Canavan Disease

Secondary Structure

To determine which approach to follow we examined the proposed run-combinations for ReProf, where prediction only from FASTA-sequence vs. prediction from PSSM generated by PSI-Blast was looked at. Additionally the prediction of the secondary structure by ReProf with PSSM was further divided into PSSM generated by using big_80 and PSSM generated by using SwissProt. For further comparison a secondary structure prediction via PSI-Pred was initiated as well as a secondary structure assignment by DSSP. As DSSP assigns the secondary structure using the atom coordinates stored in PDB, we assume that we can use the DSSP assignment as the "true secondary structure" and compare the prediction methods in terms of performance to DSSP as reference. For the evaluation of the prediction methods there were however some problems we stepped into and had to deal with. First of all the PDB entry of ACY2 regards the protein as a homo-dimer, however it only exists in that form when crystallized. Therefore to compare and create statistics between the prediction methods and DSSP the output of the DSSP assignment had to be double checked and only one part of the assignment (to get the monomer) could be used. Additionally the beginning as well as the ending of the DSSP assignment had to be extended with some no secondary structure assigned symbols to stretch the DSSP assignment data to the full length of the protein. The final statistics concerning the secondary structure prediction of Aspartoacylase (P45381|ACY2_HUMAN) is displayed in <xr id="ACY2_statistics"> Table </xr>.

<figtable id="ACY2_statistics">

Secondary Structure Prediction Statistics for ACY2
	Precision			Recall			F-Measure
Type	H	E	L	H	E	L	H	E	L
ReProf (FASTA)	0.773	0.822	0.562	0.829	0.446	0.808	0.800	0.578	0.663
ReProf (big_80)	0.878	0.889	0.644	0.793	0.675	0.890	0.833	0.767	0.747
ReProf (SwissProt)	0.853	0.937	0.62	0.780	0.711	0.849	0.815	0.809	0.717
Psi-Pred	0.914	0.970	0.647	0.780	0.771	0.904	0.842	0.859	0.754

Statistical overview of Precision, Recall and F-Measure for the prediction tools used, with DSSP as reference. H = Helix, E = Beta-Strand, L = Loop. Psi-Pred shows the best performance for ACY2. ReProf with a PSSM created by Psi-Blast using big_80 as database preforms second best but greatly outperforms (not shown) Psi-Pred in terms of speed (ReProf run locally, Psi-Pred run on offical webserver)

</figtable>

As Psi-Pred predictions when run via the official webserver take up much more time than running ReProf locally on the students lab, the decision to further use ReProf was made. More specifically ReProf with a position specific scoring matrix derived from big_80 was chosen (PSSM created with Psi-Blast, cut-off e-10 and 3 iterations). However, out of curiosity, additionally to the ReProf prediction, PSI-Pred predictions for the remaining proteins where run nevertheless.

During the mapping of Uniprot ID to PDB ID there arose some complications as not all proteins that where found contained the full sequence of the translated gene. The proteins that where used for the DSSP assignment where chosen manually to ensure that the whole sequence is contained within the protein, at least as part of the whole PDB entry. Additionally some modifications had to done again to ensure that the DSSP assignment has the same length as the predictions by ReProf and PSI-Pred. For example Q08209 mapped to 1AUI chain A covering most of translated gene, however parts of 1AUI could not be crystallized and the atom coordinates are missing from the PDB file (374 - 468). As a result those positions are fully absent from the DSSP assignment as well, and had to be filled with no predicted structure. After dealing with all those complications Precision, Recall and F-measure where calculated again in the same manner as it was done to decide on the preferred prediction method. An overview of the prediction statistics with the DSSP assignment as reference can be seen in <xr id="additional_statistics"> Table </xr>.

<figtable id="additional_statistics">

Secondary Structure Prediction Statistics for P10775, Q08209, Q9X0E6
		Precision			Recall			F-Measure
Protein	Type	H	E	L	H	E	L	H	E	L
P10775 (1DFJ_I)	ReProf	0.974	0.959	0.793	0.945	0.855	0.912	0.959	0.904	0.848
	Psi-Pred	0.976	0.980	0.630	0.814	0.873	0.938	0.888	0.923	0.754
Q08209 (1AUI_A)	ReProf	0.957	0.842	0.658	0.780	0.787	0.878	0.859	0.814	0.752
	Psi-Pred	0.895	0.971	0.594	0.723	0.557	0.944	0.800	0.708	0.729
Q9X0E6 (1O5J)	ReProf	0.973	0.971	0.526	0.947	0.829	0.833	0.960	0.894	0.645
	Psi-Pred	1.000	1.000	0.600	0.947	0.854	1.000	0.973	0.921	0.750

Statistical overview of Precision, Recall and F-Measure for the prediction tools used, with DSSP as reference. H = Helix, E = Beta-Strand, L = Loop. For P10775 (1DFJ chain I) and Q08209 (1AUI chain A) ReProf clearly shows the better performance. Psi-Pred shows better preformance for Q9X0E6.

