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
19
system (Lu et al., unpublished), the NusA-fused DmTPST was firstly obtained with maximal solubility and high purity (Table 1 and lane 2 in Fig. 3), and used for studying the enzymatic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
20
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.
21
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
22
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.
23
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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
31
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)
32
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 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and sorted shading by BOXSHADE server (http://www.ch.embnet.org/software/BOX_form.html). The black background indicated identity to each other and the gray one meant conserved substitutions. The residue colored in red is the predicted transmembrane domain calculated by PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/psiform.html) ranged from residue 6 to 28 both for human TPST1 and TPST2, and 12 to 28 for Drosophila melanogaster TPST, respectively. The pairwise sequence identity of these TPSTs for DmTPST/hTPST-1 (58%), DmTPST/hTPST-2 (56%), and hTPST-1/hTPST-2 (63%), respectively, was calculated.
34
Figure 3. Purification of homogenous recombinant DmTPST
The protein was expressed in BL21(DE3)pLysS cells and purified through His-Tag column. Lane 1 and lane 2 was crude extract and homogenous DmTPST, respecticely, and lane 3 was standard protein marker.
35
Figure 4. The protein of LC-MS-MS fingerprinting analysis was identified as DmTPST.
The excised spot from SDS-PAGE was identified as DmTPST by LC-MS-MS. The sequence (red) obtained from mass fingerprinting was mapped to the protein sequence with high confidence. The result was particularly described in Appendix 8 and Appendix 9.
36
Figure 5. Autoradiography of [35S]-labeled polyEAY catalyzed by DmTPST.
Lanes 1 to 3 were controlled reactions by the presence of DmTPST w/o PAPSS (lane 1), polyEAY (lane 2) and DmTPST (lane 3), respectively. Lane 4 using polyEAY as substrate proceeding sulfation reaction. The arrowheads indicated the [35S]sulfated polyEAY peptides and [35S]sulfate.
37
Figure 6. Effective range of DmTPST assay.
polyEAY sulfation catalyzed by the variable amount of recombinant DmTPST (from 0.5 to 20 g) was determined under the standard condition. Each point and bar represented the mean and SD, respectively, obtained from three experiments.
DmTPST amount (g)
38
Figure 7. Time course of the activity of recombinant DmTPST.
Time course effected on the activity of DmTPST. Activities of DmTPST were measured in different time (15, 30, 45, 60, 90, 120 min) under the standard condition.
Each point and bar represented of three experiments.
Time(min)
0 20 40 60 80 100 120 140
polyEAY sulfate(nmol/mg)
60 80 100 120 140 160 180 200 220
39
Figure 8. pH profile effected on the activity of recombinant DmTPST.
The pH profile of DmTPST activity. TPST activities were measured in 50 mM buffer at selected pHs (MES for pH 5.5, 6.0, 6.5, potassium phosphate for pH 6.5, 7.0, 7.5, and Tris for pH 7.5, 8.0, and 8.5) under the standard condition. Each point and bar represented the mean SD of three experiments.
pH
40
Figure 9. Kinetics of DmTPST using polyEAY as substrate.
The DmTPST kinetics activities were determined under the standard condition with various polyEAY concentration from 0.3125 to 160 M. Each point and bar represented the mean SD of three experiments. The data indicated the Km and Vmax was 11.5 ± 2.5 mM and 4.5 ± 0.2 nmole/min/mg, respectively.
Michaelis-Menten
[polyEAY] (M)
0 20 40 60 80 100 120 140 160 180
Specific Activity (nmol/min/mg)
0 1 2 3 4 5 6
41
Figure 10. Autoradiography of [35S]-labeled Drosulfakinin catalyzed by DmTPST.
Lanes 1 to 4 were controlled reactions in the absence of PAPSS (lane 1), substrate (lane 2), and DmTPST (lane 3), respectively. Lane 4 using polyEAY as substrate proceed sulfation reaction was positive control, and lane 5 was full reaction that using drosulfakinin as substrate. The arrowhead indicated the [35S]sulfated drosulfakinin peptides and [35S]sulfate.
42
Appendix
Appendix 1. Schematic representation of protein modifications related to the regulation of biological processes. (Adapted from Seo et al. 2004)
43
Appendix 2. Some common and important post-translation modifications (Mann