Fabry:Sequence-based analyses

Fabry Disease » Sequence-based analyses

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The following analyses were performed on the basis of the α-Galactosidase A sequence. Please consult the journal for the commands used to generate the results.

Transmembrane helices

α-Galactosidase A (P06280)

<figure id="fig:1r46_TM">

Human α-Galactosidase A, plot of posterior probabilities of features (source)

</figure> <figure id="fig:1r46_TMHMM">

Transmembrane helix prediction with TMHMM for human α-Galactosidase A

</figure>

organism:	Homo sapiens (Human)
pdb-id:	1r46

Since the whole sequence is labeled as "NON CYTOPLASMIC", the prediction of Polyphobius is that it contains no membrane helices (see Polyphobius output file). This result is consistent with both databases, OPM and PDBTM and the TMHMM-2.0 prediction (see <xr id="fig:1r46_TMHMM" />).
In <xr id="fig:1r46_TM"/> the posterior probabilities of cytoplasmic(green), non cytoplasmic(blue), TM helix(grey area) and signal peptide(red) are shown. The grey area indicates weak evidence for a transmembrane helix which where not predicted. The first small peak can be observed in both pictures. Considering the shown probabilities and our background knowledge, the prediction seems to be true. Only the end of the signal peptide at residue 29 is, according to our knowledge, too early and should be shifted to position 31.

D(3) dopamine receptor (P35462)

<figtable id="tab:3pbl_TM_PostProb"> Posterior probability plots Polyphobius and TMHMM

Human D(3) dopamine receptor, plot of posterior probabilities of features predicted by Polyphobius

Human D(3) dopamine receptor, plot of posterior probabilities of features predicted by TMHMM

</figtable>

organism:	Homo sapiens (Human)
pdb-id:	3pbl (only available structure)

Polyphobius predicts 7 transmembrane regions. Comparing this result to the pictures in <xr id="tab:3pbl_TM"/>, we can again see a consistent result with the two databases. The only difference between the models seems to be the number of residues on the cytosolic side of the membrane (green in the right picture) of this hydrolase/hydrolase inhibitor. Inquiring the PDBTM site, this area is marked as residues 1002-1161, which do not even belong to the 400 aa long protein. This might result from the experiment, the structure was derived from. It says, that the protein was enginered (see source1 and source2)
Besides from that, a difference in predicted and observed starting and end points could be revealed (see <xr id="tab:3pbl_start_end"/>), mainly those of PDBTM, which leads to a great difference in its mean length compared to the other methods (see <xr id="tab:3pbl_TM"/>, picture 3). Here, PDBTM throughout assigns shorter helices, since its helices always start later and end earlier, which can also be seen in <xr id="fig:bitshift" />. The boundaries Polyphobius predicted may be seen in the Polyphobius output file.
The posterior probability of the first 6 predicted helices is very similar, as one can see in <xr id="tab:3pbl_TM_PostProb"/>. The only difference is in the last TM, where the probability assigned by TMHMM is nearly 90%, whereas Polyphobius only assigns about 55%.

<figtable id="tab:3pbl_start_end"> Start and end points of each transmembrane helix predicted

TMH number	1			2			3			4			5			6			7
Method	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End
Polyphobius	26	30	55	23	66	88	22	105	126	21	150	170	25	188	212	24	329	352	20	367	386
OPM	19	34	52	25	67	91	26	101	126	21	150	170	23	187	209	22	330	251	24	363	382
PDBTM	18	35	52	17	68	84	15	109	123	15	152	166	16	191	206	14	334	347	15	368	382
Uniprot	23	33	55	23	66	88	22	105	126	21	150	170	25	188	212	22	330	351	22	367	388
TMHMM	23	32	54	23	67	89	23	104	126	23	150	172	23	192	214	23	331	353	23	368	390

</figtable>

<figtable id="tab:3pbl_TM"> Visualizations for the human D(3) dopamine receptor as a transmembrane protein. Note: Cytoplasmic side of the membrane is different in the pictures

Human D(3) dopamine receptor as depicted in OPM database (Cytoplasmic side on the bottom) (source)

Human D(3) dopamine receptor as shown in PDBTM database (Cytoplasmic side above) (source)

Human D(3) dopamine receptor, comparison of length distribution of different prediction methods

</figtable>

Voltage-gated potassium channel (Q9YDF8)

<figtable id="tab:1orq_TM_PostProb"> Posterior probability plots Polyphobius and TMHMM for Q9YDF8

Voltage-gated potassium channel, plot of posterior probabilities of features predicted by Polyphobius

