beta-(1,6)-Branched beta-(1,3)-D-glucans like schizophyllan from the basidiomycete
A variety of fungal beta-(1,3)-D-glucans are described as substances with antitumoral and immunomodulating activities [
The primary molecular structure of SCH consists of a beta-(1,3)-D-glucan main chain with single beta-(1,6)-linked glucose molecules at approximately every third glucose monomer of the backbone [
The objective of this study was the generation of recombinant monoclonal antibodies (rAbs) against the beta-D-glucan schizophyllan. Therefore, we constructed an antibody phage display library from the lymphocytes of three mice which had been immunized with proteinase K treated SCH (SCH-PK). After panning of this library for SCH-PK binding antibody phage, we were able to derive three rAbs specificity binding beta-(1,6)-branched beta-(1,3)-D-glucans with the same secondary structure as SCH.
All chemicals were purchased from Sigma Aldrich if not mentioned otherwise.
Following beta-(1,6)-branched beta-(1,3)-D-glucans were prepared from biomass-free and stabilized (5 g L−1 formic acid) culture supernatants: SCH (
In addition to this schizophyllan preparation, commercially acquired samples of schizophyllan from Contipro Biotech (Dolní Dobrouč, Czech Republik) and Actigum™ CS 11 (scleroglucan) purchased from Degussa Construction Polymers GmbH (Trostberg, Germany) were used.
SCH additionally treated with proteinase K (SCH-PK) was used as antigen for the isolation of antibodies. For its preparation, biomass-free culture supernatant of
Three mice (BALB/c, female, 7 weeks old) were immunized with SCH-PK by intraperitoneal injection. Three injections were applied in two-week intervals. Each injection consisted of 25
A volume equal to 1 × 107 of homogenized leucocytes was used to isolate the RNA with the Direct-zol MiniPrep kit (Zymo Research, Irvine, USA). The isolation of the rearranged genes for the antibody domains
The experimental protocols were carried out in accordance with the Directive 2010/63/EU of the European Parliament and the Council of the European Union of 22 September 2010 and all procedures were approved by guidelines from the Animal Committee on Ethics in the Care and Use of Laboratory Animals of TU Braunschweig, Germany (Az
For the antibody selection and ELISA experiments, schizophyllan was immobilized to Carbo-BIND multiwell plates (Corning, Corning, USA). The wells were filled with 100
The rAbs were selected by panning as described [
For further characterization of the isolated rAbs, the DNA encoding the scFv (single chain Fragment variable) was subcloned into pCSE2.6-mIgG2c-Fc-XP via NcoI and NotI (New England Biolabs, Ipswich, USA). The resulting scFv-Fc fusions (scFv fused with a murine Fc part) were produced in HEK239-6E cells (National Research Council, Biotechnological Research Institute, Montreal, Canada) as described [
The half-maximal effective concentration (EC50) values of the produced scFv-Fc were determined by titration ELISA (enzyme-linked immunosorbent assay) using serial dilutions of the scFv-Fc in blocking solution (2% (w/v) milk powder and 0.05% (w/v) Tween® 20 in PBS). ELISA was performed with SCH-PK loaded Carbo-BIND multiwell plates (100
Antigen specificity was evaluated via competition of scFv-Fc binding to immobilized SCH-PK by various soluble saccharides. Each scFv-Fc was used in the concentration that resulted in 40% of the saturation signal in the titration ELISA. They were preincubated in serial dilutions of competitors (3.2 ng mL−1 to 1 mg mL−1, blocking solution as solvent) for 1 h before they were transferred to the SCH-PK loaded Carbo-BIND plate. The further procedure was done as described for titration ELISA. The following substances were used as competitors: SCH-PK, SCH, scleroglucan
All investigations were carried out at least in three independent experiments.
The goal of this study was the generation of a rAb that binds to the beta-(1,6)-branched beta-(1,3)-D-glucan schizophyllan of
After three rounds of panning, 92 of the enriched antibody clones from each library were selected and the respective soluble scFv-fragments were tested by ELISA for binding to SCH-PK. 60 clones of the V-KAPPA library showed specific binding; 10 of those clones with the highest signal and highest difference to the signal of the negative control were sequenced. Three individual antibodies (JoJ48C11, JoJ48F1, and JoJ49D10) with high sequence similarities were identified. The heavy chain of each recombinant antibody contains the variable gene IGHV1-7
The three isolated rAbs were produced as scFv-Fc consisting of the scFv fused to a murine IgG2c Fc part. The scFv-Fc format is a bivalent antibody comparable to a full length IgG [
The rAbs were analyzed as scFv-Fc by titration ELISA. Serial dilutions from 0.095 pM to 945 nM antibody were applied on SCH-PK loaded and blank Carbo-BIND multiwell plates. The resulting data (Figure
Titration ELISA for analysis of the binding of the isolated antibodies in bivalent form (scFv-Fc). Dilution series of the antibodies from 0.095 pM to 945 nM were applied to SCH-PK coated or uncoated Carbo-BIND™ plates.
