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Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves
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    Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves
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    • Ryan S. Atlasi
      Ryan S. Atlasi
      Department of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United States
    • Ravinder Malik
      Ravinder Malik
      Department of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United States
    • Christian I. Corrales
      Christian I. Corrales
      Department of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United States
    • Laura Tzeplaeff
      Laura Tzeplaeff
      Department of Neurology, University of California, Los Angeles, Los Angeles, California 90095, United States
    • Julian P. Whitelegge
      Julian P. Whitelegge
      Department 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 States
    • Neil R. Cashman
      Neil R. Cashman
      Department of Neurology, University of British Columbia (UBC), Vancouver, British Columbia V6T 2B5, Canada
    • Gal Bitan*
      Gal Bitan
      Department of Neurology,  Brain Research Institute  and  Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, United States
      *E-mail: [email protected]. Tel. (310) 206-2082. Fax. (310) 206-1700.
      More by Gal Bitan
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    ACS Chemical Biology

    Cite this: ACS Chem. Biol. 2018, 13, 9, 2794–2807
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    https://doi.org/10.1021/acschembio.8b00729
    Published August 15, 2018
    Copyright © 2018 American Chemical Society

    Abstract

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    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.

    Copyright © 2018 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00729.

    • Supplementary Figure 1: Comparison of SOD1 aggregation under different conditions (PDF)

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    ACS Chemical Biology

    Cite this: ACS Chem. Biol. 2018, 13, 9, 2794–2807
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acschembio.8b00729
    Published August 15, 2018
    Copyright © 2018 American Chemical Society

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