Fig 1.
Expression and purification of recombinant α(2,6)-STs in E. coli.
Bacterial α(2,6)-ST genes (Pd-ST, P224-ST, and P145-ST) were cloned into a pCold II vector for recombinant expression in E. coli (A). Recombinant Pd-ST (B), P224-ST (C), and P145-ST (D) were purified using His-tag affinity chromatography and analyzed using SDS-PAGE. CL: soluble fraction of cell lysate, FT: flow-through fraction, WO: wash-out fraction, E1-3: eluted fractions. Purified α(2,6)-ST protein bands are indicated by the arrows.
Fig 2.
ST activity assays of bacterial α(2,6)-STs using G2 glycan as an accepting substrate.
(A) The conversion of AA-labeled G2 glycan to sialylated glycans by Pd-, P224-, and P145-STs was analyzed by HPLC. The peaks of mono-sialylated (S1) and di-sialylated glycans (S2) as well as G2 glycan are indicated by symbol representation suggested by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/). Blue square: GlcNAc, green circle: mannose, yellow circle: galactose, purple diamond: sialic acid. (B) Relative contents (%) of S1 (gray bar) and S2 glycan (black bar) were obtained by the calculation of integrated peak areas (100 × [The areas of corresponding glycan peaks]/[Total areas of all identified peaks]).
Fig 3.
Prolonged incubation of bacterial α(2,6)-ST reaction decreased sialylated glycans.
The time-courses of the enzyme reactions were performed at 30°C using Pd- (A and B), P224- (C and D), and P145-STs (E and F). In the bar graphs (A, C, and E), relative contents (%) of G2 (white bar), S1 (gray bar), and S2 (black bar) glycans are represented from 0–1,440 min, which were obtained by the calculation of integrated peak areas (100 × [The areas of corresponding glycan peaks]/[Total areas of all identified peaks]). In line graphs (B, D, and F), relative contents (%) of S1 (open diamond) and S2 (filled square) glycans are represented from 0–240 min.
Fig 4.
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]).
Fig 5.
Inhibition of sialidase activity of bacterial α(2,6)-STs by AP.
Sialidase activities of Pd- (A), P224- (B), and P145-STs (C) increased by 1 mM CMP addition were inhibited by supplemental addition of AP in a concentration dependent manner (0.1, 0.4, 1.5, and 5 U/μl). Relative contents (%) of lactose were calculated in the same way as in Fig 4.
Fig 6.
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.
Fig 7.
In vitro sialylation of asialofetuin by P145-ST.
(A) Lectin blot assay was performed using SNA lectin for detection of α(2,6)-sialic acid of fetuin sialylated by P145-ST (upper panel). As a control experiment, immunoblot assay was also carried out using an anti-fetuin antibody (lower panel). N-glycans obtained from asialofetuins sialylated by P145-ST reaction were analyzed using HPLC (B) and MALDI-TOF (C). S1, S2, and S3 represent mono-, di-, and tri-sialylated glycans respectively. Symbols for glycans are the same as in Fig 2. Here, ‘a’: asialofetuin as “a”: asialofetuin as a control without ST reaction, “b”: sialylated asialofetuin in the reaction condition without supplemental addition, “c”: sialylated asialofetuin in the optimized condition with the supplemental addition of CMP-NeuAc and AP.