Canine SOD1 harboring E40K or T18S mutations promotes protein aggregation without reducing the global structural stability

Recombinant canine SOD1 expression and purification

Bacterial expression plasmid vectors, pGEX4T-1 (GE Healthcare Bio-Sciences AB), for the production of canine recombinant SOD1 wild-type (WT) and E40K proteins were generated as described previously (Nakamae et al., 2015). The plasmid harboring the T18S mutation was obtained by site-directed mutagenesis of the SOD1 WT plasmid using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs Inc). The primer pair used was 5′-GTGGAAGGCTCCATTCATTTTGTGCAGAAA-3′ and 5′-TTTCTGCACAAAATGAATGGAGCCTTCCAC-3′. The presence of the T18S mutation was confirmed by DNA sequence analysis. The pGEX4T-1 expression plasmids were transformed into competent (BL21) E.coli cells (Thermo Fisher Scientific) and plated on LB agar with 100 µg/ml ampicillin at 37 °C overnight. Single colonies were inoculated into LB media with 100 µg/ml ampicillin and grown at 37 °C overnight, then diluted 1:20 in fresh LB media with 100 µg/ml ampicillin and grown at 37 °C for 2 h. Protein expression was induced by addition of 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20 °C for 20 h. Cell suspensions were centrifuged at 3,500 × g for 20 min, washed with 1 × PBS and centrifuged at 14,000 × g for 20 min. Cell pellets were stored at −30 °C. The cell pellets were suspended in lysis buffer (10 mM Tris, 1 mM EDTA and 150 mM NaCl) with 200 µg/mL lysozyme and 200 µg/mL phenylmethylsulfonyl fluoride (PMSF), then disrupted by sonication on ice and centrifuged at 14,000 × g for 20 min. The supernatant was filtered by 0.45 µm FILSTAR Syringe Filter (Hawach Scientific) and applied onto a Glutathione Sepharose 4 FF column (GE healthcare). The bound SOD1 proteins were cleaved on the Glutathione Sepharose by addition of 400 U thrombin (FUJIFILM Wako Pure Chemical Corporation) at 22 °C overnight. After removing thrombin using a HiTrap Benzamidine FF column (GE Healthcare), the Cu²⁺ and Zn²⁺-free SOD1 (apo-SOD1) were generated by incubation in 40 mM sodium phosphate (pH 7.4) containing 10 mM EDTA and concentrated by Amicon Ultra Centrifugal filters with a 10 K membrane (Millipore). The Cu²⁺ and Zn²⁺-bound SOD1 (holo-SOD1) were generated using the procedure previously described by Crisp et al. (2013) with some modification. Briefly, the apo-SOD1 was dialyzed with 100 mM Tris (pH8.0), 300 mM NaCl and 200 µM CuCl₂ for 3.5 h, and then with 100 mM Tris (pH8.0), 300 mM Tris and 200 µM ZnSO₄ for 3.5 h. The holo-SOD1 was dialyzed with 20 mM Tris and 10 mM NaCl overnight at 4 °C, and then concentrated using the Amicon Ultra Centrifugal filters. Protein concentrations were determined with the absorption at 280 nm using a UV spectrometer (Shimadzu).

The secondary structure of each protein was examined by circular dichroism (CD). The CD spectra were acquired using a Chirascan spectrophotometer (Applied Photophysics). Net charges of the SOD1 proteins were calculated using a protein analysis software (PROTEIN CALCULATOR v3.4).

SOD1 activity assay

The SOD1 activity was determined using SOD Assay Kit-WST (DOJINDO LABORATORIES) according to a protocol provided by the manufacture. Each SOD1 protein was diluted to 250 µg/mL with 40 mM sodium phosphate (pH 7.4). The assay was repeated three times.

Thioflavin T assay

For amyloid fibril formation, solutions containing 40 µM SOD1 and 10 µM thioflavin T (ThT) were shaken at a rate of 170 rpm in a 96-well plate at 37 °C. For the experiments using apo-SOD1, 10 mM EDTA was added to the above solutions. Amyloid fibril assembly was monitored by the change in ThT fluorescence at 495–505 nm (excitation wavelength of 405 nm) using GloMax Discover Microplate Reader (PromegaKK).

