performed in triplicate.
Kinetic constants determinations of TPST—Measurements of kinetic constants were
performed by varying the polyEAY concentration while keeping the PAP at a fixed
and near saturating concentrations. The apparent Km and Vmax were determined using
nonlinear regression by SigmaPlot 2001. V7.0 and Enzyme module, V1.1.
2.4 Result
Expression of recombinant human TPST2 in prokaryote expression system.
The human TPST2 is localized in the membrane of Golgi apparatus network and the
transmembrane domain is shown as Fig 8. In our experiment, the catalytic domain
of TPST2 from residue 29 to 377 was incorporated into various expression vectors.
Various expression vectors incorporated human TPST2 cDNA in the open reading
frame were examined (data not shown). Most of the expression vector could not
prevent TPST2 from inclusion body. pET-43a (Appendix 2), a vector of expressing
proteins was purchased from Novagen, was competent to express recombinant
human TPST2 in E. coli with the reducing amount of inclusion body. The
prokaryotic expression of human TPST2 was optimized to reach the maximal
soluble amount and purified to nearly homogeneity (Fig. 9). A band on the
SDS-PAGE of 100 kDa protein composed of NUS-Tag fusion protein (60 kDa) and
TPST protein (40 kDa) upon treatment in coomassie blue R350. The spots excised
from SDS-PAGE were analyzed by LC-MS/MS. (Fig. 10) There were many
peptides (colored in red) come after trypsin digestion and they indicated the peptide
sequences of human TPST2. The alignment of these peptide sequences showing
homology to human TPST2 with high scores of confidences.
Time-dependence of human TPST2-catalyzed tyrosine O-sulfation
polyEAY is a substrate of TPST2 with higher specificity activity than other
endogenous substrates as described in the previous reports. Firstly the time
dependence of the activity of the human TPST2 with polyEAY as substrate was
examined. The concentrations of PAPS and polyEAY, were both saturated in the
reactions. The tyrosine O-sulfation of polyEAY increased linearly with the incubation
time as shown in the Fig. 11. The slope in this reaction was not appreciably reduced
up to 120 mins.
Temperature effect on TPST activity
So far the catalytic activity of human TPST2 in different temperatures was not
studied clear in the past. The experiment demonstrated that human TPST2 exhibited
the activity at 25 °C was three times than that at 40 °C (Fig. 12). This catalytic reation
was almost quenched under the treatment of 45 °C for 30 mins. The human TPST2
might denature and thus lost the fuction of tyrosine O-sulfation catalysis. The activity
at 37 °C (body temperature) was also investigated and it was apparently lower than
the activity at 25 °C. It might be attributed to the reason as decribed above.
pH profile of TPST
pH affects the electricity of amino acid and further contributes to the substrate
binding affinity, enzymatic catalysis, and protein conformational structure. The pH
profiles of the recombinant human TPST2 were determined by measuring the activity
at various pH values. The pH optimum was ranged from 5.5 to 6.0 within the error
tolerance as showed in Fig. 13. The catalytic activity of human TPST2 appreciably
decreased from pH 6.0 to 6.5 and was nearly nondetectable at pH 7. pH values higher
than 6.0 might result in the instability of tyrosine O-sulfated peptide, or the
dysfunction of human TPST2 in catalysis.
Metal ion effect on TPST2
It is known to require exogenous metal ions for activity with Mn2+ and Mg2+ to
activate the highest activity of TPSTs (Mishiro et al. 2006). The data indicated that
whether there is metal ion in enzyme catalysis or not, it is not necessary for human TPST2 (Fig. 14). The concentration of Mn2+ at 25 mM performed the maximal activity and 2.5 folds higher than the absence of Mn2+.
Kinetics of TPST2 utilized polyEAY as substrate
Previous studies indicated that tyrosine sulfation was studied in the subcellular
fractions containing the enzyme activity.(William et al. 1997; Sane et al. 1993; Lin
et al. 1990) The most widely used sulfonate acceptor was EAY as a positive
control. The kinetic constants toward polyEAY, the synthetic polypeptides0
composed of Glu, Ala, and Tyr in the ratio 6:3:1, demonstrated that Km was 10.6 μM
and Vmax was 4.8 pmole/min/mg (Fig. 15). It revealed that the heterologous
expression of human TPST2 was active in the catalysis and performed the similar
kinetic constants compared to the previous studies. (Sane et al. 1993)
2.5 Discussion
Tyrosine O-sulfation is firstly discovered in 1954. (Bettelheim, F.R.1954)
Tyrosylprotein sulfotransferase (TPST) is demonstrated to catalyze tyrosine
O-sulfation by Lee and Huttner in 1983. In opposition to the researches of kinases, the
ones of TPST are extremely few. It may be attributed to some characteristics of TPST,
such as the difficulty to purify the homogenous and ample amount of TPST. TPST is
reported to be labile and is hard to purify during the process of purification (Ouyang
et al. 1998). In the previous studies, the enzyme source came from the nature
materials or mammalian cell lines, and further purified through affinity column whose
beads conjugated with its substrate or antibody.(Ramaprasad et al. 1998; Kasinathan
et al. 2005) In this research, the heterologous expression system utilizing E. coli as
host to purify the human TPST2 was optimized with high recovery. In the process of
the purifications, the inclusion body and contamination of chaperonin 60 (GroEL)
resulted in the difficulties to overcome.
