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High-Throughput Screening of Sulfated Proteins by Using a GenomeWide Proteome Microarray and Protein Tyrosine Sulfation System Bo-Yu Huang, Po-Chung Chen, Bo-Han Chen, Chen-Chu Wang, HsuanFu Liu, Yi-Zao Chen, Chien-Sheng Chen, and Yuh-Shyong Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02853 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 18, 2017
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Analytical Chemistry
High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System Bo-Yu Huang1, Po-Chung Chen2, Bo-Han Chen1, Chen-Chu Wang1, Hsuan-Fu Liu1, Yi-Zao Chen1, Chien-Sheng Chen2*, and Yuh-Shyong Yang1* 1
Bo-Yu Huang, Chen-Chu Wang, Bo-Han Chen, Hsuan-Fu Liu, Yi-Zao Chen, Prof. Yuh-Shyong Yang Department of Biological Science and Technology. National Chiao Tung University. 75 Boai Street, Hsinchu 300 (Taiwan) Fax: (+886)3-5712121 ext. 56926. E-mail:
[email protected] 2 Po-Chung Chen, Prof. Chien-Sheng Chen. Graduate Institute of Systems Biology and Bioinformatics. National Central University. 300 Jhongda Rd, Jhongli 320 (Taiwan) Fax: (+886)3-4227151 ext. 36103. E-mail:
[email protected] Abstract: Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymes activities. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.
Keywords: protein tyrosine sulfation • tyrosylprotein sulfotransferase • posttranslational modification • protein-protein interaction • proteome microarrays
receptor 5 (CCR5) serves as the receptor for several inflammatory chemokines and is a coreceptor that facilitates HIV-1 infection.9 PTS in the N-terminal region of CCR5 critically affects the binding affinity for chemokine ligands,10 and mutation of the 4 sulfotyrosine residues in CCR5 to phenylalanine inhibits HIV infection by 50%–75% in cultured cells, depending on the HIV isolate tested.11 P-selectin glycoprotein ligand-1 (PSGL-1), a PTS substrate expressed on leukocytes, plays a crucial role in the tethering and rolling of leukocytes during recruitment of the cells from blood vessels to the sites of acute inflammation following stimulation by infection. PSGL-1 is also a functional receptor for enterovirus 71 (EV71),12 and the occurrence of severe EV71 infection coupled with a high number of cases of fatal encephalitis continues to pose a major public health threat.13 Sulfation on PSGL-1 N-terminal tyrosine residues contributes to the binding of specific strains of EV71 through its capsid protein (VP1).13,14 In a mouse model of pathologic atherosclerosis, PTS was reported to function as a key contributor to monocyte/macrophage recruitment or retention,14 and lethally irradiated Ldl-/- mice were rescued by using hematopoietic progenitors lacking TPST activity because of the deletion of TPST1 and TPST2 genes.15 In the case of autoimmunity, diverse adhesion molecules and chemokine receptors are recognized to be tyrosine sulfated, and in patients with the autoimmune disease rheumatoid arthritis, enhanced TPST1 expression might cause increased sulfation of crucial tyrosine residues in chemokine receptors, which could constitutively increase their binding affinities for their ligands.16 However, in sharp contrast to the numerous methods available for detecting protein phosphorylation,17,18 few tools are currently available for PTS detection. Moreover, although the UniProt database lists nearly 500 sulfated proteins, by excluding redundant and similar proteins in distinct organisms, we determined that only approximately 157 sulfated proteins (Tables S1) have been experimentally examined to date, and this represents a minor fraction of the total predicted sulfated proteins. Thus, it is essential to identify the remaining majority of the potential TPST substrates.
