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 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>

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

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 Dynamic structural insights into the molecular mechanism of DNA unwinding by the bacteriophage T7 helicase

2020 ◽  
Vol 48 (6) ◽  
pp. 3156-3164 ◽  
Author(s):  
Jian-Bing Ma ◽  
Ze Chen ◽  
Chun-Hua Xu ◽  
Xing-Yuan Huang ◽  
Qi Jia ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Molecular Mechanism ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bacteriophage T7 ◽  
Dna Unwinding ◽  
Coupling Mechanism ◽  
Mechanical Coupling ◽  
Dna Complex ◽  
Structural Insights

Abstract The hexametric T7 helicase (gp4) adopts a spiral lock-washer form and encircles a coil-like DNA (tracking) strand with two nucleotides bound to each subunit. However, the chemo-mechanical coupling mechanism in unwinding has yet to be elucidated. Here, we utilized nanotensioner-enhanced Förster resonance energy transfer with one nucleotide precision to investigate gp4-induced unwinding of DNA that contains an abasic lesion. We observed that the DNA unwinding activity of gp4 is hindered but not completely blocked by abasic lesions. Gp4 moves back and forth repeatedly when it encounters an abasic lesion, whereas it steps back only occasionally when it unwinds normal DNA. We further observed that gp4 translocates on the tracking strand in step sizes of one to four nucleotides. We propose that a hypothetical intermediate conformation of the gp4–DNA complex during DNA unwinding can help explain how gp4 molecules pass lesions, providing insights into the unwinding dynamics of gp4.

scholarly journals Characterization of the interaction between hepatitis C virus NS5B and the human oestrogen receptor alpha

2012 ◽  
Vol 93 (4) ◽  
pp. 780-785 ◽  
Author(s):  
Julia Hillung ◽  
Elena Ruiz-López ◽  
Itxaso Bellón-Echeverría ◽  
Pilar Clemente-Casares ◽  
Antonio Mas
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Oestrogen Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Host Factors ◽  
Host Proteins ◽  
Receptor Alpha ◽  
Oestrogen Receptor Alpha

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Förster Resonance Energy Transfer. NS5B–ESR1C and ESR1C–ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B–ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugs.

Design and Synthesis of Intramolecular Resonance-Energy Transfer Probes for Use in Ratiometric Measurements in Aqueous Solution

2000 ◽  
Vol 112 (19) ◽  
pp. 3580-3582 ◽  
Author(s):  
Yasutomo Kawanishi ◽  
Kazuya Kikuchi ◽  
Hideo Takakusa ◽  
Shin Mizukami ◽  
Yasuteru Urano ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Design And Synthesis
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