Efficient Enzymatic Glycosylation of Peptides and Oligosaccharides from GalNAc and UTP

Bourgeaux, Vanessa; Cadène, Martine; Piller, Friedrich; Piller, Véronique

doi:10.1002/cbic.200600369

Cited by 21 publications

(7 citation statements)

References 32 publications

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“…They were further peracetylated as in [13]. The chemoenzymatic synthesis of glycoproteins and their purification and characterization were performed with published procedures [29,30] and (S. Pouilly, V. Bourgeaux, F. Piller and V. Piller, submitted).…”

Section: Methodsmentioning

confidence: 99%

“…A 17‐residue peptide (STPSTPSTPSTPSTPAG) was O‐glycosylated with UDP‐GalNAz and a recombinant polypeptide αGalNAc transferase, as previously described [30]. The glycopeptide was coupled to keyhole limpet hemocyanin (Sigma‐Aldrich) with the SureLINK coupling system from KPL (Gaithersburg, MD, USA) and according to the supplier’s recommendations.…”

Section: Methodsmentioning

confidence: 99%

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Metabolic glycoengineering through the mammalian GalNAc salvage pathway

Pouilly¹,

Piller²,

Piller³

2012

The FEBS Journal

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GalNAc is the initial sugar of mucin‐type O‐glycans, and is a component of several tumor antigens. The aim of this work was to determine whether synthetic GalNAc analogs could be taken up from the medium and incorporated into complex cellular O‐glycans. The cell line employed was CHO ldlD, which can only use GalNAc and Gal present in the medium for the synthesis of its glycans. All GalNAc analogs with modified N‐acyl groups (N‐formyl, N‐propionyl, N‐glycolyl, N‐azidoacetyl, N‐bromoacetyl, and N‐chloroacetyl) were incorporated into cellular O‐glycans, although to different extents. The GalNAc analogs linked to Ser or Thr could be extended by the β3‐galactosyltransferase glycoprotein‐N‐acetylgalactosamine 3β‐galactosyl transferase 1 in vitro and in vivo and by α6‐sialyltransferase α‐N‐acetylgalactosaminide‐α‐2,6‐sialyltransferase 1. At the surface of CHO ldlD cells, all analogs were incorporated into sialylated O‐glycan structures like those present on wild‐type CHO cells, indicating that the GalNAc analogs do not change the overall structure of core‐1 O‐glycans. In addition, this study shows that the unnatural synthetic GalNAc analogs can be incorporated into human tumor cells, and that a tumor antigen modified by an analog can be readily detected by a specific antiserum. GalNAc analogs are therefore potential targets for tumor immunotherapy.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Metabolic glycoengineering through the mammalian GalNAc salvage pathway

Pouilly¹,

Piller²,

Piller³

2012

The FEBS Journal

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“…The human ppGalNAcT2-gene was obtained from Geneservice Ltd., UK (IMAGE-clone #5553465). Cloning and production of soluble human ppGalNAcT2 was performed in the yeast Pichia pastoris according to Bourgeaux et al[20] with slight modifications. The coding sequence corresponding to amino acids 51 to 571 was inserted in 3′ of the α-factor signal sequence of a pPICZαB expression vector (Invitrogen, UK, Paisley) modified to introduce an N-terminal hexahistidine tag.…”

Section: Methodsmentioning

confidence: 99%

“…The N-terminal transmembrane domain was replaced by a hexahistidine tag and cloned into pPICzα, a Pichia pastoris vector used for expression and secretion. [20] Culture supernatants were assayed against different peptide substrates, and amongst the peptides tested, the previously reported GAGAPGPTPGPAGAGK sequence ( 1 )[21] was found to be the optimal substrate giving a high rate of glycosylation in 2 h in a solution-phase assay (data not shown). The lysine residue on the C terminus of 1 allowed for easy immobilisation on gold surfaces using well established chemistry (Scheme 1).…”

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confidence: 99%

Enzymatic Glycosylation of Peptide Arrays on Gold Surfaces

et al. 2008

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“…Synthesis with glycosyl transferases is a popular alternative to purely chemical methods; large-scale synthesis of glycopeptides is frequently performed with a set of enzymes enabling transferases to be reused. Bourgeaux et al (2007) have developed a system that reduces the number of enzymes from six to four. Glycosylated products were analyzed by MALDI-TOF MS.…”

Section: Carbohydrate Synthesismentioning

confidence: 99%

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Harvey

2011

Mass Spectrometry Reviews

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This review is the fifth update of the original review, published in 1999, on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2008. The first section of the review covers fundamental studies, fragmentation of carbohydrate ions, use of derivatives and new software developments for analysis of carbohydrate spectra. Among newer areas of method development are glycan arrays, MALDI imaging and the use of ion mobility spectrometry. The second section of the review discusses applications of MALDI MS to the analysis of different types of carbohydrate. Specific compound classes that are covered include carbohydrate polymers from plants, N- and O-linked glycans from glycoproteins, biopharmaceuticals, glycated proteins, glycolipids, glycosides and various other natural products. There is a short section on the use of MALDI mass spectrometry for the study of enzymes involved in glycan processing and a section on the use of MALDI MS to monitor products of the chemical synthesis of carbohydrates with emphasis on carbohydrate-protein complexes and glycodendrimers. Corresponding analyses by electrospray ionization now appear to outnumber those performed by MALDI and the amount of literature makes a comprehensive review on this technique impractical. However, most of the work relating to sample preparation and glycan synthesis is equally relevant to electrospray and, consequently, those proposing analyses by electrospray should also find material in this review of interest.

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Efficient Enzymatic Glycosylation of Peptides and Oligosaccharides from GalNAc and UTP

Cited by 21 publications

References 32 publications

Metabolic glycoengineering through the mammalian GalNAc salvage pathway

Metabolic glycoengineering through the mammalian GalNAc salvage pathway

Enzymatic Glycosylation of Peptide Arrays on Gold Surfaces

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

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