Protein tyrosine sulfation (PTS) is a posttranslational modification commonly detected in secreted proteins and cell-surface receptors.1 PTS facilitates protein-protein interactions and critically affects enzymatic activity and protein lifespan,2 and PTS is catalyzed by the enzyme tyrosylprotein sulfotransferase (TPST), a Golgi-localized type II transmembrane protein.3 In contrast to phosphorylation, which is central to intracellular signal transduction, sulfation modulates cell-cell and cellmatrix communication, and TPST functions at critical steps in generating the sulfation that forms a part of the recognition motifs for adhesion molecules, chemokines, growth factors and their receptors, and pathogens.4 PTS plays a crucial role in physiology and pathology, A proteome microarray, also known as a proteome chip, contains including in the immune responses and viral infection involved in most of the individually purified proteins in a given proteome. Proteome numerous diseases.5 For example, PTS on certain chemokine receptors microarrays have been used to discover protein-DNA, protein-lipid, might regulate chemokine receptor-ligand interactions relevant to protein-drug, and protein-peptide interactions,19 and have emerged as a inflammatory response and disease,6 and tyrosine-sulfated chemokines powerful tool for use in systems biology.20 Moreover, proteome and adhesion molecules have been proposed as potential therapeutic microarray assays, which can be completed in hours, are faster and targets for developing anti-inflammatory drugs and inhibitors.7 In the case simpler than other proteomic assays.21 Several proteome microarrays have of viral infection, PTS in polyomavirus and varicella zoster virus might been fabricated, including yeast, Escherichia coli, and human proteome facilitate modulating host-cell recognition and facilitate viral attachment microarrays.22 Chen et al.23 developed a high-throughput protein and entry; to date, 97 729 tyrosine residues, including 5091 tyrosine ACS Paragon Plusexpression Environment and purification protocol to fabricate an E. coli proteome sulfation sites, have been predicted in 1024 viruses. 8 CC chemokine microarray and used it for discovering DNA damage-recognition
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activities. Moreover, E. coli proteome microarrays were used for identifying serological biomarkers for inflammatory bowel disease and screening for the substrates of protein acetyltransferases. 24 However, compared with the development of gene chips, proteome microarray development is less mature because the techniques used to process proteins are more complex than the corresponding methods used for nucleic acids, and this includes the purification technology, chip fabrication, and analysis approaches. Figure 1 illustrates PTS on a proteome microarray by using a 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis (PTS coupled-enzyme system).25
Preparation of Drosophila melanogaster TPST (DmTPST) and human PAPSS-1 hPAPSS-1 for in vitro PTS
EXPERIMENTAL SECTION
B. Expression and purification of recombinant hPAPSS-1. A prokaryotic expression vector harboring the hPAPSS-1 cDNA was used to transform BL21(DE3) competent cells (at 37 °C) in LB broth containing 100 μg/mL ampicillin. A single colony was grown in 500 mL of LB medium containing 100 μg/mL ampicillin until the OD600 reached 0.6–0.8, and 1 mM IPTG (final concentration) was then added to induce protein expression and the cells were cultivated at 20 °C for 16 h. Bacterial cells were harvested through centrifugation and then homogenized in ice-cold HisTrap Buffer A (50 mM Tris, pH 8.0, 500 mM NaCl 2, 10% glycerol, 5 mM imidazole). The crude homogenates were centrifuged at 30 000 × g for 25 min, and the collected supernatants were individually fractionated using a HisTrap Sepharose column. The recombinant protein was eluted with 50 mL of HisTrap Buffer C (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 305 mM imidazole, 10% glycerol), and the purity of the obtained hPAPSS-1 was verified by performing SDS-polyacrylamide gel electrophoresis.
A. Expression and purification of DmTPST. A single colony of BL21(DE3)pLysS cells transformed with the DmTPST plasmid was used to inoculate (at 37 °C) LB broth containing ampicillin as the antibiotic. Cells were allowed to grow until an OD600 of 0.4–0.6, and protein expression was then induced (at 20 °C for 24 h) with 1 mM IPTG. The cultures were centrifuged at 14000 × g for 20 min, and the pellets were sonicated in the IMAC5 buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole, 10% glycerol) for DmTPST purification, which was performed using NiSO4-charged HisTrap Sepharose. Protein purity was assessed using SDS-polyacrylamide gel electrophoresis.
