Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies ThemselvesClick to copy article linkArticle link copied!
- Ryan S. AtlasiRyan S. AtlasiDepartment of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United StatesMore by Ryan S. Atlasi
- Ravinder MalikRavinder MalikDepartment of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United StatesMore by Ravinder Malik
- Christian I. CorralesChristian I. CorralesDepartment of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United StatesMore by Christian I. Corrales
- Laura TzeplaeffLaura TzeplaeffDepartment of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United StatesMore by Laura Tzeplaeff
- Julian P. WhiteleggeJulian P. WhiteleggeDepartment of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, Brain Research Institute and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, United StatesMore by Julian P. Whitelegge
- Neil R. CashmanNeil R. CashmanDepartment of Neurology, University of British Columbia (UBC), Vancouver, British Columbia V6T 2B5, CanadaMore by Neil R. Cashman
- Gal Bitan*Gal Bitan*E-mail: [email protected]. Tel. (310) 206-2082. Fax. (310) 206-1700.Department of Neurology, Brain Research Institute and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, United StatesMore by Gal Bitan
Abstract

Mutations in Cu/Zn-superoxide dismutase (SOD1) gene are linked to 10–20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations cause misfolding and self-assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. The molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization of misfolded SOD1 is particularly challenging because of its metastable nature. Antibodies against misfolded SOD1 are useful tools for this purpose, provided their specificity and selectivity are well-characterized. Here, we characterized three recently introduced antimisfolded SOD1 antibodies and compared them with two commercial, antimisfolded SOD1 antibodies raised against the fALS-linked variant G93A-SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti-SOD1 antibodies expected to be insensitive to misfolding. We asked to what extent the antibodies could distinguish between WT and variant SOD1 and between native and misfolded conformations. WT, G93A-SOD1, or E100K-SOD1 were incubated under aggregation-promoting conditions and monitored using thioflavin-T fluorescence, electron microscopy, and dot blots. WT and G93A-SOD1 also were analyzed using native-PAGE/Western blot. The new antimisfolded SOD1 and the commercial antibody B8H10 showed variable reactivity using dot blots but generally showed maximum reactivity at the time misfolded SOD1 oligomers were expected to be most abundant. In contrast, only B8H10 and the control antibodies were reactive in Western blots. Unexpectedly, the polyclonal antibodies showed strong preference for the misfolded form of G93A-SOD1 in dot blots. Surprisingly, antimisfolded SOD1 antibody C4F6 was specific for the apo form of G93A-SOD1 but insensitive to misfolding. Antibody 10C12 showed preference for early misfolded structures, whereas 3H1 bound preferentially to late structures. These new antibodies allow distinction between putative early- and late-forming prefibrillar SOD1 oligomers.
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