Abstract
Mutations in superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (fALS). Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE) as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1) is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z), immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1.
Highlights
Amyotrophic Lateral Sclerosis (ALS) is primarily characterized by loss of upper and lower motor neurons and usually arises by an unknown etiology
Antibodies to superoxide dismutase 1 (SOD1) included the following, a rabbit polyclonal peptide antiserum raised against the human peptide sequence 2436 that recognizes human SOD1 [32], a monoclonal antibody raised against the apo-form of human SOD1-G93A [33], and a rabbit polyclonal antiserum raised against a peptide representing amino acids 145-151 in human SOD1
We have investigated the utility of using blue native gel electrophoresis as a means to detect soluble multimeric forms of mutant SOD1 in cell lysates
Summary
Amyotrophic Lateral Sclerosis (ALS) is primarily characterized by loss of upper and lower motor neurons and usually arises by an unknown etiology (sporadic ALS or sALS). Between 10 and 20% of ALS cases can be linked to defined genetic causes (familial ALS or fALS), and of these inherited genetic mutations, approximately 20% are found in the Cu-Zn superoxide dismutase (sod1) gene [1]. Over 150 mutations in sod have been linked to fALS {http:// alsod.iop.kcl.ac.uk/default.aspx}. There are rare reports of frame-shift and early terminations that would produce mRNAs that would be predicted to be degraded by nonsense mediated decay pathways {http://alsod.iop.kcl.ac.uk/default.aspx}. Whether these mutations are causative of disease in these individuals is uncertain
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