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

Wear²,

Smith³

et al. 2020

Preprint

Self Cite

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. 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 Förster Resonance Energy Transfer (FRET) probes, which we used to discover antibodies with innate glycosidase activity and analyse their enzyme kinetics, including mAb 2H1, a polysaccharide lyase, and the most efficient glycosidase 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. Further the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labelled by 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 the anti-phagocytic 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.

Section: Methodsmentioning

confidence: 99%

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

Wear²,

Smith³

et al. 2020

Preprint

Self Cite

“…To allow comparison we performed a hydrogenolysis reaction optimisation study using a synthetic serotype A decasaccharide 1, which when deprotected, we have recently identified as a good antigen to be tested as part of a semi-synthetic vaccine candidate against C. neoformans infections (Scheme 1, Table 1). 4 Using either a Pearman's catalyst (20% Pd[OH]2/C, Sigma-Aldrich) or a 10% Pd/C (Sigma-Aldrich) both led to exceedingly long reaction times (5-6 days), this was in spite of using our optimised pre-tuning conditions (Table 1 Entry 1 & 2). While, in keeping with our previous observations the 5% Pd/C Evonik Noblyst ® from Strem Chemicals allowed access to the desired decasaccharide 2 in the shortest reaction times, highest yields, and no aromatic protecting group related saturation when using our pre-tuning methodology ( Figure 2, Table 1 Entry 3).…”

Section: Palladium Catalyst Analysismentioning

confidence: 99%

“…To complete the total synthesis of the serotype A decasaccharide we followed a convergent building block approach, utilizing di-and tetrasaccharide thioglycoside building blocks. 5 The synthesis of which has reported previously, 4 the use of a convergent synthesis is attractive for several reasons; as it allows quick assembly of the target GXM glycans in minimal steps; the 6-O-acetylation along the mannose backbone is preinstalled; and glycan branching is formed at an early stage, specifically the β-1,2 xylose branches and β-1,2 glucuronic acid branches.…”

Section: Total Synthesis Of Serotype a Decasaccharidementioning

confidence: 99%

See 1 more Smart Citation

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

Qiao²,

Liu³

et al. 2020

Preprint

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We report insights into our observations of the wide variability in quality of palladium catalyst from different suppliers, finding the fundamental differences can be rationalized through a combination of XRD, XPS, and TEM analysis, offering the possibility to predict a catalysts performance prior to the use of valuable synthetic material and save time consuming efforts to identify high quality palladium on carbon catalysts. The synthetic glycan accessed in this study will allow further steps towards the development of semisynthetic vaccines against cryptococcal infections.

“…Recently we have developed a synthetic glycan array containing a library of GXM structures (20). When screening this library with mAbs to C. neoformans GXM we found a decasaccharide that was recognized by a wide variety of antibodies (20). Consequently, we selected it as a good structure for the development into a FRET probe to serve as a potential assay to identify catalytic antibodies.…”

Section: Antibody Epitope Mappingmentioning

confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

Wear

Smith

et al. 2021

Self Cite

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.