News Release

Title
Elucidating a mechanism of species-specific aggregation of canine SOD1 with adegenerative myelopathy-linked mutation

Key Points•E40K mutation induces aggregation in canine SOD1, but not human SOD1;
However, the mechanism remains unknown.
•We found that the species-specific aggregation depended on a difference ofthe 117th amino acid residues, methionine and leucine in canines and humans,respectively.
•Crystal structural analysis revealed the instability of the hydrophobic core incanine SOD1 because of 117th methionine creating a cavity, but the cavity wasfilled by 117th leucine.
• Instability of canine SOD1 due to the cavity is the basis of species-specificaggregation of SOD1, and a clue for developing novel therapeutics for canine
DM.

SummaryA research group led by Professor Koji Yamanaka, Lecturer Seiji Watanabe, andGraduate Student Kei Hashimoto of the Nagoya University, Research Instituteof Environment Medicine, Tokai National Higher Education and Research System,has discovered the mechanism of species-specific aggregation of canine SOD1
with a degenerative myelopathy (DM)-linked mutation in collaboration withResearch Fellow Hiroaki Kamishina of Gifu University and Professor Yoshiaki
Furukawa of Keio University.
In DM, the E40K mutation, in which the 40th glutamate of the SOD1 protein isreplaced with lysine, is thought to cause abnormal aggregation of the SOD1protein, resulting in damage to motor neurons in the spinal cord of dogs. On theotherhand,

hereditary

amyotrophic

lateralsclerosis(ALS),

ahumanneurological disease, also causes motor neuron damage due to abnormal
aggregation of SOD1 protein, but the E40K mutation has no effect on human
SOD1, suggesting that the aggregation of canine SOD1 caused by E40K
mutation is species-specific. Therefore, this research group conducted
research to elucidate the mechanism of species-specific aggregation of canine
SOD1 caused by the E40K mutation, and discovered that canine SOD1 is less

stable and aggregates more easily than human SOD1, because canine SOD1
intrinsically has a "gap" in the highly hydrophobic region inside the protein. By
manipulating the presence or absence of this "gap," they also reproduced thespecies-specific aggregation of SOD1 protein caused by the E40K mutation.
This indicates that the inherent vulnerability of canine SOD1 derived from the"gap" is the cause of the species-specific aggregation by the E40K mutation.
The results of this research are expected to lead to the development of novel
therapies for DM in the future. This work was published online in the Journal of

Biological Chemistry on May 6, 2023.

Research Background
Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease inwhich motor neurons in the spinal cord are injured, resulting in progressive
paralysis of muscles (Awano et al. 2009 PNAS), and the replacement ofglutamic acid at position 40 of the Cu/Zn superoxide dismutase (SOD1) protein
encoded by the SOD1 gene with lysine (E40K mutation) is thought to be aprimary cause.
ALS and DM caused by mutations in the SOD1 gene share many clinicaland pathological similarities. For this reason, DM has been recognized as aspontaneous disease model of ALS. Previous studies have shown that in familialALS,

intracellularaggregationofSOD1

proteininduces

motor

neurondegeneration. On the other hand, canine SOD1 also aggregates due to E40Kmutation, but there are no E40K mutation that causes ALS, and introduction ofE40K mutation into human SOD1 does not cause aggregation (Crisp et al. 2013Exp Neurol), suggesting that the aggregation of canine SOD1 by the E40K
mutation is species-specific. If the E40K mutation causes aggregation througha mechanism unique to canine SOD1 that is completely different from that ofhuman SOD1, then it is necessary to develop a suitable treatment for theE40K-linked DM. Therefore, the aim of this study was to elucidate the molecularmechanism of the species-specific aggregation of canine SOD1 caused by theE40K mutation.

Research Results

Canine and human SOD1s have 20% differences in the amino acid sequences,and this research group hypothesized that amino acid residues that differ
between these species may be responsible for the species-specific aggregation
and aimed to identify them. Chimeric SOD1s, in which a part of canine SOD1 wasreplaced with human SOD1, were generated and verified as to whether theproteins aggregated, revealing that the 117th amino acid residue (methionine incanine and leucine in human) is important for the species-specific aggregation.
Replacement of methionine at position 117 in canine SOD1 with leucine, thehuman counterpart, markedly suppressed the E40K mutation-dependent
aggregation of canine SOD1 (Figure left upper panel). Conversely, replacementof leucine at position 117 with methionine in human SOD1 induced the E40K
mutation-dependent aggregation of human SOD1 (Figure left lower panel).
The research group subsequently examined how this 117th amino acidresidue is important for the species-specific aggregation of canine SOD1.
Previous studies have shown that many aggregated proteins are unstable instructure, and their protein structures are easily destroyed by heating. In fact,canine SOD1 was found to be less stable for heat than human SOD1, even in thewild-type. This suggests that canine SOD1 is intrinsically more prone toaggregation than human SOD1. Interestingly, when the 117th methionine
identified in this study was replaced with the human counterpart, leucine, thethermal stability of canine SOD1 was dramatically improved, whereas thethermal stability of human SOD1 was inversely reduced when the 117th leucine
in human SOD1 was replaced with the methionine. Moreover, the replacement ofmethionine with leucine reduced cytotoxicity of canine SOD1 with the E40Kmutation. These results suggest that the 117th amino acid residue, leucine,stabilizes the structure of both canine and human SOD1 proteins and isimportant for suppressing aggregation and cytotoxicity caused by the E40K
mutation.
Then, why is SOD1 stabilized by the 117th leucine? To answer thisquestion, the research group further analyzed the structure of canine SOD1 by
X-ray crystallography. As a result, methionine created a "gap" in thehydrophobic region located inside canine SOD1 (Figure right). Furthermore,
replacement of the 117th methionine with leucine filled this "gap". These
results suggest that, unlike human SOD1, canine SOD1 is structurally vulnerable
due to the "gap" created by the 117th methionine, which is the cause of thespecies-specific aggregation by the E40K mutation.
Research Summary and Future Perspective
We have elucidated the key mechanism of E40K-dependent species-specificaggregation of canine SOD1. The results of this study indicate that canine SOD1
has the intrinsic vulnerability of the hydrophobic region derived from 117th
methionine which forms a "gap" there. Stabilization of this hydrophobic region
may suppress abnormal aggregation of canine SOD1 and inhibit the onset andprogression of DM.
Publication
Journal: Journal of Biological Chemistry
Title: Intrinsic structural vulnerability in the hydrophobic core induces
species-specificaggregationofcanine

SOD1

withdegenerative

myelopathy–linked E40K mutation
Authors:
Kei Hashimoto1, 2, Seiji Watanabe1, 2, Masato Akutsu3, Norifumi Muraki3, Hiroaki
Kamishina4, 5, Yoshiaki Furukawa3, Koji Yamanaka1, 2, 6, 7Affiliations:
1. Department of Neuroscience and Pathobiology, Research Institute ofEnvironmental Medicine, Nagoya University, Nagoya, Aichi, Japan
2. Department of Neuroscience and Pathobiology, Nagoya University Graduate
School of Medicine, Nagoya, Aichi, Japan
3. Department of Chemistry, Keio University, Yokohama, Kanagawa, Japan
4. Life Science Research Center, Gifu University, Gifu, Japan
5. Kyoto AR Advanced Veterinary Medical Center, Kyoto, Japan
6. Institute for Glyco-core Research (iGCORE), Nagoya University, Aichi, Japan
7. Center for One Medicine Innovative Translational Research (COMIT), Gifu

University Institute for Advanced Study, Gifu, Japan
DOI：10.1016/j.jbc.2023.104798
Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Jou_230522.pdf