Sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates

Abstract

Bacterial sialyltransferases (STs) from marine sources were characterized using glycosphingolipids (GSLs). Bacterial STs were found to be β-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 α2-3 sialic acid (Sia) linkages, named α2-3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form α2-6 Sia linkages, named α2-6ST. All STs showed affinity to neolacto- and lacto-series GSLs, particularly in neolactotetraosyl ceramide (nLc4Cer). 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 μM) using these recombinant enzymes. Gangliosides synthesized from nLc4Cer by α2-3 and α2-6STs were structurally characterized by several analytical and immunological methods, and they were identified as IV3αNeuAcnLc4Cer(S2-3PG) and IV6αNeuAc-nLc4Cer (S2-6PG), respectively. Further characterization of these STs using lactotetraosylceramide (Lc4Cer), neolactohexaosylceramide (i antigen), and IV6kladoLc8Cer (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 ganglio-side synthesis could be a valuable tool for selectively synthesizing Sias modifications, thereby permitting the exploration of unknown functions. © The Author 2009. Published by Oxford University Press.