Preparation of E. coli K12 proteome microarrays. The high-throughput protein expression, protein purification, and protein printing methods used here were modified from published procedures.23 Briefly, E. coli K12 clones were inoculated into 96 DeepWellTM plates (Nunc) containing 2× LB medium supplemented with 30 μg/mL chloramphenicol. The plates were incubated overnight with shaking at 37 °C, following which the cultures were sampled and the OD595 values of the solutions were measured using a Synergy 2 Multi-Mode Microplate Reader (Bio-Tek). The OD595 of the solution in each well was adjusted to approximately 0.1 by using 2× LB medium and the cells were then allowed to grow until the OD595 reached 0.7–0.9, at which point protein expression was induced by adding 5 mM isopropyl β-D-thiogalactopyranoside (IPTG). After induction at 37 °C for approximately 3.5 h, cell pellets were collected by centrifuging the samples at 2240 × g at 4 °C for 5 min and then stored at 80 °C. To purify proteins, the cell pellets were thawed and resuspended in Sulfation assay through PAPSS-coupled TPST catalysis. The complete a lysis buffer at 4 °C; the 42 μL of the buffer added per well contained 5 assay mixture25 contained the following components: the TPST substrates μL of 10× CelLytic B, 1 μL of 50 mg/mL lysozyme, 0.01 μL of 50 U/mL mentioned previously in this section, 4 mM inorganic [35S]Na2SO4, 5 mM benzonase, 0.5 μL of a proteinase-inhibitor cocktail, 0.5 μL of 100 mM β-mercaptoethanol, 1 mM MgCl2, 50 mM MES (pH 6.5), 5 μg of phenylmethylsulfonyl fluoride, 18 μL of prewashed Ni-NTA Superflow hPAPSS-1, 20 μg of TPST, and 0.5 U of pyrophosphatase in a final resin (QIAGEN), and 17 μL of 50 mM NaH2PO4, 300 mM NaCl, and 40 volume of 20 μL. Assays were initiated by adding hPAPSS-1 and mM imidazole, pH 8. The plates were vigorously shaken at 4 °C for 1.5 h, performing preincubation for 15 min at 37 °C, and this was followed by after which the mixtures were transferred into 96-well filter plates (Nunc) the addition of TPST and incubation for 45 min at 37 °C. Preincubation and washed sequentially with Wash Buffers I and II (I: 50 mM NaH2PO4, with hPAPSS-1 is to produce adequate amount of PAPS, which is 300 mM NaCl, 10% glycerol, 30 mM imidazole, 0.01% Triton X-100, pH important if initial rate of TPST is to be determined. The reactions were 8; and II: 50 mM NaH2PO4, 150 mM NaCl, 30% glycerol, 30 mM terminated by heating at 95 °C for 2 min. TPST catalyzes the transfer of a imidazole, 0.01% Triton X-100, pH 8). The proteins were then eluted sulfuryl group (SO3-1) from PAPS onto tyrosine residues within a target using Elution Buffers I and II sequentially (I: 50 mM NaH2PO4, 150 mM protein on the protein microarray. Proteins that were recognized by NaCl, 30% glycerol, 500 mM imidazole, 0.01% Triton X-100, pH 7.5; antisulfotyrosine antibodies were visualized and quantified using Cy3and II: 50 mM NaH2PO4, 150 mM NaCl, 30% glycerol, 300 mM labeled antimouse antibodies (Figure 1). imidazole, 0.01% Triton X-100, pH 7.5). To ensure the purity of the E. coli K12 proteins obtained from different preparations, we determined the Identification of sulfated proteins on the E. coli proteome microarray. quality and quantity of the purified proteins using electrophoresis and The entire screening process, except for the washing steps as specified, Coomassie staining to adjust and to confirm that each proteome chip was performed at room temperature (RT). The E. coli protein chips stored 23 contains similar protein in quality and quantity for further experiments . at -80 °C were thawed at RT and blocked in 3% BSA for 1 h. After the We estimated that approximately 88% of the proteins were purified at the chip was washed thrice with PBST (PBS pH 7.4 containing 