</figtable>

Transmembrane Helices

Following the task the transmembrane helices and topology for the three given proteins plus ACY2 were predicted via Polyphobius and MEMSAT-SVM. As running the prediction with MEMSAT-SVM automatically returned the prediction results for MEMSAT-3 too, this data was incorporated in the comparison of the results as well.

P45381

ACY2 (P45381) is a protein that is located in the cytoplasma and not bound to the cell membrane therefore it should be save to expect that none of the prediction methods predicts a transmembrane helix. However Polyphobius was the only one to do so. MEMSAT-3 predicted a helix from the amiino acid position 60 to 78, even though the score is negative. MEMSAT-SVM predicted a helix ranging form amino acid 114 to 129 again with a negative score. As MEMSAT seems to test all possible combinations of helices present in the protein, ranging from the amount of 1 to n, with the possibility of 0 not tested, it could be hypothesized that MEMSAT always returns a prediction for a transmembrane helix even if the score is negative.

P35462

P35462 (PDB:3PBL) a dopamine receptor in human is a 7-helical-transmembrane protein. Prediction of the transmembrane helices was done with the aid of MEMSAT-(SVM & 3) and Polyphobius. Interestingly MEMSAT-SVM did not predict the correct amount of helices, stoping after the sixth one. MEMSAT-3 did correctly predict seven helices despite being claimed to be worse in prediction power. PolyPhobis did achieve the best prediction for that protein, have correctly predicted all 7 helices and having predicted the borders of the helices more precisely than MEMSAT. The exact numbers can be found in <xr id="P35462_tmhs"> Table </xr>

<figtable id="P35462_tmhs">

Predicted Transmembrane Helices for P35462
	Helix Positions
Method	#1	#2	#3	#4	#5	#6	#7
UniProt	33-55	66-88	105-126	150-170	188-212	330-351	367-388
PolyPhobius	30-55	66-88	105-126	150-170	188-212	329-352	367-386
MEMSAT-SVM	32-55	65-88	101-129	151-169	188-209	331-354	no prediction
MEMSAT-3	31-55	67-91	102-126	148-167	189-213	327-350	365-383

Overview of the predicted transmembrane helices for P35462 compared to the annotation in UniProt

</figtable>

Additional information:

• UniProt entry: P35462
• OMP entry: 3PBL
• PDBTM entry: 3PBL

Q9YDF8

Q9YDF8 (PDB:1ORQ/1ORS/2A0L/2KYH) a crucial part to form potassium channels is a 7-helical-transmembrane protein. Prediction of the transmembrane helices was done with the aid of MEMSAT-(SVM & 3) and Polyphobius. In this case only Polyphobius correctly predicted the number of existent helices. Both MEMSAT-3 and MEMSAT-SVM predicted only six. Additionally all three tools had great problems of predicting the right borders. Polyphobius seems to have jumped over the third helix annotated in Swissprot, completely misspredicting the borders of the fifth helix (fourth helix predicted) and predicts a (sitxth) helix where in the actual protein a intramembrane element is located at the amino acid position 196 to 208. MEMSAT-SVM and MEMSAT-3, although falsely predicting six transmembrane helices, are concerning the precision of predicted helix borders closer to the annotation in SwissProt, except for the third helix where MEMSAT seems to have fused the third and fourth annotated helix. The exact numbers can be found in <xr id="Q9YDF8_tmhs"> Table </xr>

<figtable id="Q9YDF8_tmhs">

Predicted Transmembrane Helices for Q9YDF8
	Helix Positions
Method	#1	#2	#3	#4	#5	#6	#7
UniProt	39-63	68-92	97-105	109-125	129-145	160-184	222-253
PolyPhobius	42-60	68-88	108-129	137-157	163-184	196-213	224-244
MEMSAT-SVM	43-59	72-90	101-118	128-143	163-184	221-245	no prediction
MEMSAT-3	38-60	66-90	100-119	122-141	161-184	218-242	no prediction

Overview of the predicted transmembrane helices for Q9YDF8 compared to the annotation in UniProt

</figtable>

Additional information:

• UniProt entry: Q9YDF8
• OMP entry: not clear
• PDBTM entry: see OMP

P47863

P47863 (PDB:2D57) a aquaporin in rat is a 6-helical-transmembrane protein. Prediction of the transmembrane helices was done with the aid of MEMSAT-(SVM & 3) and Polyphobius. In this case every prediction tool correctly predicted the number of existent helices. PolyPhobius and MEMSAT-SVM were slightly off predicting the borders of the helices, whereas in this case the claimed inferiority of MEMSAT-3 compared to MEMSAT-SVM can clearly be seen showing less precise border prediction. The exact numbers can be found in <xr id="P47863_tmhs"> Table </xr>

