Plasmid and DNA constructs
Plasmids for this study were constructed as shown in . Plasmids encoding human WT, A4V, G85R, and G93A SOD1 cDNAs in pVL1393 were used as templates as previously described [10
]. The plasmid encoding human CCS cDNA in pCCS-HIS was a generous gift from Dr. Valeria C. Culotta (Johns Hopkins University). The pAAV-IRES-hrGFP plasmid (Stratagene, La Jolla, CA) was used to construct a vector system that can co-express SOD1 and CCS in a single mRNA using a viral IRES (internal ribosome entry site) sequence under control of the CMV promoter. SOD1
gene (wild-type, A4V, G85R, or G93A) was inserted into multiple cloning sites of plasmids and hrGFP
was replaced with the CCS gene as a second open reading frame (ORF) allowing it to make a dicistronic mRNA for SOD1 and CCS ().
Effect of simultaneous expression of FALS hSOD1 and hCCS on SOD1 containing HMWS in AAV 293 cells
To generate SOD1 from monocistronic mRNA, the 0.5 kb human SOD1 gene, containing BamHI at the 5′ end and XhoI at the 3′ end was amplified by PCR with pVL1393-SOD1 as template and inserted into pAAV-MCS to generate the pAAV-SOD1 plasmid. The plasmid pAAV-IRES-hrGFP (Stratagene) was used as the vector for the expression of hSOD1 and hCCS from dicistronic mRNA. To replace the hrGFP gene in pAAV-IRES-hrGFP with the hCCS gene, the plasmid was digested with HindIII and BglII, resulting in a 1.1 kb fragment that contains a part of IRES and the hrGFP gene was subcloned into pSP72 (Promega, Madison, WI) to make pSP72-1.1hrGFP. The KpnI site in hrGFP was mutated into the EcoRI site using the Quick change site-directed mutagenesis kit (Stratagene). The hrGFP gene in the pSP72-1.1hrGFP plasmid was replaced with the PCR-amplified 0.8 kb CCS gene that has the NcoI site at the 5′ and the EcoRI site at the 3′ end. Primers used for hCCS gene amplification were 5′-CAT GCC ATG GCT TCG GAT TCG GGG AAC-3′ and 5′-CGG AAT TCC TAT CAA AGG TGG GCA GGG GGC TG-3′. The 1.2 kb HindIII/BglII fragment that contains a part of IRES and the CCS gene was cloned into the same restriction enzyme-treated pAAV-IRES-hrGFP plasmid to generate pAAV-IRES-CCS. Primers used for hSOD1 gene amplification were 5′-CGG GAT CCA TGG CGA CGA AGG CCG TGT GC-3′ and 5′-CCG CTC GAG GCT ATT ATT GGG CGA TCC CAA TTA C-3′. The PCR amplified 0.5 kb human wild type (WT) SOD1 gene that has BamHI at the 5′ and XhoI at the 3′ end was inserted into pAAV-IRES-CCS to generate pAAV-SOD1-IRES-CCS. Mutant SOD1 genes were inserted in the same way and confirmed by nucleotide sequencing (Macrogen USA, Rockville, MD). To generate the FLAG tag at the carboxyl terminus of SOD1, the SOD1 gene was amplified to match the in-frame of the FLAG sequence of pAAV-IRES-hrGFP. Primers used for hSOD1-FLAG gene amplification were 5′-CGG GAT CCA TGG CGA CGA AGG CCG TGT GC-3′ and 5′-CCG CTC GAG TTG GGC GAT CCC AAT TAC ACC-3′. The PCR amplified 0.5 kb human SOD1 gene that has BamHI at the 5′ and XhoI at the 3′ end was inserted into pAAV-IRES-CCS to generate pAAV-SOD1-FLAG-IRES-CCS.
Cell culture and expression of SOD1 and CCS
HEK293 or AAV 293 (Stratagene, La Jolla, CA) cells were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The recombinant pAAV plasmid, pAAV-SOD1 or pAAV-SOD1-IRES-CCS, was transfected into HEK293 or AAV293 cells in Biocoat 6-well plates (Becton Dickinson Labware, Bedford, MA) using the CaCl2 transfection method. Cells were harvested 24 hr after transfection for analysis. If required, cells were incubated with epoxomicin (100 nM; Sigma, St. Louis, MO) or 3-methyladenine (5 mM; Sigma) 24 hrs after transfection.
