Sequence-based mutation analysis of ARSA
Contents
Intro
Description of Mutations
We randomly picked 10 missense mutations from dbSNP and HGMD. The mutations are listed below, together with a pymol mutagenesis image and a description of the properties of the mutations.
| Nr. | mutation | position | reference | mutation | both | ||||||
| 1 | Asp-Asn | 29 | |||||||||
Description of Asp-Asn
Aspartic acid is an acidic amino acid while Asparagine is a hydrophilic amino acid. So the mutation changes the behaviour towards water as well as the pH. | |||||||||||
| 2 | Pro - Ala | 136 | |||||||||
Description of Pro-Ala
Proline and Alanine are both hydrophobic amino acids. So the behaviour towards water does not change. As Proline is a cyclic amino acid, it can "break" alpha-helices and is structural very important. Alanine is one the the smallest amino acids and so the mutation from Pro to Ala should have a big influence on the structure of ARSA. | |||||||||||
| 3 | Gln-His | 153 | |||||||||
Description of Gln-His
Glutamine is a hydrophilic amino acid while Histidine is a basic amino acid. So the behaviour towards water changes as well as the charge of the amino acid. Also Gln and His are very different in structure, so His needs much more space than Gln, which should have a big influence on the structure of ARSA. | |||||||||||
| 4 | Trp-Cys | 193 | |||||||||
Description of Trp-Cys
Tryptophan is a hydrophobic, aromatic amino acid while Cysteine is a hydrophilic amino acid. So the behaviour towards water changes dramatically. Also, Trp is the largest amino acid while Cys is a rather small amino acid. So the space needed for the amino acid changes also. This should have a huge influence on the structure of ARSA. | |||||||||||
| 5 | Thr-Met | 274 | |||||||||
Description of Thr-Met
Threonine is a hydrophilic amino acid while Methionine is a hydrophobic amino acid. So the behaviour towards water changes. Also, Methionine has a very long sidechain while Threonine does not. So the structure of ARSA should be altered by this mutation. | |||||||||||
| 6 | Phe -Val | 356 | |||||||||
Description of Phe-Val
Phenylalanine and Valine are both hydrophobic amino acids. So the only impact on structure could come frome the structural differences between Phe and Val. Phe has a aromatic ring and due to that needs more space than Val. | |||||||||||
| 7 | Thr-Ile | 409 | |||||||||
Description of Thr-Ile
Threonine is a hydrophilic amino acid while Isoleucine is a hydrophobic amino acid. So the behaviour towards water changes. | |||||||||||
| 8 | Asn-Ser | 440 | |||||||||
Description of Asn-Ser
Asparagine and Serine are both hydrophilic amino acids. Also they are almost of the same size. So the mutation should not have a very dramatic effect. | |||||||||||
| 9 | Cys-Gly | 489 | |||||||||
Description of Cys-Gly
Cystein and Glycine are both hydrophilic amino acids. The only difference is the size: Gly is the smallest of the amino acids while Cys is a little bigger. The effect should not be that dramatically. | |||||||||||
| 10 | Arg-His | 496 | |||||||||
Description of Arg-His
Arginine and Histidine are both basic amino acids so the only effect could come from the difference in size of the two. | |||||||||||
Substitution Matrices
To compare the different mutations here the scores for the three matrices in one table:
| Nr. | Substitution | BLOSUM62 | PAM1 | PAM250 |
|---|---|---|---|---|
| 1 | Asp(D) -> Asn(N) | 1 (worst: -4) | 36 (worst: 0) | 7 (worst: 0) |
| 2 | Pro(P) -> Ala(A) | -1 (worst: -4) | 22 (worst: 0) | 11 (worst: 0) |
| 3 | Gln(Q) -> His(H) | 0 (worst: -3) | 20 (worst: 0) | 7 (worst: 0) |
| 4 | Trp(W) -> Cys(C) | -2 (worst: -4) | 0 (worst: 0) | 1 (worst: 1) |
| 5 | Thr((T) -> Met(M) | -1 (worst: -3) | 2 (worst: 0) | 1 (worst: 0) |
| 6 | Phe(F) -> Val(V) | -1 (worst: -4) | 1 (worst: 0) | 10 (worst: 1) |
| 7 | Thr(T) -> Ile(I) | -2 (worst: -3) | 7 (worst: 0) | 4 (worst: 0) |
| 8 | Asn(N) -> Ser(S) | 1 (worst: -4) | 34 (worst: 0) | 8 (worst: 0) |
| 9 | Cys(C) -> Gly(G) | -3 (worst: -4) | 1 (worst: 0) | 4 (worst: 0) |
| 10 | Arg(R) -> His(H) | 0 (worst: -3) | 8 (worst: 0) | 5 (worst: 1) |
Secondary Structure
As one can see in the picture above, none of the mutations is in the middle of a secondary structure element. Only the mutations 1,2,4 and 5 are close to or - depending on the prediction method - at the border of secondary structure elements.
