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. 2007 Jul 24;104(30):12524-9.
doi: 10.1073/pnas.0705044104. Epub 2007 Jul 16.

Common molecular signature in SOD1 for both sporadic and familial amyotrophic lateral sclerosis

Arie Gruzman  1 , William L Wood, Evgenia Alpert, M Dharma Prasad, Robert G Miller, Jeffery D Rothstein, Robert Bowser, Ronald Hamilton, Troy D Wood, Don W Cleveland, Vishwanath R Lingappa, Jian Liu
Affiliations

Common molecular signature in SOD1 for both sporadic and familial amyotrophic lateral sclerosis

Arie Gruzman et al. Proc Natl Acad Sci U S A. .

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS ( approximately 90%) are sporadic (SALS), occurring in the absence of genetic associations. Approximately 20% of familial ALS (FALS) cases are due to known mutations in the copper, zinc superoxide dismutase (SOD1) gene. Molecular evidence for a common pathogenesis of SALS and FALS has remained elusive. Here we use covalent chemical modification to reveal an attribute of spinal cord SOD1 common to both SOD1-linked FALS and SALS, but not present in normal or disease-affected tissues from other neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and spinal muscular atrophy, a non-ALS motor neuron disease. Biotinylation reveals a 32-kDa, covalently cross-linked SOD1-containing protein species produced not only in FALS caused by SOD1 mutation, but also in SALS. These studies use chemical modification as a novel tool for the detection of a disease-associated biomarker. Our results identify a shared molecular event involving a known target gene and suggest a common step in the pathogenesis between SALS and FALS.

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Conflict of interest statement

Conflict of interest statement: V.R.L. is a founder and J.L. a consultant to Prosetta Corporation, an early stage biotechnology company.

Figures

Fig. 1.
Fig. 1.
A 32-kDa SOD1-IR protein species revealed by biotinylation of spinal cord extracts of ALS patients. (A) Schematic diagram of the detection of a 32-kDa SOD1-IR protein species by immunoblot analysis. Sulfo-NHS-LC-Biotin reacts with structurally available NH2 groups (outside the rings) from the lysine residues in the SOD1 protein resulting in the modification of the protein at the sites of the side-chains (indicated by *). The biotinylated SOD1 proteins were separated on SDS/PAGE gel and immunorecognized by the SOD1 antibody (darker ring). (B–G) Spinal cord cytosolic proteins were incubated with (+) or without (−) Sulfo-NHS-LC-Biotin, separated on SDS/PAGE gel, and immunoblotted with the SOD1 antibody. (B) The FALS sample was from an A4V mutation. Cytosolic proteins from SALS-4 subject were used for C–G. (C) Time course of the reaction. One hundred percent value was taken for the density of the 32 kDa at 150-min reaction time. (D) Sulfo-NHS-LC-Biotin dose responses. One hundred percent value was taken for the density of the 32 kDa at 100 mM biotin concentration. (E) Dose–response of the total amount of proteins used. One hundred percent value was taken at a total amount of 20 μg of proteins. (F) Temperature dependence of the reaction. The optimal temperature for this reaction lies around 25°C. (G) Conformation-specificity of the reaction. NHS-digoxigenin (1 mM) was added before the reaction can block the biotinylation reaction resulting in the disappearance of the 32-kDa protein species (two middle lanes). All error bars represent SD from three independent experiments.
Fig. 2.
Fig. 2.
Prevalence of the 32-kDa SOD1-IR protein species in both SALS and FALS spinal cords after biotinylation. Immunoblot analysis of cytosolic proteins from human spinal cord autopsy samples (A and B) incubated with (+) or without (−) Sulfo-NHS-LC-Biotin. The density of the 32-kDa SOD1-IR signal from SALS-1 spinal cord autopsy sample (indicated as SALS-std) was chosen as a 100%, which was run on all gels as the standard for spinal cord samples. (A) Spinal cords are from normal, SALS, and SOD1-linked FALS autopsy samples. Mutations in SOD1 are G93C, A4V, G127X, D101G for FALS-1,2,3,4, respectively. (B) Bar graphs of intensities of the 32-kDa signal from spinal cords for all ALS (SALS + SOD1-linked FALS, n = 23) and normal (n = 16). Error bars represent SD. *, P < 0.0001. (C) Subject with a mutation in the Senetexin gene (ALS4). (D) The spinal cord samples from one normal and one SALS subject were separated as the white and gray matter before the analysis.
Fig. 3.
Fig. 3.
The 32-kDa SOD1-IR protein species is specific for ALS pathology. Immunoblot analysis of cytosolic proteins from different brain region autopsy samples were incubated with (+) or without (−) Sulfo-NHS-LC-Biotin. The density of the 32-kDa SOD1-IR signal from spinal cord autopsy sample SALS-1 (indicated as SALS-std) was chosen as a 100%, which was run on all gels as the standard for all samples. (A) The anterior hippocampus (Hip) and primary motor cortex (MC) from AD subjects were analyzed. The spinal cord from two PD subjects (B) and four SMA patients (C) was analyzed. (D) Bar graphs of the relative intensities the 32-kDa signal from ALS spinal cords (SC) and MC, AD Hip and MC, PD putamen (PU) and MC, HD PU and MC, and SMA SC.
Fig. 4.
Fig. 4.
Antigen-specific peptide eliminates observed immunoreactivity to SOD1 in the 32 kDa. Biotinylation and immunoblot were done as described. Samples used are cytosolic proteins from muscle (SALS-2) and spinal cords (SALS-7 and FALS-4). Peptides were incubated with the primary antibody for 30 min at room temperature in 500 μl of 5% milk in PBST before incubation with the membranes. Antigen-specific peptide (DDLGKGGNEESTK) was used at 1 μg and 4 μg, respectively. Nonspecific peptide (KESNGPVKVWGSIKGGC) was used at 1 μg.
Fig. 5.
Fig. 5.
The 32 kDa is a modified SOD1 protein species. (A) The commercial purified hSOD1 protein (Std, 40 μg) and SALS spinal cord cytosolic proteins (SALS-4, 300 μg) were subjected to gel filtration without biotinylation (−Biotin). Molecular mass standards were used during elution, and only the fractions corresponding to ≈32-kDa range (between ≈45 kDa and 28 kDa, as indicated) were shown in fractions 1–10. Fractions from the SALS-4 samples were further subjected to biotinylation (+Biotin). (B) SALS muscle cytosolic proteins (300 μg) were applied to the Q-type ion exchange column, and the flow-through (fractions 1–3) and eluted fractions (–9) were analyzed. An aliquot of the same sample (total input, 1/30) before the Q-Column is also shown. (C) The Q-column flow-through material was subjected to gel filtrations and analyzed directly (−Biotin) or after biotinylation (+Biotin). All proteins were separated on SDS/PAGE and immunoanalyzed with the SOD1 antibody.
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