Atsushi Miyagawa

Oral Therapeutic Agents with Highly Clustered Globotriose for Treatment of Shiga Toxigenic Escherichia coli Infections

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

Structural Analysis of the Interaction between Shiga Toxin B Subunits and Linear Polymers Bearing Clustered Globotriose Residues

ABSTRACT We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.

Alternative methods of globotrioside production using Vero cells: a microcarrier system procedure

BackgroundGlycolipids are one component of cell membranes, and are found most prevalently at the surface of the plasma membrane. Animal cells take in amphipathic glycosides, which are later glycosylated after assimilation in biosynthetic pathways. Gycosylated glycosides are released outside of cells to the surrounding culture medium. This represents an accessible method of obtaining complex glycosides.ResultsVero cells are sensitive to Shiga toxins and are known to express the glycosides globotriaosyl ceramide (Gb3) and globotetraosyl ceramide (Gb4) on the surface of the plasma membrane. By administering amphipathic lactosides to Vero cells, the above mentioned glycolipids could be produced by the action of cellular enzymes. In our study, the optimum conditions (seeded cell number, incubated time period, 12-azidododecyl lactoside concentration and medium volume) for the production of Gb3 analogue were investigated. The 87.9 μg/100 mm dish (11.7 % yield) Gb3 analogue was produced under appropriate conditions. The large-scale culture of Vero cells using a microcarrier culture method with repetitions produced about 30 mg of the Gb3 analogue.ConclusionThe mass production of glycosides in Vero cells was carried out on a microcarrier with repeated administration of 12-azidododecyl lactoside. The results indicated that the use of both a microcarrier culture and repetition were highly effective in the production of Gb3, Gb4 and sialyl lactoside (GM3) type-oligosaccharides.