Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations.
The reactivity of asparagine residues in Cu, Zn superoxide dismutase (SOD1) to deamidate to aspartate remains uncharacterized; its occurrence in SOD1 has not been investigated, and the biophysical effects of deamidation on SOD1 are unknown. Deamidation is, nonetheless, chemically equivalent to Asn-to-Asp missense mutations in SOD1 that cause amyotrophic lateral sclerosis (ALS). This study utilized computational methods to identify three asparagine residues in wild-type (WT) SOD1 (i.e., N26, N131, and N139) that are predicted to undergo significant deamidation (i.e., to >20%) on time scales comparable to the long lifetime (>1 year) of SOD1 in large motor neurons. Site-directed mutagenesis was used to successively substitute these asparagines with aspartate (to mimic deamidation) according to their predicted deamidation rate, yielding: N26D, N26D/N131D, and N26D/N131D/N139D SOD1. Differential scanning calorimetry demonstrated that the thermostability of N26D/N131D/N139D SOD1 is lower than WT SOD1 by ~2-8 °C (depending upon the state of metalation) and <3 °C lower than the ALS mutant N139D SOD1. The triply deamidated analog also aggregated into amyloid fibrils faster than WT SOD1 by ~2-fold (p < 0.008**) and at a rate identical to ALS mutant N139D SOD1 (p > 0.2). A total of 534 separate amyloid assays were performed to generate statistically significant comparisons of aggregation rates among WT and N/D SOD1 proteins. Capillary electrophoresis and mass spectrometry demonstrated that ~23% of N26 is deamidated to aspartate (iso-aspartate was undetectable) in a preparation of WT human SOD1 (isolated from erythrocytes) that has been used for decades by researchers as an analytical standard. The deamidation of asparagine--an analytically elusive, sub-Dalton modification--represents a plausible and overlooked mechanism by which WT SOD1 is converted to a neurotoxic isoform that has a similar structure, instability, and aggregation propensity as ALS mutant N139D SOD1.
Department of Chemistry and Biochemistry, Baylor University , Waco, Texas 76706, United States.
Yunhua
Y
Shi
Nicholas R
NR
Rhodes
Alireza
A
Abdolvahabi
Taylor
T
Kohn
Nathan P
NP
Cook
Angel A
AA
Marti
Bryan F
BF
Shaw
D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D020125:Mutation, Missense; D055550:Protein Stability; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D013696:Temperature
C000606290:SOD1 protein, human; D001224:Aspartic Acid; D001216:Asparagine; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1