Development of BODIPY labelled sialic acids as sialyltransferase substrates for direct detection of terminal galactose on N- and O-linked glycans

Abstract Glycosylation is a common modification found on numerous proteins and lipids. However, direct detection of glycans on these intact biomolecules has been challenge. Here, utilizing enzymatic incorporation of fluorophore-conjugated sialic acids, dubbed as direct fluorescent glycan labeling, we report the labeling and detection of N- and O-glycans on glycoproteins. The method allows detection of specific glycans without the laborious gel blotting and chemiluminescence reactions used in Western blotting. The method can also be used with a variety of fluorescent dyes.

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Cooperativity of catalytic and lectin-like domain of T. congolense trans-sialidase modulates its catalytic activity

10.1101/2021.05.28.446113 ◽

2021 ◽

Author(s):

Mario Waespy ◽

Thaddeus Termulun Gbem ◽

Nilima Dinesh Kumar ◽

Shanmugam Solaiyappan Mani ◽

Jana Rosenau ◽

...

Keyword(s):

Enzyme Activities ◽

In Silico Analysis ◽

Sialic Acids ◽

Biological Functions ◽

Lectin Domain ◽

African Animal Trypanosomosis ◽

Terminal Galactose ◽

Donor And Acceptor ◽

Fundamental Understanding ◽

Silico Analysis

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Most work on trypanosomal TS has been done on enzymatic activities of TS from T. cruzi (causing Chagas disease in Latin America), subspecies of T. brucei, (causing human sleeping sickness in Africa) and T. congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS) lectin domain (LD) binds to several carbohydrates, such as 1,4-β-mannotriose. To investigate the influence of TconTS-LD on enzyme activities, we firstly performed in silico analysis on structure models of TconTS enzymes. Findings strongly supports the potential of domain swaps between TconTS without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear sialidase and sialic acid (Sia) transfer activities, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolysis was drastically reduced. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities favouring trans-sialylation over hydrolysis. In summary, this study provides strong evidence that TconTS-LDs play pivotal roles in modulating enzyme activity and biological functions of these and possibly other TS, revising our fundamental understanding of TS modulation and diversity.

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Principles of N-Linked Glycosylation Variations of IgG-Based Therapeutics: Pharmacokinetic and Functional Considerations

Antibodies ◽

10.3390/antib9020022 ◽

2020 ◽

Vol 9 (2) ◽

pp. 22

Author(s):

Souad Boune ◽

Peisheng Hu ◽

Alan L. Epstein ◽

Leslie A. Khawli

Keyword(s):

Modern Medicine ◽

Sialic Acids ◽

Igg Subclass ◽

Post Translational Modifications ◽

Effector Functions ◽

Terminal Galactose ◽

Complement Dependent Cytotoxicity ◽

Dependent Cell ◽

Ch2 Domain ◽

The Stability

The development of recombinant therapeutic proteins has been a major revolution in modern medicine. Therapeutic-based monoclonal antibodies (mAbs) are growing rapidly, providing a potential class of human pharmaceuticals that can improve the management of cancer, autoimmune diseases, and other conditions. Most mAbs are typically of the immunoglobulin G (IgG) subclass, and they are glycosylated at the conserved asparagine position 297 (Asn-297) in the CH2 domain of the Fc region. Post-translational modifications here account for the observed high heterogeneity of glycoforms that may or not impact the stability, pharmacokinetics (PK), efficacy, and immunogenicity of mAbs. These modifications are also critical for the Fc receptor binding, and consequently, key antibody effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Moreover, mAbs produced in non-human cells express oligosaccharides that are not normally found in serum IgGs might lead to immunogenicity issues when administered to patients. This review summarizes our understanding of the terminal sugar residues, such as mannose, sialic acids, fucose, or galactose, which influence therapeutic mAbs either positively or negatively in this regard. This review also discusses mannosylation, which has significant undesirable effects on the PK of glycoproteins, causing a decreased mAbs’ half-life. Moreover, terminal galactose residues can enhance CDC activities and Fc–C1q interactions, and core fucose can decrease ADCC and Fc–FcγRs binding. To optimize the therapeutic use of mAbs, glycoengineering strategies are used to reduce glyco-heterogeneity of mAbs, increase their safety profile, and improve the therapeutic efficacy of these important reagents.

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Detection and Characterization of earth-like Planets

International Astronomical Union Colloquium ◽

10.1017/s0252921100014810 ◽

1997 ◽

Vol 161 ◽

pp. 299-311 ◽

Cited By ~ 22

Author(s):

Jean Marie Mariotti ◽

Alain Léger ◽

Bertrand Mennesson ◽

Marc Ollivier

Keyword(s):

Direct Detection ◽

Contrast Ratio ◽

Angular Size ◽

Physical Nature ◽

Major Axis ◽

Infrared Emission ◽

Space Technology ◽

Semi Major Axis ◽

Earth Like Planets ◽

Visible Wavelengths

AbstractIndirect methods of detection of exo-planets (by radial velocity, astrometry, occultations,...) have revealed recently the first cases of exo-planets, and will in the near future expand our knowledge of these systems. They will provide statistical informations on the dynamical parameters: semi-major axis, eccentricities, inclinations,... But the physical nature of these planets will remain mostly unknown. Only for the larger ones (exo-Jupiters), an estimate of the mass will be accessible. To characterize in more details Earth-like exo-planets, direct detection (i.e., direct observation of photons from the planet) is required. This is a much more challenging observational program. The exo-planets are extremely faint with respect to their star: the contrast ratio is about 10−10at visible wavelengths. Also the angular size of the apparent orbit is small, typically 0.1 second of arc. While the first point calls for observations in the infrared (where the contrast goes up to 10−7) and with a coronograph, the latter implies using an interferometer. Several space projects combining these techniques have been recently proposed. They aim at surveying a few hundreds of nearby single solar-like stars in search for Earth-like planets, and at performing a low resolution spectroscopic analysis of their infrared emission in order to reveal the presence in the atmosphere of the planet of CO H2O and O3. The latter is a good tracer of the presence of oxygen which could be, like on our Earth, released by biological activity. Although extremely ambitious, these projects could be realized using space technology either already available or in development for others missions. They could be built and launched during the first decades on the next century.

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