A pattern-recognition protein for beta-1,3-glucan. The binding domain and the cDNA cloning of beta-1,3-glucan recognition protein from the silkworm, Bombyx mori.
(49/1612)
The beta-1,3-glucan recognition protein (betaGRP) has strong specific affinity for beta-1,3-glucan, a component of the fungal cell wall. Its interaction with beta-1,3-glucan initiates the activation of the prophenoloxidase cascade, which is an important defense system in invertebrates of many species. We cloned the cDNA of the betaGRP of the silkworm Bombyx mori. The betaGRP mRNA transcript was constitutively expressed in the hemocytes, fat body, and epithelial cells of the naive silkworm. At the same time, a bacterial or yeast challenge was indicated to intensify the transcription. Comparison of the deduced amino acid sequence with known sequences revealed that the betaGRP contained a region (Thr(264) to Pro(386)) displaying significant similarity to the catalytic regions of bacterial beta-1,3-glucanases and much higher similarity to the glucanase-like regions of Gram-negative bacteria-binding proteins found in the silkworm B. mori and the mosquito Anopheles gambiae. The region (Thr(264) to Pro(386)) of the betaGRP, however, was demonstrated not to have appreciable affinity for beta-1,3-glucan. A recombinant peptide corresponding to an N-terminal region (Tyr(1) to Ala(102)) of the betaGRP bound strongly to beta-1,3-glucan. These results indicate that the binding domain of the betaGRP for beta-1,3-glucan is located in the N-terminal region. Glucanases and the current pattern-recognition proteins that contain a glucanase-like region seem to have a common origin in their molecular evolution. (+info)
Molecular modeling study of highly branching (1-->3)-alpha-D-glucan, a model polysaccharide for cariogenic glucan, using the N-H mapping method.
(50/1612)
A systematic search for possible regular helical structures of a highly branching (1-->3)-alpha-D-glucan was done using the n-h mapping technique, combined with MM3-generated relaxed-residue energy map calculations with respect to the conformations of the backbone glycosidic linkages. The alpha-D-glucan, consisting of a (1-->3)-alpha-linked backbone with alpha-D-glucose side residues attaching to an O6 atom of every second backbone residue, was considered as a model polysaccharide of a branching part of the glucan produced by oral bacteria, which was known to be related to dental plaque formation and to contribute to dental caries. The potential energy surfaces of the trisaccharide repeating unit of the branching alpha-D-glucan indicated that (1-->6)-alpha-linked side residues did not appear to interfere significantly with the backbone stereochemistry, probably due to a further separation of the three-bond-linked side residue compared with an ordinary two-bond-linked residue. Based on the n-h maps of the branching alpha-D-glucan, the side residues, when involved in a complete helix, mostly contributed additional stabilizations to particular helical structures. It was found by checking the typical helix models that formation of hydrogen bonds involving side residues was probably a major cause of the stabilization. This hydrogen bonding was expected to increase insolubility for the glucan chain--a typical, physical property observed for the bacterial alpha-D-glucan--by introducing its backbone stereochemistry as an additional stiff feature. (+info)
GDP-fucose uptake into the Golgi apparatus during xyloglucan biosynthesis requires the activity of a transporter-like protein other than the UDP-glucose transporter.
(51/1612)
The molecular mechanisms regulating hemicelluloses and pectin biosynthesis are poorly understood. An important question in this regard is how glycosyltransferases are oriented in the Golgi cisternae, and how nucleotide sugars are made available for the synthesis of the polymers. Here we show that the branching enzyme xyloglucan alpha,1-2 fucosyltransferase (XG-FucTase) from growing pea (Pisum sativum) epicotyls was latent and protected against proteolytic inactivation on intact, right-side-in pea stem Golgi vesicles. Moreover, much of the XG-FucTase activity was membrane associated. These data indicate that XG-FucTase is a membrane-bound luminal enzyme. GDP-Fuc uptake studies demonstrated that GDP-Fuc was taken up into Golgi vesicles in a protein-mediated process, and that this uptake was not competed by UDP-Glc, suggesting that a specific GDP-Fuc transporter is involved in xyloglucan biosynthesis. Once in the lumen, Fuc was transferred onto endogenous acceptors, including xyloglucan. GDPase activity was detected in the lumen of the vesicles, suggesting than the GDP produced upon transfer of Fuc was hydrolyzed to GMP and inorganic phosphate. We suggest than the GDP-Fuc transporter and GDPase may be regulators of xyloglucan fucosylation in the Golgi apparatus from pea epicotyls. (+info)
A beta1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade.
