Glycan FRET Probes for Screening Catalytic Antibodies Against Cryptococcus Neoformans Capsule

Catalytic monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans have been identified but characterization of their Michaelis-Menten kinetics against oligosaccharides has so far not been possible. To address this, we report the design and synthesis of two glycan based Förster resonance energy transfer (FRET) probes that express a major structural unit of the cryptococcal polysaccharide. These probes allowed the kinetic analysis of four catalytic antibodies with glycosidase activity, including 2H1, an antibody which was not known previously to be catalytic. This is only the second report of an antibody with naturally occurring catalytic activity against glycans and the most efficient identified to date. The probe’s capability as a diagnostic for catalysis was demonstrated by accurately predicting glycosidase activity on the native capsule. Furthermore, we used molecular docking studies to reveal the antibody-glycan interactions, with the first structural insights into these interactions between anti-GXM mAbs and their epitopes. Through modelling we see no classical catalytic residues in the antigen binding site, signifying the possibility of further glycan hydrolyzing mechanisms yet to be discovered.