RESUMO
BACKGROUND: Pseudomonas aeruginosa intestinal carriage rates are significantly higher in immunosuppressed individuals and hospitalized patients who therefore have increased risk of infections and antibiotic-associated diarrhea. To combat intestinal dysbiosis and decolonize P. aeruginosa from gastrointestinal tract, we investigated the anti-adherence and gut microbiota modulation properties of marine prebiotic fucoidans. METHODS: Proteomic analysis of culture supernatant was performed by LC-MS/MS. Using lectin-based enzyme-linked immunosorbent assay, hemagglutinin domain interaction and inhibition with biomolecules were studied. We investigated the role of nutritional grade fucoidans in a mouse model and used 16S ribosomal RNA sequencing to examine fecal microbiota composition. RESULTS: Analysis of culture supernatant proteins indicated the secretion of two-partner secretion (TPS) family proteins, including TpsA1/CdiA2 and TpsA2/CdiA1. Lectin like activity at the N-terminal of TpsA due to a conserved hemagglutinin domain (Pfam identifier [ID] PF05860) mediates binding to mucins that carry multiple fucosylated glycans. Fucose-rich sulfated polysaccharides (fucoidans) and sulfated dextrans were found to be potent inhibitors of the recombinant N-terminal hemagglutinin domain of TpsA (TpsA-NT-HAD) binding to mucins. In a mouse model, antibiotic-induced dysbiosis was essential for P. aeruginosa gastrointestinal colonization. After prophylactic oral fucoidans supplementation, a higher proportion (60%) of the mice were decolonized over time and resisted re-colonization, this was associated with remarkable expansion of Bacteroides (post-infection day-3 abundance, 29-50%) and consequential reductions in bloom of Enterobacteriaceae and Enterococcaceae populations. In the non-supplemented group, Parabacteroides mediated recovery from dysbiosis but failed to decolonize P. aeruginosa. CONCLUSIONS: Supplementing diet with marine prebiotic fucoidans can mediate earlier recovery from dysbiosis and decolonization of P. aeruginosa from gut by inhibiting secreted virulence factor (TpsA/CdiA) interaction with mucins and promoting the growth of beneficial Bacteroides population. We suggest the prophylactic use of nutritional grade fucoidans to decolonize P. aeruginosa from gastrointestinal tract of at-risk individuals to prevent infection and transmission of colonizing P. aeruginosa.
Assuntos
Prebióticos , Pseudomonas aeruginosa , Camundongos , Animais , Mucinas , Disbiose , Bacteroides , Hemaglutininas , Cromatografia Líquida , Proteômica , Espectrometria de Massas em Tandem , Antibacterianos/farmacologia , Polissacarídeos , Modelos Animais de Doenças , LectinasRESUMO
BACKGROUND: Salmonella enterica serotype Typhimurium is a nontyphoidal and common foodborne pathogen that causes serious threat to humans. There is no licensed vaccine to prevent the nontyphoid bacterial infection caused by S. Typhimurium. METHODS: To develop conjugate vaccines, the bacterial lipid-A free lipopolysaccharide (LFPS) is prepared as the immunogen and used to synthesize the LFPS-linker-protein conjugates 6a-9b. The designed bifunctional linkers 1-5 comprising either an o-phenylenediamine or amine moiety are specifically attached to the exposed 3-deoxy-D-manno-octulosonic acid (Kdo), an α-ketoacid saccharide of LFPS, via condensation reaction or decarboxylative amidation. In addition to bovine serum albumin and ovalbumin, the S. Typhimurium flagellin (FliC) is also used as a self-adjuvanting protein carrier. RESULTS: The synthesized conjugate vaccines are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC), and their contents of polysaccharides and protein are determined by phenol-sulfuric acid assay and bicinchoninic acid assay, respectively. Enzyme-linked immunosorbent assay (ELISA) shows that immunization of mouse with the LFPS-linker-protein vaccines at a dosage of 2.5 µg is sufficient to elicit serum immunoglobulin G (IgG) specific to S. Typhimurium lipopolysaccharide (LPS). The straight-chain amide linkers in conjugates 7a-9b do not interfere with the desired immune response. Vaccines 7a and 7b derived from either unfractionated LFPS or the high-mass portion show equal efficacy in induction of IgG antibodies. The challenge experiments are performed by oral gavage of S. Typhimurium pathogen, and vaccine 7c having FliC as the self-adjuvanting protein carrier exhibits a high vaccine efficacy of 74% with 80% mice survival rate at day 28 post the pathogen challenge. CONCLUSIONS: This study demonstrates that lipid-A free lipopolysaccharide prepared from Gram-negative bacteria is an appropriate immunogen, in which the exposed Kdo is connected to bifunctional linkers to form conjugate vaccines. The decarboxylative amidation of Kdo is a novel and useful method to construct a relatively robust and low immunogenic straight-chain amide linkage. The vaccine efficacy is enhanced by using bacterial flagellin as the self-adjuvanting carrier protein.
