title: 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 (SOD1(mut)) 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 SOD1(mut) have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1(WT)). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1(mut) from both SOD1(WT) and non-toxic SOD1(mut). However, MS785 could not recognize SOD1(mut) 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 SOD1(mut), 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 SOD1(WT) from the pathogenic SOD1(mut), which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1(mut), 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.
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Takao
authors: T
authors: Fujisawa
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Namiko
authors: N
authors: Yamaguchi
authors: Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
authors: Hisae
authors: H
authors: Kadowaki
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Yuka
authors: Y
authors: Tsukamoto
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Naomi
authors: N
authors: Tsuburaya
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Atsushi
authors: A
authors: Tsubota
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Hiromitsu
authors: H
authors: Takahashi
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Isao
authors: I
authors: Naguro
authors: Department of Neurology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
authors: Yuji
authors: Y
authors: Takahashi
authors: Department of Neurology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
authors: Jun
authors: J
authors: Goto
authors: Department of Neurology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
authors: Shoji
authors: S
authors: Tsuji
authors: Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
authors: Hideki
authors: H
authors: Nishitoh
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Kengo
authors: K
authors: Homma
authors: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: ichijo@mol.f.u-tokyo.ac.jp.
authors: Hidenori
authors: H
authors: Ichijo
mesh_terms: D000595:Amino Acid Sequence; D000690:Amyotrophic Lateral Sclerosis / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D004926:Escherichia coli; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D008822:Mice, Transgenic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology; D013116:Spinal Cord / Q000378:metabolism / Q000473:pathology; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D000072105:Superoxide Dismutase-1; D014162:Transfection
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; C000606290:SOD1 protein, human; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1
keywords: Amyotrophic lateral sclerosis; Common conformational change; ELISA; ER stress; Mutant SOD1; Mutant SOD1-specific antibody