Synthesis of globopentaose using a novel β1,3‐galactosyltransferase activity of the Haemophilus influenzae β1,3‐N‐acetylgalactosaminyltransferase LgtD

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Abstract
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 β1,3‐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 β1,3‐galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galβ‐3GalNAcβ‐3Galα‐4Galβ‐4Glc) which reacts with the monoclonal antibody defining the stage‐specific embryonic antigen‐3. In vitro assays confirmed that LgtD had both β1,3‐GalT and β1,3‐GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.