One-Step Selective Labeling of Native Cell Surface Sialoglycans by Exogenous α2,8-Sialylation

Babulic, Jonathan L.; Kofsky, Joshua M.; Boddington, Marie E.; Kim, Youjin; Leblanc, Emmanuelle V.; Cook, Madeleine G.; Garnier, Cole R.; Emberley-Korkmaz, Sophie; Colpitts, Che C.; Capicciotti, Chantelle J.

doi:10.1021/acschembio.3c00475

Cited by 2 publications

(1 citation statement)

References 84 publications

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“…As B3GNT2 requires LacNAc as the acceptor substrate and is reported to act on glycolipids in addition to glycoproteins, it is feasible that B3GNT2 may also be acting on glycolipid substrates. However, glycolipids may not be extended far enough from the cell surface to be accessible for B3GNT2 transfer, analogous to what has been suggested for sialyltransferases that did not exo-enzymatically label cellular glycolipids despite having reported glycolipid substrate activity . Overall, our data suggest that the apparent glycan subclass specificity of glycosyltransferases employed in exo-enzymatic glycan editing is dependent on the cell line used, a factor that should be considered when reporting enzyme specificity.…”

Section: Resultssupporting

confidence: 62%

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs

De León González,

Boddington,

Kofsky

et al. 2024

ACS Chem. Biol.

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Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1 F383A . The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc-or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc-or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.

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Section: Resultssupporting

confidence: 62%

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs

De León González,

Boddington,

Kofsky

et al. 2024

ACS Chem. Biol.

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Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in Gram-negative bacteria

Alvarado Melendez,

de Jong,

Hartman

et al. 2024

Preprint

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Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid,N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labelling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

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One-Step Selective Labeling of Native Cell Surface Sialoglycans by Exogenous α2,8-Sialylation

Cited by 2 publications

References 84 publications

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs

Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in Gram-negative bacteria

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