In situ generated O-Glycan core 1 structure as substrate for Gal(β1–3)GalNAc β-1,6-GlcNAc Transferase

Dudziak, Gregor; Zeng, Steffen; Berger, Eric G.; Gallego, Ricardo Gutiérrez; Kamerling, Johannis P.; Kragl, Udo; Wandrey, Christian

doi:10.1016/s0960-894x(98)00464-8

Cited by 14 publications

(9 citation statements)

References 14 publications

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“…After HPLC purification, 4 was obtained in a yield of 29%. No formation of the core-6 glycopeptide (5, vide infra) could be observed, in contrast to previous studies [13] whereby, using GalNAc(a1-OBn) as acceptor substrate, both GlcNAc(b1-6)GalNAc(a1-OBn) (yield 56%) and GlcNAc (b1-6)[Gal(b1-3)]GalNAc(a1-OBn) (yield 34%) were formed. Apparently, 2 is not such a good acceptor substrate, and after incorporation of GlcNAc the glycopeptide (4) ceases to be a substrate for b-galactosidase.…”

Section: Synthesis Of Muc1a' Glycopeptidescontrasting

confidence: 90%

“…Polypeptide N-acetylgalactosaminyltransferase 3 (GalNAcT3, EC 2.4.1.41) and core-2 b6-N-acetylglucosaminyltransferase (C2GnT, EC 2.4.1.102) were expressed and purified as described [13]. b-Galactosidase from bovine testes (EC 3.2.1.23) was obtained from Oxford Glycosciences, BSA, b4-galactosyltransferase 1 (b4-GalT1, EC 2.4.1.38), and calf intestine alkaline phosphatase (CIAP, EC 3.1.3.1) from Sigma, and a3-fucosyltransferase 3 (a3-FucT3, EC 2.4.1.65) and a3-sialyltransferase 3 (ST3Gal3, EC 2.4.99.5) from CalBioChem.…”

Section: Methodsmentioning

confidence: 99%

“…It should be noted that after 1 d the formation of 4 had come to an end (data not shown). UDP, UMP and uridine concentrations were stable indicating that no UDP-GlcNAc was being metabolized after 1 d. It is suggested that the protease-inhibitor cocktail influences the activity of one or both enzymes since this restricted conversion was not observed in an identical experimental set-up, without the protease inhibitors, using GalNAc(a1-OBn) as acceptor substrate [13].…”

Section: Synthesis Of Muc1a' Glycopeptidesmentioning

confidence: 99%

“…UDP-induced deactivation of the enzyme was overcome by the addition of CIAP. For the generation of 4, rather than using an enriched b3-GalT preparation [20] which resulted in relatively low overall yield, an earlier reported "one-pot" strategy was preferred, employing both b-galactosidase and C2GnT [13,21]. In a pilot experiment using lactose as donor substrate and b-galactosidase from bovine testes, 2 was tested as acceptor substrate for galactosylation.…”

Section: Synthesis Of Muc1a' Glycopeptidesmentioning

confidence: 99%

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Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a’ peptide

Gallego¹,

Dudziak²,

Kragl³

et al. 2003

Biochimie

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Starting from a tumor-associated synthetic MUC1-derived peptide MUC1a' and using a completely enzymatic approach for the synthesis of the core-2 sialyl Lewis X glycopart, the following glycopeptide was synthesized:First, polypeptide N-acetylgalactosaminyltransferase 3 was used to site-specifically glycosylate MUC1a' to give MUC1a'-GalNAc. Then, in a one-pot reaction employing b-galactosidase and core-2 b6-N-acetylglucosaminyltransferase the core-2 O-glycan structure was prepared. The core-2 structure was then sequentially galactosylated, sialylated, and fucosylated by making use of b4-galactosyltransferase 1, a3-sialyltransferase 3, and a3-fucosyltransferase 3, respectively, resulting in the sialyl Lewis X glycopeptide. The overall yield of the final compound was 23% (3.2 mg, 1.4 µmol). During the synthesis three intermediate glycopeptides containing O-linked GalNAc, Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc, and Neu5Ac(a2-3)Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc, respectively, were isolated in mg quantities. All products were characterized by mass spectrometry and NMR spectroscopy.

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Section: Synthesis Of Muc1a' Glycopeptidescontrasting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: Synthesis Of Muc1a' Glycopeptidesmentioning

confidence: 99%

Section: Synthesis Of Muc1a' Glycopeptidesmentioning

confidence: 99%

See 2 more Smart Citations

Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a’ peptide

Gallego¹,

Dudziak²,

Kragl³

et al. 2003

Biochimie

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“…Recently, b-galactosidase from bovine testes was used in a one pot reaction together with a recombinant b-1,6-GlcNAc transferase. The galactosidase, which reversibly links galactose via a (b1-3) linkage to N-acetylgalactosamine, provides the substrate for the GlcNAc transferase in situ [54]. Since this synthesis ended up with a mixture of core 2 and core 6 (GlcNAc(b1-6)GalNAc) structures, and can only be driven towards high yields of the core 6 structure (yield Ͼ90%), we preferred the use of a crude b3-GalT preparation in combination with C2GnT.…”

Section: Discussionmentioning

confidence: 99%

Untitled

Zeng

Gallego

Dinter

et al. 1999

Glycoconjugate Journal

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Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(bet a1-3)]GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 micromol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.

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Hydrolysis and Formation of Glycosidic Bonds

Wong

2002

Enzyme Catalysis in Organic Synthesis

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In situ generated O-Glycan core 1 structure as substrate for Gal(β1–3)GalNAc β-1,6-GlcNAc Transferase

Cited by 14 publications

References 14 publications

Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a’ peptide

Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a’ peptide

Untitled

Hydrolysis and Formation of Glycosidic Bonds

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