To study the effect of oxidative stress on SOD1 mutant uptake by mitochondria, AAV293 cells were transiently transfected with A4V mutant expression vectors with or without CCS co-expression and incubated for 24 hr. These cells were then treated with 200 μM of hydrogen peroxide for 15 min prior to cellular fractionation (see below) to determine the mitochondrial levels of A4V.
Phosphate-buffered saline (PBS)-washed HEK293 or AAV293 cells transfected with plasmids for 24 hr before they were sonicated in PBS containing 1% Triton X-100 in the presence of protease inhibitor cocktail (Sigma). For fractionation, cell lysates were centrifuged at 100,000×g for 20 min using an Optima Ultracentrifuge (Beckman Coulter, Fullerton, CA), and the supernatants were used for analysis as the “nonionic detergent-soluble fraction”, and the “nonionic detergent-insoluble fraction” was obtained by dissolving the pellets in PBS containing 5% SDS with brief sonication. For preparation of total cell lysates containing both detergent-soluble and -insoluble fractions, cells were lysed in SDS buffer and sonicated. Protein concentrations were determined with the RC-DC protein assay kit (Bio-Rad, Hercules, CA) or the BCA protein assay kit (Pierce, Rockford, IL).
Mitochondrial and cytosolic fractions were prepared as described previously [35
]. Briefly, transfected cells were homogenized with a tight-fitting glass-Teflon motorized homogenizer (500 rpm, 30 strokes) in MSH buffer (210 mM mannitol, 70 mM sucrose, 5 mM Hepes, pH 7.5) supplemented with 1 mM EDTA. After 30 min incubation on ice, the lysates were centrifuged at 600×g for 8 min at 4 °C. The resultant supernatant was then centrifuged at 5,500×g for 15 min to obtain mitochondrial fractions.
Western blotting and antibodies
Proteins from extracts were loaded onto 10%-20% gradient pre-made gels and subjected to electrophoresis using either SDS-PAGE or non-reduced PAGE. Separated proteins were then transfered onto a nitrocellulose membrane (Invitrogen, Carlsbad, CA), probed with an appropriate antibody, and quantitated with the Odyssey infrared system (Lincoln, NE, USA). The results presented in the figures represent a typical observation from indicated number of independent experiments. The primary antibodies used were as follows: sheep polyclonal anti-human Cu,Zn SOD1 (574597) (1:2000; Calbiochem, La Jolla, CA, USA), rabbit polyclonal anti-human Cu,Zn SOD1 (RDI-SODabRx ) (1:5000; Fitzgerald, Concord, MA, USA), mouse monoclonal anti-mitochondrial Hsp70 (MN3-028) (1:5000; Affinity Bioreagents, Golden, Co.), mouse monoclonal anti-human CCS (2A1) (1:5000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-actin (1:10000; Sigma), and mouse monoclonal anti-Hsp70 (W27) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For ECL detection (Amersham, Piscataway, NJ, USA), horseradish peroxidase conjugated secondary antibody was used and incubation was performed at 1:10,000 dilution. For the blot and quantitative analysis of protein bands the LI-COR Odyssey (Lincoln, NE, USA) was used, Alex Fluor goat anti-rabbit 800 or goat anti-mouse 680 secondary antibodies (Invitrogen) was used at 1:10,000 dilution.
SOD activity staining
Cell extracts were electrophoresed on native PAGE gel (18%) (Invitrogen, Carlsbad, CA) for 12 hr. The gel was soaked in 20 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM riboflavin and 2 mM o-dianisidine for 1 hr. The gel was then placed in a dry tray and illuminated with fluorescent light for 10-20 minutes. The dark-yellowish color indicates the presence of SOD activity in the bands.
An unpaired two-tailed distribution Student’s t test (Microsoft Excel) was used to analyze the data and results were expressed as means ± SEM.