Self-assembly of Human Galectin-1 via dual supramole ...

Nano Res.

Electronic Supplementary Material

Self-assembly of Human Galectin-1 via dual supramole-cular interactions and its inhibition of T-cell agglutinationand apoptosis

Wenjing Qi1, Yufei Zhang1, Zdravko Kochovski2, Jue Wang1, Yan Lu2,3, Guosong Chen1 (), and Ming Jiang1

1 The State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University,

Shanghai 200438, China 2 Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin 14109, Germany 3 Institute of Chemistry, University of Potsdam, 14476 Potsdam, Germany

Supporting information to https://doi.org/10.1007/s12274-018-2169-7

Experimental

Materials. HBr (33%) in glacial acetic acid, propargylamine, silver trifluoromethanesulfonate, 1,1,4,7,7-

Pentamethyldiethylenetriamine (PMDETA), Copper(I) bromide (CuBr), 2-bromoethanol, p-toluenesulfonyl

chloride (TsCl), 4-dimethylaminopyridine (DMAP) were purchased from J&K Chemical and used as received.

D-Lactose, D-Galactose were purchased from Darui fine chemical Co., Ltd. and used without further

purification. Ion-exchange resin Dowex® 50WX2 supplied by Sigma-Aldrich Co. LLC. was washed by methanol

three times before use. Sodium azide supplied by Xiya Reagent was used as received. All anhydrous solvents

were distilled before use. Human Galectin-1 was purchased from PeproTech (USA) and used as received.

Unless specially mentioned, all other chemicals were purchased from Sinopharm Chemical Reagent Co. and

used as received. The reactions were monitored and the Rf values were determined using analytical thin-layer

chromatography (TLC). The TLC plates were visualized by immersion into 5% sulfuric acid solution in ethanol

followed by heating using heat gun.

Characterization. 1H NMR spectra were recorded with an AVANCE III HD 400 MHz spectrometer. Matrix

Assisted Laser Desorption Ionization-Time Of Flight (Maldi-TOF) Mass Spectrum was taken by a AB SCIEX 5800

instrument while 2’, 4’, 6’-Trihydroxyacetophen monohydrate (THAP) being used as matrix. Ultraviolet–vis

(UV-vis) absorption spectra were recorded by Shimadzu UV-2550 spectrophotometer with the 1 mm cuvette.

Circular dicroism spectra was taken by a JASCO-815 instrument with the 1 mm cuvette. Dynamic light scattering

(DLS) was taken by Zetasizer Nano ZS90 from Malvern Instruments (UK). Transmission electron microscopy

(TEM) and cryo-electron microscopy (Cryo-EM)were performed with an FEI Tecnai G2 and a JEOL JEM-2100

transmission electron microscopes at an accelerating voltage of 200 kV. AFM images were operated on a Bruker

Multimode VIII SPM equipped with a J scanner. Sample solution was kept on mica surface for 2 min and then

200 μL buffer was used to wash the mica surface carefully followed by drying in air. Experiments were performed

in tapping mode with SNL10 tip. Confocal laser-scanning microscpic images were taken from Nikon C2+.

Address correspondence to [email protected]

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Preparation of the protein assembly. The protein solution was prepared by dissolving Gal-1 in PBS buffer

(20 mM Na2HPO4 ·12H2O, 150 mM NaCl, pH 7.4) and stored at 4 C. The inducing ligands were also dissolved

in PBS buffer. Then the solutions of protein and ligands were filtered through a Millipore 0.45 μm membrane

before mixing. Protein assemblies were achieved in the mixture and stored at 4 C for 24h. The protein assemblies

at this condition are stable for more than six months.

Data collection by electron microscopy. For the preparation of negatively stained samples, a drop of the

sample solution (3 mL) was applied onto a copper grid and the excessive solution was blotted away. Samples

were subsequently stained with 1 wt% uranyl acetate. The grid was then air dried. Tomographic data were

acquired on a JEOL JEM-2100 equipped with a 4 k 4 k CMOS digital camera (TVIPS TemCam-F416), operated

at 200 kV. Tilt series were collected to ± 60° with a 2° angular increment and a total dose of 300 e−/Å2 for

negatively stained samples. Samples for Cryo-EM were prepared by applying 4 μL drop of sample solution to

holey carbon grids (Quantifoil R2/1) and plunge-frozen into liquid ethane with an FEI vitrobot Mark IV set at

4°C and 95% humidity. Vitrified grids were either transferred directly to the microscope cryoholder or stored in

liquid nitrogen. All grids were glow-discharged before use.

