Unique gangliosides synthesized in vitro by sialyltransferases from marine bacteria and their characterization: ganglioside synthesis by bacterial sialyltransferases.

On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcß1-4Galß1-4Glcß-Cer and NeuAcα2-6Galß1-3GalNAcß1-4Galß1-4Glcß-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcß1-4Galß1-4Glcß-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.

Sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates.

Bacterial sialyltransferases (STs) from marine sources were characterized using glycosphingolipids (GSLs). Bacterial STs were found to be beta-galacotoside STs. There were two types of STs: (1) ST obtained from strains such as ishi-224, 05JTC1 (#1), ishi-467, 05JTD2 (#2), and faj-16, 05JTE1 (#3), which form alpha2-3 sialic acid (Sia) linkages, named alpha2-3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form alpha2-6 Sia linkages, named alpha2-6ST. All STs showed affinity to neolacto- and lacto-series GSLs, particularly in neolactotetraosyl ceramide (nLc(4)Cer). No large differences were observed in the pH and temperature profiles of enzyme activities. Kinetic parameters obtained by Lineweaver-Burk plot analysis showed that #3 and #4 STs had practical synthetic activity and thus it became easily possible to achieve large-scale ganglioside synthesis (100-300 muM) using these recombinant enzymes. Gangliosides synthesized from nLc(4)Cer by alpha2-3 and alpha2-6STs were structurally characterized by several analytical and immunological methods, and they were identified as IV(3)alphaNeuAc-nLc(4)Cer(S2-3PG) and IV(6)alphaNeuAc-nLc(4)Cer (S2-6PG), respectively. Further characterization of these STs using lactotetraosylceramide (Lc(4)Cer), neolactohexaosylceramide (i antigen), and IV(6)kladoLc(8)Cer (I antigen) showed the synthesis of corresponding gangliosides as well. Synthesized gangliosides showed binding activity to the influenza A virus [A/panama/2007/99 (H3N2)] at a similar level to purified S2-3PG and S2-6PG from mammalian sources. The above evidence suggests that these STs have unique features, including substrate specificities restricted to lacto- and neolactoseries GSLs, as well as catalytic potentials for ganglioside synthesis. This demonstrates that efficient in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing Sias modifications, thereby permitting the exploration of unknown functions.

Glycosphingolipids as a possible signature of microbial communities in activated sludge and the potential contribution of fungi to wastewater treatment under cold conditions.

Seven strains of fungi were isolated from activated sludge and identified as Mucor sp., Geotrichum sp., Trichosporon sp., Candida sp., and Trichoderma sp. by 28S rDNA D2 region sequences analysis. The structures of the main ceramide monosaccharides (CMSs) from these fungi were identified as glucosylceramide (GlcCer) consisting of ceramide moieties of 9-methyl-octadeca-sphingadienine (9-Me d18:2), with 2-hydroxyhexadecanoate (h16:0) (Mucor sp. and Geotrichum sp.), 2-hydroxyoctadecanoate (h18:0) (Trichosporon sp. and Candida sp.), and 2-hydroxyoctadecenoate (h18:1) (Trichoderma sp.). Seasonal changes in glycosphingolipids in activated sludge suggest the possibility that microbial flora in activated sludge changes with the seasons, and that fungi adaptable to low temperatures dominate in the cold period, resulting in the maintenance of stable effluent quality. Mucor sp., Geotrichum sp., and Candida sp. satisfactorily reduced the BOD of synthetic sewage at 10 degrees C. These results indicate that fungi in activated sludge can contribute to wastewater treatment in cold conditions.