<figtable id="P47863_tmhs">

Predicted Transmembrane Helices for P47863
	Helix Positions
Method	#1	#2	#3	#4	#5	#6
UniProt	37–57	65-85	116-136	156-176	185-205	232-252
PolyPhobius	34-58	70-91	115-136	156-177	188-208	231-252
MEMSAT-SVM	35-56	71-89	113-136	157-178	190-205	232-252
MEMSAT-3	35-59	71-95	117-141	157-180	187-206	240-264

Overview of the predicted transmembrane helices for P47863 compared to the annotation in UniProt

</figtable>

Additional information:

• UniProt entry: P47863
• OMP entry: 2D57
• PDBTM entry: 2D57

Signal Peptides

For the prediction of signal peptides SignalP version 4.1 (webserver) was used.

P02768

Serum albumin (P02768) is a protein that is one of the main components of blood plasma. As it clearly has to to be secreted into the blood vessels it can be expected that P02768 has motives that are crucial for the delivery down the secretory pathway and therefore contains a signal peptide sequence. This is exactly what the prediction for signal peptides using SignalP shows. SignalP predicts that P02768 has signal peptide sequence and that a cleavage site exists between amino acid position 18 and 19. Looking at the plot <xr id="P02768_signalp"> (see Figure</xr>) created by SignalP v4.1 this clear signal at position 19 (0.710) can be observed.

<figure id="P02768_signalp">

Plot displaying the scores (C = cleavage, S = signal peptide, Y = combined) predicted for each aminoacid by SignalP v4.1 for P02768. A clear spike for the cleavage site at position 19 can be seen, as well as high scores for signal peptide for the first 18 amino acids. (Image Source: Maple Sirup Urine Disease Group to prevent file duplicates in the wiki)

</figure>

Additional information:

• UniProt entry: P02768
• Signal Peptide Database Entry: ALBU_HUMAN

P47863

As we know after the task to predict transmembrane helices P47863 is a aquaporin that is located within the membrane. The prediction by SignalP shows that neither a signal peptide sequence nor a cleavage site can be detected. Detailed graphical output can be seen in <xr id="P47863_signalp">Figure</xr>.

<figure id="P47863_signalp">

Plot displaying the scores (C = cleavage, S = signal peptide, Y = combined) predicted for each aminoacid by SignalP v4.1 for P47863. Neither a spike in the c-score nor high s-scores can be seen, therefore no signal peptide sequence and no cleavage site is predicted by SignalP. (Image Source: Maple Sirup Urine Disease Group to prevent file duplicates in the wiki)

</figure>

Additional information:

• UniProt entry: P47863

P11279

LAMP-1 (Lysosome-associated membrane glycoprotein 1 | P11279) is a membrane protein. It takes an important role in the autophagy process and is associated with tumor metastasis. It has one transmenbrane helix which could be a some sort of protein anchor. Taking a look at the signal peptide prediction by SignalP reveals that LAMP-1 has an assumed signal peptide sequence and a cleavage site between the amino acids 28 and 29. This is congruent with the information stored in the Signal Peptide Database [1]. A detailed graphical output of the SignalP prediction is displayed in <xr id="P11279_signalp"> see Figure</xr>.

<figure id="P11279_signalp">

Plot displaying the scores (C = cleavage, S = signal peptide, Y = combined) predicted for each aminoacid by SignalP v4.1 for P11279. A clear spike for the cleavage site at position 29 can be seen, as well as high scores for signal peptide for the first 28 amino acids. (Image Source: Maple Sirup Urine Disease Group to prevent file duplicates in the wiki)

</figure>

Additional information:

• UniProt entry: P11279
• Signal Peptide Database Entry: LAMP1_HUMAN

GO-Terms

The GO-Term prediction for Aspartoacylase executed by GO-Pet (see <xr id="P11279_signalp"> Table</xr>) is very acurate. Looking at the know enzymatic acitvity of ACY2, it can be observed that the predectied biological processes exactly reflect the chemical reaction happening.

<figtable id="P45381_gopet">

Predicted GOTerms for P45381 by GO-Pet
GO-ID	GO-Term / Description	Confidence
GO:0016787	hydrolase activity	96%
GO:0004046	aminoacylase activity	82%
GO:0019807	aspartoacylase activity	82%
GO:0016788	hydrolase activity acting on ester bonds	81%

Overview of the predicted GO-Terms for P45381

</figtable>

Prot-Fun interestingly correclty predicts that ACY2 is an enzym however mispredicting it for an isomerase. (Prob:Odds 0.084:2637 vs 0.115:0.363 for Hydrolase). Additinonally Prot-Fun can not decide on a Gene Ontology category and sorts ACY2 into the functional category of "Central intermediary metabolism".