Voltage-gated potassium channel, plot of posterior probabilities of features predicted by TMHMM

</figtable>

organism:	Aeropyrum pernix (strain ATCC 700893 / DSM 11879 / JCM 9820 / NBRC 100138 / K1)
pdb-id:	1orq (smaller resolution than 2AOL, more residues determined)

The voltage-gated potassium channel is a tetrameric protein, that causes a voltage-dependent potassium ion permeability of the membrane. It may occur in an opened or closed conformation. This state can be changed by the voltage difference across the membrane. (source) To fulfill this function, the protein needs the transmembrane helices. Of these, 7 are predicted according to the Polyphobius output file, but referring to OPM, only three helices should be predicted, according to PDBTM 4 helices are considered true. Looking carefully at the probability plot of Polyphobius in <xr id="tab:1orq_TM_PostProb"/>, one may see, that there actually are only four peaks of transmembrane helix posterior probability, but three of the less higher peaks are considered true as well.
Only 2 of all 7 helices are common among all three methods (see <xr id="tab:1orq_TM" />, picture 3), the third and fourth transmembrane helices are even only predicted by Polyphobius, Uniprot and TMHMM and only Polyphobius and Uniprot, respectively. If not predicted or shown by one of the methods, the helix is considered as membrane loop.
Here OPM has the smallest mean length of transmembrane helices (see <xr id="tab:1orq_TM" />, picture 3). Start and end points of the helices seem to be differing more than in the first described picture (see <xr id="tab:1orq_start_end" />, picture 3), although Polyphobius, Uniprot and TMHMM are quite similar. Personally, considering the features predicted by Polyphobius and TMHMM (see <xr id="tab:1orq_TM_PostProb" />), I would trust the PDBTM version the most in this case. But since in this case, the advantage of Polyphobius to include homologue data is not given, because the BLAST search did not find any homologue sequences, it might on the other hand be a false prediction. This might, to a certain degree, explain the rather diverse result.

<figtable id="tab:1orq_start_end"> Start and end points of each transmembrane helix predicted

TMH number	1			2			3			4			5			6			7
Method	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End
Polyphobius	19	42	60	21	68	88	22	108	129	21	137	157	22	163	184	18	196	213	21	224	244
OPM	0	-	-	0	-	-	0	-	-	0	-	-	20	153	172	13	183	195	19	207	225
PDBTM	32	21	52	24	57	80	0	-	-	0	-	-	21	151	171	28	209	236	0	-	-
Uniprot	25	39	63	25	68	92	17	109	125	21	137	157	25	160	184	13	196	208	32	222	253
TMHMM	23	39	61	20	68	87	23	107	129	0	-	-	23	162	184	20	199	218	20	225	244

</figtable>

<figtable id="tab:1orq_TM"> Visualizations for the Voltage-gated potassium channel as a transmembrane protein. Note: Cytoplasmic side of the membrane is different in the pictures

Voltage-gated potassium channel as depicted in OPM database (Cytoplasmic side on the bottom) (source)

Voltage-gated potassium channel as shown in PDBTM database (Cytoplasmic side above) (source)

Voltage-gated potassium channel, comparison of length distribution of different prediction methods

</figtable>

Aquaporin-4 (P47863)

<figtable id="tab:2d57_TM_PostProb"> Posterior probability plots Polyphobius and TMHMM for Q9YDF8

Aquaporin-4, plot of posterior probabilities of features predicted by Polyphobius

Aquaporin-4, plot of posterior probabilities of features predicted by TMHMM

</figtable>

organism:	Rattus norvegicus (Rat)
pdb-id:	2d57 (non-mutant)

Another homotetramer, the water-specific channel Aquaporin-4, has been analyzed. In the first two pictures in <xr id="tab:2d57_TM"/>, the protein is depicted in its tetrameric structure, while in this exercise we only examine one of the four chains. Here Polyphobius again predicted the number of transmembrane helices correctly according to PDBTM and all the other ressources(6 - see Polyphobius output file), while OPM alone shows 8 TMH structures. Taking a closer look at the results, we see that the 2 "missing" TMHs are interpreted as membrane loop in PDBTM (depicted in orange in <xr id="tab:2d57_TM"/>, picture 2) and Polyphobius, which might be due to the short length of the helical structure (10 residues each - see <xr id="tab:2d57_TM"/>). Considering the posterior probability, I would rather prefer one of the 6 helical models.
The 6 shared helices' start and end points are again quite conform among Polyphobius and OPM, PDBTM, Uniprot and TMHMM. The mean length is again around 23, except for the two databases which have a mean length of around 18.

