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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

Optimized P145-ST reaction for increase of S2 glycan generation.For increase of S2 glycan content (%), additional CMP-NeuAc (A) or AP (B) was added 30 min after the beginning of the P145-ST reaction. Further increase of S2 glycan content was achieved by the simultaneous addition of CMP-NeuAc and AP at 30 min of the reaction (C). Through two sequential additions of both CMP-NeuAc and AP at 30 and 120 min of the reaction, relative content of S2 glycan increased up to 98% (D). Arrows indicate the time points for additions of CMP-NeuAc and/or AP. Open circles and filled squares represent the relative contents (%) of S2 glycan generated by the reactions with and without the supplemental addition respectively.
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pone.0133739.g006: Optimized P145-ST reaction for increase of S2 glycan generation.For increase of S2 glycan content (%), additional CMP-NeuAc (A) or AP (B) was added 30 min after the beginning of the P145-ST reaction. Further increase of S2 glycan content was achieved by the simultaneous addition of CMP-NeuAc and AP at 30 min of the reaction (C). Through two sequential additions of both CMP-NeuAc and AP at 30 and 120 min of the reaction, relative content of S2 glycan increased up to 98% (D). Arrows indicate the time points for additions of CMP-NeuAc and/or AP. Open circles and filled squares represent the relative contents (%) of S2 glycan generated by the reactions with and without the supplemental addition respectively.

Mentions: The content of di-sialylated S2 glycan reached a maximum at 30 min in the P145-ST reaction and decreased after that (filled squares in Figs 3F and 6). The supply of CMP-Neu5Ac to the reaction at 30 min led to a further increase in S2 glycan content up to 83% at 60 min, and then it started to decrease (open circles in Fig 6A). On the other hand, the addition of AP at 30 min prevented the decline of S2 glycan content, and the content level was maintained for the tested time (open circles in Fig 6B). With simultaneous addition of both CMP-Neu5Ac and AP to the reaction at 30 min, the S2 glycan content increased up to 92% and maintained that level for 240 min (open circles in Fig 6C). To further enhance the S2 glycan content, both CMP-Neu5Ac and AP were added to the reaction at 30 and 120 min, and this resulted in the S2 glycan content reaching 98% (open circles in Fig 6D). These results show that the α(2,6)-sialylation efficiency of the P145-ST reaction can be enhanced greatly through the supplementation of CMP-Neu5Ac and dephosphorylation of the generated CMP to cytidine by AP treatment.


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)

Optimized P145-ST reaction for increase of S2 glycan generation.For increase of S2 glycan content (%), additional CMP-NeuAc (A) or AP (B) was added 30 min after the beginning of the P145-ST reaction. Further increase of S2 glycan content was achieved by the simultaneous addition of CMP-NeuAc and AP at 30 min of the reaction (C). Through two sequential additions of both CMP-NeuAc and AP at 30 and 120 min of the reaction, relative content of S2 glycan increased up to 98% (D). Arrows indicate the time points for additions of CMP-NeuAc and/or AP. Open circles and filled squares represent the relative contents (%) of S2 glycan generated by the reactions with and without the supplemental addition respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133739.g006: Optimized P145-ST reaction for increase of S2 glycan generation.For increase of S2 glycan content (%), additional CMP-NeuAc (A) or AP (B) was added 30 min after the beginning of the P145-ST reaction. Further increase of S2 glycan content was achieved by the simultaneous addition of CMP-NeuAc and AP at 30 min of the reaction (C). Through two sequential additions of both CMP-NeuAc and AP at 30 and 120 min of the reaction, relative content of S2 glycan increased up to 98% (D). Arrows indicate the time points for additions of CMP-NeuAc and/or AP. Open circles and filled squares represent the relative contents (%) of S2 glycan generated by the reactions with and without the supplemental addition respectively.
Mentions: The content of di-sialylated S2 glycan reached a maximum at 30 min in the P145-ST reaction and decreased after that (filled squares in Figs 3F and 6). The supply of CMP-Neu5Ac to the reaction at 30 min led to a further increase in S2 glycan content up to 83% at 60 min, and then it started to decrease (open circles in Fig 6A). On the other hand, the addition of AP at 30 min prevented the decline of S2 glycan content, and the content level was maintained for the tested time (open circles in Fig 6B). With simultaneous addition of both CMP-Neu5Ac and AP to the reaction at 30 min, the S2 glycan content increased up to 92% and maintained that level for 240 min (open circles in Fig 6C). To further enhance the S2 glycan content, both CMP-Neu5Ac and AP were added to the reaction at 30 and 120 min, and this resulted in the S2 glycan content reaching 98% (open circles in Fig 6D). These results show that the α(2,6)-sialylation efficiency of the P145-ST reaction can be enhanced greatly through the supplementation of CMP-Neu5Ac and dephosphorylation of the generated CMP to cytidine by AP treatment.

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