A Glycan FRET Assay for Detection and Characterization of Catalytic Antibodies to the Cryptococcus neoformans Capsule

Abstract

Classical antibody functions include opsonization, complement activation and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. In this study, we mapped the epitopes of several monoclonal antibodies (mAbs) against the capsule of <i>Cryptococcus neoformans</i> using a synthetic glycan array. From this, we designed and synthesized two glycan based Förster Resonance Energy Transfer (FRET) probes, which allowed the discovery of antibodies with innate glycosidase activity, and analysis of their enzyme kinetics. We confirmed that the mAbs mediate glycosidase activity on intact cryptococcal capsules, by reacting antibody-treated capsules with a hydroxylamine-armed fluorescent probe, which revealed the appearance of reducing ends from polysaccharide hydrolysis in the capsule. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and hydroxylamine-armed fluorescent probes as tools for investigating this activity.