Cell agglutination assays. Jurkat cells were seeded on 96-well plates at a concentration of 5×104 cells per well.

The nuclei were stained with Hoechst 33342 for 10 min and washed with PBS for three times. The cells were

then incubated with Gal-1 (500 μg/mL) and ligands (from 17 μg/mL, 1 eqv. to 340 μg/mL, 20 eqv.) and lactose

for 4 h. Automatic cell count was performed with image J. Statistical analysis was performed under GraphPad

Prism software.

Cell viability assays. Jurkat cells were seeded on 96-well plates at a concentration of 5×104 cells per well. The

cells were then incubated with Gal-1 (500 μg/mL) and ligands (5 eqv. to 20 eqv.). Then CCK-8 reagent was added

into each well and incubated for 2 h in the incubator. The absorbance was measured at 450 nm using a microplate

reader (BioTek ELx800).

Scheme S1 Synthetic routes of RnL. The synthetic details and characterizations are given at the end of the Electronic Supplementary Material

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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Figure S1 ITC results of raw and integrated data for titration of lactose (10 mM) to Gal-1 (0.2 mM, calculated as monomer) in PBS buffer at 25 ºC.

Figure S2 Above: CD spectra of Gal-1/R4L solution (black line) after 24 h incubation and free ligands (red line) as a control. Bottom: The UV-vis absorbance spectra of Gal-1/R4L solution (cyan line) after 24 h incubation and free R4L in different concentration as controls.


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Figure S3 XY tomographic slices of well-packed protein ribbons and the corresponding isosurface representations in different magnifications and angles.

Figure S4 Cryo-EM images of wide protein microribbon constituted of proteinfilaments. (well-defined packing of protein filaments in red circles)

Figure S5 Time-dependent DLS results of Gal-1/R4L solution at 4C.


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Figure S6 TEM images of negatively stained Gal-1/ R4L protein microribbons after 6 months.

Figure S7 (a) The UV-vis spectra of Gal-1/R4L solutions in different molar ratios after 24 h incubation and free ligands (blue line) as a control. (b) Absorbance difference between Gal-1/R4L solutions and the corresponding amount of ligand in each Gal-1/ R4L molar ratios: Blue column represents absorbance increase in 525 nm. Red column represents absorbance decrease in 560 nm.

Figure S8 (a) AFM image of microwires and (b) the height profile along the corresponding lines in Figure a.


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Figure S9 Structural comparison between lactose (green) and galactose (purple) molecules derived from the crystal structures of related molecules. (PDB accession code: 1W6O, 1W6M ). The D-galactopyranoside in lactose matches well with the galactose in crystals.

Figure S10 Microscopic images of agglutinated cells. Jurkat cells were seeded on 96-well plates (0.05 million/well) and incubated with (0, 31.25, 62.5, 125, 250, 500 μg/ml Gal-1. Scale bar = 50 μm.

Figure S11 TEM images of protein assemblies in DMEM (dulbecco’s modified eagle medium) at 37 °C (protein/ligand ratios is 1: 3).


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Figure S12 Illustration of automatic cell counts performed in image J to quantify the cell agglutination of Gal-1 and Gal-1/ Ligand (10 equiv.). One black dot represents one cell counted by image J.

Figure S13 (a) Cytotoxicity assay of ligands (CCK-8). (b) Cell apoptosis difference with and without Gal-1 (500 μg/mL) in the presence of ligands. Results are shown as means ± SEM as indicated. Statistical significance was calculated by One-way ANOVA (compared to PBS group). * p < 0.05, *** p < 0.001.

