1. Introduction
1.6 Tyrosylprotein sulfotransferase in Drosophila melanogaster
Most vertebrates (such as rat, cow, chicken, zebrafish, African clawed frog) and invertebrates (such as Anopheles gambiae (mosquito), and Caenorhabditis elegans) have two TPSTs. It is interesting to note that Drosophila melanogaster is so far the only species that was discovered to contain a single TPST gene (Moore, 2003).
Therefore, D. melanogaster is a good model to study TPST, which a complete elimination of protein sulfation modification can be reached by a simple knockout or knockdown of a single gene. About 75% of known human disease genes have a recognizable match in the genetic code of fruit flies (Reiter et al., 2001), and 50% of fly protein sequences have mammalian analogues. Many advantages of using D.
melanogaster as a study model include the short generation time and easy growth.
Therefore, D. melanogaster is a suitable model to study protein tyrosine sulfation by using complete genetic tools to understand physiological and pathological mechanisms. The completion of genomic database is helpful for protein identification and its function. According to speculated that there are approximately up to 1%
tyrosines of total proteins in an organism that are sulfated, but D. melanogaster
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published less substrate, such as Drosulfakinin, Vitellogenin, and Glutactin (Nichols R. et al. 1988; Baeuerle P.A. et al. 1985; Olson P.F. et al. 1990).
1.7 Contribution from this study
This was the first research focused on the identification, cloning, expression and characterization of DmTPST at protein level. Following the expression of DmTPST in a prokaryotic system, the desired tyrosine sulfated proteins were further
produced in vitro in a PAPS generating system. The homogeneous DmTPST was characterized through the PAPS generating system with polyEAY as a substrate.
Large quantity of homogeneous DmTPST was obtained that facilitated further studies and applications in protein tyrosine sulfation. Optimal reaction conditions for DmTPST catalysis and pH profile were determined. Finally, an endogenous
compound of D. melanogaster, drosulfakinin, was demonstrated to serve as substrate of recombinant DmTPST. The results indicated that recombinant DmTPST can further decipher the ignorant mechanisms and functions of protein tyrosine sulfation in Drosophila melanogaster.
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2. Materials
Adenosine 5’-triphosphate (ATP), tris[hydroxymethyl]aminomethane (Tris),
2-[N-morpholino]ethanesulfonic acid (MES), poly-(Glu6, Ala3, Tyr1) (EAY: Mr 33KDa), inorganic pyrophosphatase, and imidazole were purchased from Sigma (St.
Louis, MO, USA). Potassium phosphate (dibasic), glycine, and sodium dodecyl sulfate (SDS) were obtained from J. T. Baker (Phillipsburg, NJ, USA). Sodium [35S]sulfate (1050-1600 Ci/mmol) of 99.0% radiochemical purity was purchased from PerkinElmer (Boston, MA, USA). Taq polymerase, T4 DNA ligase, and reagents for PCR were obtained from New England Biolabs (Beverly, MA, USA). Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA, USA). Expression vector and BL21(DE3) pLysS competent cells were purchased from Novagen (Madison, WI, USA). HisTrap sepharose was obtained from GE Healthcare (Uppsala, Sweden).
Cellulose thin-layer chromatography (TLC) plates were products of Merck (Whitehouse Station, NJ, USA). All other chemicals were of the highest purity commercially available.
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3. Experimental procedures
3.1 Prediction of transmembrane domain of DmTPST
The transmembrane region and orientation of DmTPST were predicted on the website—PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/psiform.html) (Jones, 2007).
Only scores of hydrophobicity above 0 were considered significantly to be the potential transmembrane region.
3.2 Cloning of DmTPST
The Drosophila melanogaster TPST cDNA was subcloned into pET-43a
vector. The potential catalytic domain of TPSTs predicted above was amplified by PCR with specific primers designed to contain BamHI in the forward direction (5’-TGAAGAATTCGACGCCCCCAACGAGCTCTCCTC-3’) and EcoR1 restriction sites in the reverse one contain XhoI restriction (5’-TGCCCTCGAGCTCTCCCACAGCATTCGATTGGC-3’). cDNA fragments were inserted into the EcoRI/XhoI double-restriction sites and then confirmed using an ABI Prism, 346 DNA sequencer (Applied Biosystems, Foster City, CA) following the standard protocol.
