Generierung von rekombinanten Antikörpern gegen das beta-(1,6)-verzweigte beta-(1,3)-D-Glucan Schizophyllan

Schizophyllan is a homopolysaccharide consistent of a beta-(1,3)-D-glucan main chain with single beta-(1,6)-linked glucose residues at approximate every third glucose moiety. It forms a triple helical structure in aqueous solution, which is stable at temperatures up to 135 °C as well as at pH values up to pH 12 and is responsible for the pseudo plastic flow behavior of Schizophyllan solutions. Additionally, it shows bioactivities like antitumoral and Immunomodulating effects. Due to these properties it is interesting for various applications in different industries. Therefore, an anti-Schizophyllan antibody would be of great value since it could be used for quantitative analysis and for investigation of e the glucan bioactivity. After establishment of glucan immobilization onto functionalized microwell plates as well as generation of an immune library, 4 recombinant antibodies (rAbs) were successfully generated by phage display technology and produced as bivalent scFv-Fc. The rAbs JoJ48C11, JoJ48F1 and JoJ49D10 were generated from the immune library and JoJ58B9 from naive library HAL9. Immunochemical investigations of these rAbs showed for each a specificity which is not directed solely against SCH but also against other beta-glucans with the same primary and triple helical structure as Schizophyllan, like Scleroglucan, Cinerean and Fructican. For further investigation of binding-specificity, the molecular structure of JoJ48C11 as Fab and a complex structure with Laminarihexaose were solved using X-ray crystallography. Based on the derived crystal structures, computational modeling and mutagenesis studies a structural insight into Schizophyllan binding of JoJ48C11 was gained. This proved the assumed combination of the beta-(1,6)-bound glucose residues and the triple helical structure as conformational antigen epitope for JoJ48C11. In sum, this study describes the first successful generation of monoclonal antibodies via phage display from a human naive and mouse immune antibody library, which recognize a structural epitope of beta-(1,6)-branched beta-(1,3)-D-glucans like SCH. Additionally, it shows the first structural description of such an anti-beta-D-glucan antibody complex.In dieser Arbeit wird die erfolgreiche Generierung von rekombinanten Antikörpern mittels Phage-Display Technologie beschrieben, welche gegen das extrazelluläre Polysaccharid Schizophyllan des Basidiomyceten Schizophyllum commune gerichtet sind. Schizophyllan besteht aus einer beta-(1,3)-D-Glucan Hauptkette mit einzelnen beta-(1,6)-verknüpften Glucoseresten an ca. jeder dritten Glucoseeinheit. In wässriger Lösung besitzt es eine tripelhelikale makromolekulare Struktur, die stabil gegenüber hohen Temperaturen bis zu 135°C und pH-Werten bis zu pH 12 ist. Durch diese Struktur weisen Lösungen von Schizophyllan ein pseudo-plastisches Fließverhalten vor. Zusätzlich besitzt Schizophyllan eine bioaktive Wirkung durch seine immunmodulierende und antitumoralen Effekte. Dieser Eigenschaften machen Schizophyllan interessant für die Anwendungen in verschiedenen Bereichen. Daher ist ein anti-Schizophyllan Antikörper von großem Wert, dieser kann zur quantitativen Bestimmung von Schizophyllan und zur Untersuchung der Bioaktivität von Schizophyllan verwendet werden. Zur Generierung der rekombinanten Antikörper (rAb) wurden eine Immobilisierungsmethode für Glucane an Mikrowellplatten etabliert und eine Immun-Antikörpergenbibliothek erstellt. Mit deren Hilfe konnten vier Antikörper isoliert werden, welche anschließend im bivalenten scFv-Fc Format produziert wurden. Die rAb JoJ48C11, JoJ48F1 sowie JoJ49D10 wurden aus der Immun-Antikörpergenbibliothek HAL9 isoliert und JoJ58B9 aus der naiven Antikörpergenbibliothek HAL9. Immuno-chemische Untersuchungen zeigten für jeden dieser rAb eine Spezifität die nicht allein gegen Schizophyllan gerichtet ist, sondern auch gegen andere beta-Glucane mit derselben primären Struktur und makromolekularen Strukturelementen, wie Scleroglucan, Cinerean and Fructican. Zur tieferen Analyse der Bindungsspezifität wurde daraufhin die Struktur von JoJ48C11 als Fab und als Fab/Laminarihexaose Komplex über Röntgenstrukturkristallographie aufgelöst. Durch die Kristallstrukturen, Modellierung und Mutagenese Studien konnte ein genauerer Einblick in die Bindung von JoJ48C11 an Schizophyllan erhalten werden und die angenommene Kombination von tripelhelikaler Glucan Struktur mit den beta-(1,6)-gebunden als Bindungsepitop nachgewiesen werden

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

Streamlining Homogeneous Glycoprotein Production for Biophysical and Structural Applications by Targeted Cell Line Development

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP ‘master’ cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP)

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

Abstract Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a β-(1,3)-D-glucan main chain with branching β-(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 β-(1,6)-branched β-(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 β-(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 β-(1,6)-branched β-(1,3)-D-glucans by JoJ48C11 and provides another structure example for anti-carbohydrate antibodies