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

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 b1,3-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][198][199][200][201][202][203]. Here, we found that LgtD has an additional b1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galb-3GalNAcb-3Gala-4Galb-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both b1,3-GalT and b1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.

Chemoenzymatic Syntheses of Tumor‐Associated Carbohydrate Antigen Globo‐H and Stage‐Specific Embryonic Antigen 4

“…17 α was deprotected in the same manner as described for the pentasaccharide 3 , producing the tetrasaccharide 23 in 50% yield (Scheme 4b). Tetrasaccharide 23 can serve as a substrate to galactosyl transferase LgtD,[41,74] providing an alternative access to SSEA-3.…”

“…ide 23 can serve as a substrate to galactosyl transferase LgtD, [41,74] providing an alternative access to SSEA-3.…”

“…Thus, chemoenzymatic synthesis, through the combination of the power of enzymatic synthesis with the flexibility of chemical synthesis, presents a potent approach to access complex oligosaccharides. Several gangliosides such as GM3,[38] GM2,[39] GM1,[39] GD1a,[39] Gb3[40] and SSEA-3[41] have been synthesized chemoenzymatically. Herein, we report our studies on chemoenzymatic syntheses of Globo-H 1b and SSEA-4 2b , both of which contain aminopropyl side chains and can be conjugated to protein carriers for future immunological studies.…”

Auf dem Weg zur automatisierten Oligosaccharid‐ Synthese

“…Mit gentechnisch veränderten E.-coli-Stämmen konnten erfolgreich (in manchen Fällen im Multigramm-Maßstab) konjugierbare Saccharidreste von GM2 und GM3, [182] die Kohlenhydratreste verschiedener tumorassoziierter Oligosaccharide wie GD3 [183] und SLe x [181c] und Globoside wie Globotetraose, [328] Globopentaose [329] und Globo H [330] synthetisiert werden.…”

Immunosignaturing Can Detect Products from Molecular Markers in Brain Cancer

“…Antibodies make excellent biomarkers because they are stable in serum, have high specificity and affinity to their cognate antigen, are abundant, and enable retrospective studies. An activated B cell can produce 5000–20,000 antibodies per minute [8], [9] while replicating every ∼70 hr [10] with a lifespan up to 4½ months [11], [12], leading to >10 11 amplification of a specific signal per week. Antibody-based biomarkers avoid the dilution problem seen with proteomic biomarkers [13], [14] and in are not only highly abundant but can be physically captured at nano- and picomolar affinities.…”