Antibodies to the Cryptococcus neoformans Capsule have Innate Glycosidase Activity

2020 ◽  
Author(s):  
Conor Crawford ◽  
Maggie P. Wear ◽  
Daniel F. Q. Smith ◽  
Clotilde d'Errico ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Catalytic Activity ◽  
Cryptococcus Neoformans ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Biological Functions ◽  
Glycan Array ◽  
Glycosidase Activity ◽  
Antimicrobial Function ◽  
Polysaccharide Hydrolysis

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.

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

2020 ◽  
Author(s):  
Conor Crawford ◽  
Maggie P. Wear ◽  
Daniel F. Q. Smith ◽  
Clotilde d'Errico ◽  
Scott McConnell ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cryptococcus Neoformans ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Antibodies ◽  
Glycan Microarray ◽  
Glycosidase Activity ◽  
Antimicrobial Function ◽  
Complement Deposition

<p>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 <i>Cryptococcus neoformans</i>. 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 <i>C. neoformans </i>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.</p>

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

2020 ◽  
Author(s):  
Conor Crawford ◽  
Maggie P. Wear ◽  
Daniel F. Q. Smith ◽  
Clotilde d'Errico ◽  
Scott McConnell ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cryptococcus Neoformans ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Antibodies ◽  
Glycan Microarray ◽  
Glycosidase Activity ◽  
Antimicrobial Function ◽  
Complement Deposition

<p>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 <i>Cryptococcus neoformans</i>. 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 <i>C. neoformans </i>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.</p>

scholarly journals A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule

2021 ◽  
Vol 118 (5) ◽  
pp. e2016198118
Author(s):  
Conor J. Crawford ◽  
Maggie P. Wear ◽  
Daniel F. Q. Smith ◽  
Clotilde d’Errico ◽  
Scott A. McConnell ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cryptococcus Neoformans ◽  
Epitope Mapping ◽  
Catalytic Antibodies ◽  
Biological Functions ◽  
Glycan Microarray ◽  
Glycosidase Activity ◽  
Antimicrobial Function ◽  
Complement Deposition

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.

scholarly journals Glycan FRET Probes for Screening Catalytic Antibodies Against Cryptococcus Neoformans Capsule

2020 ◽  
Author(s):  
Conor Crawford ◽  
Maggie P. Wear ◽  
Daniel F. Q. Smith ◽  
Clotilde d'Errico ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Docking Studies ◽  
Catalytic Antibodies ◽  
Antigen Binding Site ◽  
Design And Synthesis ◽  
Glycosidase Activity ◽  
Structural Insights

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.

scholarly journals Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods

2020 ◽  
Vol 21 (23) ◽  
pp. 9150
Author(s):  
Yoshitomo Shiroma ◽  
Go Fujita ◽  
Takuya Yamamoto ◽  
Ryou-u Takahashi ◽  
Ashutosh Kumar ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclear Factor Κb ◽  
Screening Methods ◽  
Biological Functions ◽  
Mobility Shift ◽  
Dna Strand Exchange ◽  
Mobility Shift Assay ◽  
Dna Strand

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA–DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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