Glycobiology Advance Access published online on October 16, 2009
Glycobiology, doi:10.1093/glycob/cwp160
Sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates
a Department of Agriculture and Life Science
b Health Care Administration Center, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan
c Laboratory of Veterinary Immunology, Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, 598-8531, Japan
d Instrumental Analysis Research Center, Tokyo Medical Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
e Glycotechnology Business Unit, Plant Innovation Center, Japan Tabacco Inc., 700 Higashibara, Iwata, Shizuoka, 483-0802, Japan
* Address Correspondence to: Yasunori Kushi, Obihiro Univ. of Agri. & Vet. Med., Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. Tel.: +81 -155-49-5555; Fax: +81-155-49-5577; e-mail: ykushi{at}obihiro.ac.jp
Received on March 11, 2009; accepted on October 2, 2009
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 
2–3 sialic acid (Sia) linkages, named 2–3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form 2–6 Sia linkages, named 2–6ST. 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 the practical synthetic activity and thus it became easily possible to achieve large-scale ganglioside synthesis (100
300µM) using these recombinant enzymes. Gangliosides synthesized from nLc4Cer by 2–3 and 2–6STs were structurally characterized by several analytical and immunological methods, and they were identified as IV3NeuAc-nLc4Cer(S2–3PG) and IV6NeuAc-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 ganglioside synthesis could be a valuable tool for selectively synthesizing Sias modifications, thereby permitting the exploration of unknown functions.
Key words: bacterial sialyltransferase / gangliosides synthesis / sialylparagloboside / Ii antigens / in vitro synthesis