Analysis for the specificity in antigen recognition was performed by a competitive approach. The rAbs were incubated in solutions of different saccharides or yeast extract before they were added to SCH-PK loaded Carbo-BIND plates. For illustration, only the data of JoJ49D10 are presented in Figure
Competitive ELISA of antibody JoJ49D10 for the characterization of antigen specificity. JoJ49D10 (scFv-Fc) was preincubated in dilutions of different carbohydrates or yeast extract ranging from 10 ng mL−1 to 1 mg mL−1 before being added to SCH-PK loaded Carbo-BIND multiwell plates. The absorbance signals were normalized by division with the absorbance of respective samples without competitor.
The expected decrease of signal with increasing concentrations of SCH-PK in solution was observed for each rAb. Furthermore, a decrease of signal was also found, as expected, for nontreated SCH and the other beta-(1,6)-branched beta-(1,3)-D-glucans like scleroglucan of
Only a slight signal decrease at higher concentrations of NaOH treated SCH-PK was observed for each rAb. Due to the NaOH treatment of SCH-PK, the glucan possesses a changed macromolecular structure. The triple helix denatures into single chains when the pH exceeds 13 [
JoJ48C11 and JoJ48F1 showed a slight interaction with laminarin of
Xanthan, dextran, laminarihexaose, beta-(1,6)-D-gentiobiose, glucose, yeast extract, and glucanase treated SCH/SCH-PK did not inhibit the binding of any of the three antibodies to SCH-PK. Xanthan consists of a beta-(1,4)-D-glucan backbone with beta-(1,3)-linked beta-D-mannose-(1,4)-beta-D-glucuronic acid-(1,2)-alpha-D-mannose side chain at every second glucose and forms a double helix in aqueous solution [
The experiments showed that the triple helical structure of the glucans is the essential factor for antibody recognition. It is assumed that the spatial arrangement of the beta-(1,6)-linked D-glucose residues is an important part of the antibody binding epitope. The beta-(1,6)-branching ratio of the glucan also affects the reactivity of the antibody binding. Fructican possesses a higher degree of branching points but does not influence the reactivity of the antibodies compared to the different types of schizophyllan and scleroglucan as well as cinerean. Laminarin shows not only a low occurrence of triplex structure but also a lower degree of branching which could be also responsible for the weak antibody binding. This leads to the additional conclusion that besides the triplex the beta-(1,6)-branching degree also influences antibody reactivity.
With the gained datasets the EC50 values of the competing glucans (except for NaOH treated SCH-PK and laminarin) were calculated from the signal reduction and compared as value for the binding strength of the antibodies to the glucans (all data shown in Table
EC50 values of competitors which are recognized by the recombinant antibodies.
Competitors | EC50 values [ | ||
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JoJ48C11 | JoJ48F1 | JoJ49D10 | |
SCH-PK |
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SCH |
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Scleroglucan |
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Scleroglucan (PK-treated) |
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Cinerean |
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Fructican |
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Scleroglucan |
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SCH (Contipro biotech) |
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Actigum CS11 (Scleroglucan) |
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In summary, the results suggest that the generated rAbs recognize a conformational epitope requiring an intact triple helical secondary structure of beta-(1,6)-branched beta-(1,3)-D-glucans with an optimal branching degree of one beta-(1,6)-bound D-glucose attached to approximately every three glucose units of the main chain. As a consequence, the rAbs not only are specific for SCH but also bind to similar beta-(1,6)-branched beta-(1,3)-D-glucans with triple helical structure.
Reports about the generation of an antibody against SCH already exist. Tabata et al. reported the generation of an antiserum from immunized rabbits [
Investigations of monoclonal antibodies generated with other beta-(1,3)-D-glucans also exist with binding specificities to more than one glucan [
In this report the successful generation of three recombinant glucan antibodies is described. They were selected for binding to the beta-(1,6)-branched beta-(1,3)-D-glucan schizophyllan of
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors would like to thank Doris Meier and Wolfgang Graßl for their technical assistance. The authors appreciate the support and help of Michael Hust in antibody selection, data analysis, and writing of the manuscript.