Structural and Functional Analysis of Hypothetical Proteins in Mouse Hippocampus from Two-Dimensional Gel Electrophoresis Leila Afjehi-Sadat,† Jae-Won Yang,† Arnold Pollak,† Dae-Won Kim,‡ Soo-Young Choi,‡ and Gert Lubec*,† Department of Pediatrics, Medical University of Vienna, Vienna, Austria, and Department of Biomedical Sciences, Hallym University, Chunchon, South Korea Received September 6, 2006
Protein profiling in five individual mouse strains showed strain-specific expression of three hypothetical proteins (HPs). As functional and structural assignment of HPs were based on predictions and low identity to known structures, HPs were identified by MALDI-TOF/TOF, and their proposed tentative function was determined by enzyme assays. Three identified HPs were extracted from gels and renatured, and pyridoxal phosphate phosphatase, inorganic pyrophosphate phosphatase, and antioxidant activities were revealed, findings in agreement with functional predictions. Keywords: hypothetical proteins • enzyme activity • 2D gel electrophoresis • recovery of enzyme activity • mass spectrometry
Introduction Protein profiling in cells and tissues very often leads to the detection of hypothetical proteins (HPs), structures that have been predicted from nucleic acid sequences only, without experimental proof of their existence at the protein level.1 Most publications provide proteomic information on partial protein sequence only or carry out bioinformatic predictions of functions.2-8 In many instances, these predictions are based upon some identity to known proteins, conformation, or upon the presence of functional domains.9 The presence of a functional domain, however, does not unambiguously assign a function to an HP, and indeed, experimental validation is needed. Based upon literature and own experience, a large part of HPs detected by two-dimensional gel electrophoresis with subsequent mass spectrometrical identification presents with low identity to known enzymes or shows the presence of enzyme domains including active sites.3 A classical way would be to go back to the nucleic acid sequence, express the gene to construct a recombinant protein, and assay the predicted enzyme activity.10-13 The aim of this study was to bypass generation of a recombinant protein and measure enzyme activities directly from the protein spots punched from the gel. Based upon literature from the 1980s, we tried this approach following the principles of Hager and Burgess14 who showed that proteins could be recovered from SDS-gels by elution and subsequent renaturation in the presence of guanidine hydrochloride. Performing protein hunting in mouse hippocampus,15-20 we unambiguously detected and characterized several hypothetical * Corresponding author. Prof. Dr. Gert Lubec, Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel: + 43-1-40 400 3215. Fax: + 43-1-40 400 3194. E-mail:
[email protected]. † Medical University of Vienna. ‡ Hallym University. 10.1021/pr060453o CCC: $37.00
2007 American Chemical Society
proteins by mass spectrometry. The application of bioinformatic tools predicted low identities to proteins that showed enzyme activities for which reliable enzyme assays were commercially available. The corresponding enzyme activities were observed, enabling the use of this method to experimentally verify predicted function of hypothetical proteins with enzyme activity. This protein chemical approach may represent a step forward in functional proteomics with a series of applications.
Experimental Section Animals. Mice studied were FVB/NHim (FVB), C57BL/6JHim (C57), 129S2/SvHim (129Sv), Balb/cHim (Balb), and Him:OF1 (OF) (n ) 10 for each strain) males, about 20 weeks old. Animals were housed in standard transparent laboratory cages in a temperature-controlled colony room (22.1 °C). They were maintained on a 12 h artificial light/dark cycle (with lights on at 6:00 a.m.) and provided with food and water ad libitum. Mice were killed by neck dislocation, and hippocampi were dissected. Tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C until used for analysis. All animal experiments were carried out in accordance with the European Community Council Directive (96/609/EEC) on animal welfare and approved by the local animal committee (confirmation number: LF1-TVG-17/002-2004). All efforts were made to minimize animal suffering and the number of animals used. Two-Dimensional Gel Electrophoresis (2-DE). Hippocampal tissue was powdered and suspended in 1.0 mL of sample buffer consisting of 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO), 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma), 65 mM DTT (1,4-dithioerythritol; Merck), 1 mM EDTA (Merck), protease inhibitors complete (Roche, Basel, Switzerland), and 1 mM phenylmethylsulfonyl chloride. The suspension was sonicated for approximately 30 s. After homogenization, samples Journal of Proteome Research 2007, 6, 711-723
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Table 1. Mass-Spectrometrical Analysis of HPs in Mouse Hippocampus MASCOT combined results
acc. no
protein name
Q9CXN1 13 days embryo head cDNA, RIKEN full-length enriched library, clone:3110052N05 product:hypothetical HAD-like structure containing protein, full insert sequence
Q9GZP4
AD039
Q8CHP8 RIKEN cDNA 1700012G19
TMWa TPIb OPIc OMWd score
expectation value PMe
28730
4.7e-011
5.7
5.2
28883
152
17
MASCOT MS/MS results
no. of sequence significant coverage MS/MS 76%
7
24178
5.5
5.6
24391
296
3.6e-025
9
52%
2
34541
5.2
5.7
34975
114
3e-007
8
34%
2
MS/MS peptide sequences TFFLEALR
LLLDGAPLIAIHK LEFEISEDEIFTSLTAAR DGLALGPGPFVTALEYATDTK KLEFEISEDEIFTSLTAAR AVLVDLNGTLHIEDAAVPGAQEALKR ALPEFTGVQTQDPNAVVIGLAPEHFHYQLLNQAFR FSNVYHLSIHISK FVESDADEELLFNIPFTGNVK AVVVGFDPHFSYMK FIFDCVSQEYGINPER
a
b
c
d
Theoretical molecular weight. Theoretical isoelectric point. Observed isoelectric point. Observed molecular weight. e Matched peptides.
hundred micrograms of protein was applied on immobilized pH 3-10 nonlinear gradient strips at their basic and acidic ends. Focusing was started at 200 V, and voltage was gradually increased to 8000 V over 31 h and then kept constant for a further 3 h (approximately 150 000 Vh totally). After the first dimension, strips (18 cm) were equilibrated for 15 min in the buffer containing 6 M urea, 20% glycerol, 2% SDS, and 2% DTT and then for 15 min in the same buffer containing 2.5% iodoacetamide instead of DTT. After equilibration, strips were loaded onto 9-16% gradient sodium dodecylsulfate polyacrylamide gels for second-dimensional separation. Immediately after the second dimension run, gels were fixed for 18 h in 50% methanol and 10% acetic acid and subsequently stained with Colloidal Coomassie Blue (Novex, San Diego, CA) for 6 h on a rocking shaker.
Figure 1. Partial images from the 2D reference maps of mouse hippocampus in two mouse strains (C57 and 129Sv). These maps represents identified hypothetical proteins (HP Q9CXN1, Q9GZT4, and Q8CHP8) in mouse hippocampus. Identified proteins are designated by their Swiss-Prot accession number. The names of the proteins are listed in Table 1. 2D Western blot of HP Q8CHP8 was performed with 30 µg of protein and based upon 9-16% gradient SDS-PAGE as second-dimensional separation developed membrane with anti-PLPP (1:5000) antibody.
were left at room temperature for 1 h and centrifuged at 14 000g for 1 h. The supernatant was transferred into an Ultrafree-4 centrifugal filter unit (Millipore, Bedford, MA), for desalting and concentrating proteins. Protein content of the supernatant was determined by the Bradford protein assay system.21 The standard curve was generated using bovine serum albumin, and absorbance was measured at 595 nm. Samples prepared from each individual mouse (n ) 10 per strain) were subjected to 2-DE as described elsewhere.22 Seven 712
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Molecular masses were determined by running standard protein markers (Bio-Rad Laboratories, Hercules, CA) covering the range 10-250 kDa. pI values 3-10 were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Excess dye was washed from the gels with distilled water, and the gels were scanned with an Image-Scanner (Amersham Bioscience). Electronic images of the gels were recorded using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) and Microsoft PowerPoint (Microsoft Corp., Redmond, WA) software. MALDI-TOF and MALDI-TOF/TOF-Mass Spectrometry (MS). Spots were excised with a spot picker (PROTEINEER sp, Bruker Daltonics) and placed into 96-well microtiter plates, and in-gel digests and sample preparation for MALDI analysis were performed by an automated procedure (PROTEINEER dp, Bruker Daltonics).23,24 Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM ammonium bicarbonate. After washing, gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. The dried gel pieces were reswollen with 40 ng/µL trypsin (Promega, Madison, WI) in enzyme buffer (consisting of 5 mM octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate) and incubated for 4 h at 30 °C. Peptide extraction was performed with 10 µL of 1% trifluoroacetic acid (TFA) in 5 mM OGP. Extracted
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Figure 2. (a) LIFT-TOF/TOF spectrum of m/z 996.55, assignment of the identified sequence 183-190, TFFEALR; spectrum of m/z 2199.108, assignment of the identified sequence 62-80, KLEFEISEDIFTSLTAAR; and spectrum of m/z 1373.836, assignment of LLLDGAPLAIHK of the identified sequence 133-145 to HP Q9CXN1. (b) LIFT-TOF/TOF spectrum of m/z 2700.462, assignment of the identified sequence 9-29, AVLVDLNGTLHIDAAVPGAQ; spectrum of m/z 2071.020, assignment of the identified sequence 63-80, LEFEISEDEIFTSLTAAR; and spectrum of m/z 3920.954, assignment of the identified sequence 98-124, ALPEFTGVQTQDPNAVVIGLAPEHFHYQ to HP Q9CXN1. Amino acids in bold show the matched MS/MS sequence from selected MS/MS peptides.
peptides were directly applied onto a target (AnchorChip, Bruker Daltonics) that was loaded with R-cyano-4-hydroxycinnamic acid (Bruker Daltonics) matrix thinlayer. The mass spectrometer used in this work was an Ultraflex TOF/TOF (Bruker Daltonics) operated in the reflector mode for MALDITOF peptide mass fingerprint (PMF) or LIFT mode for MALDITOF/TOF with a fully automated mode using the FlexControl software. An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M + H]+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1-17 and clip 18-39). Each spectrum was produced by accumulating data from 200 consecutive laser shots for PMF. Samples which were analyzed by PMF from MALDI-TOF were additionally identified using LIFT-TOF/TOF MS/MS from the same target using two MS/MS modes: laser-induced dissociation (LID) and collision-induced dissociation (CID). In the LID-MS/MS mode using a long-lifetime N2 laser, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation in the TOF1 stage. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analyzed in the reflector with high sensitivity. In addition to the LID-MS/MS mode, a high-energy
CID mode was adapted for distinguishing leucine and isoleucine by their different side-chain fragmentation. Argon gas was used as a collision gas, and about 1500 shots were summed to achieve spectra. PMF and LIFT spectra were interpreted with the Mascot search engine (versions 2.0 and 2.1; Matrix Science Ltd, London, U.K.). Database searches, through Mascot, using combined PMF and MS/MS data sets, were performed via BioTools (versions 2.2 and 3.0) software. A mass tolerance of 25 ppm, MS/MS tolerance of 0.5 Da, and 0 or 1 missing cleavage site were allowed, and oxidation of methionine residues was considered. The probability score calculated by the software was used as criterion for correct identification (http://www.matrixscience.com/help/scoring-help.html). Expression and Purification of Human Pyridoxal Phosphate Phosphatase (PLPP). To produce human brain PLP phosphatase monoclonal antibodies, a recombinant fusion protein was cloned and expressed in bacteria. The pET15b bacterial expression vector used contained six consecutive histidine residues and an 891 bp PLP phosphatase gene at the amino-terminus of inserts to help purify fusion proteins. The recombinant protein was expressed in Escherichia coli BL21 (DE3) and purified using a Ni2+-nitrilotriacetic acid Sepharose column (Qiagen), according to the manufacturer’s instructions. Salts in the purified protein were removed by PD10 column chromatography (Amersham, Sweden).10 Journal of Proteome Research • Vol. 6, No. 2, 2007 713
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Figure 4. LIFT-TOF/TOF spectrum of m/z 1612.789, assignment of the identified sequence 167-180, AVVVGFDPHFSYMK, and spectrum of m/z 1973.903, assignment of the identified sequence 239-254, FIFDCVSQEYGINPER to HP Q8CHP8. Amino acids in bold show the matched MS/MS sequence from selected MS/MS peptides.
Figure 3. LIFT-TOF/TOF spectrum of m/z 1544.847, assignment of the identified sequence 146-158, FSNVYHLSIHISK, and spectrum of m/z 2384.161, assignment of the identified sequence 8799, FVESDADEELLFNIPFT to HP Q9GZP4. Amino acids in bold show the matched MS/MS sequence from selected MS/MS peptides.
Sample Preparation and 2D Gel Electrophoresis of Purified Pyridoxal Phosphate Phosphatase (PLPP). Purified human PLPP mixed with 1.0 mL sample buffer was run using the abovementioned system. For 2-DE, different concentrations of PLPP ranging from 5 to 50 µg were applied on immobilized pH 3-10 nonlinear gradient strips at their basic and acidic ends. Production of Monoclonal Antibody for HP Q8CHP8. The production of a monoclonal antibody against HP Q8CHP8 was performed as previously described.25,26 Briefly, the purified enzyme was mixed with an equal volume of complete Freund’s adjuvant and was injected intraperitoneally into a BALB/c mouse (6-8 weeks old). Two booster injections with incomplete Freund’s adjuvant at 3-week intervals were followed by a final injection without adjuvant at 3 days before the cell fusion experiment. Feeder layer cells were prepared 1 day before fusion. For fusion, prepared spleen cells and SP2/o-Ag-14 mouse myeloma cells were combined and collected in a 50 mL disposable tube by centrifugation at 650g for 5 min. After removing the supernatant completely, the cell pellet was mixed by tapping the tube with fingers. Then, 1 mL of 50% polyethylene glycol (PEG) was added over a period of 1 min with constant swirling. The fusion reaction was stopped after 90 s by slowly adding 10 mL of Dulbecco’s modified Eagle medium 714
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(DMEM). Positive clones were first selected by immunodotblot analysis and confirmed by Western blotting.10 Western Blot Analysis of HP Q8CHP8. Two-dimensional gelseparated proteins from mouse hippocampus were electrotransferred onto polyvinylidene difluoride (PVDF) membranes (MilliPore, Bedford, MA). After incubation in blocking solution (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, and 5% non-fat dry milk), membranes were incubated with antibodies against HP Q8CHP8 for 2 h at room temperature. After three washes for 10 min with washing solution (0.3% Tween 20 in TBS), membranes were incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG for 1 h at room temperature. Membranes were washed three times for 10 min, and antigen-antibody complexes were visualized by an ECL reagent (Western Lightning, Perkin-Elmer Life Sciences, Boston, MA) on an X-ray film according to the manufacturer’s protocol. Blots were blocked by skimmed milk (TBS-T) and then incubated with antibody (1:5000 in TBS-T). Detection was performed using an HRP-conjugated secondary anti-rabbit antibody (Bio-Rad) and Chemiluminescence Substrate (SuperSignal West Pico, Pierce). Bioinformatic Characterization of Hypothetical Proteins. Bioinformatic database searches that were performed for the characterization of HPs were the following: InterPro (http:// www.ebi.ac.uk/InterProScan/), TIGR (http://tigrblast.tigr.org/ web-hmm/), and Pfam (http://pfam.wustl.edu/) databases for detecting of domains; ScanProsite (http://www.expasy.org/ prosite/) for motif predictions; STRING (http://string.embl.de/) databases for prediction of interacting partners; and SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) and PSORT II (http://psort.ims.u-tokyo.ac.jp/form2.html) for prediction of
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Identification and Function of HPs in Mouse Hippocampus Table 2. Sequence Alignment Search Results of HPs in Mouse Hippocampus protein name
acc. no.
13 days embryo head cDNA, RIKEN full-length enriched library, clone:3110052N05 product:hypothetical HAD-like structure containing protein, full insert sequence
Q9CXN1
AD039 (human)
RIKEN cDNA 00012G19
Q9GZP4
Q8CHP8
BLASTp
PSI-blast
RPS-BLAST
FASTA
Q9H008
Q632B0
gnl|CDD|40783
Q9H008
Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (human) 270 AA Score: 166 bits (420)
4-nitrophenylphosphatase (Bacillus cereus)
Hydrolase, haloacid dehalogenase-like hydrolase.
Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (human)
E-value: 7e-40 Identities: 100/257 (38%) Gaps: 6/257 (2%)
E-value: 3e-69 Identitiy: 22%
94.4% aligned Score: 40.0 bits (92) E-value: 4e-04
Q2UPJ7_ASPOR
O43396
gnl|CDD|46092
Thioredoxin-like protein] [Aspergillus oryzae]
Domain of Unknown Function (DUF1000)
223 AA Score: 92.0 bits (227)
Thioredoxin-like protein 1 (32 kDa thioredoxin-related protein) (human) 289 AA Score: 62.0 bits (149)
E-value: 1e-17 Identities: 56/175 (32%) Gaps: 8/175 (4%)
E-value: 1e-09 Identities: 47/142 (33%) Gaps: 18/142 (12%)
98.7% aligned Score: 199 bits (507) E-value: 2e-52
Q96GD0 Pyridoxal phosphate phosphatase
gi|44888310|sp|Q96GD0 Pyridoxal phosphate phosphatase
gnl|CDD|40783 Hydrolase, haloacid dehalogenase-like hydrolase
254 aa Score: 259
CD-Length: 197 residues
CD-Length: 152 residues
(PLP phosphatase) (human) 296 AA
296 AA
Score: 247 bits (631) E-value: 3e-64 Identities: 140-312 (44%)
Score: 255 bits (652) E-value: 1e-67 Identities: 144/312 (46%)
CD-Length: 197 residues 94.4% aligned Score: 40.0 bits (92)
Gaps: 20/312 (6%)
Gaps: 20/312 (6%)
E-value: 4e-04
signal peptides and subcellular localization. Local sequence alignment results were based on different BLAST database searches and FASTA (http://www.ebi.ac.uk/fasta33/). PSI- and BLASTp were linked at BLAST Web site (http://www.ncbi.nlm.nih.gov/BLAST/). ClustalW (http://www.ebi.ac.uk/clustalw/) was used for multiple sequence alignment searches. Threedimensional (3D) structure prediction was based on SWISSMODEL database search results (http://swissmodel.expasy.org/ ). HMMTOP (http://www.enzim.hu/hmmtop) and TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) databases were used for prediction of transmembrane segments and helices. PFP-Pred27 (http://202.120.37.186/bioinf/fold/PFPPred.htm) was used to identify protein fold pattern. DISULFIND (http://disulfind.dsi.unifi.it/) database was used for predicting disulfide-bonded cysteines. GRAVY-index values were obtained from ProtParam tool (http://www.expasy.org/tools/protparam.html). Recovery of Protein Activity. After samples were separated on 2D gels, proteins of interest were selected and excised. Protein elution from the gel matrix was performed by a Nanosep centrifugal device (300K molecular weight cutoff (MWCO); Pall Life Sciences, Ann Arbor, MI) in the presence of elution buffer (0.25 M Tris-HCl buffer, pH 6.8, and 0.1% (w/v)
270 aa initn: 573; init1: 317; opt: 605 Z-score: 759.4 bits: 148.2 E(): 5.4e-34 Smith-Waterman score: 605 40.856% identity (68.872% similar) in 257 aa overlap (7-257:11-267) Q5KI01 Thiol-disulfide exchange i
326 aa initn: 152; init1: 98; opt: 236 Z-score: 307.0 bits: 64.5 E(): 8.6e-09 Smith-Waterman score: 236 34.965% identity (62.937% similar) in 143 aa overlap (40-178:155-295) PLPP_RAT Q8VD52 Pyridoxal phosphate phosphatase 309 aa initn: 856; init1: 478; opt: 548 Z-score: 651.8 bits: 128.8 E(): 5.4e-28 Smith-Waterman score: 879 47.284% identity (69.968% similar) in 313 aa overlap (11-321:2-292)
SDS). The eluted protein was concentrated using a new Nanosep centrifugal device equipped with a 3K-MWCO, and 6 M guanidine in renaturation buffer (100 mM KCl, 12.5 mM MgCl2, 25 mM HEPES (pH 7.6), 0.1 mM EDTA, 20 mM β-mercaptoethanol, and 10% (v/v) glycerol) was treated in a Nanosep centrifugal device. The solution was then exchanged and permitted to renature with renaturation buffer. Inorganic Pyrophosphate (PPi) Phosphatase Assay. PPi phosphatase activity of the recovered protein was measured by a fluorometric assay kit (Molecular Probes, Invitrogen Co.) based on formation of the resorufin product to detect PPi.28 Protein was serially diluted for the assay, and a standard curve was determined using PPi (at 530 and 540 nm absorbance) to calculate enzyme activity according to the manufacturer’s protocol. Antioxidant Activity Assay of HP Q9GZP4. Antioxidant activity of the recovered protein was measured by a colorimetric assay kit (Cayman Chemical) based on the crocin bleaching (oxidation) method.29,30 The assay relies on the ability of an antioxidant in the sample to inhibit the oxidation of ABTS (2,2′-azino-di-[3-ethylbenzthiazoline sulfonate]) to ABTS+ by metmyoglobin. The capacity Journal of Proteome Research • Vol. 6, No. 2, 2007 715
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Figure 5. Multiple sequence alignment of HP Q9CXN1 with predicted HAD superfamily domains are demonstrated. Three typical motifs (I, II, and III) with their conserved amino acids within “core” domain and “capping” domain of HP Q9CXN1 are shown. The conserved substitutions (:), semiconserved substitutions (.), and identical residues (green) are marked.
of the antioxidants of HP Q9GZP4 to inhibit ABTS oxidation as compared to Trolox (water-soluble tocopherol analogue) was 716
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quantified as millimolar Trolox equivalent (at 630 nm absorbance).
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Identification and Function of HPs in Mouse Hippocampus Table 3. Computer-Based Functional Characterization of HPs in Mouse Hippocampusa
protein name
acc. no.
identified mouse strain
13 days embryo head cDNA, RIKEN full-length enriched library, clone:3110052N05 product:hypothetical HAD-like structure containing protein, full insert sequence
Q9CXN1
C57Bl/6J
gene name
motif
subcellular localizationb
Haloacid dehalogenase-like Hydrolase: (PF00702, IPR005834)
N-myristoylation site
cytoplasmic
HAD-superfamily subfamily IIA hydrolase, hypothetical 2 (IPR006355, TIGR01458) HAD-superfamily hydrolase, subfamily IIA (IPR006357, TIGR01460)
N-glycosylation site
domain
Hdhd2 (haloacid dehalogenasehydrolase domain containing 2)
literature availablec
Protein kinase C phosphorylation site
Casein kinase II phosphorylation site AD039 (human)
Q9GZP4
129S2/Sv
RP5-886K2.4
DUF1000: IPR010400
N-myristoylation site
PF06201
Strausberg et al., 2002 nuclear
N-glycosylation site Protein kinase C phosphorylation site Casein kinase II phosphorylation site RIKEN cDNA 1700012G19
Q8CHP8
C57Bl/ 6J
1700012G19Rik
Haloacid dehalogenase-like Hydrolase: (PF00702, IPR005834) HAD-superfamily subfamily IIA hydrolase, hypothetical 2 (IPR006355, TIGR01458) HAD-superfamily hydrolase, subfamily IIA (IPR006357, TIGR01460)
Amidation site
cytoplasmic
availabled
N-myristoylation site N-glycosylation site
Protein kinase C phosphorylation site
Casein kinase II phosphorylation site a No signal peptide was predicted (all proteins nonsecretory proteins). b Limitations and bias of PSORT II database are documented in ref 53. c http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi?db)gene&cmd)Display&dopt)gene_pubmed&from_uid)76987. d http://www.ncbi.nlm.nih.gov/entrez/ query.fcgi?db)gene&cmd)Display&dopt)gene_pubmed&from_uid)67078.
Table 4. Structural Predictions of HPs in Mouse Hippocampus
protein name
acc. no.
