The authors have declared that no competing interests exist.
Conceived and designed the experiments: MDCC JFM. Performed the experiments: MDCC. Analyzed the data: MDCC JFM. Contributed reagents/materials/analysis tools: JFM. Wrote the paper: MDCC JFM.
Aging in the world population has increased every year. Superoxide dismutase
2 (Mn-SOD or SOD2) protects against oxidative stress, a main factor influencing
cellular longevity. Polymorphisms in SOD2 have been associated with the development
of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s
disease, as well as psychiatric disorders, such as schizophrenia, depression
and bipolar disorder. In this study, all of the described natural variants
(S10I, A16V, E66V, G76R, I82T and R156W) of SOD2 were subjected to
Although aging is a multifactorial process, there is significant evidence that shows that oxidative stress is one of the main factors that influences cellular longevity. Interest in the factors that determine longevity has grown recently because the life expectancy of the world population is increasing. Additionally, in many countries, the main causes of death are currently comorbidities connected to age and oxidative stress.
Superoxide dismutases (SODs) protect against oxidative stress and have
three forms: Cu-Zn SOD (SOD1), located in the cytosol; Mn-SOD (SOD2), located
in the mitochondrial matrix; and extracellular SOD (SOD3)
The first 24 amino acids of Mn-SOD are the mitochondrial targeting sequence
(MTS), which guides and docks the Mn-SOD protein to mitochondria.
Polymorphisms in SOD2 have been associated with the development of neurodegenerative
diseases, such as Alzheimer’s
In this study, we collected the natural variants of SOD2 for
As a result, a database was generated for biologists and clinicians to
explore SOD2 nsSNPs and the resulting changes in structure and function. This
database is freely available at
The sequence and natural variants of Mn-SOD were retrieved from the UniProt database.
The functional effects of non-synonymous single-nucleotide substitutions
(nsSNPs) were predicted using the following programmes: PhD-SNP
The mutant (E66V, G76R, I82T and R156W) models were built using the MHOLline
workflow
ConSurf was used for high-throughput characterisation of the functional
regions in the protein
The natural variants listed in the database come from UniProt. For each SNP, we provide predictions of the function effects using SNPeffect, PolyPhen-2, PhD-SNP, PMUT, SIFT, SNAP, SNPs&GO and nsSNPAnalyzer.
The database is web-accessible and can show the following in a comparative table: mutant name; a visualisation of the aligned structures and the predicted functional effects.
The protein sequence and the natural variants of Mn-SOD were retrieved
from the UniProt database
Position | Mutation | Feature identifier |
10 | S10I (S-15I) | VAR_019363 |
16 | A16V (A-9V) | VAR_016183 |
66 | E66V | VAR_019364 |
76 | G76R | VAR_025898 |
82 | I82T | VAR_007165 |
156 | R156W | VAR_019365 |
The Mn-SOD variants were subjected to a variety of
Non synonymous SNP analysis programs | |||||||||||
Natural Variant | nsSNP Analyzer | PhD-SNP | PMUT | Polyphen-2 | SIFT | SNAP | SNPs&GO | TANGO Aggregation Tendency | WALTZ Amyloid Propensity | LIMBO Chaperone Binding Tendency | FoldX Protein Stability |
Unknown | Neutral | Neutral | Benign | Tolerated | Non-neutral | Disease | Not Affected | Not Affected | Not Affected | Unknown | |
Unknown | Neutral | Pathological | Benign | Tolerated | Neutral | Neutral | Not Affected | Not Affected | Not Affected | Unknown | |
Disease | Disease | Neutral | Possibly damaging | Tolerated | Neutral | Neutral | Not Affected | Not Affected | Not Affected | Slightly Enhanced | |
Disease | Neutral | Pathological | Benign | Tolerated | Non-neutral | Disease | Not Affected | Not Affected | Not Affected | Reduced | |
Neutral | Neutral | Neutral | Benign | Affect Protein Function | Non-neutral | Disease | Not Affected | Not Affected | Decreased | Not Affected | |
Neutral | Disease | Pathological | Benign | Affect Protein Function | Neutral | Disease | Not Affected | Not Affected | Not Affected | Slyghtly Reduced |
The SNPeffect workflow evaluates aggregation tendency (TANGO), amyloid propensity (WALTZ), chaperone binding tendency (LIMBO) and protein stability (FoldX). The natural variant E66V slightly enhances the protein stability, in contrast with the G76R variant, which reduces the protein stability. The I82T variant decreases the chaperone binding tendency, and the R156W variant slightly reduces the protein stability.