0.05% Tween expected molecular weight with yields of more than 0.2 mg/ml, of which 20), the sulfation assay reaction buffer was applied to the chip to entirely around 50% were seen as the predominant band. The high throughput cover the chip surface. After incubation for 2 h with gentle shaking on a protein expression, protein purification, and protein printing were rocker, the chip was rinsed once with 4 mL of Tris-buffered saline (TBS) described previously23. The purified proteins were stored at -80 °C before containing 0.05% Tween 20 (TBS-T), following which the chip was printing. To print the proteome chips, the frozen proteins were thawed and soaked in 4 mL of TBS-T and washed for 10 min at 25 °C with gentle each protein was then spotted in duplicate on each aldehyde slide by using horizontal agitation; this washing step was repeated twice. After a SmartArrayer 136 (CapitalBio) at 4 °C. There are 4256 proteins encoded discarding the TBS-T, the chip was incubated for 1.5 h with the primary by the E. coli K12 strain to printing. After protein printing, the chips were antibody (antisulfotyrosine) diluted 1:1000 in 3 mL of PBS, and then incubated for 8 h at 4 °C to allow protein immobilization on the slides, ACS Paragon Pluswashed Environment for 10 min at 25 °C with gentle horizontal agitation; the washing and then stored at -80 °C until use in the chip assay. step was repeated twice. Next, the TBS-T was discarded and the chip was
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Analytical Chemistry
incubated for 40 min with the secondary antibody (Cy3-labeled antimouse IgG; Jackson ImmunoResearch Laboratories) diluted 1:100000 in 3 mL of PBS. Lastly, the chip was washed at 25 °C as in the previous washing step, rinsed briefly in sterile water and quickly spun to dryness at 2000 rpm, and then scanned (at 536 nm) using a GenePix array scanner (GenePix Pro 6.0 or GenePix 4200AL, Molecular Devices, PA). All the data presented in this report were triplicated with different proteome chips to confirm their reproducibility. Data processing and selection of protein hits. Data were normalized using ProCAT (protein chip analysis tool) with Perl 5.0. The local cutoff was set as the mean plus 3 standard deviations. A binomial test was used to select hits between sample groups; hits with a P value lower than 0.05 were considered specific hits for each group. The list of resultant hits was verified by “eyeballing” the original images. Each quantified sample array image was exported from Genepix (Molecular Devices, CA) as a text file for preprocessing. The goal of preprocessing is to yield a feature of interest from each protein spot in the array that minimizes technical variability and maximizes the signal of interest. The ratio of the mean signal over the mean background signal for each protein spot was determined to be the best method of preprocessing. This method has the advantage that all features are normalized to their background signals. Thus, if a protein spot signal is artificially high due to an artifact on the slide the ratio will account for it. Furthermore this preprocessing method also normalizes the features across all arrays, as the ratio is a standardized metric. The ratio represents the fold change of the signal above background and can be interpreted as the degree of tyrosine sulfation proteins to each spotted protein. Verification of identified sulfated proteins by using a radioactive assays and western blotting
min, covered with Saran wrap (after removing excess solution from the surface), and used for exposing X-ray films in a dark room. Several exposure periods were tested, and the signal from the unknown sample was compared with that of the standard to estimate the protein concentration (Figure 4a).
RESULTS AND DISCUSSION As depicted in Figure 1, 4256 E. coli K12 proteins immobilized on a proteome microarray24 were treated with a PTS coupled-enzyme system that included TPST and PAPS synthase (PAPSS). 25 The sulfated proteins were detected using antisulfotyrosine antibodies and visualized and quantified using Cy3-labeled goat antimouse antibodies. The results are shown in Figures 2 and S1. Figure 2 shows the E. coli proteome microarray composed of 4256 Nterminal GST-fusion proteins immobilized on nitrocellulose-coated glass slides (FAST slides, Whatman). Each protein was printed in duplicate on the chip23 and all the proteome chip data presented in this research were triplicated. Our results indicated that highly reproducible data were obtained even when different proteome chips were used. For protein sulfation experiments, one proteome chip was treated with the complete PTS coupled-enzyme system (Figure 2a and Figure 2b top) and the other was treated under similar conditions but in the absence of TPST (Figure 2c and Figure 2b bottom). Pairs of sulfated proteins were detected only after treatment with the complete PTS coupled-enzyme system. Additional images are presented in Figure S1. Comparison between two proteome chips using the same proteins is shown in Figure S2. a) R1
R1
OH
O
NH
O
NH2
N
P
TPST N
O -O
O
(sulfated tyrosylprotein)
NH2
PAPS synthetase
O
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(tyrosylprotein)
SO42-+ATP
S
O-
O
R2
R2
-O
S
NH
H N
H N
A. Radioactive assay. Protein sulfation was verified by monitoring the radioactive isotope [ 35S] attached to the proteins through the PAPSScoupled TPST assay reaction.26 Briefly, [35S]PAPS, the sulfuryl donor in the sulfation reaction, was generated in situ by hPAPSS-1 and coupled to recombinant DmTPST. The desired samples (selected proteins from the E. coli protein microarray) were examined under the standard assay condition. The standard assay mixture contained 50 mM MES at pH 6.5, 5 mM β-mercaptoethanol, 4 mM inorganic [35S]SO42-, 1 mM MgCl2, 1 mM ATP, 1 μg of recombinant hPAPSS-1, 1 U of pyrophosphatase, and the protein samples 3μg at various concentrations. In the positive control, the samples were replaced with 20 μM PSGL-1 peptide (ATEYEYLDYDFL). The sulfated products were separated on a cellulose thin-layer chromatography plate and the [ 35S] autoradiography counts were obtained (Figure 3b).
O O
O
N
N
N
O
N
O
O
-O
O-
P
O
O
O-O
O
OH
-O
P O
O
OH
P O-
O
(PAPS)
O-
(PAP)
b) PAP
PAPS
TPST GST
Protein
Step1
GST
Protein
Step2
TPST
S
S GST
Protein
Step3
GST
Protein
Step4
S GST
Protein
Step5
E. coli proteome microarrays Step6
Figure 1. In vitro and in situ posttranslational sulfation and identification of sulfated
B. Dot blotting. Nitrocellulose membranes were prepared by drawing proteins on E. coli proteome microarrays. a) A PTS coupled-enzyme system is used to catalyze the sulfation of proteins immobilized on the proteome microarray. b) Procedures a grid with a pencil to indicate the region to be blotted. Next, a pipette fit for in situ protein sulfation and analysis of the sulfated proteins: Step 1, Protein with a narrow-mouth pipette tip was used to spot 2 μL of each sample immobilization on a chip: 4256 E. coli K12 GST-fusion proteins are printed on a onto the membranes at the center of the grid. To minimize the area that the proteome microarray. Step 2, PAPS generation: PAPS synthase catalyzes the synthesis of solution penetrated (typically 3–4 mm in diameter), the samples were the sulfation donor. Step 3, In situ sulfation: target proteins are sulfated through in situ TPST catalysis. Step 4, Antisulfotyrosine antibody binding: target proteins are recognized applied slowly, and subsequently the membranes were allowed to dry. by an antibody against tyrosine sulfation sites. Step 5, Visualization: sulfated proteins are Nonspecific sites were blocked by soaking the membranes in 5% BSA in visualized using a Cy3-labeled goat