Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases

Watson, David; Wakarchuk, Warren W.; Gervais, Christian; Durocher, Yves; Robotham, Anna; Fernandes, Steve M.; Schnaar, Ronald L.; Young, N. Martin; Gilbert, Michel

doi:10.1007/s10719-015-9624-4

Cited by 18 publications

(17 citation statements)

References 22 publications

(27 reference statements)

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“…Although a native Leg5,7Ac 2 glycosyltransferase has yet to be identified, several mammalian and bacterial sialyltransferases have been tested for catalyzing the transfer of Leg5,7Ac 2 from CMP‐Leg5,7Ac 2 to galactosides. Porcine ST3Gal I, human ST6Gal I, Pasteurella multocida sialyltransferase 1 (PmST1), and Neisseria meningitides MC58 α2‐3‐sialyltransferase showed reasonable activity in forming Leg5,7Ac 2 ‐glycosides. Nevertheless, these enzymatic syntheses relied on a complex process to produce CMP‐Leg5,7Ac 2 either from UDP‐GlcNAc by multiple enzymes in vitro with chemical acetylation of the 4‐amino group or from Leg5,7Ac 2 produced de novo using Escherichia coli engineered with combined biosynthetic pathways from Saccharomyces cerevisiae , Campylobacter jejuni , and Legionella pneumophila …”

Section: Figurementioning

confidence: 99%

“…[8] Leg5,7Ac 2 is activated by ac ytidine 5'-monophosphate-Leg5,7Ac 2 (CMP-Leg5,7Ac 2 )s ynthetase [3] to provide CMP-Leg5,7Ac 2 as the glycosyltransferase donor for the synthesis of desired structures. Although an ative Leg5,7Ac 2 glycosyltransferase has yet to be identified, several mammalian and bacterial sialyltransferases have been tested for catalyzing the transfer of Leg5,7Ac 2 from CMP-Leg5,7Ac 2 to galactosides.P orcine ST3Gal I, human ST6Gal I, [9] Pasteurella multocida sialyltransferase 1( PmST1), [10] and Neisseria meningitides MC58 a2-3-sialyltransferase [11] showed reasonable activity in forming Leg5,7Ac 2 -glycosides.Nevertheless,these enzymatic syntheses relied on ac omplex process to produce CMP-Leg5,7Ac 2 either from UDP-GlcNAc by multiple enzymes [12] in vitro with chemical acetylation of the 4-amino group [10] or from Leg5,7Ac 2 produced de novo using Escherichia coli engineered with combined biosynthetic pathways from Saccharomyces cerevisiae, Campylobacter jejuni, [13] and Legionella pneumophila. [14] Herein we show that aversatile library of a2-3-and a2-6linked Leg5,7Ac 2 -glycosides can be produced readily from chemically synthesized 2,4-diazido-2,4,6-trideoxymannose (6deoxyMan2,4diN 3 )a sachemoenzymatic synthon in highly efficient one-pot multienzyme (OPME) sialylation systems [15] using commercially available enzymes with downstream chemical derivatization.…”

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confidence: 99%

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A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di‐N‐acetyllegionaminic Acid‐Containing Glycosides

Santra

Xiao

et al. 2018

Angew Chem Int Ed

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A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

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Section: Figurementioning

confidence: 99%

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confidence: 99%

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di‐N‐acetyllegionaminic Acid‐Containing Glycosides

Santra

Xiao

et al. 2018

Angew Chem Int Ed

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“…Sia containing C7 fluoride, Neu5Ac-7F was transferred to the glycoprotein asialo-A1AT by ST6Gal1 [ 121 ]. Further expanding the donor variety, legionaminic acid was used as a donor substrate by porcine ST3Gal1 and human ST6Gal1 for several acceptors [ 132 ]. When analyzing the yield from chemoenzymatic glycan synthesis, it is important to note that many of these studies were done using a one-pot multienzyme (OPME) system, in which SiaT and CMP-Sia synthase were added together, and the apparent efficiency of the reactions reflects the combined efficiency of SiaT and nucleotide sugar synthase [ 131 ].…”

Section: Manipulating Sialyltransferases At a Molecular Levelmentioning

confidence: 99%

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

2021

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Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals.

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“…(Figure 5B). We based our assumption on the close stereochemical structural similarity of Neu5Ac and legionaminic acid, and on evidence for the reverse scenario, i.e., that mammalian and engineered bacterial sialyltransferases are able to use CMP-legionaminic acid instead of CMP-Neu5Ac as donor substrate 62,63,64 .…”

Section: Glycosylation Sites Of Gkflaa1mentioning

confidence: 99%

Novel Serine/Threonine-O-glycosylation with N-Acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid by Maf glycosyltransferases

Khairnar

Sunsunwal

Babu³

et al. 2020

Preprint

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Some bacterial flagellins are O-glycosylated on surface-exposed Serine/Threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid, and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called Motility associated factors (Maf).We report here two new glycosidic linkages previously unknown in any organism, Serine/Threonine-O-linked N-Acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and Serine/Threonine-O-linked 3-Deoxy-D-manno-octulosonic acid (Ser/Thr-O-KDO), both catalysed by Geobacillus kaustophilus Maf (putative flagellin sialyltransferase) and Clostridium botulinum Maf (putative flagellin legionaminic acid transferase). We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were co-expressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. The glycosylation of G. kaustophilus flagellin with KDO, and that of C. botulinum flagellin with Neu5Ac and KDO indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering. Significance StatementGlycosylation, the modification of proteins with sugars, is one of the most common posttranslational modifications observed in proteins. While glycosylation is versatile, the most common forms of glycosylation are N-glycosylation, where the N atom of Asparagine is modified with a glycan, and O-glycosylation where the O atom of serine or threonine residues is modified with a glycan. Here, we report a novel type of O-glycosylation in the bacterial flagellin proteins of two Gram-positive bacteria, Geobacillus kaustophilus and Clostridium botulinum. We demonstrate for the first time that the enzyme flagellin Maf glycosyltransferase is capable of transferring the monosaccharides, N-acetylneuraminic acid 3 and 3-Deoxy-D-manno-octulosonic acid, on to serine and threonine residues of these proteins.

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Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases

Cited by 18 publications

References 22 publications

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di‐N‐acetyllegionaminic Acid‐Containing Glycosides

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di‐N‐acetyllegionaminic Acid‐Containing Glycosides

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

Novel Serine/Threonine-O-glycosylation with N-Acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid by Maf glycosyltransferases

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