title: 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 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.
authors.affiliation: Department of Agriculture and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. ykushi@obihiro.ac.jp
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kushi
authors.forename: Hisashi
authors.initials: H
authors.lastname: Kamimiya
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hiratsuka
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nozaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fukui
authors.forename: Mayumi
authors.initials: M
authors.lastname: Yanagida
authors.forename: Makoto
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Kimihide
authors.initials: K
authors.lastname: Nakamura
authors.forename: Shinobu
authors.initials: S
authors.lastname: Watarai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kasama
authors.forename: Hitomi
authors.initials: H
authors.lastname: Kajiwara
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D005732:Gangliosides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010776:Photobacterium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012799:Sialyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005732:Gangliosides; D011994:Recombinant Proteins; D012799:Sialyltransferases