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.
Highlights
Schizophyllan (SCH) is a homopolysaccharide produced by the basidiomycete Schizophyllum commune[1]
In order to obtain detailed information about the binding of SCH and other β-(1,3)-branched β-(1,6)-D-glucans by JoJ48C11, we resorted to an X-ray crystallographic structure analysis
Initial phasing of the JoJ48C11 Fab fragment was performed by molecular replacement with the structure of a chimeric antibody Fab fragment against CD25 (PDB entry 1MIM) as a search model[27]
Summary
Schizophyllan (SCH) is a homopolysaccharide produced by the basidiomycete Schizophyllum commune[1]. We have determined the crystal structure of the antigen binding fragment (Fab) of anti-SCH antibody JoJ48C11, both in the apo form and in complex with the oligosaccharide laminarihexaose. These structures allow modelling of an antibody/SCH complex which suggests that all three chains of the helical glucan engage in interactions with the paratope of JoJ48C11. These interactions involve several protruding β-(1,6)-bound glucose residues of SCH, explaining the specificity of the antibody. The model was validated by introducing point mutations into JoJ48C11, corroborating its correctness
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