Abstract
Greater than 160 missense mutations in copper-zinc superoxide dismutase-1 (SOD1) can cause amyotrophic lateral sclerosis (ALS). These mutations produce conformational changes that reveal novel antibody binding epitopes. A monoclonal antibody, clone C4F6 - raised against the ALS variant G93A of SOD1, has been identified as specifically recognizing a conformation shared by many ALS mutants of SOD1. Attempts to determine whether non-mutant SOD1 adopts a C4F6-reactive conformation in spinal tissues of sporadic ALS (sALS) patients has produced inconsistent results. To define the epitope recognized by C4F6, we tested its binding to a panel of recombinant ALS-SOD1 proteins expressed in cultured cells, producing data to suggest that the C4F6 epitope minimally contains amino acids 90–93, which are normally folded into a tight hairpin loop. Multiple van der Waals interactions between the 90–93 loop and a loop formed by amino acids 37–42, particularly a leucine at position 38, form a stable structure termed the β-plug. Based on published modeling predictions, we suggest that the binding of C4F6 to multiple ALS mutants of SOD1 occurs when the local structure within the β-plug, including the loop at 90–93, is destabilized. In using the antibody to stain tissues from transgenic mice or humans, the specificity of the antibody for ALS mutant SOD1 was influenced by antigen retrieval protocols. Using conditions that showed the best discrimination between normal and misfolded mutant SOD1 in cell and mouse models, we could find no obvious difference in C4F6 reactivity to spinal motor neurons between sALS and controls tissues.Electronic supplementary materialThe online version of this article (doi: 10.1186/2051-5960-2-55) contains supplementary material, which is available to authorized users.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons
Robust expression of D90A, along with G93A and Wild-type SOD1 (WT) superoxide dismutase-1 (SOD1) was indicated by immunostaining with the control hSOD antibody that can detect all variants of hSOD1 (Figure 1a-c)
In this study we sought to more precisely define the epitope of the anti-hSOD1 C4F6 antibody and elucidate the basis for its ability to recognize many different ALS mutants of SOD1
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Co-expression of WT SOD1 with mutant SOD1 almost invariably accelerates the onset of paralysis with evidence that WT SOD1 has been induced to aggregate with mutant SOD1 [9,10,11,12,13] These studies point to WT SOD1 as a potential pathogenic link between fALS and sALS and more importantly, implicate SOD1 as a target for therapeutic intervention in the majority of ALS cases. Through the course of these studies, conformation-specific antibodies to SOD1 have emerged as critical reagents to distinguish misfolded, presumably toxic SOD1, from protein that achieves a more native conformation. The epitope recognized by these antibodies has not been completely characterized
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