Co-expression of WT and mutant hSOD1 in cultured HEK293FT cells reduced the level of detergent-insoluble mutant SOD1 proteins that accumulate in 24 hours (). HEK293FT cells transfected with A4V, G85R or G93A SOD1 alone formed detergent-insoluble aggregates that sedimented upon ultracentrifugation, whereas cells expressing both WT and mutant hSOD1 produced little or no detergent-insoluble SOD1 protein (, upper panel). Instead, both WT and mutant hSOD1 proteins were found only in soluble fractions (, lower panel). To control for non-specific effects of co-transfection, such as reduced mutant protein expression that may have caused a reduction in aggregation, we co-expressed the mutant SOD1 constructs (A4V, G85R and G93A SOD1) with an expression plasmid for GFP and performed the detergent extraction and centrifugation assay. In each case, the expression of GFP did not affect the aggregation of mutant SOD1 (, upper panel). In these cell culture experiments, a large fraction of the mutant SOD1 remains fully soluble in detergent (, lower panels). The levels of soluble SOD1 protein provide a good indication of protein expression, indicating that all mutant proteins were expressed at high levels relative to non-transfected control cells. Immunoblot data from at least four experiments for each set of WT and mutant hSOD1 co-transfections was quantified and analyzed statistically (; see Methods - Estimation of aggregation propensity and statistical analysis for an explanation of the methodology used to quantify relative aggregation propensities), providing clear evidence that in our cell culture model the co-expression of WT SOD1 modulates mutant hSOD1 aggregation.
To examine the effects of WT hSOD1 on mutant SOD1 aggregation over time, we extended the interval between transfection and harvest to 48 hours. Interestingly we found that WT hSOD1 differentially affected the aggregation of the different SOD1 mutants (A4V, G85R and G93A SOD1; ). As compared to cells expressing A4V SOD1 alone, cells co-transfected with vectors for WT and A4V hSOD1 continued to accumulate less detergent-insoluble mutant protein (, upper panel). However, aggregation was not blocked as these cells contained significantly more detergent-insoluble SOD1 than cells transfected with WT hSOD1 (). The levels of detergent-soluble SOD1 protein in extracts from cells co-transfected with WT and A4V hSOD1 indicated relatively high levels of expressed protein (, lower panel). However, because the WT and A4V hSOD1 proteins could not be distinguished by SDS-PAGE and the low amount of detergent-insoluble SOD1 in the co-transfected cells, we could not determine whether the insoluble hSOD1 is limited to mutant protein. When WT hSOD1 was co-expressed with G85R hSOD1, it was possible to differentiate the WT and mutant hSOD1 proteins by SDS-PAGE; the G85R variant migrates anomalously in SDS-PAGE, running slightly faster than the expected size (Hayward et al. 2002
;Wang et al. 2003
;Wang et al. 2006
). In cells co-expressing WT with G85R hSOD1, we observed significant accumulation of detergent-insoluble mutant protein at 48 hours. More interestingly, WT hSOD1 was clearly detected in the detergent-insoluble fraction ( upper panel, and F). We estimate the relative ratio of G85R to WT hSOD1 in the insoluble fraction to be about 4 to 1. In cells co-expressing WT and G93A hSOD1 for 48 hours, a significant amount of SOD1 was detected in the detergent-insoluble fraction ( and E). To determine whether WT hSOD1 was present in the detergent-insoluble fraction, we analyzed these fractions by hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometry (FTMS). FTMS analysis revealed the presence of both WT and G93A hSOD1 in both the detergent-insoluble and -soluble fractions (). In the detergent-insoluble fractions, however, the amount of WT hSOD1 was about 10 fold less than G93A hSOD1 (,); while in the soluble fractions, the levels of WT and G93A hSOD1 were similar (,). Overall, these results are consistent with the discoveries of detergent-insoluble WT hSOD1 in spinal cords of transgenic mice co-expressing WT and mutant hSOD1 (Deng et al. 2006
). We interpret our data in cell culture as evidence that WT hSOD1 primarily slows the rate of mutant SOD1 aggregation, but ultimately when aggregates form, the WT protein may inefficiently co-aggregate with some mutant SOD1 variants.
Fig. 2 Comparison of SOD1 molecular mass profiles from soluble (S1) and insoluble (P2) extracts of HEK293FT cells co-expressing human WT and G93A hSOD1. SOD1 was recovered from spinal cord extracts, solubilized and purified by reverse-phase chromatography for (more ...)
To control for the effects of co-transfection and for the possibility that SOD1 proteins of differing sequences might interfere with aggregation, we also co-transfected G85R SOD1 with WT, A4V, and G93A hSOD1 constructs. In these experiments we took advantage of the anomalous migration of G85R hSOD1 to examine how the co-expression of two different SOD1 mutants might affect their aggregation. Cells co-expressing G85R SOD1 with either A4V or G93A SOD1 produced detergent-insoluble forms of each SOD1 mutant (, upper panel). As described above, the presence of mutant hSOD1 protein in the S1 fraction for each transfection indicated that only a portion of the total protein adopted the insoluble conformation (, lower panel). Quantification of multiple independent experiments demonstrated that whether expressed alone or in combination with the A4V or G93A SOD1 variants, the propensity of G85R SOD1 to aggregate was not significantly altered (). These data suggest that the apparent reduction in aggregation caused by the co-expression of WT with mutant hSOD1 is not due to some non-specific effect of co-transfection or some non-specific effect of interactions between two different SOD1 subunits, but rather appears to be due to a specific property of the WT hSOD1 protein.
