Metabolically incorporated photocrosslinking sialic acid covalently captures a ganglioside–protein complex

Bond, Michelle; Whitman, Chad M.; Kohler, Jennifer J.

doi:10.1039/c0mb00069h

Cited by 40 publications

(58 citation statements)

References 25 publications

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“…NEU3 activity on cells may be a result of incorporation of SiaDAz(2me) into ganglioside substrates. 33,34 Interestingly, we found that NanA, AUNA, and NEU4, each of which showed no detectable activity toward SiaDAz(2me) in our cell-free assay, cleaved between 50 and 70 % of SiaDAz(2me) on cells surfaces. Of all the sialidases tested, only STNA was completely inactive toward SiaDAz(2me) on BJAB K20 cell surfaces, with 100 % of SiaDAz(2me) remaining after STNA treatment.…”

Section: Resultsmentioning

confidence: 69%

Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities

et al. 2015

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Sialic acid-mediated interactions play critical roles on the cell surface, providing impetus for the development of methods to study this important monosaccharide. In particular, photocrosslinking sialic acids incorporated onto cell surfaces have allowed covalent capture of transient interactions between sialic acids and sialic acid-recognizing proteins via crosslinking. However, natural sialic acids also present on the cell surface compete with photocrosslinking sialic acids in binding events, limiting crosslinking yields. In order to improve the utility of one such photocrosslinking sialic acid, SiaDAz, we examined a number of sialidases, enzymes that remove sialic acids from glycoconjugates, to find one that would cleave natural sialic acids but remain inactive toward SiaDAz. Using this sialidase, we improved SiaDAz-mediated crosslinking of an anti-sialyl Lewis X antibody and of endoglin. This protocol can be applied generally to sialic acid-mediated interactions and will facilitate identification of sialic acid binding partners.

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Section: Resultsmentioning

confidence: 69%

Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities

et al. 2015

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“…Lysates incubated with UDP-GlcNDAz produced a product whose mass corresponded to the intact O-GlcNDAz-modified peptide (m∕z ¼ 1; 309) and an additional product whose mass was consistent with loss of N 2 from the O-GlcNDAz-modified peptide (m∕z ¼ 1; 281). Loss of N 2 likely occurs during the mass spectrometry ionization step, a phenomenon we have observed with other diazirine-containing molecules (19). When OGTcontaining lysates were incubated with an equimolar mixture of UDP-GlcNAc and UDP-GlcNDAz, masses corresponding to both O-GlcNAc-and O-GlcNDAz-modified peptides were observed.…”

Section: Resultsmentioning

confidence: 94%

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Boyce

Wands

et al. 2012

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

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O-linked β- N -acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

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“…[8, 19, 21, 27] However, attempts to produce SiaDAz-containing glycoconjugates in other cell lines resulted in variable outcomes. To assess the cell line-dependence of SiaDAz incorporation, we cultured fifteen cell lines with 100 μM Ac 4 ManNDAz for 48–72 h. We isolated the membrane fractions from these cells, released the glycoconjugate-bound sialic acids, and quantified the amounts of Neu5Ac and SiaDAz.…”

Section: Resultsmentioning

confidence: 99%

Cellular metabolism of unnatural sialic acid precursors

et al. 2015

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Carbohydrates, in addition to their metabolic functions, serve important roles as receptors, ligands, and structural molecules for diverse biological processes. Insight into carbohydrate biology and mechanisms has been aided by metabolic oligosaccharide engineering (MOE). In MOE, unnatural carbohydrate analogs with novel functional groups are incorporated into cellular glycoconjugates and used to probe biological systems. While MOE has expanded knowledge of carbohydrate biology, limited metabolism of unnatural carbohydrate analogs restricts its use. Here we assess metabolism of SiaDAz, a diazirine-modified analog of sialic acid, and its cell-permeable precursor, Ac4ManNDAz. We show that the efficiency of Ac4ManNDAz and SiaDAz metabolism depends on cell type. Our results indicate that different cell lines can have different metabolic roadblocks in the synthesis of cell surface SiaDAz. These findings point to roles for promiscuous intracellular esterases, kinases, and phosphatases during unnatural sugar metabolism and provide guidance for ways to improve MOE.

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Metabolically incorporated photocrosslinking sialic acid covalently captures a ganglioside–protein complex

Abstract: When photoirradiated, an unnatural sialic acid analog can covalently capture the complex formed by ganglioside GM1 and cholera toxin subunit B.

Cited by 40 publications

References 25 publications

Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities

Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Cellular metabolism of unnatural sialic acid precursors

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