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

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.

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

10.26434/chemrxiv.12144699 ◽

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

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.

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Antibodies to the Cryptococcus neoformans Capsule have Innate Glycosidase Activity

10.26434/chemrxiv.12144699.v2 ◽

2020 ◽

Cited By ~ 1

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 Cryptococcus neoformans 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.

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Glycan FRET Probes for Screening Catalytic Antibodies Against Cryptococcus Neoformans Capsule

10.26434/chemrxiv.12144699.v1 ◽

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 Cryptococcus neoformans 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.

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Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

Nature Communications ◽

10.1038/s41467-021-23892-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Praveen Bawankar ◽

Tina Lence ◽

Chiara Paolantoni ◽

Irmgard U. Haussmann ◽

Migle Kazlauskiene ◽

...

Keyword(s):

Catalytic Activity ◽

Sex Determination ◽

Ubiquitin Ligase ◽

Mammalian Cells ◽

E3 Ubiquitin Ligase ◽

Human Cells ◽

Biological Functions ◽

Mrna Metabolism ◽

Mrna Methylation ◽

Core Components

AbstractN6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

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Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression.

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)43160-8 ◽

1990 ◽

Vol 31 (3) ◽

pp. 375-384

Author(s):

S Marcovina ◽

S Fantappie ◽

A Zoppo ◽

G Franceschini ◽

AL Catapano

Keyword(s):

Monoclonal Antibodies ◽

Epitope Mapping ◽

Immunochemical Characterization ◽

Human Apolipoprotein ◽

Apolipoprotein A

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Synthesis, Characterization of Chitosan-Aluminum Oxide Nanocomposite for Green Synthesis of Annulated Imidazopyrazol Thione Derivatives

Polymers ◽

10.3390/polym13071160 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1160

Author(s):

Abir S. Abdel-Naby ◽

Sara Nabil ◽

Sarah Aldulaijan ◽

Ibtisam M. Ababutain ◽

Azzah I. Alghamdi ◽

...

Keyword(s):

Catalytic Activity ◽

Aluminum Oxide ◽

Reaction Medium ◽

Thiol Group ◽

Docking Studies ◽

Significant Loss ◽

Gram Negative Bacteria ◽

Organic Catalyst ◽

Group 1

Chitosan-aluminum oxide nanocomposite was synthesized, characterized, and used as a green heterogeneous catalyst to synthesize novel imidazopyrazolylthione derivatives. Nanocomposite polymeric material was characterized by EDS-SEM and XRD. The powerful catalytic activity, and its base character of the nanocomposite, was used to synthesize imidazopyrazolylthione (1) in a good yield compared to traditional cyclocondensation synthesis. Using the nanocomposite catalyst, substitution of the thiol group (1) afforded the corresponding thiourea (2) and the corresponding ester (3). The efficiency of the nanocomposite over the traditional base organic catalyst, Et3N and NaOH, makes it an effective, economic, and reproducible nontoxic catalyst. Moreover, the heterogeneous nanocomposite polymeric film was easily isolated from the reaction medium, and recycled up to four times, without a significant loss of its catalytic activity. The newly synthesized derivatives were screened as antibacterial agents and showed high potency. Molecular docking was also performed for a more in-depth investigation. The results of the docking studies have demonstrated that the docked compounds have strong interaction energies with both Gram-positive and Gram-negative bacteria.

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Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

International Journal of Molecular Sciences ◽

10.3390/ijms22020734 ◽

2021 ◽

Vol 22 (2) ◽

pp. 734

Author(s):

Paul K. Varghese ◽

Mones Abu-Asab ◽

Emilios K. Dimitriadis ◽

Monika B. Dolinska ◽

George P. Morcos ◽

...

Keyword(s):

Catalytic Activity ◽

Large Scale ◽

Magnetic Beads ◽

Enzymatic Reaction ◽

Oculocutaneous Albinism ◽

Transmission Electron ◽

Hill Kinetics ◽

Rate Limiting ◽

Human Tyrosinase

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19–469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

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