title: Enhanced Bacterial α(2,6)-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate.
abstract: Bacterial α(2,6)-sialyltransferases (STs) from Photobacterium damsela, Photobacterium sp. JT-ISH-224, and P. leiognathi JT-SHIZ-145 were recombinantly expressed in Escherichia coli and their ST activities were compared directly using a galactosylated bi-antennary N-glycan as an acceptor substrate. In all ST reactions, there was an increase of sialylated glycans at shorter reaction times and later a decrease in prolonged reactions, which is related with the inherent sialidase activities of bacterial STs. These sialidase activities are greatly increased by free cytidine monophosphate (CMP) generated from a donor substrate CMP-N-acetylneuraminic acid (CMP-Neu5Ac) during the ST reactions. The decrease of sialylated glycans in prolonged ST reaction was prevented through an inhibition of sialidase activity by simple treatment of alkaline phosphatase (AP), which dephosphorylates CMP to cytidine. Through supplemental additions of AP and CMP-Neu5Ac to the reaction using the recombinant α(2,6)-ST from P. leiognathi JT-SHIZ-145 (P145-ST), the content of bi-sialylated N-glycan increased up to ~98% without any decrease in prolonged reactions. This optimized P145-ST reaction was applied successfully for α(2,6)-sialylation of asialofetuin, and this resulted in a large increase in the populations of multi-sialylated N-glycans compared with the reaction without addition of AP and CMP-Neu5Ac. These results suggest that the optimized reaction using the recombinant P145-ST readily expressed from E. coli has a promise for economic glycan synthesis and glyco-conjugate remodeling.
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Korea.
authors.forename: Ji-Yeon
authors.initials: JY
authors.lastname: Kang
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, Korea.
authors.forename: Se-Jong
authors.initials: SJ
authors.lastname: Lim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, Korea.
authors.forename: Ohsuk
authors.initials: O
authors.lastname: Kwon
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, Korea.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
authors.affiliation: College of Pharmacy, Chung-Ang University, Seoul, Korea.
authors.forename: Ha Hyung
authors.initials: HH
authors.lastname: Kim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, Korea.
authors.forename: Doo-Byoung
authors.initials: DB
authors.lastname: Oh
mesh_terms: D000469:Alkaline Phosphatase / Q000494:pharmacology; D003568:Cytidine Monophosphate / Q000378:metabolism*; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000494:pharmacology; D009439:Neuraminidase / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D010776:Photobacterium / Q000201:enzymology*; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D000469:Alkaline Phosphatase; D009439:Neuraminidase; D003568:Cytidine Monophosphate; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase