dbPTM: an information repository of protein post-translational modification

dbPTM: an information repository of protein

post-translational modification

Tzong-Yi Lee

, Hsien-Da Huang

*, Jui-Hung Hung

, Hsi-Yuan Huang

,

Yuh-Shyong Yang

and Tzu-Hao Wang

51Institute of Bioinformatics, National Chiao Tung University, Hsin-Chu 300, Taiwan,2Department of Biological Science

and Technology, National Chiao Tung University, Hsin-Chu 300, Taiwan,3Institute of Biochemical Engineering, National Chiao Tung University, Hsin-Chu 300, Taiwan and4Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Lin-Kou Medical Center, Tao-Yuan 333, Taiwan

Received August 15, 2005; Revised and Accepted October 11, 2005

10ABSTRACT

dbPTM is a database that compiles information on protein post-translational modifications (PTMs), such as the catalytic sites, solvent accessibility of amino acid residues, protein secondary and tertiary

15structures, protein domains and protein variations.

The database includes all of the experimentally validated PTM sites from Swiss-Prot, PhosphoELM and O-GLYCBASE. Only a small fraction of Swiss-Prot proteins are annotated with experimentally

20verified PTM. Although the Swiss-Prot provides rich

information about the PTM, other structural proper-ties and functional information of proteins are also essential for elucidating protein mechanisms. The dbPTM systematically identifies three major types

25of protein PTM (phosphorylation, glycosylation

and sulfation) sites against Swiss-Prot proteins by refining our previously developed prediction tool, KinasePhos (http://kinasephos.mbc.nctu.edu.tw/). Solvent accessibility and secondary structure of

30residues are also computationally predicted and are

mapped to the PTM sites. The resource is now freely available at http://dbPTM.mbc.nctu.edu.tw/.

INTRODUCTION

Protein post-translational modification (PTM) is an extremely

35important cellular control mechanism because it may alter

proteins’ physical and chemical properties, folding, confor-mation, distribution, stability, activity and consequently, their functions (1). Examples of the biological effects of protein

modifications include phosphorylation for signal transduction,

40

attachment of fatty acids for membrane anchoring and association, and glycosylation for changing protein half-life, targeting substrates, and promoting cell-cell and cell-matrix interactions. With the accelerating progress in proteomics, biological knowledgebases containing a wealth of

informa-45

tion, in particular protein modifications, are playing crucial roles in cell regulation research (2).

The Swiss-Prot knowledge base (3) includes as much modi-fication information as is available with consistency and struc-ture, allowing easy retrieval by biologists. Phospho.ELM (1),

50

which was developed as part of the ELM (Eukaryotic Linear Motif) resource, is a new resource containing experimentally verified phosphorylation sites that were manually curated from the literature. O-GLYCBASE (4) is a database of glycopro-teins, most of which include experimentally verified O-linked

55

glycosylation sites. The RESID protein modification database is a comprehensive collection of annotations and structures for protein modifications and cross-links including pre-, co-and post-translational modifications (5). The RESID database provides modification information, literature citations, Gene

60

Ontology (GO) cross-references, protein sequence database feature table annotations, structure diagrams and molecular models. Each RESID entry presents a protein with a chemic-ally unique modification and indicates how the modification is currently annotated in the Swiss-Prot (6).

65

In this study, we collect the known PTM information from external biological data sources. Since only a small fraction of Swiss-Prot proteins are annotated with experimentally verified PTMs, we also developed computational tools to comprehens-ively identify phosphorylation sites, glycosylation sites and

70

sulfation sites against the Swiss-Prot proteins. Protein struc-tural properties and functional information, such as the solvent accessibility of residues, protein variations, non-synonymous *To whom correspondence should be addressed. Tel:+886 3 5712121, ext. 56952; Email: [email protected]

Correspondence may also be addressed to Tzu-Hao Wang. Tel:+886 3 3281200, ext. 8984; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
The Author 2006. Published by Oxford University Press. All rights reserved.

The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]

single nucleotide polymorphism (SNP), protein tertiary struc-tures and protein functional domains, are provided for researchers who are investigating the protein PTM mechan-isms. Web query interface and graphical visualization were

5designed and implemented to facilitate access to the database

content.

DATA GENERATION

The data generation flow of the dbPTM is briefly depicted in Figure 1. The data generation flow comprises the three major

10components: integration of external known PTM databases,

learning and prediction of PTM sites, and structural or func-tional annotations. The experimentally validated PTM data sources were extracted from Swiss-Prot (3), Phospho.ELM (1) and O-GLYCBASE (4). The experimentally verified PTM

15sites were used to generate computer models to further identify

putative PTM sites against the Swiss-Prot proteins. Additional structural properties and functional information, such as pro-tein tertiary structures, propro-tein secondary structures, solvent accessibility of residues, protein functional domains, protein

20

variations and non-synonymous SNP, are also annotated to the Swiss-Prot proteins. The detailed data generation flow is described below.

Integration of external known PTM databases

Three external biological databases related to protein

25

PTM information, Swiss-Prot (3), Phospho.ELM (1) and O-GLYCBASE (4), are integrated into the proposed resource. Both the experimentally validated PTM sites and the putative PTM sites, which are annotated as ‘by similarity’, ‘potential’ or ‘probable’ in the ‘MOD_RES’ fields, have been extracted

30

from the Swiss-Prot database (3). As summarized in Table 1, release 46.0 of Swiss-Prot contributes 11 025 experimental validated PTM sites within 4921 proteins, and 72 308 putative PTM sites within 31 026 proteins. The Phospho.ELM entries store information about substrate proteins with the exact

35

positions of residues are known to be phosphorylated by cellular kinases. A total of 1703 experimentally verified phos-phorylation sites within 556 proteins were obtained from Phospho.ELM version 2 (1). O-GLYCBASE (4) Version 6.00 provides 242 glycoproteins containing 2765 experimentally

Table 1. The list of the integrated external data sources

Databases Description Statistics

Swiss-Prot (3,12) Experimental PTMs 11 025 PTM sites within 4921 proteins

Putative PTMs 72 308 PTM sites within 31 026 proteins

Proteins 176 469 Proteins

Protein variants 32 101 Variants corresponding to 6115 proteins PhosphoELM (1) Experimental phosphorylation sites 1703 PTM sites within 556 proteins

O-GLYCBASE (4) Experimental glycosylation sites 2353 PTM sites within 185 glycoproteins

RESID (5) PTMs 373 PTM types

InterPro (14) Protein domain 1 113 928 Entries corresponding to 161 988 Swiss-Prot entries

Ensembl (13) Human variations 23 378 Non-synonymous SNPs within 7230 Swiss-Prot human proteins

PDB (15) Protein structures 31 721 Entries corresponding to 6806 Swiss-Prot proteins

verified O-linked, N-linked and C-linked glycosylation sites. Moreover, 185 glycoproteins in O-GLYCBASE are corres-ponded to Swiss-Prot proteins, which have 2353 experiment-ally verified glycosylation sites.

5Learning and prediction of PTM sites

To provide the PTM information of the PTM un-annotated proteins available from Swiss-Prot, we integrated several computational tools for identifying the PTMs of the Swiss-Prot proteins. Our previous work, namely KinasePhos (7),

10incorporated the profile hidden Markov model (HMM) to

identify kinase-specific phosphorylation sites with87% pre-diction accuracy (8), which was compared with several phos-phorylation prediction tools, such as NetPhos (9), DISPHOS (10) and rBPNN (11) (see Supplementary Table S1).

Kinase-15Phos is integrated and used to fully detect the kinase-specific

phosphorylation sites against the Swiss-Prot proteins. To reduce the number of false positive predictions by KinasePhos, we set the predictive parameters as the values when the prediction specificity is 100% (8).

20 As depicted in Supplementary Figure S1, the

KinasePhos-like method, which is similar to KinasePhos for phosphoryla-tion sites, was designed and implemented to learn models for the prediction of the sulfation sites, N-linked glycosylation sites and C-linked glycosylation sites (Table 2). We used

25the 144 known sulfation sites of tyrosine, 1790 N-linked

glyc-osylation sites of asparagine and 49 C-linked glycosyla-tion sites of tryptophan to evaluate the performance of the KinasePhos-like prediction tools. The result suggests that all three KinasePhos-like tools exhibited high prediction

pre-30cision, sensitivity and specificity (Supplementary Table S2).

Structural and functional annotations

In order to provide more effective information about protein structural and functional annotations relevant to protein PTM, a variety of biological databases, such as Swiss-Prot (12),

35Ensembl (13), InterPro (14), PDB (15) and RESID (5), are

integrated.

Protein variation is the change of amino acids in poly-peptides. As summarized in Table 1, Swiss-Prot contributes 32 101 protein variants corresponding to 6115 proteins,

40where 47 variant residues are located at the PTM sites and

267 variant residues are located surrounding 236 PTM sites ( 4  +4 AA). Furthermore, single amino acid polymorph-ism (SAP) is the amino acid variation corresponding to the genetic variation as the definition of non-synonymous SNP

45

in genomic sequence. The amino acid variants may have an impact on protein folding, active sites, or the overall solubility and stability of a protein. SAP is the type of variation most frequently related to human diseases (12). Therefore, when the amino acid variations occur in the PTM sites or the

surround-50

ing residues, they may affect the recognition of PTM sites by catalytic kinases. A total of 23 378 human non-synonymous SNPs located at 7230 Swiss-Prot human proteins were obtained from the variation part of Ensembl database (13).

InterPro provides 1 113 928 entries corresponding to

55

161 988 Swiss-Prot proteins. We found that about 65% of Swiss-Prot annotated PTM sites are located at InterPro annotated protein domains. The RESID (5) protein modifica-tions database is integrated into dbPTM to provide PTM related information, such as mass difference, chemical

60

formula, enzymatic activities, literature citations, GO cross-references, structure diagrams and molecular models.

The latest version of PDB contains 31 721 tertiary structures corresponding to 6806 Swiss-Prot protein entries (Table 1). For the proteins with known tertiary structures, the DSSP (16)

65

program was used to extract the true secondary structure and solvent accessibility for those 6808 Swiss-Prot proteins. Solvent accessibility of amino acids residues is important for both the structure and function of proteins, especially the PTMs studied in this investigation. Protein secondary structure

70

is the regular arrangement of amino acid residues in a segment of a polypeptide chain, where each amino acid is assigned a structure state, helix (H), strand (E) or coil (C). There are 1124 experimentally verified PTMs have the true secondary structure and solvent accessibility.

75

However, only 4% of Swiss-Prot proteins have the known tertiary structures. For proteins without known tertiary structures, two previously published tools, RVP-net (17) and PSIPRED (18), were applied to predict the solvent accessib-ility and the secondary structure, respectively (see Table 2).

80

RVP-net (17) presents a feed-forward type neural network which can predict a real value ranging from 0 to 100% of accessible surface areas (ASAs) for amino acid residues, based on their neighborhood information. We applied the RVP-net program (17) to fully predict the real-valued ASA

85

for the amino acid residues of all Swiss-Prot proteins. By selecting a suggested threshold (17) (i.e. 25%), the residues with larger ASA values are viewed as surface residues. DATA STATISTICS

The statistics of the experimentally verified PTMs and the

90

putative PTMs compiled in the dbPTM resource are shown in the Table 3. For instance, dbPTM contains 14 057 known PTM sites and 772 154 putative PTM sites. The parameters of the predictive tools, KinasePhos, KinasePhos-like Sulfation and KinasePhos-like Glycosylation—for the prediction of

95

phosphorylation sites, sulfation sites and glycosylation sites, respectively—are set as the values when the predictive spe-cificity is set to 100% during the parameter optimization of the trained models (8). The numbers of putative phosphoryla-tion and sulfaphosphoryla-tion sites, where the ASA of the substrates are

100

>25% (defined as the residue locating at the protein surface), are 652 756 and 13 315, respectively. There are a total of 33 887 predicted N-linked glycosylations of asparagine and C-linked glycosylations of tryptophan.

Table 2. The list of the integrated annotated tools

Tools Description

KinasePhos (7) Identifying kinase-specific phosphorylation sites KinasePhos-like sulfation Identifying sulfation sites

KinasePhos-like N-linked glycosylation

Identifying N-linked glycosylation sites KinasePhos-like C-linked

glycosylation

Identifying C-linked glycosylation sites DSSP (16) Calculating the secondary structure and solvent

accessibility of residues

RVP-net (17) Predicting the solvent accessibility of residues PSIPRED (18) Predicting the protein secondary structures Weblogo (15) Generating sequence logo for PTM substrates

INTERFACE

To facilitate the use of the dbPTM resource, we developed a website for users to browse and search for content. As depicted in Supplementary Figure S2, the user can select a particular

5type of PTM for browsing the information. When clicking on

a PTM entry, it pops up a window showing the solvent accessibility of the residues, the secondary structures and the flanking sequence of the PTM site.

The search pages allow users to query the database using the

10Swiss-Prot ID and protein name. The interface also presents

structural properties and functional information corresponding to the resulting proteins, such as the solvent accessibility of residues, non-synonymous variations, protein domains and protein secondary structures. Furthermore, the positional

15relationships among the PTMs, protein structural properties

and protein functional information are graphically displayed (Figure 2).

Generally, a 3D presentation is an effective manner for revealing the PTM information corresponding to the protein

20tertiary structures. For these purposes, we developed a protein

structure viewer for the visualization of protein tertiary struc-tures and especially of the port-translational modification resi-dues. As shown in Supplementary Figure S3, the visualization tool provides a comprehensive view of the whole protein

25structure and marks residues that are annotated as the PTM

sites. This visualization tool is implemented as a client-side tool based on OpenGL’s pipeline.

The visualization of the protein structures and the annotated residues are provided by two different ways according to

30

different users’ platforms. For users in MS Windows, the users can download the installable package of the Silver. After the Silver is installed, the protein tertiary structures and the PTM sites can be graphically and directly provided, as shown in Supplementary Figure S3. Alternatively, for users

35

in other platforms such as Mac OS X, Linux and Solaris, the user can download the PDB structure and the Rasmol (http:// www.umass.edu/microbio/rasmol/) scripts for the labeling of the PTM sites.

CONCLUSIONS

40

The proposed resource not only integrates the experimentally validated PTM information, but it also computationally annot-ates the Swiss-Prot proteins for putative phosphoryla-tion, glycosylation and sulfation sites. Furthermore, the PTM related protein structural properties and functional

45

information, such as solvent accessibility of amino acid resi-dues, protein variations, protein secondary structures, protein tertiary structures and protein domains, are provided to facil-itate the research of protein PTMs.

Table 3. The data statistics of the dbPTM database

PTM types Substrates No. of

known PTMs No. of putative PTMs Total

Phosphorylation Serine, threonine, tyrosine, aspartate, histidine or cysteine 3367 5852

Serine, threonine and tyrosine (predicted in this resource, ASA> 0%) 1 346 067 661 975 (ASA> 25%) Serine, threonine and tyrosine (predicted in this resource, ASA> 25%) 652 756

Glycosylation N-linked, O-linked and C-linked glycosylation 4586 55 059

N-linked asparagines and C-linked tryptophane (predicted in this resource, ASA> 0%)

43 894 94 132 (ASA> 25%) N-linked asparagines and C-linked tryptophane (predicted in this resource,

ASA> 25%)

33 887

Sulfation Serine, threonine and tyrosine 144 413

Tyrosine (predicted in this resource, ASA> 0%) 189 457 13 872 (ASA> 25%)

Tyrosine (predicted in this resource, ASA> 25%) 13 315

Lipidation GPI-anchor, N-terminal myristoylation and palmitoylation 520 4688 5208

Acetylation N-terminal of some residues and side chain of lysine or cysteine 1019 1580 2599 Amidation Generally at the C-terminal of a mature active peptide after oxidative cleavage of

last glycine

1554 523 2077

Methylation Generally of N-terminal phenylalanine, side chain of lysine, arginine, histidine, ralinenes or glutamate and C-terminal cysteine

455 1105 1560

Hydroxylation Generally of ralinenes, aspartate, raline or lysine 816 515 1331

Pyrrolidone carboxylic acid

N-terminal glutamine which has formed an internal cyclic lactam 567 408 975 Gamma-carboxyglutamic

acid

4-Carboxyglutamate 343 263 606

Trimethylation N6-methylated lysine, N6,N6,N6-trimethyllysine, N,N,N-trimethylalanine 158 294 452

Blocked Unidentified N- or C-terminal blocking group 108 10 118

FAD O-8alpha-FAD tyrosine, Pros-8alpha-FAD histidine, S-8alpha-FAD cysteine and Tele-8alpha-FAD histidine

12 77 89

S-nitrosylation S-nitrosocysteine 5 59 64

Formylation Of the N-terminal methionine 35 27 62

Deamidation Deamidated asparagin and deamidated glutamine (needs to be followed by a G) 33 18 51

Citrullination Citrulline 7 41 48

Others 328 1274 1134

Total 14 057 772 154 786 211

One of the prospective goals for dbPTM is to integrate more efficient prediction tools for other types of PTM in addition to phosphorylation, sulfation and N- and C-linked glycosylation. Other protein sequence databases besides the Swiss-Prot

5protein database can also be considered and annotated for

post-translation modifications by the proposed resource.

AVAILABILITY

The dbPTM resource will be regularly maintained and updated. The resource is now freely available at http://dbPTM.mbc.

10nctu.edu.tw/.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

The authors would like to thank Shih-Yee M. Wang (University

15of Illinois at Chicago) for English editing, the National Science

Council of the Republic of China for financially supporting this research under Contract No. NSC 94-2213-E-009-025 (to H.-D.H.), and Chang Gung Memorial Hospital for the Research Grant CTRP1006 (to T.-H.W.). Funding to pay the Open

20Access publication charges for this article was provided by

Chang Gung Memorial Hospital.

Conflict of interest statement. None declared.

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