Antibodies to the Cryptococcus neoformans Capsule have Innate Glycosidase Activity

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.

A synthetic glycan array containingCryptococcus neoformansglucuronoxylomannan capsular polysaccharide fragments allows the mapping of protective epitopes

“…13,14 The acetylation is known to be important for the antibody-antigen interactions and virulence with an estimated average of 2 acetates per mannose triad (serotype A and D). 14,15 This heterogeneity of the GXM severely complicates its structural analysis as well as structure-activity investigations, e.g., its use in conjugate vaccines, where the stated heterogeneity has shown to lead to irreproducible effects in vaccination in murine models. 16,17 Also, reducing ends are not obviously available in the GXM CPS, 18 why CPS-based conjugate vaccines candidates have been constructed using methods, e.g., cyanogen bromide activation, resulting in complex cross-linked conjugates.…”

Illuminating glycoscience: synthetic strategies for FRET-enabled carbohydrate active enzyme probes

“…Recently, Casadevall and Oscarson reported the design and synthesis of oligosaccharide FRET probes to characterise the hydrolytic activity of four catalytic monoclonal antibodies, effectively demonstrating their innate glycosidase activity. 67 This was undertaken as part of a strategy targeting glycoconjugate vaccine candidates against the opportunistic fungi Cryptococcus neoformans, where the capsule polysaccharide glucuronoxylomannan (GXM) dominates. Based on their prior work developing synthetic glycan arrays of GXM structures, 68 the group synthesised GXM decasaccharide targets 55 and 56, equipped with FRET capability, using a coumarin/DNP pairing (Fig.…”