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3.3 Protein expression and purification of DmTPST
A single colony of BL21(DE3)pLysS consisted of DmTPST plasmid was used to inoculate in the LB broth with ampicillin as the antibiotic at 37°C. Growth was continued to an ODA600 of 0.4–0.6 and then induced with 1 mM isopropyl-thio-β -D-galactoside (IPTG) for 24-hr incubation at 20°C. The cultures were centrifuged at 14000g for 20 minutes, and the pellet was sonicated in IMAC5 buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole, and 10% glycerol) for DmTPST.
Further the HisTrap sepharose charged with NiSO4 was utilized to the DmTPST purification. The homogeneous proteins were determined by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970).
3.4 Mass analysis
The in-gel digestion of interested proteins was performed by the conventional protocol. MALDI-TOF was carried out to study the identification of excised proteins.
The PMF data was analyzed by MASCOT based on the NCBInr database.
3.5 A assay of DmTPST enzymatic activity
For the determination of DmTPST activity from bacterial expression, we detected radiation of 35S using [35S]PAPS as donor and transferred sulfate group to
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substrate, such as polyEAY. The couple-enzyme (human PAPS synthetase 1 and DmTPST) radioactive assay was newly established for the measurement of DmTPST
activity. The complete assay mixture contained the following components: sulfate acceptor DmTPST substrate (polyEAY), 4 mM inorganic Na2[35S]SO4, 5 mM 2-mercaptoethanol, 1 mM MgCl2, 50mM MES (pH6.5), 5 g recombinant human
PAPS synthetase 1 (hPAPSS1), DmTPST, and 0.5 U pyrophosphatease in a final volume of 20 l. Assays were initiated by the addition of the hPAPSS1 and incubated
for 15 min at 37°C followed by the addition of DmTPST incubation for 45 min at 37°C. The reactions were terminated by heating at 95°C for 2 min. The supernatant was collected and analyzed by spotting 1 l aliquot of the reaction mixture on a cellulose thin-layer chromatography (TLC) plate and developed with n-butanol/pyridine/formic acid/water (5:4:1:3; by volume) as the solvent system. The dried plate was exposed with Kodak BioMax MR film which provide the optimal resolution for 35S autoradiography.
3.6 Using enzymatic activity assay characterize DmTPST
According to TPST enzymatic activity assay control DmTPST amounts (from s to 120 mins). Finally, calculated the kinetics when polyEAY as substrate. The
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DmTPST enzymatic activity of different substrates was changed from polyEAY to
drosulfakinin proceeding TPST enzymatic activity assay.
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4. Results
4.1 Expression of recombinant D. melanogaster TPST in prokaryote expression
system.
The expression vector, pET-43a, harboring DmTPST cDNA was competent to express recombinant TPST in E. coli. The prokaryotic expression of TPST was optimized to reach the maximal soluble amount and purified to nearly homogeneity (Fig. 3). A 96-kDa protein showed on the SDS-PAGE was composed of NusA-tag fusion protein (60 kDa) and DmTPST (36 kDa) upon treatment in coomassie blue R250. The spot excised from SDS-PAGE was analyzed by LC-MS/MS (Fig. 4). Two peptides (colored in red) come after trypsin digestion the alignment of these peptide sequences showed homology to DmTPST with high scores of confidences. The purification table revealed the purification-related information of DmTPST (Table.
1).
4.2 Sulfation of polyEAY using a PAPSS and TPST coupled system
Autoradiography on the cellulose TLC plate demonstrated that the DmTPST activity could be determined under the enzymatic activity assay condition (Fig. 5).
[35S]-labeled EAY produced only in the presence of DmTPST and complete PAPS regenerating system (lane 4). The [35S]-labeled polyEAY could not be produced in the
16
absence of PAPSS (lane 1), polyEAY (lane 2) or DmTPST (lane 3), respectively.
These results indicated that PAPS produced through PAPSS catalytic reactions could be used for the sulfation of polyEAY catalyzed by DmTPST.
4.3 Determination of the effective range and time course of DmTPST enzyme
catalysis
The linearly effective range of DmTPST amount in the standard assay ranged within 5 g as shown in Fig. 6. Accordingly, 4 g DmTPST was used in further experiments as standard assay. The time dependence of DmTPST activity with polyEAY as substrate was examined. The concentrations of sulfate and polyEAY, were both saturated in the reactions. The tyrosine O-sulfation of polyEAY increased linearly with the incubation time from 15 to 120 minutes as shown in Fig. 7.
