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Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells.

Kudelka MR, Antonopoulos A, Wang Y, Duong DM, Song X, Seyfried NT, Dell A, Haslam SM, Cummings RD, Ju T - Nat. Methods (2015)

Bottom Line: Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS).Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus.Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

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The chaperone Cosmc and active T-synthase are required for production of Core 1- and 2-based Bn-O-glycans. (a) T-synthase activity was measured and reported relative to mannosidase control enzyme for LS174T colorectal cells, LOX melanoma cells, and Jurkat T cells with or without a functional Cosmc. Enzymes were assayed in triplicate (n = 2), and a representative experiment is shown, mean ± SD of triplicates. (b–d) The same cells were incubated with 50µM Ac3GalNAc-Bn for 3 days and the media was analyzed for Bn-O-glycans for LS174T (b), LOX (c), and Jurkat (d) ± Cosmc. Only major glycans are annotated (composition) for LS174T (b) for clarity; highly fucosylated minor species as shown in Supplementary Fig. 17b were also observed. Spectra for each graph (b–d) are off-set, but scaled to same absolute intensity for each cell; representative profiles are shown (n = 2).
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Figure 2: The chaperone Cosmc and active T-synthase are required for production of Core 1- and 2-based Bn-O-glycans. (a) T-synthase activity was measured and reported relative to mannosidase control enzyme for LS174T colorectal cells, LOX melanoma cells, and Jurkat T cells with or without a functional Cosmc. Enzymes were assayed in triplicate (n = 2), and a representative experiment is shown, mean ± SD of triplicates. (b–d) The same cells were incubated with 50µM Ac3GalNAc-Bn for 3 days and the media was analyzed for Bn-O-glycans for LS174T (b), LOX (c), and Jurkat (d) ± Cosmc. Only major glycans are annotated (composition) for LS174T (b) for clarity; highly fucosylated minor species as shown in Supplementary Fig. 17b were also observed. Spectra for each graph (b–d) are off-set, but scaled to same absolute intensity for each cell; representative profiles are shown (n = 2).

Mentions: To confirm that Bn-α-GalNAc can only be utilized by glycosyltransferases involved in mucin-type O-glycan biosynthesis, but not other irrelevant or unknown pathway(s), we performed CORA on cells with mutant or wild-type Cosmc. Only cells with functional Cosmc and active T-synthase secreted Bn-O-glycans when administered Ac3GalNAc-Bn (Fig. 2). Furthermore, cells incubated with the isomer Ac3GlcNAc-β-Bn (Supplementary Fig. 1b) secreted no Bn-O-glycans or only the simple trisaccharide Neu5Ac-Gal-GlcNAc-Bn (Supplementary Fig. 11), indicating that Ac3GalNAc-α-Bn is specific for mucin-type O-glycans. These demonstrate that CORA faithfully reports the O-glycome and that modification in cells requires active T-synthase.


Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells.

Kudelka MR, Antonopoulos A, Wang Y, Duong DM, Song X, Seyfried NT, Dell A, Haslam SM, Cummings RD, Ju T - Nat. Methods (2015)

The chaperone Cosmc and active T-synthase are required for production of Core 1- and 2-based Bn-O-glycans. (a) T-synthase activity was measured and reported relative to mannosidase control enzyme for LS174T colorectal cells, LOX melanoma cells, and Jurkat T cells with or without a functional Cosmc. Enzymes were assayed in triplicate (n = 2), and a representative experiment is shown, mean ± SD of triplicates. (b–d) The same cells were incubated with 50µM Ac3GalNAc-Bn for 3 days and the media was analyzed for Bn-O-glycans for LS174T (b), LOX (c), and Jurkat (d) ± Cosmc. Only major glycans are annotated (composition) for LS174T (b) for clarity; highly fucosylated minor species as shown in Supplementary Fig. 17b were also observed. Spectra for each graph (b–d) are off-set, but scaled to same absolute intensity for each cell; representative profiles are shown (n = 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697867&req=5

Figure 2: The chaperone Cosmc and active T-synthase are required for production of Core 1- and 2-based Bn-O-glycans. (a) T-synthase activity was measured and reported relative to mannosidase control enzyme for LS174T colorectal cells, LOX melanoma cells, and Jurkat T cells with or without a functional Cosmc. Enzymes were assayed in triplicate (n = 2), and a representative experiment is shown, mean ± SD of triplicates. (b–d) The same cells were incubated with 50µM Ac3GalNAc-Bn for 3 days and the media was analyzed for Bn-O-glycans for LS174T (b), LOX (c), and Jurkat (d) ± Cosmc. Only major glycans are annotated (composition) for LS174T (b) for clarity; highly fucosylated minor species as shown in Supplementary Fig. 17b were also observed. Spectra for each graph (b–d) are off-set, but scaled to same absolute intensity for each cell; representative profiles are shown (n = 2).
Mentions: To confirm that Bn-α-GalNAc can only be utilized by glycosyltransferases involved in mucin-type O-glycan biosynthesis, but not other irrelevant or unknown pathway(s), we performed CORA on cells with mutant or wild-type Cosmc. Only cells with functional Cosmc and active T-synthase secreted Bn-O-glycans when administered Ac3GalNAc-Bn (Fig. 2). Furthermore, cells incubated with the isomer Ac3GlcNAc-β-Bn (Supplementary Fig. 1b) secreted no Bn-O-glycans or only the simple trisaccharide Neu5Ac-Gal-GlcNAc-Bn (Supplementary Fig. 11), indicating that Ac3GalNAc-α-Bn is specific for mucin-type O-glycans. These demonstrate that CORA faithfully reports the O-glycome and that modification in cells requires active T-synthase.

Bottom Line: Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS).Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus.Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

Show MeSH
Related in: MedlinePlus