Specific Synthesis of Neurostatin and Gangliosides O-Acetylated in the Outer Sialic Acids Using a Sialate Transferase

Abstract

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides’ outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients.

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells

“…The 9-O-acetylated forms of GD2 and GD3 were generated by enzymatic in vitro synthesis using NeuD from C. jejuni, which allows the site-selective introduction of an O-acetyl group at position C9 of a terminal α2,8-linked sialic acid [28]. The O-acetylation of GD3 (Sigma-Aldrich, 345752) and GD2 (Sigma-Aldrich, 345743) was performed according to Romero-Ramirez and co-workers [29]. The reaction was stopped by adding an equal volume of methanol and the gangliosides were purified on Chromabond C18 columns (Macherey-Nagel, Düren, Germany), dried under a nitrogen stream, and then dissolved in chloroform/methanol (1:2, v/v).…”

Sphingolipids: promising lipid-class molecules with potential applications for industry. A review

“…Gangliosides can also undergo O-acetylation at C9 in a sialic acid unit via action of the enzyme, possessing O-acetyltransferase activity. Such acetylated glycosphingolipids are potential therapeutic drugs, against brain tumor development ( Romero-Ramırez et al, 2012).…”