Decreased Metallation and Activity in Subsets of Mutant Superoxide Dismutases Associated with Familial Amyotrophic Lateral Sclerosis

Abstract

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause ϳ2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALSrelated SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133⌬) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.Amyotrophic lateral sclerosis (ALS, 1 Lou Gehrig's disease) is an age-dependent, degenerative disorder of motor neurons in the spinal cord and brain. Progressive dysfunction of both upper and lower motor neurons causes death from respiratory paralysis, usually within 5 years. Investigation of the causes of familial ALS, which comprises ϳ10% of cases, may contribute insights relevant to the pathophysiology of sporadic ALS and of other motor neuron diseases (1, 2).A subset of autosomal dominant ALS is caused by over 90 mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) (3, 4) (see an updated list of all mutations on the World Wide Web at www.alsod.org). SOD1 is a 32-kDa homodimeric enzyme that functions as an antioxidant, converting two molecules of superoxide anion (O 2 . ) to O 2 and H 2 O 2 .This redox cycle involves alternate reduction (reaction 1) and reoxidation (reaction 2) of the catalytic copper ion by O 2 . .REACTIONS 1 and 2 SOD1 contains an eight-stranded ␤-barrel motif, an intrasubunit disulfide bond, and a zinc binding site that contribute to its extreme thermochemical stability (Fig. 1). The mutant residues are scattered throughout the protein, including some residues important for copper or zinc coordination, others located near the dimer interface or at either pole of the ␤-barrel, and several in the charged loop near the C terminus that may guide O 2 . to the active site. Although most are missense substitutions, some are predicted to truncate the C terminus of the protein, including the charged loop. No null mutations have been described. Mutant SOD1 most likely causes motor neuron death by gain of an unknown toxic property rather than by deficiency of dismutase a...

Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction

“…Transgenic mice overexpressing either WT SOD1 (line B6SJL-TgN(SOD1)2Gur) or G93A SOD1 (line B6SJL-TgN(SOD1-G93A)1Gur) (4) were obtained from the Jackson Laboratory (Bar Harbor, ME), whereas those overexpressing G85R SOD1 (7) bred to homozygosity were provided by Dr. Zuoshang Xu. WT or ALS-related mutant SOD1 enzymes (A4V, L38V, G41S, H46R, H48Q, G72S, D76Y, G85R, D90A, G93A, D124V, D125H, E133⌬, and S134N) containing biologically incorporated metal ions were isolated from a baculoviral expression system (18). Protein concentrations were estimated using a dimeric molar extinction coefficient at 280 nm of 10,800 M Ϫ1 cm Ϫ1 (32) or 13,800 M Ϫ1 cm Ϫ1 for D76Y SOD1 (33).…”

“…We previously purified 14 different biologically metallated ALS mutant SOD1s and observed that one group of "WT-like" mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133⌬) bound copper in a fully active coordination environment remarkably similar to that of normal SOD1 despite causing a lethal phenotype (18). The other six "metal-binding region" mutants (H46R, H48Q, G85R, D124V, D125H, and S134N) were clearly distinguished from WT SOD1 according to decreased metal ion contents, altered visible absorption spectra, or decreased specific activities.…”

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

“…Therefore, an impaired maturation process would lead to the accumulation of immature SOD1 species, which are prone to oligomerization. Recent studies reported a higher propensity for deficient protein maturation in vivo or in-cell culture models for some fALS mutants 39,40 . However, most of the studies addressing the properties of fALS SOD1 mutants and their effects on the maturation process have been performed in vitro, therefore, far from physiological conditions [41][42][43] .…”

Mutant SOD1 detoxification mechanisms in intact single cells

“…Three distinct bands of SOD1 activity are observed in samples from EGFP-SOD1 wt -and EGFP-SOD1 G93A -transfected cells, representing the dimers of endogenous untagged SOD1, EGFP-SOD1 dimers, and the respective heterodimers. In contrast to the 'wt-like' SOD1 G93A , in cells transfected with the dismutase-dead mtSOD1 G85R , 23 only endogenous SOD1 wt activity could be detected. CON: untransfected control cells.…”

“…In contrast to EGFP-SOD1 G93A , enzymatic activity was lost by EGFP-SOD1 G85R , as observed before for the untagged SOD1 G85R mutant. 23 The finding that our SOD1 fusion proteins still dimerize and -if not abolished by the respective mutation -exhibit normal dismutase activity, indicates that the EGFP tag does not preclude proper maturation, dimerization and copper loading of SOD1. This was further confirmed by the presence of the intramolecular disulfide bond in EGFP-SOD1 fusion proteins that is essential for activity and stability ( Figure 1c).…”