Mutation-dependent polymorphism of Cu,Zn-superoxide dismutase aggregates in the familial form of amyotrophic lateral sclerosis

Abstract

More than 100 different mutations in Cu,Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (fALS). Pathogenic mutations facilitate fibrillar aggregation of SOD1, upon which significant structural changes of SOD1 have been assumed; in general, however, a structure of protein aggregate remains obscure. Here, we have identified a protease-resistant core in wild-type as well as fALS-causing mutant SOD1 aggregates. Three different regions within an SOD1 sequence are found as building blocks for the formation of an aggregate core, and fALS-causing mutations modulate interactions among these three regions to form a distinct core, namely SOD1 aggregates exhibit mutation-dependent structural polymorphism, which further regulates biochemical properties of aggregates such as solubility. Based upon these results, we propose a new pathomechanism of fALS in which mutation-dependent structural polymorphism of SOD1 aggregates can affect disease phenotypes.

FIGURE 1.

MALDI-TOF mass spectrometric analysis of a core structure in SOD1 aggregate and experimental identification of SOD1 sequences with high aggregation propensities. A, peptides corresponding to the mass peaks detected are mapped on the primary sequence of SOD1. Numbers indicated above the primary sequence of SOD1 (shown as open bars) represent the amino residue numbers. Peptides were identified either by MS/MS analysis (thick bars) or mass (thin bars). Details of peptide identification are summarized in supplemental Table S1. B, three-dimensional structure of an SOD1 dimer with copper and zinc ions bound (Protein Data Bank code 1HL5). Regions A–C are colored red, blue, and green, respectively. C, after overnight agitation of 100 μm SOD1 fragments (ex1–2, 2–3, 3–4, and 4–5 as shown in A) at 37 °C, a sample solution was ultracentrifuged at 110,000 × g for 30 min to separate a supernatant (s) and a pellet (p) fraction, and these fractions were analyzed by a 15% SDS-polyacrylamide gel stained with Coomassie Brilliant Blue. D, fluorescence intensity was measured in the samples containing 25 μm ThT and 3 μm SOD1 fragments before (0 h, open bars) and after (13 h, filled bars) 1,200 rpm agitation at 37 °C. E and F, electron micrograms of the pellet fraction containing (E) ex1–2 and (F) ex4–5 aggregates.

FIGURE 2.

Core regions in aggregates of WT and fALS-mutant SOD1 proteins identified by MALDI-TOF mass analysis. SOD1^G37R, SOD1^G85R, and SOD1^G93A indicate aggregates extracted from lumbar spinal cords of the corresponding transgenic mice, whereas the others are SOD1 aggregates prepared from the recombinant proteins with indicated fALS mutations. SOD1 aggregates were treated with Pronase, and the peptides resistant to digestion by Pronase were identified by MALDI-TOF mass spectrometry and mapped on the SOD1 primary sequence. Numbers indicated above the primary sequence of SOD1 (shown as an open bar) represent the amino residue numbers. Identification of mass peaks was performed based upon the observed mass values, and peptides shown as thick bars were further confirmed by MS/MS analysis. Details of peptide identification are summarized in supplemental Table S1 and supplemental Fig. S3, L–O.

FIGURE 3.

Identification of SOD1 Cys residues that are modified with Alexa555. Alexa555-modified SOD1 (WT, A4V, G37R, L126X, or I149T) was digested with lysyl endopeptidase, and mass peaks from the following Cys-containing peptides modified with Alexa555 are shown: A, Ala⁴–Lys²³ (Cys⁶) with an Alexa555; B, Gly³⁷–Lys⁷⁰ (Cys⁵⁷) with an Alexa555; C, His⁷¹–Lys¹²² (Cys¹¹¹) with an Alexa555; and D, Thr¹³⁷–Gln¹⁵³ (Cys¹⁴⁶) with an Alexa555. In each panel, proteolytic digestion was performed using either soluble (left) or aggregated (right) SOD1 modified with Alexa555. Observed mass was shown in each spectrum, and calculated mass values of Alexa555-modified peptides are as follows: 3070.4 in Ala⁴–Lys²³ (3098.5 for A4V), 4548.2 in Gly³⁷–Lys⁷⁰ (4647.3 for G37R), 6434.3 in His⁷¹–Lys¹²², and 2543.1 in Thr¹³⁷–Gln¹⁵³ (2531.1 for I149T). All of these mass peaks were not observed in unmodified SOD1 samples, confirming that the mass peaks shown in A–D are derived from Alexa555-modified peptides.

FIGURE 4.

Electron micrograms of (A) WT and (B–K) fALS-mutant SOD1 aggregates. A bar in each panel represents 50 nm.

FIGURE 5.

Distinct solubility of SOD1 aggregates with different fALS mutations in the presence of Sarkosyl. A, 2 μg of WT or L126X SOD1 aggregates were prepared in an NNE buffer with or without a detergent; either 1% Nonidet P-40 (NP-40), 1% Sarkosyl, or 1% SDS. After incubation at 37 °C for an hour, samples were ultracentrifuged at 110,000 × g for 30 min, and a supernatant (s) and a pellet (p) fraction were analyzed by a 12.5% SDS-polyacrylamide gel. B, temperature dependence of the WT or L126X aggregate solubility in the presence of 1% Sarkosyl. Experimental conditions were the same as described in A except the incubation temperature. C, effects of temperature on the aggregate solubility were further tested in all of the other fALS mutant SOD1 proteins, and fractions of solubilized SOD1 were estimated from its relative band intensities between supernatant and pellet on an SDS-polyacrylamide gel and plotted against incubation temperature. D, SOD1 aggregates were extracted from lumbar spinal cords of transgenic mice expressing G37R, G85R, and G93A SOD1 and incubated for an hour at indicated temperature in NNE buffer containing 1% Sarkosyl and 5 mm DTT. After ultracentrifugation at 110,000 × g for 30 min, supernatant (s) and pellet (p) fractions were probed by Western blotting analysis using sheep anti-SOD1 polyclonal antibody (Calbiochem). Percentages indicated below each gel image represent the fraction of solubilized SOD1 that is estimated from band intensities. E, T_½ was obtained by fitting the data in C with a sigmoidal function (see “Experimental Procedures”) and plotted against fibril types defined in this study.

FIGURE 6.

Our proposed model to describe mutation-dependent structural polymorphism of SOD1 aggregates. In secondary structural representation of SOD1 (left), regions A–C are colored red, blue and green, respectively. Rearrangement of these regions results in the formation of a core, and at least three different combinations of interactions among regions A–C are possible (middle). An exact alignment of β-sheets in the aggregates remains unknown; therefore, alignment of regions A–C in each schematic representation (middle) is still speculative. Interactions among aggregation core regions determine overall morphologies (right) and biochemical properties of SOD1 aggregates. Inset, average disease duration is plotted against average onset of disease. Data were taken from Refs. , . Type I/III, II, and IV aggregates are colored by black, red, and blue, respectively.