Gaucher Disease: Task 03 - Sequence-based predictions

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Revision as of 23:09, 27 May 2013 by Gerkej (talk | contribs) (Human Lysosome-associated membrane glycoprotein 1)

Secondary Structure

In this task secondary structure is predicted using ReProf and PsiPred and compared to DSSP structure assignment. ReProf uses a fasta sequence or a PSI-BLAST PSSM for prediction, PsiPred a fasta sequence and DSSP server needs a PDB file in order to use the 3D coordinates of atoms. The predictions were made for the four proteins, including the Gaucher's disease-causing protein, listed below. If several PDB structures are available, the one covering the most UniProt sequence most similarly was chosen. For glucosylceramidase the structure 1OGS was used (as in the task 2).

Uniprot PDB
Entry Protein name Origin Length Entry Method Resolution (Å) Chain Positions
P10775 Ribonuclease inhibitor pig 456 2BNH X-ray 2.30 A 1-456
Q9X0E6 Divalent-cation tolerance protein CutA bacterium Thermotoga maritima 101 1VHF X-ray 1.54 A 2-101
Q08209 Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform, EC=3.1.3.16 human 521 1AUI X-ray 2.10 A 1-521
P04062 Glucosylceramidase/acid-beta-glucosidase, EC=3.2.1.45 human 536 1OGS X-ray 2.00 A/B 40-536


The script of the Phenylketonuria group filter_secStruc.pl was used to extract the secondary structures in the three letter code: E, H and L. For DSSP irregular regions are encoded as "-". Then, precision of two output secondary structure string was calculated using the second script of the Phenylketonuria group, SecStrucComparison.jar.

First different PSSMs after different PSI-BLAST runs (all combinations: against big_80/swissprot database, 2/3 iterations, E-value 2E-3/10E-10/10E-20) were tested on the shortest protein, Q9X0E6, then the run parameters yielding the best precision compared to PsiPred and DSSP were chosen. The best parameters were: big_80, 3 iterations and evalue cutoff 10E-10, which were then applied to create PSSMs for the other proteins. (The table where the results for all parameters are summarized can be seen here: /mnt/home/student/kalemanovm/master_practical/Assignment3_SequenceBasedPredictions/SecondaryStructure/reprof_out/parsedSecStr/README.Q9X0E6.psiblast_param.precision.)

TODO: What features are predicted? Discuss the results for your protein and the example proteins. Using the predictions, what could you learn about your protein and the example proteins? Compare to the available knowledge in UniProt, PDB, DisProt, OPM, PDBTM, Pfam...

Disorder

Transmembrane Helices

Four Proteins, including the Gaucher's disease causing Protein, where analysed under reference by transmembrane helices. The used prediction tools differ in their analysing features. While Polyphobius only differs between residues being part of a transmembrane helix or being inside/outside of the cytoplama, Memsat-SVM also predicts re-entrant helices and pore-linig helices. Due to the fact that pore-lining helices are also transmembrane helices, this kind of helices is detected of both prediction tools. In case of re-entrant helices both programms differ. In general a membrane helix crosses the membrane, so that both ends of the helix lie on different sides of the membrane. In contrast, the re-entrant helix leads bot its ends to the same side of the mebrane. Memsat-SVM can predict re-entrant helices, but Polyphobius treats this helices as a general membrane helices, which crosses the membrane (seen for Q9YDF8), or ignores it (seen for P47863). In case of re-entrant helices predictions also the C-terminal or the N-terminal may be predicted on different membrane sides, as well as some helices may be predicted to lie in a different direction within the membrane, because of an re-entrant helix.

Human Glucosylceramidase

This Protein is not a membrane protein and is located on the extracellular side of the membrane as documented in OPM. For the same reason there exist no entry in the PDBTM, as this databse only contains membrane proteins. The prediction of Polyphobius causes to the same result. Additionally Polyphobius predicted also the signal peptide (including the N/H/C-region). MemsatSVM detected a false positive transmembrane helix. As the Glucosylceramidase cleaves lipids of cell membranes, the ative site of the enzym may be mistaken for a trensmembrane helix.

