Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a.
We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.
Blixt
Ola
O
Carbohydrate Synthesis and Protein Expression Core D, Consortium for Functional Glycomics, The Scripps Research Institute, Department of Molecular Biology, CB-248A, 92037 La Jolla, USA. olablixt@scripps.edu
Vasiliu
Daniela
D
Allin
Kirk
K
Jacobsen
Nathan
N
Warnock
Dawn
D
Razi
Nahid
N
Paulson
James C
JC
Bernatchez
Stéphane
S
Gilbert
Michel
M
Wakarchuk
Warren
W
D004926:Escherichia coli / Q000201:enzymology; D005678:G(M2) Ganglioside / Q000138:chemical synthesis; D005700:Galactosyltransferases / Q000378:metabolism; D005732:Gangliosides / Q000138:chemical synthesis*; D007790:Lactosylceramides / Q000138:chemical synthesis; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism; D013379:Substrate Specificity
D005732:Gangliosides; D007790:Lactosylceramides; D005678:G(M2) Ganglioside; C026226:ganglioside, GD3; C019403:ganglioside, GD2; C047989:trisialosyllactosylceramide; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; D012799:Sialyltransferases