Prediction of effect
SNAP
We ran snap using the following command:
snapfun -i ARSA.fasta -m mutants.txt -o snap.out
output:
nsSNP Prediction Reliability Index Expected Accuracy
----- ------------ ------------------- -------------------
D29N Non-neutral 7 96%
Q153H Neutral 0 53%
T274M Non-neutral 6 93%
T409I Non-neutral 1 63%
C489G Non-neutral 5 87%
W193C Non-neutral 3 78%
F356V Neutral 1 60%
N440S Non-neutral 2 70%
R496H Neutral 1 60%
P136A Non-neutral 4 82%
In order to analyze all possible combinations of amino acid substitutions from the above mutated positions, we used the Generate Mutants tool on http://rostlab.org/services/snap/submit to create all possible exchanges from the following pattern: referenceAminoAcidPosition* . Then we again executed snap:
snapfun -i ARSA.fasta -m all_mutants.txt -o snap_all.out
Next, we wrote a perl script to parse and summarize the SNAP output in the following table, which shows which amino acid substitutions are Non-neutral or Neutral:
| ref\mutation | A | R | N | D | C | Q | E | G | H | I | L | K | M | F | P | S | T | W | Y | V |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D29 | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | |
| Q153 | Non-neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Non-neutral | Neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | |
| T274 | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | |
| T409 | Neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | |
| C489 | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | |
| W193 | Non-neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | |
| F356 | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Neutral | Neutral | Neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Neutral | Neutral | |
| N440 | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | |
| R496 | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Neutral | Non-neutral | Neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Neutral | Non-neutral | |
| P136 | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral | Non-neutral |
SIFT
SIFT predicts the effect of amino acid substitutions by building a multiple alignment and then calculating the probability of each possible substitution. The score in the SIFT-output is the probability of the substitution. SIFT predicts a substitution as damaging if this probability is <= 0.05 and as tolerated if the probability is > 0.05. The median conservation in the output measures the diversity of the sequences used in the multiple alignment. It should be between 2.75 and 3.25. Higher values indicate that the sequences were too closely related. <ref>http://sift.jcvi.org/www/SIFT_help.html</ref> We used SIFT with the UniProt-TrEMBL 2009 Database and uploaded a file containing our chosen mutations:
D29N P136A Q153H W193C T274M F356V T409I N440S C489G R496H
As median conservation we used the standard parameter 3.00 and we excluded all sequences with a sequence identity higher than 90%.
| Mutation NR | Substitution | predicted | score | median conservation | comment |
|---|---|---|---|---|---|
| 1 | D29N | AFFECT PROTEIN FUNCTION | 0.00 | 3.04 | |
| 2 | P136A | AFFECT PROTEIN FUNCTION | 0.00 | 3.07 | |
| 3 | Q153H | TOLERATED | 0.29 | 3.04 | |
| 4 | W193C | AFFECT PROTEIN FUNCTION | 0.04 | 3.04 | |
| 5 | T274M | AFFECT PROTEIN FUNCTION | 0.00 | 3.04 | |
| 6 | F356V | TOLERATED | 0.81 | 3.04 | |
| 7 | T409I | AFFECT PROTEIN FUNCTION | 0.02 | 3.48 | low confidence |
| 8 | N440S | TOLERATED | 0.07 | 3.08 | |
| 9 | C489G | AFFECT PROTEIN FUNCTION | 0.00 | 3.56 | low confidence |
| 10 | R496H | TOLERATED | 0.28 | 3.56 |
PolyPhen
PolyPhen predicts wether a mutation is damaging or not by using a Naïve-Bayes-approach. The score is the posterior probability that the mutation is damaging.<ref>http://genetics.bwh.harvard.edu/pph2/dokuwiki/overview</ref> We used PolyPhen with standard parameters. The results are shown below.
