glicoproteínas/química , Animais , Dissacarídeos/genética , Filogenia , Especificidade da EspécieRESUMO
Insects, yeasts and plants generate widely different N-glycans, the structures of which differ significantly from those produced by mammals. The processing of the initial Glc2Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 in the endoplasmic reticulum shows significant similarities among these species and with mammals, whereas very different processing events occur in the Golgi compartments. For example, yeasts can add 50 or even more Man residues to Man(8-9)GlcNAc2, whereas insect cells typically remove most or all Man residues to generate paucimannosidic Man(3-1)GlcNAc2N-glycans. Plant cells also remove Man residues to yield Man(4-5)GlcNAc2, with occasional complex GlcNAc or Gal modifications, but often add potentially allergenic beta(1,2)-linked Xyl and, together with insect cells, core alpha(1,3)-linked Fuc residues. However, genomic efforts, such as expression of exogenous glycosyltransferases, have revealed more complex processing capabilities in these hosts that are not usually observed in native cell lines. In addition, metabolic engineering efforts undertaken to modify insect, yeast and plant N-glycan processing pathways have yielded sialylated complex-type N-glycans in insect cells, and galactosylated N-glycans in yeasts and plants, indicating that cell lines can be engineered to produce mammalian-like glycoproteins of potential therapeutic value.
Assuntos
Polissacarídeos/biossíntese , Animais , Catálise , Humanos , Insetos/metabolismo , Plantas/metabolismo , Leveduras/metabolismoRESUMO
There is an urgent need for discovery of novel antimicrobials and carbohydrate-based anti-adhesive strategies are desirable as they may not promote resistance. Discovery of novel anti-adhesive molecules from natural product libraries will require the use of a high throughput screening platform. Avian egg white (EW) provides nutrition for the embryo and protects against infection, with glycosylation responsible for binding certain pathogens. In this study, a microarray platform of 78 species of avian EWs was developed and profiled for glycosylation using a lectin panel with a wide range of carbohydrate specificities. The dominating linkages of sialic acid in EWs were determined for the first time using the lectins MAA and SNA-I. EW glycosylation similarity among the different orders of birds did not strictly depend on phylogenetic relationship. The interactions of five strains of bacterial pathogens, including Escherichia coli, Staphylococcus aureus and Vibrio cholera, identified a number of EWs as potential anti-adhesives, with some as strain- or species-specific. Of the two bacterial toxins examined, shiga-like toxin 1 subunit B bound to ten EWs with similar glycosylation more intensely than pigeon EW. This study provides a unique platform for high throughput screening of natural products for specific glycosylation and pathogen interactions. This platform may provide a useful platform in the future for discovery of anti-adhesives targeted for strain and species specificity.
Assuntos
Clara de Ovo , Microbiologia de Alimentos , Glicoproteínas/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Interações Hospedeiro-Patógeno/fisiologia , Aglutininas/química , Aglutininas/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Aves , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/análise , Glicosilação , Maackia/química , Filogenia , Análise Serial de Proteínas/métodos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismoRESUMO
The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26-Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Conexinas/metabolismo , ortoaminobenzoatos/farmacologia , Animais , Conexina 26 , Corantes Fluorescentes/química , Glicoconjugados/farmacologia , Hepatócitos/metabolismo , Humanos , Glândulas Mamárias Humanas/metabolismo , ortoaminobenzoatos/análise , Proteína beta-1 de Junções ComunicantesRESUMO
[structure: see text] A new type of glycocluster based on polyhedral oligosilsesquioxanes (POSS) has been efficiently prepared from unprotected mannoside and lactoside employing a convergent approach of thiol-radical addition reaction. The versatility of this approach was demonstrated by functionalization of mannosides and lactosides of different-length spacers.