The human and mouse WT SOD1 protein share 83.6% identity at the level of amino acid sequence (Supplemental Fig. 1
); 25 amino acid differences in the 153 residue protein. Thus we next sought to investigate whether these differences in protein sequence would affect the ability of WT SOD1 to modulate the aggregation rate of mutant SOD1. HEK293FT cells co-transfected with WT mouse SOD1 (mSOD1) and mutant hSOD1 proteins (A4V, G85R and G93A SOD1) showed a significant reduction in the amount of detergent-insoluble SOD1 aggregates produced in 24 hours (). Interpretation of the immunoblots of cells co-transfected with WT mSOD1 and G85R hSOD1 was complicated by the fact that these proteins migrated to very near the same position in SDS-PAGE. However, these proteins could be resolved in gels exposed for short intervals, allowing for the detection of both WT mSOD1 and G85R hSOD1 in the detergent-soluble protein fraction (, right lower panel). Despite a significant reduction in the amount of insoluble G85R hSOD1 in these co-transfected cells, aggregation was not blocked and it was possible to demonstrate that the detergent-insoluble fraction contained only G85R hSOD1 (, right upper panel). Quantification of the relative aggregation propensity of the hSOD1 mutants in cells co-transfected with WT mSOD1 revealed a significant reduction in the amount of detergent-insoluble mutant hSOD1 protein that accumulated in 24 hours (). Thus, WT mSOD1 has the same capacity as WT hSOD1 to slow the rate of mutant hSOD1 to aggregate.
When we extended the interval between transfection and harvest to 48 hours, we observed that A4V, G85R and G93A hSOD1, when expressed with WT mSOD1, were able to form detectable amounts of detergent-insoluble SOD1 aggregates (). We observed that in measures of aggregation propensity, which compensates for any changes in the expression of mutant hSOD1 that may occur when co-transfected with WT mSOD1, the presence of WT mSOD1 had no significant effect on aggregation of mutant hSOD1 (). Interestingly, WT mSOD1, unlike WT hSOD1, did not seem to co-aggregate with any of the mutants even after the longer 48 hour interval (, upper panel). In co-transfections of A4V or G93A hSOD1 mutants with WT mSOD1, the amount of mSOD1 detected in the insoluble fraction was not different from that of cells transfected with mSOD1 alone (; p > 0.05, n = 4 independent replications). Moreover, we did not detect mSOD1 in detergent-insoluble fractions of cells co-expressing WT mSOD1 and G85R hSOD1 at a level greater than that of cells expressing mSOD1 alone (p > 0.20, n = 3 independent replications). Thus, although WT mSOD1 possesses an ability that is similar to WT hSOD1 in modulating the aggregation of mutant proteins, it lacks a feature that allows for co-sedimentation with mutant hSOD1.
The differing ability of WT mSOD1 and hSOD1 to co-aggregate with mutant hSOD1 is a finding that appears to be consistent with a recent report suggesting that a specific cysteine residue in hSOD1 may mediate the co-aggregation of WT and mutant hSOD1 (Cozzolino et al. 2008
). The cysteine residue at position 111 of hSOD1 has been identified as a potential mediator of disulfide cross-linking between mutant and WT hSOD1 (Cozzolino et al. 2008
). Mouse SOD1 encodes serine at position 111, and thus could not generate such disulfide linkages. To directly test this hypothesis, we used a previously described cDNA hSOD1 that encodes serine as position 111 (C111S hSOD1) (Karch and Borchelt 2008
) in co-transfection with G85R hSOD1. The mutant C111S hSOD1 is not an ALS mutation; in most species the position equivalent to 111 encodes serine. Previous studies have established that this mutant does not spontaneously aggregate (Cozzolino et al. 2008
;Karch and Borchelt 2008
). In C111S and G85R hSOD1 co-transfections, cells were harvested after 48 hours, which was the interval needed to observe WT and G85R hSOD1 co-aggregation. Consistent with previous studies, all C111S hSOD1 fractionated to the detergent-soluble fraction (, compare upper and lower panels). However, in cells co-transfected with C111S and G85R hSOD1 and harvested 48 hours later, we found both proteins in the detergent-insoluble fraction Quantification of the aggregation propensity of each SOD1 protein was performed as previously described, showing significant accumulation of aggregated C111S hSOD1 in the co-transfected cells (). This finding suggests that the co-aggregation of WT hSOD1 with mutant protein is not dependent upon a disulfide linkage between cysteine 111 of WT protein and cysteine residues of mutant hSOD1.