<figtable id="tab:2d57_start_end"> Start and end points of each transmembrane helix predicted

TMH number	1			2			3			4			5			6			7			8
Method	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End	Length	Start	End
Polyphobius	25	34	58	22	70	91	0	-	-	22	115	136	22	156	177	21	188	208	0	-	-	22	231	252
OPM	23	34	56	19	70	88	10	98	107	25	112	136	23	156	178	15	189	203	10	214	223	22	231	252
PDBTM	17	39	55	18	72	89	0	-	-	18	116	133	20	158	177	18	188	205	0	-	-	18	231	248
Uniprot	21	37	57	21	65	85	0	-	-	21	116	136	21	156	176	21	185	205	0	-	-	21	232	252
TMHMM	23	33	55	23	70	92	0	-	-	23	112	134	23	154	176	23	189	211	0	-	-	23	231	253

</figtable>

<figtable id="tab:2d57_TM"> Visualizations for the Aquaporin-4 as a transmembrane protein. Note: Cytoplasmic side of the membrane is different in the pictures

Rat's Aquaporin-4 as depicted in OPM database (Cytoplasmic side on the bottom) (source)

Rat's Aquaporin-4 as shown in PDBTM database (Cytoplasmic side above)(source)

Aquaporin-4, comparison of length distribution of different prediction methods

</figtable>

Comparison Polyphobius, OPM, PDBTM

<figure id="fig:bitshift">

Average start and end points of transmembrane helices, Polyphobius compared to OPM, PDBTM, Uniprot and TMHMM

</figure>

In <xr id="fig:bitshift" /> the predicted transmembrane helices of Polyphobius have been used as basis and the start and end point of each TMH in the models of OPM, PDBTM, Uniprot and TMHMM (see <xr id="tab:3pbl_start_end" />, <xr id="tab:1orq_start_end" /> and <xr id="tab:2d57_start_end" />) have been compared to it. The average of each model has been calculated for each helix Polyphobius and OPM, respectively each of the other models, shared. It becomes obvious, that the transmembrane helices of OPM tend to be shifted to the left, compared to the Polyphobius helices, while the PDBTM helices usually are nested inside the Polyphobius membranes due to later starting and earlier end points. The great strength of Polyphobius becomes apparent, when examining the result for 1orq, where no homologue data could be found. (see Voltage-gated potassium channel ). Here all models are very different from Polyphobius' prediction, except for the Uniprot prediction. Taking a closer look, we found out, that the Uniprot prediction among few other methods relies on the Phobius and TMHMM algorithms (see)
Since on average, the hydrophobic belt in a membrane is about 3.5 to 5nm wide and a helix needs approximately 15 to 20 amino acids to span this width, mean lengths of 18 to 23 aas seem reasonable. Nonetheless, especially TMHMM, which seems to predict helices of length 23 in almost all cases, and also Polyphobius and Uniprot tend to have longer helices than that. This fact might be either due to the type of membrane the proteins are located at, or a bias in the algorithms.
What also stood out, was the fact that the cutoff for still counting the structure as an helical structure in the OPM database is really low, considering that an α-helix on average needs 3.6 amino-acid residues per turn. Contrariwise OPM predicts the least number of transmembrane helices in the Voltage-gated potassium channel protein, which cannot be due to the length of the predicted structure.
Summing up, the method to prefer depends on what demand you have in your result. If you want to make sure your predicted helices are transmembrane helices for sure, you want to choose PDBTM's models, since in our example, they did never assign the label TMH to a structure as the only method. But then, if you want to cover all possible structures, you should choose Polyphobius, since there has not been any transmembrane helix it did not at all predict, considering the two databases.

Signal peptides

Prediction of the presence and location of signal peptide cleavage sites in amino acid sequences.

