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Enhanced Bacterial α(2,6)-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate.

Kang JY, Lim SJ, Kwon O, Lee SG, Kim HH, Oh DB - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Korea.

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.

No MeSH data available.


Related in: MedlinePlus

Sialidase activity analysis of bacterial α(2,6)-STs.(A) Removal of SA from AA-labeled 6’-sialyllactoses by Pd-, P224-, and P145-STs was analyzed using HPLC. Symbols for galactose and Neu5Ac are the same as in Fig 2, while glucose is represented by a blue circle. (B) The effects of nucleotides (CMP, CDP, CTP, cytidine, ADP, and ATP) on the α(2,6)-sialidase activity of P145-ST were examined. Reaction mixtures containing 1 mM of each nucleotide were incubated for 2 h. (C) Changes of P145-ST sialidase activities were also analyzed with addition of various concentrations (0.01, 0.05, 0.1, 0.5, 1, and 10 mM) of CMP (filled circle), CDP (triangle), CTP (reverse triangle), and cytidine (filled circle). The reaction mixtures were incubated for 2 h at 37°C. Relative contents (%) of lactose generated by sialidase activity were obtained by the calculation of integrated peak areas (100 × [The areas of lactose peaks]/[Total areas of lactose and sialyllactose peaks]).
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pone.0133739.g004: Sialidase activity analysis of bacterial α(2,6)-STs.(A) Removal of SA from AA-labeled 6’-sialyllactoses by Pd-, P224-, and P145-STs was analyzed using HPLC. Symbols for galactose and Neu5Ac are the same as in Fig 2, while glucose is represented by a blue circle. (B) The effects of nucleotides (CMP, CDP, CTP, cytidine, ADP, and ATP) on the α(2,6)-sialidase activity of P145-ST were examined. Reaction mixtures containing 1 mM of each nucleotide were incubated for 2 h. (C) Changes of P145-ST sialidase activities were also analyzed with addition of various concentrations (0.01, 0.05, 0.1, 0.5, 1, and 10 mM) of CMP (filled circle), CDP (triangle), CTP (reverse triangle), and cytidine (filled circle). The reaction mixtures were incubated for 2 h at 37°C. Relative contents (%) of lactose generated by sialidase activity were obtained by the calculation of integrated peak areas (100 × [The areas of lactose peaks]/[Total areas of lactose and sialyllactose peaks]).

Mentions: Among three α(2,6)-STs, only Pd-ST had been proven experimentally to have α(2,6)-sialidase activity [14]. In this study, we examined whether P224- and P145-STs have sialidase activity using an AA-labeled 6`-sialyllactose as a substrate. After incubation with each of the bacterial α(2,6)-STs, HPLC analysis showed that some 6`-sialyllactose was converted to lactose (Fig 4A). In a 2 h reaction to compare them under the same condition, P224-ST showed the strongest sialidase activity (generation of ~70% lactose), while Pd- and P145-ST generated ~19% and ~13% lactose.


Enhanced Bacterial α(2,6)-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate.

Kang JY, Lim SJ, Kwon O, Lee SG, Kim HH, Oh DB - PLoS ONE (2015)

Sialidase activity analysis of bacterial α(2,6)-STs.(A) Removal of SA from AA-labeled 6’-sialyllactoses by Pd-, P224-, and P145-STs was analyzed using HPLC. Symbols for galactose and Neu5Ac are the same as in Fig 2, while glucose is represented by a blue circle. (B) The effects of nucleotides (CMP, CDP, CTP, cytidine, ADP, and ATP) on the α(2,6)-sialidase activity of P145-ST were examined. Reaction mixtures containing 1 mM of each nucleotide were incubated for 2 h. (C) Changes of P145-ST sialidase activities were also analyzed with addition of various concentrations (0.01, 0.05, 0.1, 0.5, 1, and 10 mM) of CMP (filled circle), CDP (triangle), CTP (reverse triangle), and cytidine (filled circle). The reaction mixtures were incubated for 2 h at 37°C. Relative contents (%) of lactose generated by sialidase activity were obtained by the calculation of integrated peak areas (100 × [The areas of lactose peaks]/[Total areas of lactose and sialyllactose peaks]).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4521712&req=5

pone.0133739.g004: Sialidase activity analysis of bacterial α(2,6)-STs.(A) Removal of SA from AA-labeled 6’-sialyllactoses by Pd-, P224-, and P145-STs was analyzed using HPLC. Symbols for galactose and Neu5Ac are the same as in Fig 2, while glucose is represented by a blue circle. (B) The effects of nucleotides (CMP, CDP, CTP, cytidine, ADP, and ATP) on the α(2,6)-sialidase activity of P145-ST were examined. Reaction mixtures containing 1 mM of each nucleotide were incubated for 2 h. (C) Changes of P145-ST sialidase activities were also analyzed with addition of various concentrations (0.01, 0.05, 0.1, 0.5, 1, and 10 mM) of CMP (filled circle), CDP (triangle), CTP (reverse triangle), and cytidine (filled circle). The reaction mixtures were incubated for 2 h at 37°C. Relative contents (%) of lactose generated by sialidase activity were obtained by the calculation of integrated peak areas (100 × [The areas of lactose peaks]/[Total areas of lactose and sialyllactose peaks]).
Mentions: Among three α(2,6)-STs, only Pd-ST had been proven experimentally to have α(2,6)-sialidase activity [14]. In this study, we examined whether P224- and P145-STs have sialidase activity using an AA-labeled 6`-sialyllactose as a substrate. After incubation with each of the bacterial α(2,6)-STs, HPLC analysis showed that some 6`-sialyllactose was converted to lactose (Fig 4A). In a 2 h reaction to compare them under the same condition, P224-ST showed the strongest sialidase activity (generation of ~70% lactose), while Pd- and P145-ST generated ~19% and ~13% lactose.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, Korea.

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.

No MeSH data available.


Related in: MedlinePlus