4.4 pH profile of DmTPST
pH affects the electricity of amino acid and further contributes to the substrate binding affinity, enzymatic catalysis, and protein conformational structure. The pH profile of the recombinant DmTPST was determined by measuring the activity at various pH values. The pH values from 5.5 to 8.5 were shown in Fig. 8. The optimal pH was 6.5 that showed the highest catalytic activity of DmTPST.
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4.5 Kinetics of DmTPST with polyEAY as substrate
The kinetic constants toward polyEAY, which is synthetic polypeptides composed of Glu, Ala, and Tyr in the ratio 6:3:1, demonstrated that Km and Vmax was 11.5 M and 4.5 nmole/min/mg, respectively (Fig. 9). It revealed that the expression of DmTPST was active in the catalysis and performed the similar kinetic constants compared to the previous studies. (Table 2.)
4.6 Sulfation of endogenous substrate, drosulfakinin, by DmTPST
Drosulfakinin, composed of 14 amino acid residues, is a known endogenous substrate in Drosophila melanogaster of DmTPST. The result from Fig. 10 revealed that the DmTPST was competent to catalyze not only the synthetic peptide polyEAY (lane 4),
but the endogenous substrate drosulfokinin. (lane 5).
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5. Discussion
Tyrosine sulfation was discovered in 1950s in bovine fibrinogen (Bettelheim, 1954), and afterwards, the tyrosylprotein sulfotransferase (TPST) was identified to be responsible for this post-translational modification in 1982 (Huttner, 1982). Since the discovery of the tyrosine O-sulfation, little about the the enzyme mechanisms have been elucidated. This may be attributed to the lack of TPST related information, such as the difficulty of sourcing the homogeneous enzyme and ample amount of TPST, limited information of enzyme characteristics (kinetics), unstable sulfate groups on the substrate, and lack of sensitive detecting methods for the sulfated tyrosine.
Drosophila melanogaster was chosen as the source of animal study due to easy
growth, short generation span, solved genomic database, well-established transgenic tools, and more importantly, D. melanogaster only has a single TPST gene (Moore, 2003). The amino acid sequence of TPST in D.melanogaster shares 58% and 56%
with human TPST1, and TPST2, respectively (Fig. 2). Approximately 75% of known human disease genes have a recognizable match in the genetic code of D.
melanogaster, and 50% of D. melanogaster protein sequences have mammalian
analogues (Reiter et al., 2001), which makes D. melanogaster an appropriate animal model for pathological studies on TPST.
According to the successful development of TPST expression in prokaryotic
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system (Lu et al., unpublished), the NusA-fused DmTPST was firstly obtained with maximal solubility and high purity (Table 1 and lane 2 in Fig. 3), and used for studying the enzymatic characterization. The purification yield of DmTPST showed higher than that of hTPST2 and although the protein sequence of DmTPST and hTPST2 has a similarity approximately 60%. The distinct characteristics between
human and D. melanogaster TPST need to study further. The NusA-DmTPST possessed high homogeneity in our study, however, the ratio of DmTPST in this fusion protein was merely 35% and the total molecular weight was close to 100 kDa.
The NusA protein obviously performed no interference with the enzymatic activity of DmTPST and rendered high solubility to facilitate DmTPST folding. Overall, this
purification procedure of DmTPST was simple with stable material source, great quantity, and homogeneous DmTPST in this study.
By the facilitation of coupled enzyme reaction (hPAPSS1 and DmTPST), the productive rate of tyrosine sulfation was faster than that of the conventional reaction which utilized PAPS directly as sulfate donor as shown in Fig. 1 (Liu et al., unpublished). The approach avoided the contamination of PAPS from PAP (Rens-Domiano and Roth, 1989; Miller and Waechter, 1979). PAPS is extremely costly and it tends to hydrolyze easily to form PAP, a known competitive inhibitor of sulfotransferases (Lin and Yang, 1998). In this experimental design, hPAPSS1
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generated saturated PAPS from inorganic sulfate, and this scheme could obviously prevent the background from the hydrolysis of PAPS. Moreover, the production of protein tyrosine sulfation by this method was extremely efficient than previous studies and it might potentially apply to spectrometric analysis in additional to radioactive assay (Liu et al., unpublished; Mishiro et al., 2006).
In this study, DmTPST properties including the DmTPST amount (Fig. 6), time dependence of the activities of DmTPST (Fig. 7), pH profile (Fig. 8), and kinetic
parameters of DmTPST (Fig. 9), were examined. The optimal DmTPST dosage and reaction time was 5 g and 2 hours, respectively, which located in the linear range. In
the pH-dependent experiment, DmTPST displayed an optimal activity at pH 6.5 (Fig.