Assuntos
Lipopolissacarídeos/química , Vacinas contra Salmonella/química , Vacinas Conjugadas/química , Animais , Lipídeo A , Camundongos , Camundongos Endogâmicos BALB C , Vacinas contra Salmonella/imunologia , Vacinas Conjugadas/imunologiaRESUMO
More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Ramnose/metabolismo , Células A549 , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Escherichia coli/metabolismo , Glicolipídeos/metabolismo , Caranguejos Ferradura/metabolismo , Humanos , Lectinas/metabolismo , Polissacarídeos Bacterianos/metabolismo , Percepção de Quorum/efeitos dos fármacos , Peixe-ZebraRESUMO
Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore, in silico computer modeling and in vitro binding assays suggest critical roles for the following basic amino acids located within heparin binding regions (HBRs) of EDN 34QRRCKN39 (HBR1), 65NKTRKN70 (HBR2), and 113NRDQRRD119 (HBR3) and in particular Arg35, Arg36, and Arg38 within HBR1, and Arg114 and Arg117 within HBR3. Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN.
Assuntos
Aminoácidos Básicos/metabolismo , Neurotoxina Derivada de Eosinófilo/metabolismo , Glicosaminoglicanos/metabolismo , Sequência de Aminoácidos , Aminoácidos Básicos/química , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Neurotoxina Derivada de Eosinófilo/química , Neurotoxina Derivada de Eosinófilo/genética , Eosinófilos/metabolismo , Heparina/metabolismo , Humanos , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
A large set of glycome information was obtained from egg white proteins of 88 samples from Galloanserae (63 Anseriformes and 25 Galliformes). The data were obtained on whole N-glycan structures and types of sialic acids of these egg whites by glycoblotting-based high-throughput and quantitative glycomics. The results revealed clear trends and complexity patterns as well as diversity among taxonomic groups. It is well-known that chicken, a representative domesticated poultry involved in Galliformes, can become an influenza host. However, our data demonstrate that duck, wild goose, and swan of Anseriformes are representative migratory birds that are known as natural hosts of the influenza virus. Hierarchical clustering analysis of the expression pattern of N-glycome (total of 61 N-glycan peaks) revealed that the members of Galloanserae can be classified into two major groups and five submajor clusters (clusters 1-5) on the basis of simple m/z values obtained by MALDI-TOF MS. It is clear that expression patterns of N-glycomes in the five clusters are influenced significantly by the features such as the body size of the birds, rather than by the difference of the family. On the other hand, quantitative analysis showed that the total amounts of sialic acids in egg whites of Galliformes were distinctly larger than those of Anseriformes. However, it was also revealed in Anseriformes that Neu5Gc and KDN, in addition to common Neu5Ac, were expressed significantly in both N- and O-glycans of glycoproteins and glycosphingolipids, suggesting the influence of their lifestyles and diet. This is the first report that KDN exists in egg white. These results and the environmental factors are discussed preliminarily with respect to their evolutionary lineage.
Assuntos
Evolução Biológica , Aves , Clara de Ovo/química , Ácido N-Acetilneuramínico/química , Polissacarídeos/química , Animais , Cromatografia Líquida de Alta Pressão , Técnicas In VitroRESUMO
This study demonstrates the utilization of a flow chemistry system for continuous glycan hydrolysis and saccharide labeling to assist with the existing methods in glycan structural analysis. Acidic hydrolysis of glycans could be accelerated in a flow system. Aldoses and α-ketoacid-type saccharides were effectively labeled with naphthalene-2,3-diamine (NADA) at 60 °C for 10 min to form the fluorescent naphthimidazole (NAIM) and quinoxalinone (QXO) derivatives, respectively. The NADA-labeled derivatives improved the structural determination and composition analysis for their parent saccharides by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), liquid chromatography mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). Furthermore, this protocol was applied to determine the SA-Gal-Glc sequence of GM3-sugar out of six possible permutations.