According to PhD-SNP, variants S10I, A16V, G76R and I82T are neutral, whereas variants E66V and R156W cause disease.
The PMUT analysis indicates that the natural variants S10I, E66V and I82T are neutral and that A16V, G76R and R156W are pathological.
The PolyPhen-2 results show that, of the six variants, only E66V may cause damage and that all of the others are benign.
According to SIFT (Sorting Intolerant from Tolerant), tolerance was predicted for the natural variants S10I, A16V, E66V and G76R. I82T and R156W were predicted to affect protein function. The SNAP analysis indicates that variants S10I, G76R and I82T are non-neutral and that A16V, E66V and R156W are neutral.
According to SNPs&GO, variants S10I, G76R, I82T and R156W cause disease, and A16V and E66V are neutral.
The nsSNPAnalyzer results demonstrate that variants S10I and A16V are unknown and variants E66V and G76R cause disease. In contrast, I82T and R156W are neutral.
The SNP analysis, shown in
The natural variants were substituted into the wild-type sequence for comparative
modelling. These sequences were submitted to the MHOLline workflow
A) mutation E66V (E42V), RMSD: 0.21; B) mutation G76R (G52R), RMSD: 0.38; C) mutation I82T (I58T), RMSD: 0.45; D) mutation R156W (R132W), RMSD: 0.16.
Two subunits are represented as a backbone in green and blue. Four mutation sites are shown in a sphere representation: E66V, G76R, I82T and R156. The manganese binding site is shown in ball-stick form.
An alignment between the native and mutant structures was performed using
TM-Align
Pos. | Variant | TM-Align | ||
Align | RMSD | TM-Score | ||
66 | E66V (E42V) | 1LUV | 0.21 | 0.99834 |
76 | G76R (G52R) | 1LUV | 0.38 | 0.995 |
82 | I82T (I58T) | 1LUV | 0.45 | 0.995 |
156 | R156W (R132W) | 1LUV | 0.16 | 0.995 |
To analyse the three-dimensional effects of the S10I and A16V mutations,
which are located in the signal peptide,
A) S10I (S-15I) mutation highlighted in red. B) This mutation disrupts the alpha helix, RMSD: 2.02. C) A16V (A-9V) mutation highlighted in red. D) This mutation disrupts the alpha helix, RMSD: 1.94.
Pos. | Variant | I-Tasser | TM-Align | |||
C-score | TM-score | RMSD | RMSD | TM-Score | ||
10 | S10I (S-15I) | 0.18 | 0.69±0.12 | 5.9±3.7 | 2.02 | 0.90520 |
16 | A16V (A-9V) | 0.15 | 0.69±0.12 | 5.9±3.7 | 1.94 | 0.91721 |
The ConSurf
Mn-SOD is represented as a spacefill model, where the residue conservation scored is colour-coded onto the surface. The backbone model represents the other chain of a Mn-SOD dimer, chain B. The colour-coding bar shows the colouring scheme: conserved amino acids are coloured bordeaux, residues with average conservation are white, and variable amino acids are turquoise.
The colour-coding bar shows the colouring scheme: conserved amino acids are coloured bordeaux, residues of average conservation are white, and variable amino acids are turquoise. SNP positions are marked by an asterisk.
The conservation analysis of ConSurf used the evolutionary conservation
scores of the residues to identify functional regions from proteins with known
three-dimensional structures. The degree of conservation of the amino acid
sites among the nine homologues with similar sequences (
The SOD2 database currently contains all of the natural variants listed in UniProt. For each SNP, we provide the predictions of functional effects, indicated as Disease/Pathological or Neutral/Tolerated, from SNPeffect, PolyPhen-2, PhD-SNP, PMUT, SIFT, SNAP, SNPs&GO and nsSNPAnalyzer.
The database interface (
The database is curated by humans and will be updated as new natural variants are discovered.
The SOD2 database allows a user to quickly retrieve and rapidly analyse the predicted effects of protein variants. In addition to predicting the effects of variants, an alignment of the wild-type and mutant structures can be visualised using the database.
The major feature that distinguishes the SOD2 database from other databases
is that this database can use predictions from several algorithms for all
of the known natural variants of Mn-SOD. Furthermore, the user has access
to an alignment of the wild type and mutant structures and can thus visualise
the damage that a SNP can cause. Our ultimate goal is to turn the database
into a toolbox for researchers studying this protein. The