Clickable Galactose Analogues for Imaging Glycans in Developing Zebrafish

Daughtry, Jessica L.; Cao, Wendy; Ye, Johnny; Baskin, Jeremy M.

doi:10.1021/acschembio.9b00898

Cited by 17 publications

(11 citation statements)

References 32 publications

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“…Protein-based reporter reagents have enabled the study of glycobiology but rarely probe nonaccessible glycan core structures. Thus far, the forays made into developing chemical tools have yielded an arsenal of monosaccharide analogs: for instance, of ManNAc/Sia ( 8 , 56 – 58 ), GlcNAc ( 18 – 20 , 35 ), Fuc ( 59 ), Gal ( 60 , 61 ), and GalNAc/GlcNAc ( 10 , 15 , 17 , 62 , 63 ). Probes are typically selected based on their labeling intensity, which in turn, is often a function of poor glycan specificity.…”

Section: Discussionmentioning

confidence: 99%

Metabolic precision labeling enables selective probing of O-linked N -acetylgalactosamine glycosylation

Debets

Tastan

Wisnovsky

et al. 2020

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

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Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)–linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine–4-epimerase (GALE) like conventional GalNAc–based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan–specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, “bump-and-hole” (BH)–GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

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Section: Discussionmentioning

confidence: 99%

Metabolic precision labeling enables selective probing of O-linked N -acetylgalactosamine glycosylation

Debets

Tastan

Wisnovsky

et al. 2020

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

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“…However, few documents disclose the roles of Gal including its analogues in the glycosylation changes in development and disease progression due to the strict substrate specificity. Recently, Baskin’s group developed 6-alkynyl UDP-Gal for imaging N-glycans in developing zebrafish in vivo ( Daughtry et al, 2020 ). In addition, they did not find any obviously metabolic labeling of cell-surface glycans using peracetylated 6-azide or 6-alkynyl Gal, probably resulting from the limited tolerance of GALK to unnatural substrates, or the corresponding UDP-Gal analogues are not recognized by transporters for Golgi import.…”

Section: Introductionmentioning

confidence: 99%

The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

et al. 2021

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Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac46AzGal in a dose- and time-dependent manner. It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.

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“…Baskin and co‐workers have just disclosed 6‐alkynyl uridine diphosphate galactose derivatives, followed by click chemistry tagging, to visualize glycosylation during zebrafish development. Together with the work presented here, it constitutes the first examples of the use of galactose reporters with MOE to study the roles of galactose in different biological settings …”

Section: Figurementioning

confidence: 91%

A Sweet Galactose Transfer: Metabolic Oligosaccharide Engineering as a Tool To Study Glycans in Plasmodium Infection

Kitowski

Bernardes

2020

ChemBioChem

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The introduction of chemical reporter groups into glycan structures through metabolic oligosaccharide engineering (MOE) followed by bio‐orthogonal ligation is an important tool to study glycosylation. We show the incorporation of synthetic galactose derivatives that bear terminal alkene groups in hepatic cells, with and without infection by Plasmodium berghei parasites, the causative agent of malaria. Additionally, we demonstrated the contribution of GLUT1 to the transport of these galactose derivatives, and observed a consistent increase in the uptake of these compounds going from naïve to P. berghei‐infected cells. Finally, we used MOE to study the interplay between Plasmodium parasites and their mosquito hosts, to reveal a possible transfer of galactose building blocks from the latter to the former. This strategy has the potential to provide new insights into Plasmodium glycobiology as well as for the identification and characterization of key glycan structures for further vaccine development.

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Clickable Galactose Analogues for Imaging Glycans in Developing Zebrafish

Cited by 17 publications

References 32 publications

Metabolic precision labeling enables selective probing of O-linked N -acetylgalactosamine glycosylation

Metabolic precision labeling enables selective probing of O-linked N -acetylgalactosamine glycosylation

The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

A Sweet Galactose Transfer: Metabolic Oligosaccharide Engineering as a Tool To Study Glycans in Plasmodium Infection

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