Gravy index
no. of trarnsmembrane helices
no. of trarnsmembrane segments
no. of disulfidebonded cystines
13 days embryo head cDNA, RIKEN full-length enriched library, clone:3110052N05 product: hypothetical HAD-like structure containing protein, full insert
Q9CXN1
-0.748
HMMTOP: 0
4 (length 1-2 aa)
2
predicted fold pattern
(TIM)-barrel (Alpha/Belta)
3D structure model
1VJRA (PDB ID)
33,12% identity P(N): 7e-24 AD039 (human)
Q9GZP4
-0.151
HMMTOP: 0
0
4
Immunoglobin like (All-beta)
1XOYA (PDB ID) 31,98% identity P(N): 4e-10
RIKEN cDNA 1700012G19
Q8CHP8
0.004
HMMTOP: 1
1 (length 8 aa)
8
Cupredoxins (All-belta)
1PW5A (PDB ID) 27,82% identity P(N): 3e-13
Protein was serially diluted for the assay, and a standard curve was determined using Trolox to calculate enzyme activity according to the available protocol. Pyridoxal Phosphate Phosphatase (PLPP). PLPP activity of the recovered protein was based on the pyridoxal 5′-phosphatase (PLP) radio-enzymatic assay (REA) (Bu ¨ hlmann, Switzerland; ALPCO Co.) in plasma.31 The PLP standard curve was adapted to our experimental conditions.
Results Identification of HPs in Mouse Hippocampus. Mouse hippocampal protein extracts were applied on 2-DE gel and visualized by Coomassie blue staining. Representative gels of hippocampal protein extracts from the strains C57 and 129Sv are shown in (Figure 1). No HPs were observed in the other strains. Spots were analyzed by MALDI/MS and MS/MS folJournal of Proteome Research • Vol. 6, No. 2, 2007 717
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Figure 6. Predicted 3D structure of HP Q9CXN1 based on SWISSMODEL server. As template protein served the crystal structure of 4-nitrophenylphosphatase (TM1742) from Thermotoga maritime with about 33% identity to target HP Q9CXN1 and expection value of 7e24.
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lowing in-gel trypsinization, and identification was carried out by matching the peptide masses with theoretical peptide masses of all proteins in the Swiss-Prot database. Internal standards were used to correct the measured peptide mass, thus, reducing the windows of mass tolerance and increasing the confidence of identification. This proteomic approach revealed strain-specific expression of three mouse hippocampal HPs. Swiss-Prot/TrEMBL accession numbers, protein names, relative theoretical molecular weight, pI values (theoretical and observed), and MALDI-TOF/TOF results (MS and MS/MS) are given in Table 1. MS/MS results are shown in Figures 2-4 confirming MALDITOF/TOF results. Bioinformatic Characterization of Hypothetical HAD-like Structure Containing Protein, Full Insert Sequence (Q9CXN1). HP Q9CXN1 is the gene product of mouse haloacid dehalogenase hydrolase domain containing 2. Different pairwise (BLASTp, PSI-, RPS-BLAST, and FASTA) (Table 2), multiple sequence
Figure 7. Multiple sequence alignment of HP Q9GZP4 with predicted DUF 1000 domain is shown. The conserved substitutions (:), semiconserved substitutions (.), and identical residues (green) are marked.
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alignment searches (ClustalW) (Figure 5), and domain- (InterPro, Pfam, and TIGR) (Table 3) and motif (PROSITE (Table 3), PFP-Pred (Table 4)) searches were done to find orthologs, paralogs to this protein, and also to predict the conserved activity and functional characterization of HP Q9CXN1. Structural information such as number of transmembrane helices (HMMTOP) and segments. number of disulfide-bonded cysteines (PredictProtein), and also three-dimensional structure (SWISS-MODEL) were obtained from different database searches (Table 4). Database search results reveal that several conserved amino acids within three short typical motifs for the haloacid dehalogenase (HAD) superfamily are present in HP Q9CXN1 (Figure 5). The HAD superfamily was discovered by computer analysis of bacterial HADs,32 catalyzing the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways and typically showing low sequence identity
