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. 2008 May 16;283(20):13528-37.
doi: 10.1074/jbc.M800564200. Epub 2008 Mar 3.

A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis

Celeste M Karch  1 , David R Borchelt
Affiliations

A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis

Celeste M Karch et al. J Biol Chem. .

Abstract

One of the mechanisms by which mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS) is proposed to involve the accumulation of detergent-insoluble, disulfide-cross-linked, mutant protein. Recent studies have implicated cysteine residues at positions 6 and 111 as critical in mediating disulfide cross-linking and promoting aggregation. In the present study, we used a panel of experimental and disease-linked mutations at cysteine residues of SOD1 (positions 6, 57, 111, and 146) in cell culture assays for aggregation to demonstrate that extensive disulfide cross-linking is not required for the formation of mutant SOD1 aggregates. Experimental mutants possessing only a single cysteine residue or lacking cysteine entirely were found to retain high potential to aggregate. Furthermore we demonstrate that aggregate structures in symptomatic SOD1-G93A mice can be dissociated such that they no longer sediment upon ultracentrifugation (i.e. appear soluble) under relatively mild conditions that leave disulfide bonds intact. Similar to other recent work, we found that cysteines 6 and 111, particularly the latter, play interesting roles in modulating the aggregation of human SOD1. However, we did not find that extensive disulfide cross-linking via these residues, or any other cysteine, is critical to aggregate structure. Instead we suggest that these residues participate in other features of the protein that, in some manner, modulate aggregation.