antimouse antibody. Step 6, Scanning and data TBS-T for 0.5–1h at RT. The reaction chamber used for labeling was a processing: the proteome microarray is scanned at 536 nm (GenePix) and the data are 10-cm Petri dish. First, the membranes were incubated for 30 min at RT processed using ProCAT (protein chip analysis tool); protein hits are selected using a binomial test. with anti-sulfation antibodies dissolved in BSA/TBS-T (1:1000 dilution in TBST with 0.1% BSA), and then washed thrice (5 min each) with TBS-T. Next, the membranes were incubated for 30 min at RT with HRPconjugated secondary antibodies (1:10000 dilution in TBST), and then Paragon washed thrice with TBS-T (1 × 15 min, 2 × 5 min) andACS once with TBS (5 Plus Environment min). Lastly, the membranes were incubated with an ECL reagent for 1
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a)
Complete
b)
c) A B C D E F GH I J K LMN O P
- TPST
a b c d e f g h i j k l m n o p A B C D E F GH I J K LMN O P a b c d e f g h i j k l m n o p
Figure 2. Images of E. coli proteome microarrays. E. coli proteome microarrays composed of 4256 N-terminal GST-fusion proteins immobilized on nitrocellulose-coated glass slides (FAST slides, Whatman). a) An E. coli proteome-microarray chip containing 48 microarrays that was treated with the complete PTS coupled-enzyme system. b) Image of a single microarray. Each protein was duplicated on the chip, and detection of the pair was a prerequisite for the identification of sulfated proteins. The bottom rows of the microarray (Lines m, n, o, and p) contained control proteins. Io, Jo: BSA, PTS negative control; Co, Do and Eo, Fo: PSGL-1 and PolyEAY, respectively, PTS positive controls. Minimal amount of positive control proteins (5g) were used. Other control proteins were not used for this experiment. Table S2 presents the complete list of control proteins. c) A proteome microarray that was treated with the PTS coupled-enzyme system lacking TPST.
Figures 2 and S1 show protein arrays containing controls that were used to demonstrate how the sulfated proteins were visualized. Every protein array block contained 192 protein points and control proteins (Table S2); the target proteins and positive and negative controls were duplicated on the chip. Each microarray block contained 3 control proteins: BSA (negative control) and PolyEAY and PSGL-1 (positive controls). Following fluorescence scanning at 536 nm, the normalized ProCAT data were analyzed using a binomial test (P < 0.05). The tyrosine-sulfated proteins in pairs could be visualized by a chip scanner (Figures 2b and S1b), and an analysis of the proteome microarray revealed 875 TPST substrates (Table 1), which are listed in Tables S3. To confirm the proteome microarray-based identification of tyrosinesulfated proteins, we performed dot blot analysis on 11 randomly selected proteins (Figure 3a). Whereas 6 of the proteins (aas, aidB, dpiA, fiu, hemY, rhsC) were identified as TPST substrates through ProCAT analysis, the other 5 (accB, btuC, cysK, emrK, iclR) were not identified as substrates in the PTS coupled-enzyme system. The results obtained for the 11 proteins in the dot blot analysis (Figure 3a) were identical to those from the proteome microarray analysis. However, the antisulfotyrosine antibody used in the dot blot analysis was the same as that used in the proteome microarray screening to identify tyrosine-sulfated proteins. Thus, we further examined the PTS of the selected proteins by employing a distinct method that involved the use of radioactive [ 35S] sulfate (Figure 3b). The [35S] PAPS that was produced from [ 35S] sulfate and ATP through PAPSS catalysis was used as the radioactive sulfated donor for the PTS catalyzed by TPST.26 Only the proteins that were identified as TPST substrates through proteome microarray screening and dot blot analysis were labeled with [ 35S] sulfate when the complete PTS coupledenzyme system was used (Figure 3b).
