Short chain regioselectively hydrolyzed scleroglucans induce maturation of porcine dendritic cells.

Branched beta-1,3/1,6-glucans (scleroglucan) were produced by cultivation of Sclerotium rolfsii ATCC 15205. Regioselective hydrolysis at the beta-1,3-linkage of the cell-free and purified polysaccharide was performed in borosilicate glass bottles at pH 5, 121 degrees C, and 1 bar for 72 h. The mixture was divided into four molar mass fractions by stepwise cross-flow filtration using different cutoffs. In vitro studies revealed that scleroglucan hydrolysates with a low molar mass of less than 5 kDa significantly stimulated the activation and maturation of porcine monocyte derived dendritic cells (MoDC) by upregulation of CD40 and CD80/86 as well as by reduction of antigen uptake. MoDC treated with low molar mass scleroglucan showed a considerable increase in the amounts of secreted proinflammatory cytokine tumor necrosis factor alpha and stimulated the proliferation of lymphocytes. Therefore, scleroglucan molecules of low molecular weight are able to induce activation and maturation of porcine DC, which are key initiators of inflammatory and adaptive immune responses, and could provide improved protection against infectious diseases.

Production and structural analysis of the polysaccharide secreted by Trametes (Coriolus) versicolor ATCC 200801.

Trametes versicolor ATCC 200801 secretes 4.1 g L(-1) of exopolysaccharide (EPS) when synthetic minimal medium and low-shear bioreactor cultivation technique are used. Structural and compositional analyses by thin layer chromatography, gas chromatography-mass spectrometry, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy yielded predominantly glucose and small amounts of galactose, mannose, arabinose, and xylose. The main EPS is composed of beta-1,3/beta-1,6-linked D-glucose molecules which is identical with Schizophyllan but does not possess a triple helical arrangement as secondary structure. Two molar mass fractions were detected by size exclusion chromatography yielding weight-average molecular weights of 4,100 and 2.6 kDa. Protein content varies between 2-3.6% (w/w). The exopolysaccharide is different in the nature of the glycosidic linkage, composition of monosaccharides, protein content, and weight-average molecular weight compared to the well-known polysaccharopeptide (PSP) and polysaccharopeptide Krestin (PSK).

Structural insights into antigen recognition of an anti-ß-(1,6)-ß-(1,3)-D-glucan antibody.

Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a ß-(1,3)-D-glucan main chain with branching ß-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH antibody JoJ48C11 recognizes SCH and related ß-(1,6)-branched ß-(1,3)-D-glucans, but details governing its specificity are not known. Here, we fill this gap by determining crystal structures of the antigen binding fragment (Fab) of JoJ48C11 in the apo form and in complex with the unbranched ß-(1,3)-D-glucose hexamer laminarihexaose 3.0 and 2.4 Å resolution, respectively. Together with docking studies, this allowed construction of a JoJ48C11/triple-helical SCH complex, leading to the identification of eight amino acid residues of JoJ48C11 (Tyr27H, His35H, Trp47H, Trp100H, Asp105H; Asp49L, Lys52L, Trp90L) that contribute to the recognition of glucose units from all three chains of the SCH triple helix. The importance of these amino acids was confirmed by mutagenesis and ELISA-based analysis. Our work provides an explanation for the specific recognition of triple-helical ß-(1,6)-branched ß-(1,3)-D-glucans by JoJ48C11 and provides another structure example for anti-carbohydrate antibodies.

Generation of Recombinant Antibodies against the beta-(1,6)-Branched beta-(1,3)-D-Glucan Schizophyllan from Immunized Mice via Phage Display.

beta-(1,6)-Branched beta-(1,3)-D-glucans like schizophyllan from the basidiomycete Schizophyllum commune excite various immunostimulatory effects and have been clinically tested as adjuvants. Some of the glucans are also applicable in food or petrol industry due to their viscosity and temperature stability in aqueous solution. Antibodies against these glucans could be used as tool for analysis of glucan preparations or for further research of its bioactivity. Therefore, an immune phage display library was constructed from mice immunized with schizophyllan. Three recombinant monoclonal antibodies were isolated from this library by affinity selection (panning) on schizophyllan. The half-maximal effective concentration (EC50) values for those antibodies varied between 16.4 ng mL-1 and 21.3 ng mL-1. The clones showed binding specificity not only for schizophyllan but also for other beta-(1,6)-branched beta-(1,3)-D-glucans of similar macromolecular structure. Denaturation of the secondary structure led to a reduced antibody binding, indicating an epitope requiring the correct conformation of the triple helical structure of the glucans.