Comparison of TMH for Glucosylceramidase (P04062, human)
Prediction Assignment
Memsat SVM Polyphobius OPM PDMTM
# of TMH 1 - - -
TMH Topology 456-471 - - -
N-terminal extracellular extracellular extracellular -
C-terminal cytoplasmic extracellular extracellular -
Signal peptide 1-34 1-40 - -
Re-entrant Helix - - - -
Pore-lining Helix 1 - - -
Graphical position
Cartoon P04062.png
Graphik P04062.png
1ogs.png
-
more information P04062 1OGS 1OGS is not in the PDBTM

Aeropyrum pernix Voltage-gated potassium channel

Comparison of TMH for Voltage-gated potassium channel (Q9YDF8, Aeropyrum pernix)
Prediction Assignment
Memsat SVM Polyphobius OPM PDMTM
# of TMH 6 7 5 4
TMH Topology 43-59
72-90
101-118
128-143
163-184

221-245
42-60
68-88
108-129
137-157
163-184
196-213
224-244
25-46
55-78
86-97
100-107
117-148
27-50
55-75
88-107
118-142
N-terminal cytoplasmic extracellular cytoplasmic cytoplasmic
C-terminal cytoplasmic cytoplasmic cytoplasmic cytoplasmic
Signal peptide - -
Re-entrant Helix 188-217 -
Pore-lining Helix 1 -
Graphical position
Cartoon Q9YDF8.png
Graphik Q9YDF8.png
1ors-Q9YDF.png
1ors lm.png
more information Q9YDF8 1ORS 1ORS

Human Lysosome-associated membrane glycoprotein 1

Both predictions have results similar to the assignments of OPM and PDBMT. All predicted transmembrane helices differ in their position only by a few residues. The Protein consists of 6 transmembrane helices and 2 re-entrant helices. Polyphobius skips the re-entrant helices prediction but, predicts the remaining membrane helices well. MemsatSVM predicts the re-entrant helices similar to the re-entrant helices of the database entrys. Unfortunately MemsatSVM predicts the placing inside the membrane wrong. Instead of the C- and N-terminal situated in the cytoplasm, MemsatSVM places the both ends in the extracellular region. The two assignments

Comparison of TMH for Lysosome-associated membrane glycoprotein 1 (P47863, human)
Prediction Assignment
Memsat SVM Polyphobius OPM PDMTM
# of TMH 6 6 8 (per chain) 8 (per chain)
TMH Topology 35-56
71-89

113-136
157-178
190-205

232-252
34-58
70-91

115-136
156-177
188-208

231-252
34-56
70-88
98-107
112-136
156-178
189-203
214-223
231-252
39-55
72-89
95-106
116-133
158-177
188-205
209-222
231-248
N-terminal extracellular cytoplasmic cytoplasmic cytoplasmic
C-terminal extracellular cytoplasmic cytoplasmic cytoplasmic
Signal peptide 1-20
Re-entrant Helix 93-109
209-225
95-106
209-222
Pore-lining Helix 4
Graphical position
Cartoon P47863.png
Graphik P47863.png
2d57.png
2d57 lm.png
more information P47863 2D57 2D57

Human D3 dopamine receptor

Comparison of TMH for D3 dopamine receptor (P35462, human)
Prediction Assignment
Memsat SVM Polyphobius OPM PDMTM
# of TMH 6 7 7 7
TMH Topology 32-55
65-88
101-129
151-169
188-209
331-354
30-55
66-88
105-126
150-170
188-212
329-352
367-386
34-52
67-91
101-126
150-170
187-209
330-351
363-386
35-52
68-84
109-123
152-166
191-206
334-347
368-382
N-terminal extracellular extracellular extracellular extracellular
C-terminal extracellular cytoplasmic cytoplasmic cytoplasmic
Signal peptide 1-29 -
Re-entrant Helix - - -
Pore-lining Helix 1
Graphical position
Cartoon P35462.png
Graphik P35462.png
3pbl.png
3pbl lm.png
more information P35462 3PBL 3PBL