Transmission electron microscopy

Samples containing SOD1 proteins were adsorbed onto carbon-coated 200-mesh copper grids (EM Japan Co., Ltd.) and negatively stained with 2% w/v phosphotungstic acid. Transmission electron microscopy (TEM) was performed using JEM-2100F operating at 200 kV (JEOL Ltd.).

Structural stability of SOD1

The structural stability of SOD1 was examined by monitoring thermal denaturation using CD. Far-UV CD spectra were acquired in the wavelength range of 195–260 nm using a 1 mm pathlength cuvette. Thermal denaturation was examined by scanning the temperature from 20 to 95 °C at 1 °C/min and monitoring θ₂₂₀. The SOD1 proteins were diluted to 10 µg/mL with 40 mM sodium phosphate (pH 7.4).

The GdnHCl-induced unfolding was examined by monitoring the CD. The SOD1 proteins were diluted to 110 µg/mL with 5 mM HEPES (pH 7.4) containing various concentrations of GdnHCl. The solutions were incubated at 37 °C for 1 h and then CD values at 220 nm were recorded. Each examination was performed three times individually.

Statistical analysis

Multiple group means were compared by one-way ANOVA with Tukey’s multiple comparison tests for pair-wise comparisons.

Production of canine holo- and apo-SOD1

For the production of canine SOD1 proteins, we used a bacterial expression system and a one-step affinity-chromatography. SDS-PAGE of the purified SOD1 demonstrated a single homogeneous band at 16 kDa (Fig. 2A and Fig. S1), consistent with the molecular size predicted from the amino acid sequence. We produced Cu²⁺/Zn²⁺-unbound SOD1 (“apo-SOD1”) and -bound SOD1 (“holo-SOD1”) using a dialysis procedure described in the Materials & Methods section. As show in Fig. 2B and Data S1, enzymatic activity of holo-SOD1 was ∼100-fold higher than that of apo-SOD1, suggesting that the major population of apo-SOD1 (∼99%) was deprived of Cu²⁺/Zn²⁺ by the procedure.

A far-UV CD spectra of holo-SOD1 represented a negative minimum at 208 nm (Fig. 2D), indicating the formation of an anti-parallel β-sheet structure (Higurashi et al., 1999). No difference in the CD spectra was found between holo-SOD1 and apo-SOD1, consistent with previous reports in human SOD1 (Benatar, 2007). Overall, the structural and enzymatic characteristics of canine SOD1 were in good agreement with those of human SOD1.

The E40K and T18S mutations did not affect global fold of SOD1

Next, we produced canine SOD1 harboring the E40K or T18S mutation. No significant difference in enzymatic activity or CD spectrum were found between WT and the mutant SOD1 (Figs. 2B–2D), indicating that the E40K and T18S mutations did not significantly affect the global fold of canine SOD1.

The E40K and T18S mutations promote the production of worm-like fibrils

To characterize aggregation properties of the canine SOD1, we performed a Thioflavin-T (ThT) assay. The holo-SOD1 did not give rise to a ThT-positive aggregate, even in the presence of mutations (Data S2). Similarly, apo-SOD1 without mutation did not give rise to a ThT-positive aggregate (Figs. 3A and 3D). However, apo-SOD1 harboring E40K or T18S mutation produced ThT-positive aggregates after 0–200 min shaking (Figs. 3B–3D).

We examined morphology of the ThT-positive aggregates using negatively stained electron microscopy. As shown in Figs. 3E and 3F, we observed curvy fibrous aggregates, typically termed “worm-like fibrils” (Singh et al., 2012), in the E40K and T18S SOD1 aggregates (Figs. 3E and 3F). By contrast, we did not observe any aggregates in WT apo-SOD1 or holo-SOD1 (Fig. S2). Thus, the E40K and T18S mutations promote formation of ThT-positive amyloid-like aggregates.

The T18S mutation and the E40K mutation did not reduce the global structural stability

To characterize the mechanism of aggregation, structural stabilities of SOD1 were examined using thermal and GdnHCl denaturation assays and far-UV CD. Interestingly, thermal denaturation curves of WT, E40K, and T18S SOD1 were almost superimposable, both in the apo- and holo-SOD1 (Fig. 4 and Data S3). Furthermore, GdnHCl denaturation curves of the three SOD1 were also superimposable (Fig. 5 and Data S4). Thus, the T18S and the E40K mutation did not reduce the structural stability of SOD1, at least at the level of secondary structure.