Synthetic procedures and characterizations:

Synthesis of EG4OTs [S1]. Tetraethylene glycol (3.9 g, 20.0 mmol), triethylamine (1.0 g, 10.0 mmol) were mixed

in 30 mL dry DCM at 0 °C. Tosyl chloride (0.9 g, 5.0 mmol) was then added slowly to the mixed solution within

30 min and the mixture was allowed to stir at room temperature for 3 h. After filtration, solvent was removed

under reduced pressure and the crude product was purified by column chromatography using silica and

petroleum ether/ethyl acetate gradient (v: v = 3:1→1:1). The product EG4OTs was obtained as a colourless solid

(1.5 g, 83 %). 1H NMR (400 MHz, CDCl3): δ 7.81-7.79 (m, 2H, Ar), 7.35-7.33 (m, 2H, Ar), 4.18-4.15 (m, 2H, CH2OTs),

3.72-3.61 (m, 14H, 7×CH2), 2.45 (s, 3H, CH3).


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Figure S13 1H NMR of EG4OTs in CDCl3.

Synthesis of EG4N3 [S1]. To a stirred solution of EG4OTs (1.5 g, 4.2 mmol) in a mixture of H2O/acetone (1/1,

v/v, 70 mL), NaN3 (1.3 g, 20 mmol) was added and the mixture refluxed for 24 h. The acetone was then removed

under reduced pressure and the remaining aqueous phase was extracted with CH2Cl2 (3 30 mL), brine (1

30 mL), dried over MgSO4, filtered and concentrated in vacuo. The raw product was purified by column

chromatography using silica and petroleum ether/ethyl acetate gradient (v: v = 3:1→1:1). The product EG4N3

was obtained as a colourless solid (0.8 g, 86%). 1H NMR (400 MHz, CDCl3): δ 3.61-3.54 (m, 12H, 6×CH2),

3.49-3.47 (m, 2H, CH2OH), 3.41 (t, 2H, J = 5.1 Hz, CH2N3).

Figure S14 1H NMR of EG4N3 in CDCl3.


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Synthesis of R4N [S2]. RhB (1.4 g, 3.0 mmol), EG3N3 (640.0 mg, 3.0 mmol) and DMAP (20.0 mg, 1.6 mmol)

were mixed in 50 mL dry DCM. The mixture were incubated in iced water for 30min under N2 atmosphere. Then

EDC (0.9 g, 4.5 mmol) was added into the solution and the mixture was stirred at room temperature for 6 h. The

solvent was removed under reduced pressure and the crude product was purified by column chromatography

using silica and DCM/MeOH gradient (v: v = 50:1→10:1). The product R4N was obtained as red oil (960 mg, 47 %). 1H NMR (400 MHz, CD3OD-d4): δ 8.34 (dd, J = 7.8, 1.5 Hz, 1H), 7.88-7.79 (m, 2H), 7.43 (dd, J = 7.4, 1.5 Hz, 1H),

7.15 – 6.99 (m, 6H), 4.12 – 4.09 (m, 2H), 3.71 – 3.58 (m, 14H), 3.50 (dd, J = 5.9, 3.5 Hz, 2H), 3.43 – 3.38 (m, 4H), 1.31

(t, J = 7.1 Hz, 12H).

Figure S15 1H NMR of R4N in CD3OD-d4.

Synthesis of 2,2´,3,3´,4´,6,6´-Hepta-O-acetyl-α-lactosyl bromide (Lac(Ac7)-Br) [S3]: Octa-O-acetyl-lactose

(10.0 g, 14.7 mmol) was dissolved in DCM (100 mL) and cooled to 0°C. HBr (33%) in glacial acetic acid (13.3 mL,

73.0 mmol) was added slowly via dropping funnel to the exclusion of moisture. The reactions mixture was

warmed to room temperature over a period of half an hour. After detection of complete conversion of starting

material by TLC the solution was diluted with DCM (100 mL) and poured in ice water. The organic layer was

separated and washed with water (2 100 mL), sat. NaHCO3 solution (100 mL), and brine (100 mL) prior to

drying MgSO4. After filtration, solvent was removed under reduced pressure and the crude product was

purified by column chromatography using silica and petroleum ether/ethyl acetate gradient (v: v = 3:1→1:1).

