Synthesis of globopentaose using a novel beta1,3-galactosyltransferase activity of the Haemophilus influenzae beta1,3-N-acetylgalactosaminyltransferase LgtD.
We have previously described a bacterial system for the conversion of globotriaose (Gb3) into globotetraose (Gb4) by a metabolically engineered Escherichia coli strain expressing the Haemophilus influenzae lgtD gene encoding beta1,3-N-acetylgalactosaminyltransferase [Antoine, T., Bosso, C., Heyraud, A. Samain, E. (2005) Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli. Biochimie 87, 197-203]. Here, we found that LgtD has an additional beta1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galbeta-3GalNAcbeta-3Galalpha-4Galbeta-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both beta1,3-GalT and beta1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.
Randriantsoa
Mialy
M
Centre de Recherches sur les Macromolécules Végétales, BP 53, 38041 Grenoble Cedex 9, France.
Drouillard
Sophie
S
Breton
Christelle
C
Samain
Eric
E
D001426:Bacterial Proteins / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D005915:Globosides / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006193:Haemophilus influenzae / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005915:Globosides; D009844:Oligosaccharides; C035968:globotetraose; C462020:LgtD protein, Haemophilus influenzae; D017350:N-Acetylgalactosaminyltransferases