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.
Highlights
Sialic acid (SA) represents a family of negatively charged mononucleotides with nine-carbon backbones that include the most common N-acetylneuraminic acid (Neu5Ac) and its derivatives
Solvents for high-performance liquid chromatography (HPLC) including acetonitrile and water were purchased from Burdick and Jackson (Muskegon, MI, USA). 2-Aminobenzoic acid (AA), sodium cyanoborohydride, acetic acid, tetrahydrofuran, triethylamine, trifluoroacetic acid (TFA), cytidine monophosphate (CMP)-NeuAc, adenosine 50-triphosphate (ATP), cytidine 50-triphosphate (CTP), cytidine 50-monophosphate (CMP), GDP-galactose, bovine β(1,4)galactosyltransferase, asialofetuin and other reagents were purchased from Sigma-Aldrich
The coding regions including catalytic domains were synthesized with codon optimization for E. coli expression; 16–497 amino acids of P. damsela α(2,6)STs [7], 18–514 amino acids of Photobacterium sp. strain JT-ISH-224 [10], and 16–497 amino acids of P. leiognathi JT-SHIZ-145 [8] were designated as Pd, P224, and P145-ST respectively
Summary
Sialic acid (SA) represents a family of negatively charged mononucleotides with nine-carbon backbones that include the most common N-acetylneuraminic acid (Neu5Ac) and its derivatives It usually locates at the non-reducing terminal of glycans with four linkage types to penultimate residues [Neu5Ac-α(2,3)-Gal, Neu5Ac-α(2,6)-Gal, Neu5Ac-α(2,6)-GalNAc, and Neu5Ac-α (2,8)-Neu5Ac]. Many efforts have been made to ensure homogeneous SA capping in the manufacturing of therapeutic glycoproteins because non-sialylated glycoproteins do not show effective in vivo efficacies owing to a short half-life. While both α(2,3)- and α(2,6)-SA are important for in vivo half-life, it has been elucidated that there are several different biological and pathophysiological roles which are determined by their specific linkages. It has been reported that a large amount of recombinant human α(2,6)-sialyltransferase (hST6Gal1) produced from HEK293F cells is required for the in vitro reaction to generate bi-α (2,6)-sialylated glycan attached to Fc [4]
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