Fig 1.
A: Schematic presentation of recombinant TconTS-LD fusion proteins expressed in bacteria. Fusion tags flanking TconTS-LD are: His: poly histidine tag, MBP: maltose binding protein tag, TEV: tobacco etch virus protease cleavage site, 3C: human rhinovirus 3C protease cleavage site, SNAP: SNAP-tag, Strep: Strep-tag. B: Homology model of TconTS2-LD comprising the α-helix calculated using TconTS2 amino acid sequence and crystal structure of Trypanosoma cruzi TS (PDB code: 3b69) as template employing the software Yasara. C: The molecular electrostatic surface of the homology model (B) was calculated using the ESPPME method of Yasara structure. Red colour indicates a positive potential, blue a negative and grey a neutral. A yellow ellipse indicates the groove encompassing the proposed binding site. D:SDS-PAGE of purified TconTS-LD proteins. After expression in E. coli Rosetta pLacI, 1–2 μg double affinity purified recombinant TconTS-LD, containing and lacking the α-helix, were loaded in each lane of an 10% SDS polyacrylamide gel as indicated. After electrophorese, the gel was stained using Coomassie Brilliant Blue.
Table 1.
Bacterial and eukaryotic expressed recombinant TconTS constructs.
Fig 2.
Summary of TconTS-LDs binding to glycans as determined by glycan array analysis.
TconTS-LDs binding to the glycan arrays was determined as described under Methods. Black bars indicate glycans bound by the TconTS-LDs. The presence and absence of the α-helix in TconTS-LD constructs is indicated with “+” and “-“, respectively. Further binding data (S2 Fig) and all glycans on the arrays (S1 Table) are available as Supporting Information.
Fig 3.
STD NMR experiments of TconTS2-LD.
STD NMR experiments with 5.5 μM TconTS2-LD were performed as described under Methods. Off-resonance (black lines) and STD NMR (red lines) spectra are shown. A: In the presence of 1.73 mM for 3α,6α-mannotriose; B: In the presence of 3.45 mM lactose; C: In the presence of 1.73 mM 3α,6α-mannotriose and 1.73 mM lactose. D: STD NMR effects for the signals indicated were determined as ratios between the intensities at the indicated ppm in the off-resonance spectra and corresponding STD NMR spectra using the software TopSpin 3.2. M1, M2, M3 and M4 stand for the NMR signals of 3α,6α-mannotriose, and L1, L2, L3 and L4 for those of lactose, for which the STD NMR effects are shown either for the single ligands (spectra shown in A and B) or for the mixture (spectra shown in C). n.d.: not determined, since in the ligands mixture the STD effect for M3 could not be determined from the spectra.
Fig 4.
Binding specificity of TconTS2-LD.
A: TconTS2-LD concentration dependent binding to immobilised huS2-Fc (5μg/mL). B: Competitive inhibition of TconTS2-LD binding to huS2-Fc in the presents of serially diluted high-mannose N-glycans. Undiluted inhibitor solution was set to 1.0. The maximum increase in relative fluorescence units (RFU) over time was determined as described under Methods. Data points are means ± standard deviation of triplicates.
Fig 5.
Cleavage of N-glycans from TconTS1 and TconTS2.
100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoHf glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti-Strep-tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.
Fig 6.
Size exclusion chromatography on Superdex200 column and detection at E280nm was used, MWs of different peaks were determined and assigned to oligomeric (1), dimeric (2) and monomeric (3) TconTS1 as described under Methods. A: Oligomerisation pattern of TconTS1 (solid line) or TconTS2 (dashed line), 300 μg protein was loaded. B: Effect of TconTS1 deglycosylation on enzyme oligomerisation. 100 μg TconTS1 were deglycosylated using 4000 units EndoHf glycosidase in phosphate buffer pH 7.4 for 4 hours and directly applied to the column (dashed line). As control 100 μg TconTS1 was treated correspondingly without the addition of enzyme was loaded (solid line).
Fig 7.
Contact site between TconTS-CD and LD.
Homology model of TconTS1 was calculated using the crystal structure of TcTS (PDB: 3b69) as template and the software Yasara. Molecular surface of TconTS1 was calculated using the surface module of Yasara Structure. Illustrated are the parts of TconTS-CD (yellow) and LD (orange), which are in close contact to each other. The α-helix connecting both domains is shown in blue.