Glycan FRET Probes for Screening Catalytic Antibodies Against Cryptococcus Neoformans Capsule

Abstract

Catalytic monoclonal antibodies (mAbs) against the capsule of <i>Cryptococcus neoformans</i> 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.

Variation in the Structure and Activity of Commercial Palladium on Carbon Catalysts

“…A key focus in our laboratory is directed towards the synthesis of glycans from C. neoformans, [5,6] in particular the major component of its capsular polysaccharide, GXM. From native sources the GXM is highly heterogenous and to date large components of the biosynthesis are unknown -meaning synthetic glycans are currently the only viable means to access structures in any meaningful purity.…”

Synthetic Strategies for FRET-Enabled Carbohydrate Active Enzyme Probes

“…Recently, Casadevall and Oscarson reported the design and synthesis of oligosaccharide FRET probes to characterize the hydrolytic activity of four catalytic monoclonal antibodies, effectively demonstrating their innate glycosidase activity [63]. 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.…”

Clues to a Pd/C catalyst’s efficiency can be found on its surface: Identification of an efficient catalyst for the global hydrogenolysis of ether protecting groups

“…A major bottleneck we have encountered in accessing pure synthetic glycans is in the final palladium catalyzed hydrogenolysis or global deprotection reactions, where we experienced long reaction times, poor yields, and saturation of aromatic protecting groups to their corresponding ethers. 1 This was especially prominent in our synthesis of glycans related to structures from C. neoformans, 2 which we carry out in order to develop vaccine candidates. The glucuronoxylomannan (GXM) from native sources is highly heterogenous and large components of the biosynthesis are unknown, meaning synthetic glycans are currently the only viable means to access structures in any meaningful purity.…”