Assuntos
Glicosídeos/química , Manosídeos/química , Oligossacarídeos/síntese química , Compostos de Organossilício/química , Técnicas de Química Combinatória , Glicoproteínas/efeitos dos fármacos , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/química , Oligossacarídeos/farmacologiaRESUMO
N-Glycans in glycoprotein can be liberated either from glycoproteins or from their glycopeptides with glycoamidases. The latter approach is preferable, because it requires a smaller amount of the enzyme, and yields N-glycans in excellent yields. Moreover it alleviates the necessity of removing from the reaction mixture the detergents needed to denature the glycoproteins. On the other hand, this approach necessitates removal of interfering peptidic materials, because some of the peptide peaks often overlap with the peaks of carbohydrate chains in high-performance anion-exchange chromatography (HPAEC). These peptidic materials also hinder labeling of N-glycans by reductive amination. We have tried to remove the interfering peptidic materials by several different methods--octadecyl (C18) silica cartridge, cation-exchange resin column, and graphitized carbon cartridge. Unfortunately, none of these could completely remove the interfering peptidic materials. Therefore, we resorted to modify the amino groups of the peptidic materials with sodium 2,4,6-trinitro-benzene-1-sulfonate (TNBS) to render them more hydrophobic, so that they can be retained more strongly on the C18 or graphitized carbon cartridges. In the model study presented here, we were able to obtain N-glycans for HPAEC analyses without any interfering materials by a combination of TNBS reaction and graphitized carbon treatment.
Assuntos
Glicopeptídeos/química , Polissacarídeos/análise , Resinas de Troca de Cátion , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Polissacarídeos/isolamento & purificação , Reprodutibilidade dos TestesRESUMO
To facilitate the use of oligosaccharides as analytical tools in biological studies, we have designed, synthesized, and conjugated to maltosaccharides a novel series of homologous small fluorescent moieties that differ in formal charge. These moieties are amide derivatives of anthranilic acid: uncharged N-(2-aminobenzoyl)glycinamide (ABGlyAmide; 2), acidic N,N-dimethyl-N(')-(2-aminobenzoyl)ethylenediamine (ABGlyDIMED; 3), and basic N-(2-aminobenzoyl)glycine (ABGly; 1). Routes for synthesis and optimal reaction conditions for glycoconjugation by conventional reductive amination are presented, as is the compatibility of these adducts with common analytical and preparative chromatographic methods, including RP-HPLC and HPAEC-PAD. These novel anthranilic acid derivatives confer both fluorescence and defined charge to oligosaccharides, and so enhance the repertoire of chromatographic and analytical methods for which anthranilic acid can be used. Furthermore, because glucosaccharides have rigid solution structure, these small fluorescent adducts with different formal charge are ideal tools for molecular sizing studies of membrane pores.
Assuntos
Oligossacarídeos/química , ortoaminobenzoatos/química , Ácidos/química , Adsorção , Aminas/química , Configuração de Carboidratos , Concentração de Íons de Hidrogênio , Íons/química , Estrutura Molecular , Oligossacarídeos/isolamento & purificação , Oxirredução , Soluções/química , Análise Espectral , Eletricidade Estática , ortoaminobenzoatos/síntese químicaRESUMO
We propose that a C-type lectin receptor, SIGNR-1 (also called Cd209b), helps to condition dendritic cells (DCs) in the gastrointestinal lamina propria (LPDCs) for the induction of oral tolerance in a model of food-induced anaphylaxis. Oral delivery of BSA bearing 51 molecules of mannoside (Man(51)-BSA) substantially reduced the BSA-induced anaphylactic response. Man(51)-BSA selectively targeted LPDCs that expressed SIGNR1 and induced the expression of interleukin-10 (IL-10), but not IL-6 or IL-12 p70. We found the same effects in IL-10-GFP knock-in (tiger) mice treated with Man(51)-BSA. The Man(51)-BSA-SIGNR1 axis in LPDCs, both in vitro and in vivo, promoted the generation of CD4(+) type 1 regulatory T (Tr1)-like cells that expressed IL-10 and interferon-γ (IFN-γ), in a SIGNR-1- and IL-10-dependent manner, but not of CD4(+)CD25(+)Foxp3(+) regulatory T cells. The Tr1-like cells could transfer tolerance. These results suggest that sugar-modified antigens might be used to induce oral tolerance by targeting SIGNR1 and LPDCs.