protein is dependent on the methods used. The amounts of proteins used for sulfation detection were about 5g , 3g, and 3g for proteome chip (Figure 2), dot blot analysis (Figure 3a) and radioactive labeling (Figure 3b), respectively. It has been shown that the detection limit is at 6.25 pg using Cy dyes on protein microarray.29 The limit of detection using HRP in Western blot was shown to be at 60 pg.30 The limit of detection of various radio-TLC techniques was around 100 pg.31 In this report, we determine the quantity and purity of each protein with electrophoresis following Coomassie staining, and the amount of protein used for the determination of sulfation was significantly higher than those of reported detection limits in the literature. Next, we analyzed and compared the potential tyrosine sulfation sites and their surrounding amino acids among all 4256 proteins (Table 1). Previously, acidic amino acids, aspartic acid (D) and glutamic acid (E), were observed to be present near the tyrosine sulfation sites in all identified TPST protein substrates.27,28 Here, in all 875 tyrosine sulfation substrates identified, we detected at least one tyrosine that was within 5 amino acids upstream or downstream from an acidic amino acid. By contrast, in 3150 (97%) of the 3381 proteins that were identified as negative for PTS by using the PTS coupled-enzyme system, no tyrosine was present within +5 or -5 amino acids from an acidic amino acid. This observation strongly indicates that PTS requires the presence of an acidic amino acid within 5 amino acids from the sulfation site, which agrees with previous proposals based on a limited number of identified sulfated proteins.27,28 a)
b)
[35S] Sulfated protein
PAPS
Figure 3. a) Dot blot analysis performed on 11 proteins randomly selected from the E. coli proteome microarray. Detailed experimental procedures are presented in Supporting Information. b) Verification of PTS through [35S] sulfate radioactive labeling. The radioactive PTS coupled-enzyme system is described in a published report26 and in Supporting Information.
Table 1. Analysis of the presence of glutamic acid and aspartic acid within 5 amino acids upstream or downstream from potential tyrosine sulfation sites. [a]
ACS Paragon Plus Environment
The minimum amount of protein required for the detection of sulfated
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Analytical Chemistry
[a] Data obtained from the images of the proteome microarrays following sequence analysis; the data were processed using ProCAT and protein hits were selected using a binomial test.
Notably, 231 proteins (3%) identified as negative for PTS in the microarray analysis contained at least one tyrosine that was surrounded by acidic amino acids but were not TPST substrates. This observation indicated that in addition to the acidic amino acids surrounding potential tyrosine sulfation sites, other sequence features might affect substrate selection by TPST. Among the 875 sulfated proteins identified (Table 1), 29 proteins contain only one tyrosine sulfation site where an acidic amino acid is present in the -5 to +5 region around the potential sulfated tyrosine. Therefore, we analyzed the composition of the amino acids surrounding tyrosine residues in the 29 proteins and the 231 proteins identified as negative for PTS (Figure 4). Our analysis showed that most of the proteins contained more than one tyrosine surrounded by acidic amino acids (Figure 5). Here, we could not identify either the number of tyrosine residues sulfated in a protein or the exact location of the sulfation sites except in the case of proteins that contained only one potential tyrosine sulfation site. Thus, the comparison presented in Figure 4 shows the difference in amino acid composition in the -5 to +5 region around potential tyrosine sites between these 2 groups of proteins. Our results revealed notable variations. PTS is recognized to occur preferentially at sites that are surrounded by acidic amino acids. However, our results showed that in TPST substrates (Figure 4a), D was predominantly present near the tyrosine sulfation site; by contrast, in proteins that were not identified as TPST substrates (Figure 4b), E was the predominantly detected acidic acid and was evenly spread in the -5 to +5 region around the target tyrosine.
Thus, we determined the number of potential tyrosine sulfation sites in each of the 875 proteins identified as TPST substrates, and our results showed that the majority of the proteins contained 3–6 potential tyrosine sulfation sites (Figure 5). Certain widely recognized TPST substrates that contain multiple tyrosine sulfation sites include CCR5, 12 CCR8,14 and PSGL-1,34 which have been shown to be involved in key physiological and pathological mechanisms. The data presented in Figure 5 indicate that most of the identified proteins might contain more than one tyrosine sulfation site. The number of PTS sites in a given protein has been reported to potentially influence the protein’s biological functions, 32 but few previous studies have specifically investigated this. By using the PTS coupled-enzyme system, we determined here that 875 proteins were sulfated, which is a substantial increase compared with the number of previously identified sulfated proteins (approximately 157), and further that each of these proteins might harbor multiple sulfation sites. Our results support the view that acidic amino acids are present within a distance of 5 amino acids from all sulfation sites. However, we did not experimentally identify the sulfation in proteins that contain multiple potential sites of tyrosine sulfation (Figure 5). The requirement of the presence of acidic amino acids around multiple tyrosine sulfation sites remains poorly examined,35,36 and this is our current focus.
