Acceptor-mediated regioselective enzyme catalyzed sialylation: chemoenzymatic synthesis of GAA-7 ganglioside glycan

Tseng, Hsin-Kai; Su, Yung-Yu; Chang, Ting-Wei; Liu, Hsin-Chien; Li, Pei-Jhen; Chiang, Pei‐Yun; Lin, Chun Cheng

doi:10.1039/d1cc00653c

Cited by 12 publications

(24 citation statements)

References 37 publications

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“…Trisaccharide GalNAcβ3LacβPropN 3 ( 20 ) was synthesized from LacβPropN 3 ( 13 ) and N -acetyl-D-galactosamine (GalNAc) in 90% yield using a one-pot four-enzyme GalNAc-activation and transfer system containing NmLgtA [ 35 ] and uridine 5′-diphosphate N -acetylgalactosamine (UDP-GalNAc) biosynthetic enzymes including Bifidobacterium longum strain ATCC55813 N -acetylhexosamine-1-kinase (BLNahK) [ 47 ], Pasteurella multocida N -acetylglucosamine uridylyltransferase (PmGlmU) [ 48 ], and PmPpA. GalNAcβ3Lac was the oligosaccharide component of ganglioside GAA-7 which showed excellent neuritogenic activity to neuron-like rat adrenal pheochromocytoma (PC12) cells [ 49 , 50 ].…”

Section: Resultsmentioning

confidence: 99%

A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

Zhang

Bai

et al. 2021

Molecules

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Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.

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Section: Resultsmentioning

confidence: 99%

A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

Zhang

Bai

et al. 2021

Molecules

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“…Thus, the fucosylation site was viably controlled on di- N -modified trilactosamines. This acceptor-mediated regioselective enzyme-catalyzed glycosylation was further applied to efficiently synthesize a series of naturally occurring myeloglycans and linear HMOs with fucoses at the indicated sites, which have never been chemically synthesized. Recently, microarrays containing synthetic fucosylated glycans have been employed to investigate the interactions of glycans with E-selectin and other fucose binding proteins. , However, the binding results between synthetic and isolated natural glycans are contradictory .…”

Section: Discussionmentioning

confidence: 99%

Exploring Regioselective Fucosylation Catalyzed by Bacterial Glycosyltransferases through Substrate Promiscuity and Acceptor-Mediated Glycosylation

Tseng,

Wang,

et al. 2023

ACS Catal.

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Fucose in fucosylated glycans is primarily assembled at multiple sets of N-acetyllactosamine (LN) and lacto-N-biose (LNB) units, which are known as Lewis antigens. The synthesis of these compounds is difficult due to the diversity of Lewis antigens on complex glycans. A promising strategy is to perform enzymatic fucosylation with oligosaccharide backbones because the method is stereoselective and synthetically efficient. However, purification is difficult with this method because heterogeneous glycans are produced. Herein, we developed a general strategy to perform sitespecific fucosylation on poly LN or LN/LNB hybrid backbones. We found that trifluoroacetyl glucosamine (GlcNTFA) can function as a GlcNAc substituent and be incorporated into the LN/LNB chain. The TFA group was readily converted to a butyloxycarbonyl (Boc) group, and the resulting GlcNBoc can be accepted by bacterial galactosyltransferases and provides steric hindrance to prevent fucosyltransferase recognition. Thus, enzymatic fucosylation can only occur specifically at GlcNAc sites within GlcNHBoc-containing poly LN/LNB. We demonstrated the feasibility of this strategy by efficiently synthesizing myeloglycans (dodecasaccharides) and internal/terminal fucosylated human milk oligosaccharides. This approach offers a solution to bypass complex chemical glycosylations and control the selectivity of enzymatic fucosylations. Thus, the method shows significant potential for the future synthesis of branched glycans, such as N-glycans and O-glycans.

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“…For example, C7 mono-fluorinated or di-fluorinated sialic acid is recognized by PmST1, Psp2,6ST, as well as Cst-I and Cst-II from C. jejuni , which were used to sialylate glycoprotein A1AT to assess their stabilizing effects [ 121 ]. Neu5Gc methylated in the C8 hydroxyl group was attached to terminal GalNAc through α2,6 linkage by PmST1 and Psp2,6ST, whereas replacement of the C5 acetamide on terminal GalNAc with a smaller azide enabled α2,3 sialylation [ 122 ]. Legionaminic acid, a sialic acid with C7 acetamide and no C9 hydroxyl group, was efficiently transferred by Psp2,6ST(A235M) onto fluorescently labelled LacNAc [ 123 ].…”

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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Acceptor-mediated regioselective enzyme catalyzed sialylation: chemoenzymatic synthesis of GAA-7 ganglioside glycan

Abstract: Herein, we applied PmST1 (a sialyltransferase) to achieve acceptor-mediated regioselective sialylation (AMRS) on the nonreducing end GalNH2 or GalAz (N-azidogalactosamine). Thus, C5 and C8-modified sialic acid was efficiently assembled on...

Cited by 12 publications

References 37 publications

A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

Exploring Regioselective Fucosylation Catalyzed by Bacterial Glycosyltransferases through Substrate Promiscuity and Acceptor-Mediated Glycosylation

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

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