The product was obtained as colourless solid (9.46 g, 92%). 1H NMR (400 MHz, CDCl3): δ 6.55 (d, J = 4.0 Hz,

1H, H-1), 5.58 (t, J = 9.6 Hz, 1H, H-3), 5.38 (dd, J = 3.5, 1.2 Hz, 1H, H-2´), 5.15 (dd, J = 10.4, 7.9 Hz, 1H, H-3´), 4.99

(dd, J = 10.4, 3.4 Hz, 1H, H-4´), 4.78 (dd, J = 10.0, 4.1 Hz, 1H, H-2), 4.54 – 4.51 (m, 2H, H-1´, H6´a), 4.23 – 4.08 (m,

4H, H-5, H-6a, H6b, H-6´b), 3.93 – 3.86 (m, 2H, H-4, H5´), 2.18 – 2.08 (m, 21H, 7 x OAc). 13C NMR (101 MHz,

CDCl3): δ 170.4-168.96 (OCOCH3), 100.79 (C-1´), 86.41(C-1), 74.96 (C-4), 72.99 (C-5´), 70.99 (C-5), 70.85 (C-3´),

70.79 (C-2), 69.60 (C-3), 69.03 (C-2´), 66.63 (C-4´), 61.06 (C-6´), 60.89 (C-6), 20.79, 20.64, 20.49 (OCOCH3).


Nano Res.

Figure S16 1H NMR of Lac(Ac7)-Br in CDCl3.

Figure S17 13C NMR of Lac(Ac7)-Br in CDCl3.

Synthesis of propargyl 2,2´,3,3´,4´,6,6´-hepta-O-acetyl-β-lactoside (Lac(Ac7)-yn) [S4]: Lactosyl bromide (5.00 g,

7.15 mmol) and propargyl alcohol (970 μL, 14.3 mmol) were dissolved in dry DCM (50 mL) and stirred for one

hour at room temperature. Then silver carbonate (2.95 g, 10.7 mmol) was added and the reaction mixture was

stirred until complete conversion of starting material was detected by TLC. The reaction mixture was diluted

with DCM (50 mL) and washed with water (2 50 mL), sat. NaHCO3 solution (50 mL), and brine (50 mL) prior

to drying over MgSO4. After filtration, solvent was removed under reduced pressure and the crude product

was purified by column chromatography using silica and petroleum ether/ethyl acetate gradient (v: v = 3:1→

1:1). The product was obtained as colourless solid (3.43 g, 71%). 1H NMR (400 MHz, CDCl3): δ 5.37 (dd, J = 3.5,

1.2 Hz, 1H, H-4´), 5.25 (t, J = 9.3 Hz, 1H, H-3), 5.13 (dd, J = 10.4, 7.9 Hz, 1H, H-2´), 5.06 – 4.91 (m, 2H, H-3´, H-2),

4.76 (d, J = 7.9 Hz, 1H, H-1), 4.59 – 4.46 (m, 2H, H-1´, H-6a), 4.36 (d, J = 2.4 Hz, 2H, -OCH2C≡), 4.18 – 4.07 (m, 3H,

H-6b, 2 x H-6´), 3.96 – 3.79 (m, 2H, H-5´, H-4 ), 3.66 (ddd, J = 9.9, 4.9, 2.1 Hz, 1H, H-5), 2.48 (d, J = 2.4 Hz, 1H,

-C≡CH), 2.14 – 2.02 (m, 21H, 7 x OAc). 13C NMR (101 MHz, CDCl3): δ 170.35-169.04 (OCOCH3), 101.03 (C-1´),

97.86 (C-1), 76.12 (C-4), 75.47 (C≡CH), 72.74 (C-5´), 72.67 (C-5), 71.29 (C-3´), 70.97 (C-2), 70.69 (C-3), 69.08 (C-2´),

66.60 (C-4´), 61.81 (C-6´), 60.82 (C-6), 55.86 (CH2C≡) ,20.854-20.50 (OCOCH3).


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Figure S18 1H NMR of Lac(Ac7)-yn in CDCl3.

Figure S19 13C NMR of Lac(Ac7)-yn in CDCl3.