Assuntos
Glicômica/métodos , Polissacarídeos/metabolismo , Reologia , Coloração e Rotulagem , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , 2-Naftilamina/metabolismo , Glucose/química , Glucose/metabolismo , Hidrólise , Imidazóis/química , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/químicaRESUMO
For a detailed examination of the interaction of rhamnose containing derivatives with recombinant horseshoe crab plasma lectin (rHPL), two di-rhamno-di-lipids (an α-1,2- and an α-1,3-linked) were synthesized via a new simple method. The N-iodosuccinimide/triflic acid mediated glycosylation of the methyl (R)-3-hydroxydecanoate with phenyl-1-thio-rhamnobioside donors afforded the mono-lipid disaccharides. Removal of the methyl ester group followed by esterification of the mono-lipids with a second (R)-3-hydroxydecanoate unit resulted in fully protected di-lipid derivatives, transformation of which into the target compounds was accomplished in two steps. This method allows the synthesis of both regioisomers in only 6 steps starting from the corresponding free disaccharides. Both synthetic di-rhamnolipids were biologically active for lectin binding differential binding preference between two isomeric di-rhamno-di-lipids. The rHPL lectin favours the α-1,3-linked di-rhamno-di-lipids over its α-1,2-linked regioisomer.
Assuntos
Glicolipídeos/química , Glicolipídeos/síntese química , Técnicas de Química Sintética , Ésteres/química , Glicosilação , EstereoisomerismoRESUMO
The glycan part of glycoproteins is known to be involved in the structure and modulatory functions of glycoproteins, serving as ligands for cell-to-cell interactions, and as specific ligands for cell-to-microbe interactions. It is believed that intraspecies and interspecies variations in glycosylation exist. As an approach to better understand glycan diversity, egg whites (EW) from four different quail species are studied by the well-established glycoblotting procedure, a glycan enrichment and analysis method. N-Glycans were classified and the profiles were established for quail egg white samples which showed 21 relevant glycan peaks; 18 peaks were expressed significantly, and 10 glycan peaks are found to be abundant in certain species. The result establishes glycan profiles for Blue Scaled, Bobwhite, Japanese, and Mountain Quail egg whites and shows a unique difference among glycan expressions, particularly, high mannose in Japanese Quail and tetra-antennary glycan structure for other quail species.
Assuntos
Proteínas do Ovo/análise , Clara de Ovo/química , Glicoproteínas/análise , Polissacarídeos/análise , Codorniz/genética , Animais , Western Blotting/métodos , Proteínas do Ovo/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Polissacarídeos/metabolismo , Codorniz/classificação , Codorniz/metabolismoRESUMO
PURPOSE: Multidrug resistance (MDR) continues to be a major obstacle for successful anticancer therapy. One of the principal factors implicated in MDR is the over expression of P-glycoprotein (Pgp), the product of the MDR1 gene. METHODS: Here we explore the possibility of using the transcription inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) to inhibit Sp1-regulated MDR1 gene expression and restore doxorubicin and paclitaxel sensitivity to multidrug resistant human cancer cells in vitro and in vivo. RESULTS: We found that M4N acted synergistically with doxorubicin and paclitaxel in inhibiting the growth of the cells in culture allowing significant dose reductions of both drugs. We observed no such synergism when M4N was used in combination with cisplatin, another chemotherapeutic agent, but not a Pgp substrate, as analyzed by the combination index and isobologram methods. Analysis of MDR1 mRNA and Pgp levels revealed that at sublethal doses, M4N inhibited MDR1 gene expression in the multidrug resistant NCI/ADR-RES cells and reversed the MDR phenotype as measured by Rhodamine-123 retention. In addition, M4N was found to inhibit doxorubicin-induced MDR1 gene expression in drug sensitive MCF-7 breast cancer cells. CONCLUSIONS: M4N and maltose-tri-O-methyl nordihydroguaiaretic acid (maltose-M3N), a water-soluble derivative of NDGA, were also able to reverse the MDR phenotype of the tumor cells in a xenograft model system and combination therapy with M4N or maltose-M3N and paclitaxel was effective at inhibiting growth of these tumors in nude mice.
