A glycan FRET assay for detection and characterization of catalytic antibodies to theCryptococcus neoformanscapsule

Abstract

Classic 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. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans. From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

The Inflammasome NLRC4 Protects against Cryptococcus gattii by Inducing the Classic Caspase-1 to Activate the Pyroptosis Signal

“…C. neoformans is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on a capsule outside of the cell wall, which is mainly composed of the antiphagocytic glucuronoxylomannan [ 5 ]. Cryptococcus can not only resist phagocytosis by macrophages but also survive and even proliferate in cells.…”

Defining the Qualities of High-Quality Palladium on Carbon Catalysts for Hydrogenolysis

“…Our optimization study used a synthetic decasaccharide 2 ( Scheme 1 ). 11 Representing a challenging substrate as it assumes a branched tertiary structure 9 and contains 25 groups that need to be reduced under a hydrogen atmosphere, including benzyl ethers, naphthylmethyl ethers, and azides.…”

“…To overcome these selectivity issues, we introduced a catalyst pretuning methodology (dimethylformamide (DMF):H 2 O, 37% HCl) that increases catalyst selectivity toward hydrogenolysis rather than hydrogenation through amine poisoning. The catalyst pretreatment inhibits these unwanted saturation by-products and gives access to pure synthetic oligosaccharides. ,, This methodology successfully tackled an issue we faced (catalyst selectivity) but another key question was how and why different palladium on carbon (Pd/C) catalysts lead to such variable results. Pd/C catalysts remain a “black box” and force extensive testing on complex materials to identify efficient catalysts, defined under the parameters of short reaction times, high isolated yields, and its selectivity toward hydrogenolysis over hydrogenation.…”

Synthetic glucuronoxylomannans enable molecular insights into antibody structure-function and fungal capsule architecture

“…Consequently, over the past three decades great effort has been directed towards the total synthesis of these oligosaccharides (Figure 2). 16,[28][29][30][31][32][33][34] Recently, we reported a convergent synthetic strategy that allowed assembly of protected structures of GXM glycans up to an 18-mer, 28 with a limitation of this study being that problems were encountered in the global deprotections of larger structures, which we attributed to the highly branched nature of these synthetic glycans. 35 New methods were developed to overcome these issues, 35,36 and now in this work we report a library of twenty-six synthetic GXM oligosaccharides.…”

“…11 The molecular complexity of GXM is further increased by the presence a heterogenous 6-Oacetylation pattern present on the mannose backbone that is often important for antibody binding and virulence. 16,17 It is important to note that unlike in bacterial serotypes, GXM motifs occur together in differing abundances as heteropolymers. Due to this complexity several questions remain about the capsular biosynthesis, assembly and structure.…”