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Figures

FIGURE 1.
FIGURE 1.
SOD1 aggregation of fALS cysteine mutants in transfected cells. Mutants were expressed in HEK293FT cells, and aggregate levels were determined as described under “Experimental Procedures.” UT, untransfected cells provide a negative control. WT, cells transfected with vectors for WT SOD1, which does not aggregate. SOD1-G85R (G85R), a robustly aggregating fALS mutant, provides a positive control. SDS-PAGE was performed in the presence of a reducing agent in an 18% Tris-glycine gel. Immunoblots were probed with an antiserum specific for human SOD1 (hSOD1). A, P2, detergent-insoluble protein fraction (20 μg). B, S1, detergent-soluble protein fraction (5 μg). C, relative aggregation is a function of the amount of SOD1 found in the pellet fraction as compared with the supernatant (see “Experimental Procedures”). The graph represents the mean (±S.E. (error bars)) of at least three different experiments (see Table 1 for a summary of statistical data). All fALS mutants were statistically different from wild-type SOD1: *, p < 0.001. C111S was not statistically different from wild-type SOD1. Open arrowhead, dimer-sized SOD1 molecules. Closed arrowhead, monomeric SOD1 molecules.
FIGURE 2.
FIGURE 2.
Role of cysteines 6 and 111 in SOD1 aggregation. Mutants were expressed in HEK293FT cells, and aggregate levels were determined as described under “Experimental Procedures.” SDS-PAGE was performed in the presence of a reducing agent in an 18% Tris-glycine gel. Immunoblots were probed with m/hSOD1 antiserum. A, P2, detergent-insoluble protein fraction (20 μg). B, S1, detergent-soluble protein fraction (5 μg). C, quantification of the ratio of SOD1 in pellet fractions relative to supernatant was assessed as a measure of relative aggregation. The data represents the mean (±S.E. (error bars)) of at least three different experiments. Mutants where the ratios were statistically different from wild-type SOD1 are marked by * (p < 0.0001). Two mutants were not statistically different from wild-type SOD1: C6G/C111Y/C146R (GCYR) and C6G/C57S/C111Y (GSYC). Closed arrowhead, monomeric SOD1 molecules. UT, untransfected cells; CSYR, C57S/C111Y/C146R; GSCR, C6G/C57S/C146R.
FIGURE 3.
FIGURE 3.
Cysteine residues are not required for SOD1 aggregate formation. Mutants were expressed in HEK293FT cells, and aggregate levels were determined as described under “Experimental Procedures.” SDS-PAGE was performed in the presence of a reducing agent in an 18% Tris-glycine gel. Immunoblots were probed with m/hSOD1 antiserum. A, P2, detergent-insoluble protein fraction (20 μg). B, S1, detergent-soluble protein fraction (5 μg). C, quantification of relative aggregation potential (mean ratio ± S.E. (error bars)). Mutants G85R and C6G/C57S/C111Y/C146R (FSYR) were significantly different from the aggregation potential of WT SOD1: *, p < 0.0009. Open arrowhead, monomeric SOD1 molecules. UT, untransfected cells; GSYR, C6G/C57S/C111Y/C146R.
FIGURE 4.
FIGURE 4.
Role of cysteine 111 in mutant SOD1 aggregation. Mutants were expressed in HEK293FT cells, and aggregate levels were determined as described under “Experimental Procedures.” SDS-PAGE was performed in the presence of a reducing agent in an 18% Tris-glycine gel. Immunoblots were probed with m/hSOD1 antiserum. A, P2, detergent-insoluble protein fraction (20 μg). B, S1, detergent-soluble protein fraction (5 μg). C, relative aggregation ratios (mean ratio ± S.E. (error bars)) of at least three different experiments are graphed. All fALS mutants were statistically different from wild-type SOD1: *, p < 0.0005. The ratios of insoluble to soluble SOD1 for all fALS mutants that were combined with mutated cysteine 111 were not statistically different from wild-type SOD1: G85R/C111S, C6F/C111S, C6G/C111S, and C6G/C111Y. +, C6G was significantly different from C6G/C111S and from C6G/C111Y (p < 0.009). C6F did not differ from C6F/C111S. #, G85R was statistically different from G85R/C111S (p < 0.004). Open arrowhead, dimer-sized SOD1 molecules. Closed arrowhead, monomeric SOD1 molecules. UT, untransfected cells.
FIGURE 5.
FIGURE 5.
Symptomatic G86R mice form detergent-insoluble species in spinal cord tissue. Spinal cords from WT and G86R mice were extracted in buffers containing Nonidet P-40 as described under “Experimental Procedures.” SDS-PAGE was performed in the presence of a reducing agent in an 18% Tris-glycine gel. Immunoblots were probed with m/hSOD1 antiserum. Experiments were replicated twice; a representative example is shown. A, P2, detergent-insoluble protein fraction (20 μg). B, S1, detergent-soluble protein fraction (5 μg). UT, untransfected cells.
FIGURE 6.
FIGURE 6.
Mouse SOD1-G86R aggregates in cell culture. Mutants were expressed in HEK293FT cells, and aggregate levels were determined as described under “Experimental Procedures.” SDS-PAGE was performed in the presence of a reducing agent in an 18% Tris-glycine gel. Immunoblots were probed with m/hSOD1 antiserum. Experiments were replicated three times; a representative example is shown. A, P2, detergent-insoluble protein fraction (20 μg). B, S1, detergent-soluble protein fraction (5 μg). UT, untransfected cells.
FIGURE 7.
FIGURE 7.
Intermolecular disulfide bonding by SOD1 mutants in expressed cultured cells. FALS mutants (A4V, G85R, and G93A) were expressed in HEK293FT cells and detergent-extracted in buffers containing iodoacetamide (see “Experimental Procedures”). A and B, SDS-PAGE was performed in the absence of reducing agent in a 4–20% Tris-glycine gel. Immunoblots were probed with m/hSOD1 antiserum. Experiments were replicated four times; a representative example is shown. A, P2, detergent-insoluble protein fraction (20 μg). B, S1, detergent-soluble protein fraction (5 μg). C and D, the same sets of samples as depicted in A and B were analyzed by SDS-PAGE in the presence of a reducing agent. C, P2, detergent-insoluble protein fraction (20 μg). D, S1, detergent-soluble protein fraction (5 μg). Open arrowhead, dimer-sized SOD1 molecules. Closed arrowhead, monomeric SOD1 molecules. UT, untransfected cells.
FIGURE 8.
FIGURE 8.
Disulfide bonds are not sufficient to maintain SOD1 aggregate structure. A, spinal cord tissue from asymptomatic WT and symptomatic G93A SOD1 transgenic mice was extracted in 0.5% SDS detergent and iodoacetamide. B, standard extraction in Nonidet P-40 and centrifugation of the same tissues utilized in A. Samples were run in the presence or absence of reducing agent (β-mercaptoethanol (BME)), as noted, in 4–20% Tris-glycine gels. Immunoblots were probed with m/hSOD1 antiserum. S1, detergent-soluble (5 μg). P2, detergent-insoluble (20 μg).
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