Assuntos
Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Masoprocol/análogos & derivados , Masoprocol/farmacologia , Monossacarídeos/farmacologia , Paclitaxel/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Masoprocol/administração & dosagem , Camundongos , Camundongos Nus , Monossacarídeos/administração & dosagem , Paclitaxel/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
To evaluate the molecular interaction of recombinant horseshoe crab plasma lectin (rHPL) with Pseudomonas aeruginosa PAO1, multivalent rhamnobioside derivatives were designed. Eight rhamnoclusters with three or four α(1-3)-rhamnobiosides attached to different central cores, such as methyl gallate, pentaerythritol, and N-Boc Tris, through either an ethylene glycol or a tetraethylene glycol linker, were assembled in two consecutive azide-alkyne cycloaddition click reactions. The synthetic method embraced the preparation of two α(1-3)-rhamnobiosides with different linker arms and their conjugation, in stoichiometric or substoichiometric amounts, to propargyl ether-functionalized tri- or tetravalent scaffolds. A divalent derivative and two self-assembling rhamnobiosides were also prepared. The different architectures and valences of the rhamnoclusters provided an opportunity to evaluate the impact of topology and valency on the binding properties toward rHPL. Inhibitory ELISA data showed that all covalently linked rhamnoclusters could inhibit P.â aeruginosa PAO1 recognition activity of rHPL with high efficacy. Trivalent rhamnobiosides showed a stronger inhibitory effect on P.â aeruginosa PAO1 binding, and the more flexible clusters on a pentaerythritol or a Tris core were superior to the less flexible methyl gallate-based clusters. Interestingly, the length of the linker arms had a very low impact on the binding ability of the rhamnoclusters. Herein, the two trivalent derivatives on an N-Boc protected Tris central core were the best inhibitors. The self-assembling amphiphilic rhamnobioside derivatives were found to display no multivalent effect.
Assuntos
Caranguejos Ferradura/metabolismo , Lectinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Alcinos/química , Animais , Azidas/química , Catálise , Química Click , Cobre/química , Difusão Dinâmica da Luz , Ácido Gálico/análogos & derivados , Ácido Gálico/química , Lectinas/química , Lectinas/genética , Ligação Proteica , Pseudomonas aeruginosa/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
An affinity labeling reagent, uridine 5'-(6-amino-{2-[(7-bromomethyl-2-naphthyl)methoxycarbonylmethoxy]ethoxy}acetyl-6-deoxy-alpha-D-galactopyranosyl) diphosphate (1a), was designed on the basis of 3D docking simulation and synthesized to investigate the functional role of Trp310 residue located in the small loop near the active site of human recombinant galactosyltransferase (betaGalT-1). Mass spectrometric analysis revealed that the Trp310 residue of betaGalT1 can be selectively modified with the naphthylmethyl group of compound 1a at the C-3 position of the indole ring. This result motivated us to synthesize novel uridine-5'-diphosphogalactose (UDP-Gal) analogues as candidates for mechanism-based inhibitors for betaGalT-1. We found that uridine 5'-(6-O-[10-(2-naphthyl)-3,6,9-trioxadecanyl]-alpha-d-galactopyranosyl) diphosphate (2) is the strongest inhibitor (K(i) = 1.86 microM) against UDP-Gal (Km = 4.91 microM) among compounds reported previously. A cold spray ionization time-of-flight mass spectrometry study demonstrated that the complex of this inhibitor and betaGalT-1 cannot interact with an acceptor substrate in the presence of Mn2+.
