Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic amyotrophic lateral sclerosis (ALS) model mice

Abstract

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.

FIGURE 1.

A, immunoblots of native and denatured hSOD1 captured by the antibody raised against the peptide corresponding to amino acids 131–153 in hSOD1. Antibodies were immobilized on protein-A-Sepharose at 2 mg/g wet gel, and incubated with native hSOD1 or denatured hSOD1 (treated with 6 m guanidinium-Cl and 10 mm EDTA). Two consecutive incubations were done with native hSOD1 to control for trace amounts of denatured hSOD1 in the preparation. After washing, the captured SOD1 was released by boiling in SDS-PAGE sample buffer and visualized on Western immunoblots. The captured denatured hSOD1 sample was diluted 5 times more than the native sample. The capacity to bind denatured SOD1 was 1,500-fold higher than the capacity to bind native SOD1. B, quantification of hSOD1 present in 20,000 × g pellets. Pellets from three experiments each of G93A and G127X were solubilized in 1/5 of the centrifuged volume. Dilutions of supernatants and solubilized pellets were visualized on Western immunoblots and quantified. The proportions hSOD1 in pellets were 5.4 ± 1.2% and 5.5 ± 0.3%, respectively.

FIGURE 2.

Two-dimensional gel patterns of proteins captured by abSepharoses from mouse spinal cord extracts. A, G93A mouse spinal cord extract captured with 131–153-abSepharose. B, G127X mouse spinal cord extract captured with antibodies raised against the neopeptide in the C-terminal end of G127X coupled to Sepharose. Proteins identified with MALDI-TOF/MS are shown. As controls for the C-terminal 131–153 antibody, extracts from non-transgenic C57Bl6 control spinal cords and transgenic G127X mice were captured with 131–153-abSepharose in panels C and D, respectively. In panel E, IgG purified from rabbits before immunization was coupled to IgG and the Sepharose matrix, and incubated with G93A spinal cord extract to test for nonspecific binding of proteins to IgG and the Sepharose matrix. Proteins that bound non-specifically to the 131–153 antibody are marked with an asterisk. As a control for the G127X-specific antibody, it was incubated with spinal cord extracts from G93A mice (panel F). Minute amounts of Hsc70 that could be detected, as well as contaminating tubulin β5, β-actin, and keratins, are marked on the gel.

FIGURE 3.

Two-dimensional gel patterns of rat spinal cord extract proteins binding to immobilized holo- and apo-hSOD1. A, proteins bound to ethanolamine-blocked, B, wild-type holo-hSOD1-bound C, wild-type apo-hSOD1-bound, and D, G93A apo-hSOD1-bound Sepharose gels (see “Experimental Procedures”) were visualized by two-dimensional electrophoresis and silver staining. Proteins identified with MALDI-TOF/MS are shown. In A, C, and D, 10–14.5% gels were used for the second dimension and a 12.5% gel in B.

FIGURE 4.

Quantification of Hsc70 bound to hSOD1. Hsc70 bound to misfolded SOD1 in spinal cord extracts from wild-type hSOD1, G93A hSOD1, and G127X hSOD1 transgenic mice was quantified by comparison with the total amount of Hsc70 in dilutions of a G93A spinal cord extract. The figures are the means of results from four mice.