Signal Peptides

For the following proteins, the signal peptides as well as its cleavage sides were predigted with SignalP:

  • Glucosylceramidase (P04062, human)
  • Serum albumin (P02768, human)
  • Aquaporin 4 (P11279, rat)
  • Lysosome-associated membrane glycoprotein 1 (P47863, human)

The four eukaryotic proteins were also looked up in the Signal Peptide Database to campare the entry with the results of the prediction.

Glucosylceramidase (P04062)

For the Glucosylcerbrosidase, the prediction of SignalP differs from the database entry.

In the database the protein has a signal peptide of 39 residues. A signal peptide is characterized with high hydrophobicity in its core region followed by the cleavage side[1]. Escpecially the residues 18-23 and 27-34 indicate with its higher hydrohobicity to a signal peptide (green area in the hydrophobicity image).

MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASG


However, the prediction of SignalP results no signal peptide. On the visualisation of the different scores below, the green signal peptide score shows the most possible prediction for an signal peptide. The green line is higher for the first 39 residues than for the later residues. But the calculated D-score of the detected peptide lies with 0.37 below the threshold (0.5). The peptide is neglected as signal. These residues are not only defined as signal peptide by the database, but were also detected, with a light deviation, by the transmembranehelix predictors MemsatSVM(residues 1-34) and Polyphobius(residues 1-40).


Serum albumin (P02768)

The signal peptide consists of residues 1-18 and is predicted of SignalP as well as documented in the Signal Peptide Databse

MKWVTFISLLFLFSSAYS

The images below show an clearly prediction of the signal peptide. A high S-core for the signal peptide region with a D-score of 0.85 far over th threshold. The cleavage side is predicted between the residue 18 and 19. The database shows a high hydrophobicity for the residues 6-14 which marks the region as signal peptide as well.

Aquaporin 4 (P11279)

For Aquaporin the Scores are even higher than for Serum albimum. The signal peptide consists of 28 residues as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASA

The Database shows a large hydrophobic region of 17 residues. At the end of the protein a transmembrane helix with a length of 23 residues ending in cytoplasm is documented in the Aquaporin 4 entry. The SignalP prediction gives a D-score of 0.95 for the detected signal peptide. The cleavage side is predicted between between the residues 28 and 29 (ASA-AM).

Lysosome-associated membrane glycoprotein 1 (P47863)

The Rat protein has no entry in the Signal Peptide Database, as no signal peptide exists for it. The visualised results of the prediction show on the first sight, that the Protein does not have a signal peptide. All scores are lower than 0.21, which is far below the threshold for signal peptides.


GO Terms

Discussion

Other available methods

Prediction of Tool
secondary structure GOR
disorder DISOPRED2
transmembrane helices MEMSAT3
TMHMM
PredictProtein
DAS
HMMTOP
TMpred
signal peptides PrediSi
Polyphobius
MemsatSVM
SIGCLEAVE
ANTHEPROT
Signal Find Server
SPD
SPEPlip
SOSUIsignal
GO terms

What else can/is be predicted from protein sequence alone

  • Fold recognition (profile based pGenTHREADER and rapid GenTHREADER)
  • Fold domain recognition (pDomTHREADER)
  • Protein domain prediction (DomPred)
  • Homology modelling (BioSerf v2.0)
  • Function prediction (eukaryotic function: FFPred v2.0)
  • Prediction of TM topology and helix packing (SVM-based MEMPACK)

http://bioinf.cs.ucl.ac.uk/psipred/

Which predictions can be improved considerably by structure-based approaches