Assuntos
Marcadores de Afinidade/síntese química , Galactosiltransferases/antagonistas & inibidores , Galactosiltransferases/química , Açúcares de Uridina Difosfato/síntese química , Marcadores de Afinidade/química , Sítios de Ligação , Desenho de Fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triptofano/química , Açúcares de Uridina Difosfato/químicaRESUMO
To evaluate the ability of insect cells to produce complex-type N-glycans, beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity in several insect cell lines was analyzed. For this purpose, we developed a simple and highly sensitive assay for beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity, which is based on time-resolved fluorometry of europium. Bovine serum albumin (BSA) modified with GlcNAc (GlcNAc(44)-BSA) was used as the acceptor. GlcNAc(44)-BSA was coated on a 96-well microplate, and after incubation with the enzyme sample in the presence of UDP-Gal, Eu-labeled RCA(120) (Ricinus communis aggutin I), was added. RCA(120) binds to the Galbeta(1 --> 4)GlcNAc structure in the product, and the bound Eu-RCA(120) was measured by the fluorescence of europium. When bovine beta4Gal-T-I was used as a standard reference enzyme, a linear relationship between enzyme activity and fluorescent signal was obtained over the range of 0-1000 microUnits (IU). Using this system, we were able to measure a low but significant level of beta4GalT activity in Trichoplusia ni cells ('High Five'). In contrast, no endogenous beta4GalT activity was detected in a Spodoptera frugiperda (Sf-9) cell line. However, Sf-9 cells stably transfected with the bovine beta4GalT-I gene and 'High Five' cells infected with a baculovirus containing the same gene produced activity levels that were comparable to or greater than those found in Chinese hamster ovary cells. We also showed that the beta4GalT activity level observed in the baculovirus-infected T. ni cells under the control of immediate early promoter was highly dependent on the post-infection time, suggesting that galactosylation level may also be variable during the infection period.
Assuntos
Insetos/citologia , N-Acetil-Lactosamina Sintase/metabolismo , Animais , Células CHO , Bovinos , Linhagem Celular , Cricetinae , Európio , Fluorometria , Cinética , N-Acetil-Lactosamina Sintase/genética , Polissacarídeos/biossíntese , TransfecçãoRESUMO
Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.
Assuntos
Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/metabolismo , Lectinas/metabolismo , Ramnose/metabolismo , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Dicroísmo Circular , Caranguejos Ferradura/metabolismo , Klebsiella pneumoniae/metabolismo , Lectinas/química , Lectinas/genética , Lipopolissacarídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/metabolismoRESUMO
Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.
Assuntos
Proteína Catiônica de Eosinófilo/metabolismo , Heparina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteína Catiônica de Eosinófilo/química , Proteína Catiônica de Eosinófilo/genética , Eosinófilos/enzimologia , Expressão Gênica , Heparina/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , TermodinâmicaRESUMO
UNLABELLED: Signaling of the receptor for advanced glycation end products (RAGE) has been implicated in the development of injury-elicited vascular complications. Soluble RAGE (sRAGE) acts as a decoy of RAGE and has been used to treat pathological vascular conditions in animal models. However, previous studies used a high dose of sRAGE produced in insect Sf9 cells (sRAGE(Sf9))and multiple injections to achieve the therapeutic outcome. Here, we explore whether modulation of sRAGE N-glycoform impacts its bioactivity and augments its therapeutic efficacy. We first profiled carbohydrate components of sRAGE produced in Chinese hamster Ovary cells (sRAGE(CHO)) to show that a majority of its N-glycans belong to sialylated complex types that are not shared by sRAGE(Sf9). In cell-based NF-κB activation and vascular smooth muscle cell (VSMC) migration assays, sRAGE(CHO) exhibited a significantly higher bioactivity relative to sRAGE(Sf9) to inhibit RAGE alarmin ligand-induced NF-κB activation and VSMC migration. We next studied whether this N-glycoform-associated bioactivity of sRAGE(CHO) is translated to higher in vivo therapeutic efficacy in a rat carotid artery balloon injury model. Consistent with the observed higher bioactivity in cell assays, sRAGE(CHO) significantly reduced injury-induced neointimal growth and the expression of inflammatory markers in injured vasculature. Specifically, a single dose of 3 ng/g of sRAGE(CHO) reduced neointimal hyperplasia by over 70%, whereas the same dose of sRAGE(Sf9) showed no effect. The administered sRAGE(CHO) is rapidly and specifically recruited to the injured arterial locus, suggesting that early intervention of arterial injury with sRAGE(CHO) may offset an inflammatory circuit and reduce the ensuing tissue remodeling. Our findings showed that the N-glycoform of sRAGE is the key determinant underlying its bioactivity and thus is an important glycobioengineering target to develop a highly potent therapeutic sRAGE for future clinical applications. KEY MESSAGE: The specific N-glycoform modification is the key underlying sRAGE bioactivity Markedly reduced sRAGE dose to attenuate neointimal hyperplasia and inflammation Provide a molecular target for glycobioengineering of sRAGE as a therapeutic protein Blocking RAGE alarmin ligands during acute injury phase offsets neointimal growth.