Figure 5. Potential sulfation sites of the 875 proteins identified as TPST substrates on proteome microarrays. The graph shows the number of tyrosine residues present within a distance of 5 amino acids from an acidic amino acid. Figure 4. Sequence analysis of the region around tyrosine residues surrounded by acidic amino acids in sulfated and nonsulfated proteins. a) Composition of amino acids of the 29 proteins selected from the 875 sulfated proteins identified through proteome microarray screening. Each of these 29 proteins contains only one tyrosine sulfation site where an acidic amino acid is present in the -5 to +5 region around the potential sulfated tyrosine. b) Composition of amino acids of the 231 proteins that were identified as non-TPST substrates in the proteome microarray screening, but contain tyrosine residues surrounded by acidic amino acids in the -5 to +5 region. Sequence analysis graphs were developed using WebLogo.32
CONCLUSION
We used a combination of the PTS coupled-enzyme system and proteome microarrays and demonstrated that tyrosine-sulfated proteins can be identified efficiently. We increased the number of recognized TPST substrates from approximately 157 identified previously to 875 in this study. It is intriguing to compare phosphorylation, which regulates intracellular processes, with PTS, which is typically directed to the cell In addition to the amino acid sequence, the secondary and tertiary exterior where it modulates cell-cell interaction and ligand-receptor structures of the sulfation site may be critical for PTS. Tyrosine sulfation interactions.37 Both are common posttranslational protein modifications sites are expected to be present on the protein surface, particularly at loop and both play critical roles in several diseases (such as cancer, regions.27 The crystal structure of TPST33 suggests that TPST is unlikely inflammation, and infectious diseases), but PTS is rarely studied. Thus, to catalyze sulfation at tyrosine residues buried within a protein or identifying the potential proteins that are subject to PTS is one of the localized at certain structures. This is because the active site of TPST is fundamental approaches that can facilitate related biomedical research. too narrow to accommodate a secondary structure containing an E, which Currently, we are using the E. coli proteome and TPST from Drosophila is more hydrophobic than D because E contains an extra carbon in its side as an example. By replacing the TPST enzyme, we can examine the chain, and this might serve as a crucial factor in TPST selection of its substrate specificities of various TPSTs. Similar procedures can be sulfation site based on the neighboring acidic amino acids. Tyrosine applied to identify sulfated proteins in the yeast and human proteomes. We ACS Paragon Environment residues present near sulfation sites were reported to possibly affect PTS Plusexpect such approaches to contribute considerably to our understanding of in proteins containing multiple potential tyrosine sulfation sites. 14,12,34 important human diseases related PTS.
Analytical Chemistry
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ACKNOWLEDGEMENTS Funding support was provided by the Ministry of Science and Technology (105-2311-B-009-001; 104-2627-M-009-002; 102-2311-B-009-004-MY3).
Chem. 1995, 270, 22677–22680. (35) C. Seibert, T. P. Sakmar, Biopolymers 2008, 90, 459–477. (36) G. Hortin, K. F. Fok, P. C. Toren, A. W. Strauss, J. Biol. Chem. 1987, 262, 3082–3085. (37) E. M. Danielsen, EMBO J. 1987, 6, 2891–2896
Supporting Information. Protein list and proteome chip images.
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Analytical Chemistry
For TOC only PAP
PAPS
TPST
TPST GST
Protein
Step1
GST
S
S
Protein
Step2
GST
Protein
Step3
GST
Protein
Step4
S GST
Protein
Step5
E. coli proteome microarrays (4256 E. coli K12 GST-fusion proteins are printed on a proteome microarray)
Complete
-TPST
ACS Paragon Plus Environment
