Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo

Ilyushin, D G; Смирнов, Иван; Belogurov, Alexey A.; Dyachenko, Igor A.; Жармухамедова, Т. Ю.; Novozhilova, T. I.; Bychikhin, E. A.; Serebryakova, Marina V.; Kharybin, Oleg N.; Мурашев, А. Н.; Anikienko, K. A.; Nikolaev, E. N.; Пономаренко, Н. А.; Genkin, Dmitry; Blackburn, G. Michael; Masson, Patrick; Gabibov, Alexander

doi:10.1073/pnas.1211118110

Cited by 73 publications

(60 citation statements)

References 42 publications

(47 reference statements)

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“…These activities of protein-independent polySia have great potential for the development of novel carbohydrate-dependent therapeutics. Likewise, improving the efficacy of therapeutic proteins (e.g., erythropoietin and buturylcholinesterase) using polySia was demonstrated by chemical conjugation and in vitro approaches (7,8,44). Clinical trials of polySia-conjugated recombinant proteins are ongoing, indicating the pharmaceutical relevance of polySia (reviewed in ref.…”

Section: Discussionmentioning

confidence: 99%

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Engineering of complex protein sialylation in plants

Kallolimath

Castilho

Strasser

et al. 2016

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

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Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.

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Section: Discussionmentioning

confidence: 99%

“…1, 2, 5, and 6). Notably, chemical or in vitro polySia engineering has made a substantial impact on pharmacokinetic properties of recombinant glycoprotein therapeutics (7,8). These examples demonstrate a manifold functional impact of polySia and highlight the great potential for the development of novel polySia-dependent therapeutics.…”

mentioning

confidence: 99%

Engineering of complex protein sialylation in plants

Kallolimath

Castilho

Strasser

et al. 2016

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

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“…A combination of both pathways also was observed for cl.15. The selected BChE mutants were of high interest because they revealed a previously unreported target (the BChE acylbinding loop) for the creation of catalytic bioscavengers based on human BChE, which are used extensively for protection against nerve agent poisoning and postexposure treatment (12,13).…”

Section: -Stenotrophomonasmentioning

confidence: 99%

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Terekhov

Смирнов

Stepanova

et al. 2017

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

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189

172

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Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-tocell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slowgrowing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3, 4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5, 6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate. By contrast, sophisticated custom sorters demonstrated the screening of precise monodisperse droplets of water-in-oil emulsions generated by microfluidic technology (1, 7). However, it is not always convenient to use custom devices, and the use of oil as a continuous phase limits the sorting rate. Alternatively, compartmentalization in microfluidic double emulsion (MDE) enables uHTS of >10,000 events/s using commercially available cell sorters (8, 9). Furthermore, biocompatible oil and water phases provide viability and proliferation of Escherichia coli cells (10) inside the microenvironment of a double emulsion.Here, we propose an MDE-FACS platform that combines the benefits of previously reported systems based on compartmentalization in MDE and FACS selection together with modern omics (Fig. 1). We succeeded in assembling this platform using commercially available parts, which included straightforward microfluidics (Fig. S1) for MDE generation, multiparametric FACS for uHTS, next-generation sequencing (NGS) for bioinformatic predictions, and mass spectrometry for proteome and secretome analysis. We demonstrated this idea with several single-cell methods (Fig. S2), including the selection of di...

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“…4 For instance, chemical conjugation with suitable glycans has been exploited for the production of therapeutic enzymes with improved activity and reduced in vivo metabolism. 5 Neoglycoproteins, which contain unnatural linkages between protein and saccharides, can also provide carbohydrate antigens and immunogens from which immunodiagnostic and therapeutic reagents can be derived. 4 For example, conjugation of antigenic proteins from Mycobacterium tuberculosis with arabino-mannane polysaccharides has been proposed for developing highly immunogenic glycoconjugate vaccines against tuberculosis.…”

Section: Introductionmentioning

confidence: 99%

Chemoenzymatic synthesis of neoglycoproteins driven by the assessment of protein surface reactivity

et al. 2014

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In this paper a series of 2-iminomethoxyethyl mannose-based mono-and disaccharides have been synthesized by a chemoenzymatic approach and used in coupling reactions with 3-amino groups of lysine residues in a model protein (ribonuclease A, RNase A) to give semisynthetic neoglycoconjugates. In order to study the influence of structure of the glycans on the conjugation outcomes, an accurate characterization of the prepared neoglycoproteins was performed by a combination of ESI-MS and LC-MS analytical methods. The analyses of the chymotryptic digests of the all neoglycoconjugates revealed six Lys-glycosylation sites with a the following order of lysine reactivity: Lys 1 [ Lys 91 y Lys 31 > Lys 61 y Lys 66. A computational analysis of the reactivity of each lysine residue has been also carried out considering several parameters (amino acids surface exposure and pK a , protein flexibility). The in silico evaluation seems to confirm the order in lysine reactivity resulting from proteomic analysis.

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Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo

Cited by 73 publications

References 42 publications

Engineering of complex protein sialylation in plants

Engineering of complex protein sialylation in plants

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Chemoenzymatic synthesis of neoglycoproteins driven by the assessment of protein surface reactivity

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