Comparison of results
To compare the results of the different prediction methods we created the table below. If a mutation was predicted to have an effect, a "X" was set, if a mutation was predicted to have no effect, a "-" was set. For PolyPhen "X" means "damaging" or "probably damaging", a "/" means "possibly damaging" and a "-" means "benign".
| Mutation NR | Substitution | SNAP | SIFT | PolyPhen | |
|---|---|---|---|---|---|
| HumDiv | HumVar | ||||
| 1 | D29N | X | X | X | X |
| 2 | P136A | X | X | X | X |
| 3 | Q153H | - | - | / | / |
| 4 | W193C | X | X | X | / |
| 5 | T274M | X | X | X | X |
| 6 | F356V | - | - | - | - |
| 7 | T409I | X | X | X | - |
| 8 | N440S | X | - | / | - |
| 9 | C489G | X | X | X | X |
| 10 | R496H | - | - | - | - |
Multiple sequence alignments
First, we downloaded the HSSP file for ARSA to get all proteins, which are homologuous to it. Then we downloaded all mammalian protein sequences from Uniprot. This was achieved by searching for the term taxonomy:40674, which codes for all mammalian protein sequences. We saved all sequences in one multiple fasta file. Then we extracted all homologuous mammalian proteins to human ARSA by mapping the ids from the HSSP file to sequence ids in the multi fasta file. This yielded 75 homologuous mammalian sequences to human ARSA.
Next, we calculated a multiple sequence alignments of these proteins (including ARSA) with Muscle. The Jalview image of the alignment is shown below.
The following table shows the conservation of the original amino acid in the reference sequence and their mutations at the respective positions.
| pos | conservation - reference | conservation - mutant |
|---|---|---|
| 29 | 0.86 | 0 |
| 153 | 0.14 | 0 |
| 274 | 0.87 | 0 |
| 409 | 0.35 | 0.16 |
| 489 | 0.80 | 0.05 |
| 193 | 0.13 | 0 |
| 356 | 0.15 | 0 |
| 440 | 0.15 | 0 |
| 496 | 0.14 | 0.01 |
| 136 | 0.93 | 0 |
PSI-BLAST
blastpgp -i ARSA.fasta -d /data/blast/nr/nr -e 10E-6 -j 5 -Q psiblast.mat -o psiblast_eval10E_6.it.5.new.txt
Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
A R N D C Q E G H I L K M F P S T W Y V A R N D C Q E G H I L K M F P S T W Y V
29 D -5 -5 -2 8 -7 -3 -1 -4 -4 -6 -7 -4 -6 -7 -5 -3 -4 -7 -6 -6 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.49 1.56
153 Q 3 2 -1 4 -4 -1 -1 -2 0 -2 -3 -3 4 -2 -3 -1 -2 -3 -2 -2 26 10 3 23 0 3 3 3 2 2 1 1 13 2 1 3 2 0 1 2 0.53 1.48
274 T -3 -4 -3 -4 -2 -4 -4 -5 -5 -4 -4 -4 -3 -5 -4 1 8 -6 -5 -3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 92 0 0 0 1.94 1.62
409 T -1 0 0 -1 -2 -1 -1 0 -1 -1 -1 0 -1 -1 3 0 1 6 0 -1 5 5 5 4 1 3 4 8 1 3 6 5 1 2 13 6 8 11 3 4 0.26 0.95
489 C 2 -1 1 -4 8 -4 -4 -2 -1 -1 -2 -3 -1 -4 -4 0 0 5 -1 -3 15 4 8 0 36 0 0 2 1 3 3 1 1 0 0 6 5 9 2 0 0.99 1.22
440 N -5 -3 6 5 -6 -2 -1 -4 -3 -6 -6 -3 -6 -6 2 -2 -3 -6 -6 -5 0 1 46 36 0 1 2 0 0 0 0 1 0 0 10 1 1 0 0 0 1.48 1.67
356 F -3 -1 -5 -5 -3 0 -1 -6 1 3 0 -1 0 2 -6 -3 -2 -3 5 3 1 4 0 0 1 5 4 0 3 18 8 5 2 8 0 1 2 0 20 20 0.59 1.62
193 W -2 4 2 3 -5 0 0 -2 0 -3 -4 1 -3 -1 -2 -1 -2 1 1 -3 3 25 11 16 0 4 5 3 2 2 1 7 0 2 2 4 2 2 5 2 0.46 1.45
136 P -3 -5 -5 -5 -6 -4 -4 -5 -5 -6 -6 -4 -6 -7 9 -4 -4 -7 -6 -5 1 0 0 0 0 0 0 0 0 0 0 0 0 0 98 0 0 0 0 0 3.03 1.61
496 R -3 1 0 -3 -4 1 1 -1 1 -3 1 1 -2 2 4 0 -3 -1 -1 -3 1 7 4 1 0 5 10 4 3 1 16 9 0 9 20 8 1 1 1 1 0.34 0.96
References
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