α-Galactosidase A (P06280)

<figure id="fig:1r46_SP">

Human α-Galactosidase A, plot of scores for signal peptide cleavage site (source)

</figure>

organism:	Homo sapiens (Human)
pdb-id:	1r46
Signal-peptide:	1-31
Signal-peptide sequence:	MQLRNPELHLGCALALRFLALVSWDIPGARA

As already described in section Transmembrane helices the human α-Galactosidase A does not have transmembrane helical structures and from our background knowledge we know that the protein has a 31 residues long signal peptide at the N-terminus. The protein's pdb structure is available only without signal peptide, since those residues are cleaved off. Therefore no picture is provided here.
In <xr id="fig:1r46_SP"/> the signal peptide prediction of SignalP 4.0 is shown. The cleaved site is clearly indicated after residue 31, since the S-score (green) drops and the C- (red) and Y-score (blue) not only have a peak at this position, but attain their maximum at this point (see SignalP output file )

Serum Albumin (P02768)

organism:	Homo sapiens (Human)
pdb-id:	1ao6
Signal-peptide:	1-18
Signal-peptide sequence:	MKWVTFISLLFLFSSAYS

For this protein an 18 residues long signal peptide is predicted (see SignalP output file) and also reported in various sources (e.g. peptide website, Uniprot). The signal peptide of each chain of the homodimeric protein is shown in <xr id="tab:1ao6_SP"/> in the last picture.
According to the Polyphobius prediction, no transmembrane helix is present, but the signal peptide is also predicted. Those findings underlines the truth of the predicted signal peptide.
The rather high hydrophobicity (see <xr id="tab:1ao6_SP"/>, Figure 1) indicates an export of the protein to another cellular compartment. Examining the Uniprot website, we found that Serum Albumin is secreted into the cell surroundings, for this a membrane needs to be passed and the high hydrophobicity is needed.

<figtable id="tab:1ao6_SP"> Visualizations for the human Serum Albumin protein's signal peptide prediction.

Human Serum Albumin hydropathy plot (source)

Human Serum Albumin plot of scores for signal peptide cleavage site (source)

Human Serum Albumin, signal peptide of both chains depicted in red

</figtable>

Lysosome-associated membrane glycoprotein 1 (P11279)

organism:	Homo sapiens (Human)
pdb-id:	NA
Signal-peptide:	1-28
Signal-peptide sequence:	MAAPGSARRPLLLLLLLLLLGLMHCASA

<figtable id="tab:P11279_SP"> Visualizations for the human Lysosome-associated membrane glycoprotein's signal peptide prediction.

Human Lysosome-associated membrane glycoprotein 1 hydropathy plot (source)

Human Lysosome-associated membrane glycoprotein 1 plot of scores for signal peptide cleavage site (source)

</figtable>

As the name implies, LAMP1 has a transmembrane helix (see Polyphobius prediction) and is associated to the plasma membrane, where it presents carbohydrate ligands to selectins. Moreover, the protein shuttles between lysosomes, endosomes, and the plasma membrane ([www.uniprot.org/uniprot/P11279 source]) and thus needs its signal peptide (residues 1-28), which has been predicted by Polyphobius and SignalP (see output). In <xr id="tab:P11279_SP"/> the hydropathy plot and a plot of the different scores used for prediction by SignalP are shown. Again, they underline the predictions of both programs.
Unfortunately, no pdb structure is available.

Aquaporin-4 (P47863)

<figure id="fig:2d57_SP">

Aquaporin-4, plot of scores for signal peptide cleavage site (source)

</figure>

organism:	Rattus norvegicus (Rat)
pdb-id:	2d57
Signal-peptide:	none
Signal-peptide sequence:	---

In <xr id="fig:2d57_SP"/> only a small peak of the C-score at position 41 can be observed, which by far does not reach the threshold of 0.5 for being predicted a cleavage site. All other scores are constantly low, thus no signal peptide cleavage site is predicted. As already mentioned in section Transmembrane helices, Polyphobius correctly predicts no signal peptide, but 6 transmembrane helices. This predicted (and observed) structure is in conformity with the function of a water-specific channel the porin fulfills.

GO terms

GOPET

Searching the GOPET annotation tool with the AGAL_HUMAN sequence revealed 5 GOIds, which are displayed in <xr id="tab:GOPET"/>. On a first glance, since we already know the name and function of the protein, it is a bit surprising, that alpha-galactosidase activity is only the third entry with 96% confidence. In our already carried out information gathering we learned that α-galactosidase A is a hydrolase thus the first three entries were not surprising. Considering that our enzyme mainly is a glycosidase, the both entries on top of the list make perfekt sense.
Again a bit surprising was the last entry. α-N-Acetylgalactosaminidase is actually used for enzyme replacement therapy, which we mention on our main page. The structure of both enzymes is similar to each other, but this still does not explain the association of this GO term to the AGAL protein.