(52/1612)
Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. We report the purification and molecular cloning of a cDNA for a 53-kDa beta1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. This protein is constitutively expressed in fat body and secreted into hemolymph. The protein contains a region with sequence similarity to several glucanases, but it lacks glucanase activity. It binds to the surface of and agglutinates yeast, as well as gram-negative and gram-positive bacteria. Beta1,3-glucan-recognition protein in the presence of laminarin, a soluble glucan, stimulated activation of prophenoloxidase in plasma, whereas laminarin alone did not. These results suggest that beta1,3-glucan-recognition protein serves as a pattern recognition molecule for beta1,3-glucan on the surface of fungal cell walls. After binding to beta1,3-glucan, the protein may interact with a serine protease, leading to the activation of the prophenoloxidase cascade, a pathway in insects for defense against microbial infection. (+info)
Macrophage-induced lung inflammation contributes substantially to respiratory failure during Pneumocystis carinii pneumonia. We isolated a P. carinii cell wall fraction rich in glucan carbohydrate, which potently induces TNF-alpha and macrophage-inflammatory protein-2 generation from alveolar macrophages. Instillation of this purified P. carinii carbohydrate cell wall fraction into healthy rodents is accompanied by substantial increases in whole lung TNF-alpha generation and is associated with neutrophilic infiltration of the lungs. Digestion of the P. carinii cell wall isolate with zymolyase, a preparation containing predominantly beta-1,3 glucanase, substantially reduces the ability of this P. carinii cell wall fraction to activate alveolar macrophages, thus suggesting that beta-glucan components of the P. carinii cell wall largely mediate TNF-alpha release. Furthermore, the soluble carbohydrate beta-glucan receptor antagonists laminariheptaose and laminarin also substantially reduce the ability of the P. carinii cell wall isolate to stimulate macrophage-inflammatory activation. In contrast, soluble alpha-mannan, a preparation that antagonizes macrophage mannose receptors, had minimal effect on TNF-alpha release induced by the P. carinii cell wall fraction. P. carinii beta-glucan-induced TNF-alpha release from alveolar macrophages was also inhibited by both dexamethasone and pentoxifylline, two pharmacological agents with potential activity in controlling P. carinii-induced lung inflammation. These data demonstrate that P. carinii beta-glucan cell wall components can directly stimulate alveolar macrophages to release proinflammatory cytokines mainly through interaction with cognate beta-glucan receptors on the phagocyte. (+info)
Purification and partial characterization of a novel glucanhydrolase from Lipomyces starkeyi KSM 22 and its use for inhibition of insoluble glucan formation.
(54/1612)
A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single protein (100 kDa) showed either dextranolytic or amylolytic activity. We referred to the glucanhydrolase as a DXAMase. The DXAMase was produced in a starch medium and it was 3.75-fold more active for hydrolysis of the purified insoluble-glucan of Streptococcus mutans than Penicillium funiculosum dextranase. Aggregation of S. mutans cells with dextran and adherence to glass were eliminated by incubating with the DXAMase. The addition of DXAMase (0.1 IU/ml) to the mutansucrase reaction digest with sucrose reduced the formation of insoluble-glucan about 80%. Also the DXAMase (0.5 IU/ml) removed 80% of the pre-formed sucrose-dependent adherent film. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent. (+info)
Glycine prevents the phenotypic expression of streptococcal glucan-binding lectin.
(55/1612)
Glycine has been used extensively in bacterial cell surface research. Some researchers employ glycine in growth media so as to increase the transformability of streptococci during electroporation. Others have found that glycine, similar to wall antibiotics, 'weakens' peptidoglycan. It is now shown that when glycine is incorporated into the growth medium, Streptococcus sobrinus exhibits a diminished ability to aggregate with high molecular weight alpha-1,6-glucan. Growth of the bacteria in either a rich or a chemically defined medium results in a cell population with full lectin (glucan-binding) fidelity. Incorporation of glycine, but not serine or other amino acids, at concentrations of 100-200 mM gives rise to bacteria with lowered lectin activities. Bacteriolytic enzymes were able to lyse bacteria from glycine-grown cultures more readily than from cultures without the glycine supplement. The bacteria produce glucan-binding proteins, including glucosyltransferases, but they do not readily aggregate with added dextran. Furthermore, SDS-PAGE gels of supernatants of growth media (+/-glycine) are similar, suggesting the bacteria do not produce a unique set of proteins. Western blotting with a fluorescein isothiocyanate-labeled dextran probe reveals normal amounts of glucan-binding proteins in glycine-grown streptococci. Glycine may be acting as a type of antibiotic, reducing wall integrity upon which glucan promoted cellular aggregation depends. (+info)
Biochemical characterization and molecular cloning of an alpha-1,2-fucosyltransferase that catalyzes the last step of cell wall xyloglucan biosynthesis in pea.
(56/1612)
Pea microsomes contain an alpha-fucosyltransferase that incorporates fucose from GDP-fucose into xyloglucan, adding it preferentially to the 2-O-position of the galactosyl residue closest to the reducing end of the repeating subunit. This enzyme was solubilized with detergent and purified by affinity chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clone was isolated that encodes a 565-amino acid protein with a predicted molecular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. The purified transferase migrates at approximately 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight ( approximately 250 kDa), indicating that the active form is an oligomer. The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP. The enzyme has a neutral pH optimum and does not require divalent ions. Kinetic analysis indicates that GDP-fucose and xyloglucan associate with the enzyme in a random order. N-Ethylmaleimide, a cysteine-specific modifying reagent, had little effect on activity, although several other amino acid-modifying reagents strongly inhibited activity. (+info)