Synthesis of propargyl-β-lactoside (Lac-yn) [S5]: Lac(Ac7)-yn (3.43 g, 5.08 mmol) was dissolved in methanol

(30 mL), and sodium methoxide (3 M in methanol, 5 drops) was added. The reaction mixture was allowed to

stir at room temperature for 1 h. Dowex 50WX2 H+ resin was added to adjust the pH to 7. The mixture was

then filtered through Celite and the solvent was evaporated. The product was obtained as colourless solid (1.73 g,

90%). 1H NMR (400 MHz, D2O): δ 4.68 (d, J = 8.0 Hz, 1H, H-1), 4.48 (t, J = 2.6 Hz, 2H, -OCH2C≡), 4.46 (d, J = 7.8 Hz,

1H, H-1’), 3.99 (dd, J = 12.3, 2.1 Hz, 1H, H-6a), 3.93 (d, J = 3.4 Hz, 1H, H-4), 3.83 (d, J = 4.8 Hz, 1H, H-6b), 3.82 –

3.79 (m, 1H, H-6’), 3.79 – 3.75 (m, 1H, H-6’), 3.74 (d, J = 2.1 Hz, 1H, H-4’), 3.68 (d, J = 3.2 Hz, 1H, H-3), 3.68 – 3.64

(m, 2H, H-5, H-5’), 3.55 (dd, J = 10.0, 7.8 Hz, 1H, H-3’), 3.35 (m, 2H, H-2, H-2’), 2.92 (t, J = 2.4 Hz, 1H, -C≡CH); 13C NMR (101 MHz, D2O): δ 102.90, 100.35, 78.73, 78.22, 76.35, 75.32, 74.82, 74.32, 72.57, 72.48, 70.92, 68.51, 61.400,

59.96, 56.57


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Figure S20 1H NMR of Lac-yn in D2O

Figure S21 13C NMR of Lac-yn in D2O

Synthesis of R4L. R4N (221 mg, 0.33 mmol), Lac-yn (150 mg, 0.39 mmol) and N,N,N',N'',N''-

Pentamethyldiethylenetriamine (PMDTA) (11.5 mg, 0.08 mmol) were dissolved in dry DMF (5 mL) and

followed by bubbling Ar for 30 min. Then CuBr (16.7 μL, 0.08 mmol) was added accompanied by bubbling Ar

for another 20 min. The mixture was then heated to 50C for 12 h. Then the solvent was removed under reduced

pressure and the crude product was purified by column chromatography using silica and DCM/MeOH

gradient (v:v = 50:1→10:1). The product was obtained as red solid (200 mg, 58%). MALDI-TOF Mass Spectrum

(M/z): [R4L – Cl]+ calcd. for C51H70N5O17, 1024.53; found, 1024.96. 1H NMR (400 MHz, CD3OD-d4): δ 8.36 (dd, J =

7.8, 1.4 Hz, 1H), 8.04 (s, 1H), 7.86 (ddd, J = 17.2, 7.6, 1.5 Hz, 2H), 7.45 (dd, J = 7.5, 1.4 Hz, 1H), 7.26 – 6.90 (m, 6H),

4.75 (d, J = 12.3 Hz, 1H), 4.54 (dd, J = 5.5, 4.5 Hz, 2H), 4.41 (d, J = 7.8 Hz, 1H), 4.37 (d, J = 7.5 Hz, 1H), 4.24 – 4.02 (m,

2H), 3.93 (dd, J = 12.1, 2.4 Hz, 1H), 3.90 – 3.82 (m, 4H), 3.81 – 3.75 (m, 1H), 3.70 (q, J = 7.3 Hz, 9H), 3.62 – 3.50 (m,

10H), 3.49 – 3.41 (m, 7H), 3.37 (s, 1H), 1.33 (t, J = 7.1 Hz, 12H).


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Figure S22 1H NMR of R4L in CD3OD-d4.

Synthesis of R1L, R2L, R3L, R5L and R1G. The four compounds were synthesized according the same procedure

as R4L and were characterized by MALDI-TOF Mass Spectrum.

Figure S23 The MALDI-TOF Mass Spectrum of R1L. [R1L – Cl]+ = 892.98; found 892.05.


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Figure S27 The MALDI-TOF Mass Spectrum of R1G. [R1G – Cl]+ = 730.30; found 730.46.


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