<figtable id="tab:GOPET"> The results of the GOPET search

Result for GOPET search
GOid	Aspect	Confidence	GO term
GO:0016798	Molecular Function Ontology (F)	98%	hydrolase activity acting on glycosyl bonds
GO:0004553	Molecular Function Ontology (F)	98%	hydrolase activity hydrolyzing O-glycosyl compounds
GO:0016787	Molecular Function Ontology (F)	97%	hydrolase activity
GO:0004557	Molecular Function Ontology (F)	96%	alpha-galactosidase activity
GO:0008456	Molecular Function Ontology (F)	89%	alpha-N-acetylgalactosaminidase activity

</figtable>

ProtFun2.0

EC=3.2.1.22(EC 3.-.-.- Hydrolase)
Predicted: EC 6.-.-.-(Ligase)

############## ProtFun 2.2 predictions ##############

>gi_4504009_

# Functional category                  Prob     Odds
  Amino_acid_biosynthesis              0.283   12.847
  Biosynthesis_of_cofactors            0.339    4.708
  Cell_envelope                     => 0.652   10.690
  Cellular_processes                   0.057    0.783
  Central_intermediary_metabolism      0.400    6.343
  Energy_metabolism                    0.151    1.678
  Fatty_acid_metabolism                0.032    2.448
  Purines_and_pyrimidines              0.506    2.082
  Regulatory_functions                 0.013    0.083
  Replication_and_transcription        0.047    0.175
  Translation                          0.211    4.807
  Transport_and_binding                0.549    1.339

# Enzyme/nonenzyme                     Prob     Odds
  Enzyme                            => 0.805    2.811
  Nonenzyme                            0.195    0.273

# Enzyme class                         Prob     Odds
  Oxidoreductase (EC 1.-.-.-)          0.176    0.845
  Transferase    (EC 2.-.-.-)          0.195    0.564
  Hydrolase      (EC 3.-.-.-)          0.244    0.769
  Lyase          (EC 4.-.-.-)          0.029    0.608
  Isomerase      (EC 5.-.-.-)          0.010    0.321
  Ligase         (EC 6.-.-.-)       => 0.141    2.776

# Gene Ontology category               Prob     Odds
  Signal_transducer                    0.090    0.419
  Receptor                             0.014    0.083
  Hormone                              0.002    0.318
  Structural_protein                   0.004    0.127
  Transporter                          0.024    0.222
  Ion_channel                          0.010    0.169
  Voltage-gated_ion_channel            0.003    0.127
  Cation_channel                       0.010    0.215
  Transcription                        0.047    0.367
  Transcription_regulation             0.026    0.204
  Stress_response                      0.049    0.552
  Immune_response                      0.012    0.136
  Growth_factor                        0.006    0.412
  Metal_ion_transport                  0.009    0.020

//

Other programs and ressources

A simple internet search for "protein sequence prediction" reveals a confusingly high number of different ressources and databases. Clicking on the first entry leads to Rostlab's Predict protein. This program seems to predict nearly anything:

1. Multiple sequence alignment
2. ProSite sequence motifs
3. low-complexity retions (SEG)
4. Nuclear localisation signals
5. and predictions of
   1. secondary structure
   2. solvent accessibility
   3. globular regions
   4. transmembrane helices
   5. coiled-coil regions
   6. structural switch regions
   7. B-value
   8. disordered regions
   9. intra-residue contacts
   10. protein protein and protein/DNA binding sites
   11. sub-cellular localization
   12. domain assignment
   13. beta barrels
   14. cysteine predictions and disulphide bridges

Performing a search reveals that the tool gives you an overview of all the above mentioned features. On a first glance it seemed rather confusing, due to the vast amount of informations provided. Anyhow I believe giving the output some more attentiveness, it is a good tool to gather a lot of information in one place. A very good feature is that the output is presented in many different ways which can be switched at any time. A quick look at the properties we examined in the task, shows, that Predict Protein probably does not distinguish very well between signal peptides and transmembrane helices, since for the α-Galactosidase A it predicts a TM, where there actually is signal peptide (position 11-28; see section Transmembrane helices and Signal peptides)
Searching the same term again at the NCBI homepage (see search) reveals a huge mass of reviews on that topic, mainly on predicting secondary structure and function out of the protein sequence, but also on less common topics like predicting the protein solubility (see here and here) , or more recently emerged whole genome sequence approaches (see).
A really different topic was brought up by Smialowski P, Martin-Galiano AJ, Cox J and Frishman D. who give an overview of techniques to predict experimental properties of proteins from sequence (see) and thus predict experimental success in cloning, expression, soluble expression, purification and crystallization
Summarizing, it is very hard to get along in the vast amount of prediction tools and methods and one can spend a lot of time on finding out which one fits best to the requirements of one's topic.