A systematic immunoprecipitation approach reinforces the concept of common conformational alterations in amyotrophic lateral sclerosis-linked SOD1 mutants

Abstract

Mutations in the Cu, Zn superoxide dismutase (SOD1) gene are one of the causative agents of amyotrophic lateral sclerosis (ALS). Although more than 100 different mutations in SOD1 have been identified, it is unclear whether all the mutations are pathogenic or just single nucleotide polymorphisms (SNPs) unrelated to the disease. Our previous systematic analysis found that all pathogenic SOD1 mutants (SOD1mut) have a common property, namely, an association with Derlin-1, a component of the endoplasmic reticulum-associated degradation machinery. For the proposed mechanism, we found that most pathogenic SOD1mut have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1WT). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1mut from both SOD1WT and non-toxic SOD1mut. However, MS785 could not recognize SOD1mut that has mutations in the MS785 epitope region. Here, we developed a new diagnostic antibody, which could compensate for this shortcoming of MS785. We hypothesized that in ALS-causative SOD1mut, the DBR-neighboring region [SOD1(30–40)] may also be exposed. We then generated MS27, a monoclonal antibody against SOD1(30–40). We found that MS27 could distinguish SOD1WT from the pathogenic SOD1mut, which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1mut, without exception, were immunoprecipitated with a combination of MS785 and MS27. The MS785–MS27 combination could be developed as a novel mechanism-based biomarker for the diagnosis of ALS.