Assuntos
Artrite/metabolismo , Artrite/patologia , Neointima/metabolismo , Receptores Imunológicos/metabolismo , Animais , Artrite/tratamento farmacológico , Biomarcadores/metabolismo , Células CHO , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Movimento Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Glicosilação , Humanos , Ligantes , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , NF-kappa B/metabolismo , Neointima/tratamento farmacológico , Ratos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Células Sf9RESUMO
The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.
Assuntos
Amilose/química , Proteínas Fúngicas/química , Rhizopus/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Calorimetria/métodos , Carboidratos/química , Escherichia coli/metabolismo , Glucana 1,4-alfa-Glucosidase/química , Hidrólise , Cinética , Ligantes , Microscopia de Força Atômica/métodos , Mutagênese Sítio-Dirigida/métodos , Polissacarídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Amido/químicaRESUMO
MALDI-MS provides a rapid and sensitive analysis of large biomolecules such as proteins and nucleic acids. However, oligo- and polysaccharides are less sensitive in MS analysis partly due to their neutral and hydrophilic nature to cause low ionization efficiency. In this study, four types of oligosaccharides including aldoses, aminoaldoses, alduronic acids and α-keto acids were modified by appropriate tags at the reducing termini to improve their ionization efficiency. Bradykinin (BK), a vasoactive nonapeptide (RPPGFSPFR), containing two arginine and two phenylalanine residues turned out to be an excellent MS signal enhancer for maltoheptaose, GlcNAc oligomers and oligogalacturonic acids. In the MALDI-TOF-MS analysis using 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix, the GalA4-BK and GalA5-BK conjugates prepared by reductive amination showed the detection limit at 0.1 fmol, i.e. â¼800-fold enhancement over the unmodified pentagalacturonic acids. The remarkable MS enhancement was attributable to the synergistic effect of the basic arginine residues for high proton affinity and the hydrophobic property phenylalanine residues for facile ionization. A tetrapeptide GFGR(OMe) and an arginine linked phenylenediamine (H(2) N)(2) Ph-R(OMe) were thus designed to act as potent tags of oligosaccharides in MS analysis. Interestingly, concurrent condensation and lactonization of α2,8-linked tetrasialic acid (SA4) was carried out with (H(2) N)(2) Ph-R(OMe) to obtain a quinoxalinone derivative, which showed > 200-fold enhancement over unmodified SA4 in the MALDI-TOF-MS analysis.
Assuntos
Oligossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Arginina/química , Bradicinina/química , Fenilenodiaminas/químicaRESUMO
To understand the roles of species-specific carbohydrates, systematic studies of interspecific glycan analyses are imperative. An extensive series of glycomics studies on approximately 180 kinds of bird eggs have demonstrated that 60-70% of the birds, which are closely related in phylogeny, express the alpha-Galp-(1-->4)-Galp structure on their egg glycoproteins. This prompted us to investigate the glycosylation profiles of eggs from an evolutionarily related organism, a sea turtle (reptilian). We performed N-glycosylation profiling of turtle egg yolk by using HPLC mapping in conjunction with mass spectrometric methods and thereby demonstrated that the alpha-Galp-(1-->4)-Galp groups are displayed on approximately 38% of total N-glycans. Our findings suggest that the ability to express the galabiose structure was acquired at an early stage of diversification in amniotes.
Assuntos
Dissacarídeos/análise , Gema de Ovo/química , Glicômica/métodos , Tartarugas/metabolismo , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Dissacarídeos/biossíntese , Glicosilação , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
So far, there have been only a few matrices reported for detection of polysaccharides with molecular weight higher than 3000 Daltons by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this work, we found that 2',4',6'-trihydroxyacetophenone (THAP) is a good matrix for MALDI time-of-flight MS analysis of polysaccharides with broad mass range. Large polysaccharides, dextrans, glycoproteins and polysialic acids have been successfully detected by MALDI-MS with THAP as matrix.