8), which was similar to that of TPST in human liver and rat submandibular salivary
glands (Lin and Roth, 1990; William et al., 1997). The result of Fig. 8 also indicated that potassium phosphate was inhibitory to the DmTPST catalyzed sulfation of polyEAY.
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substrate polyEAY, the Km and Vmax was individually 3.4 mM and 176 pmol/min/mg;
the kcat was thus 7.0 × 10-3.(Liu et al., unpublished)(Table. 2). In this study, the Km, Vmax, and kcat of DmTPST towards polyEAy as substrates was 11.5 mM, 4.5 nmol/min/mg, and 1.6 × 10-1, respectively (Fig. 9). Obviously, the Km values were similar regardless of the TPST acquired from diverse sources, species and assayed by different methods. However, the distinct Vmax measured from coupled-enzyme reaction was higher than that of previous method for approximately 10 folds, because the method made some modifications. Consequently the detection of polyEAY sulfate reached to nanomolarr range (weird) and contributed to the discovery of sulfated peptides in the future.
In the study, commercial polyEAY consisted of Glu, Ala, Tyr with random synthesis followed the ratio of 6:3:1. Consequently, standard substrate was urgent to be utilized in the assay. The D.melanogaster endogenous substrate, Drosulfokinin, was selected to analyze in the DmTPST activity assay (Fig. 10). The data demonstrated that recombinant DmTPST could not only catalyze synthetic peptide (polyEAY) but endogenous substrate (drosulfokinin). The recombinant DmTPST will be used further in the aspects of substrate examination, substrate screening, and proteomic application.
In conclusion, we first purified DmTPST from prokaryotic system and showed
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the various purification characteristics as compared to human TPST. Furthermore, the combination of PAPS generating system facilitated to increase the catalytic rate of DmTPST, and define DmTPST optimal condition and kinetic parameters. This will be
beneficial to not only the aspects of fundamental researches but apply to Drosophila protein sulfation in biological study.
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Tables
Table 1. Purification of NusA-DmTPST from E. coli.
Step
Total Activity Total Protein Specific Activity Yield Purification
(pmole/min) (mg) (pmole/min/mg) (%) fold
Crude extract 37325 1420 26 . 3 100 1
Ni-NTA column 9720 12 . 4 783 . 8 26 29 . 8
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Table 2. Comparison of coupled enzyme assay-obtained kinetic characterization of DmTPST with previous radiometric assay
a polyEAY was synthesized followed the ratio of Glu : Ala : Tyr = 6 : 3 : 1.
b PSGL-1 was P-selectin glycoprotein ligand-1 N-terminal peptide (ATEYEYLDYDFL).
Enzyme assay Enzyme Source substrate
Kinetics
References
Vmax Km kcat
(pmol.min-1.mg-1) (µM) (min-1)
Coupled-enzyme TPST assay DmTPST E. coli polyEAYa 4459 ± 214 12 ± 2.5 1.6 × 10-1 The present study hTPST2 E. coli polyEAYa 176 ± 15 3.4 ± 1.2 7.0 × 10-3 Liu et al., unpublished hTPST2 E. coli PSGL-1b 3200 ± 170 24 ± 3.5 1.1 × 10-1 Lu et al., unpublished Traditional PAP35S assay hTPST2 E. coli polyEAYa 4.8 ± 0.5 11 ± 3.0 4.8 × 10-4 Liu et al., unpublished
hTPST1 293T cell PSGL-1b 3.95 9.67 1.7 × 10-4 Mishiro et al. (2006) hTPST2 293T cell PSGL-1b 71.43 26.89 3.0 × 10-3 Mishiro et al. (2006)
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Figures
Figure 1. Scheme for the determination of TPST activity. Isotope-based analysis (35S) was used for the DmTPST assay using PAPS as the sulfuryl group donor.
Biosynthesis of PAPS was catalyzed by PAPSS from ATP and SO4
as shown in Step A. Step B showed the reaction catalyzed by TPST using tyrosylprotein as the sulfuryl group acceptor.
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Figure 2. Bioinformatic analysis of protein sequence identity and transmembrane domain for human and Drosophila melanogaster.
The sequence pairwise alignment was performed by ClustalW
The sequence pairwise alignment was performed by ClustalW