The formation of inclusion body includes solubility limitation, protein size,
type of promoter, and improper disulfide formation. (Hartley et al. 1988; Marston et
al. 1986) The choice of vector and expression host can significantly increase the activity and amount of target protein present in the soluble fraction. In the previous
study, the truncated form of human TPST2 comprising the catalytic domain was
secreted from stably transfected Chinese hamster ovary (CHO) cells (Ouyang et
al.1998). According to the topological analysis of primary sequence of TPST2, the
N-terminal transmembrane domain in TPST2 was truncated to prevent hydrophobic
domain from interfering in this study. Moreover, the fusion protein, Nus•Tag, on the
expression vector was utilized to enhance the solubility of target proteins (Davis et
al. 1999; Harrison et al. 2000). Furthermore, the contamination of chaperonin 60
(GroEL) was found to co-elute with TPST2 in this study. Common features of
chaperone action are transient interaction with non-native species in the prevention
of aggregation and promotion of correct folding and assembly (Young et al. 2004;
Bukau et al. 2006; Anken et al. 2005). The existence of the GroEL represented that
TPST was not easy to fold or not fold into the correct stage. This interaction
between TPST2 and GroEL was interfered through Triton X-100 to be the
competence of the hydrophobic force and separate each other. This purification
procedure in the study is simple, straightforward, and can produce great quantities
and homogeneous sources of TPST2 (Fig. 9).
The homogeneous human TPST2 was measured further to understand the
characterizations and mechanism of action. The catalytic activity of zebrafish TPST
with N-terminal PSGL-1 peptide as substrate indicated the optimal activity ranged
from 28 to 37 °C (Mishiro et al. 2004). The temperature profile of human TPST2 with
polyEAY as substrate in this study was different from the catalytic activity of
zebrafish TPST (Fig. 12). The activity of TPST2 decreased with the increasing
temperature and approached to inactive while the temperature was higher than 45 °C.
Furthermore, previous research revealed that the TPST under the treatment of
detergent possessed the half life of 48 hours at 4 °C (Niehrs et al. 1990). The catalytic
specificity of human TPST2 is unknow in this studies.
Previous studies had revealed that TPST1 and TPST2 are localized in the Golgi
apparatus and the catalytic domain is situated in the lumen, which is an acidic
environment (Baeuerle et al 1987; Lee et al.1985). The recombinant human TPST2
indicated that the activity is adaptable under acidic environment and become labile
while the pH value was higher than 6.0 (Fig. 13). It might be resulted from the
influence of the affinity of PAPS or substrate binding sites. The information from
previous studies also indicated that the optimal pH of human TPST2 expressed from E.
coli was the same as that from 293T cells, but was different from human
saliva.(Mishiro et al. 2006; Kasinathan et al. 2005) It might infer that human TPST2
possessed the isoforms so that resulting in the different optimal pH towards the
catalytic activity.
Membrane lysates of Golgi apparatus have revealed the stimulatory effects of
Mn2+ on the activity of TPST.(Mishiro et al. 2006; Kasinathan et al. 2005) The
catalytic activity of human TPST from salivary and PC12 cells is stimulated by the
divalent cations, such as Mn2+, Ca2+ and Mg2+, and is inhibited by EDTA.(Kasinathan
et al. 2005) On the contrary, tyrosine O-sulfation of the endogenous membrane proteins in A431 cell is not inhibited by EDTA.(Liu et al. 1986) The stimulatory
effect and mechanism of metal inons, however, was still not clear so far. When MnCl2
was up to 40 mM in the reaction mixture, the enzyme activity was apparently
decreased (Fig. 14). High concentration of Mn2+ might affect the structure and render
the protein denaturation. The appropriate concentration of Mn2+ might also stabilize
the sulfonate groups while catalysis and further to reduce the activation energy. It
could be comparable to the role of Mg2+ in the kinase catalysis.
According to the previous studies, the Km of platelet TPST for polyEAY as
substrate was 3.7 μM and the Vmax was 0.09 pmol/min (Sane et al. 1993). In our study,
the kinetic constants indicated that the Km and Vmax were 10.5 μM and 4.8
pmole/min/mg, respectively (Fig. 15). This difference might result from that the
polyEAY is synthetized with distinct ratio of components (Glu, Ala, and Tyr), and the
various composition of the sequences also led to the different catalytic efficiency.
According to these characterizations of recombinant human TPST2, the NusA
protein fused TPST2 expressed from E. coli was similar to that either from natual
materials or eukaryotic expression. The NusA protein obviously did not affect the
catalytic activity of human TPST2 and render the high solubility to facilitate TPST
folding.
In summary, we first purified TPST from prokaryote systems (E. coli) with
catalytic activity. By means of this purification procedure, the time-and-effort-saving,
inexpensive, high quality and quantity platform was established to express and purify
homogenous human TPST2 with only one chromatography step for further
biochemical characterization. The catalytic mechanism of substrate specificity, for
example PSGL-1(Fig. 16), metal ion effect, and the regulatory residues will be
examined. Furthermore, the crystal structure and antibody will be pursued to study in
advanced for either the physiological or pathological functions and regulations.
Table 1a. Some common and important post-translational modifications.
a Adapted from Mann et al. 2003.
Table 2. Specific effects of tyrosine O-sulfationa
a Adapted from Liu et al. 2008
Table 3a. Conservation of tyrosine sulfation sites in human chemokine receptors.
Sulfation sites with scores of 2.5 or higher are in black. Sites with intermediate scores between 1.5 and 2.5 are in gray.
a Adapted from Liu ,et al. 2008
Table 4. TPSTs purified from many source.
Kasinathan C.,et al. Int. J. Biol. Sci. 2007 Statherin; EAY
Human Saliva Human Saliva (TPST)
Kasinathan C.,et al. Alcohol. 1998 EAY
rat liver rat TPST
Ramaprasad P.,et al. Gen Pharmacol. 1998 EAY
Rat Liver Rat Liver (TPST)
Ouyang Y. et al.Proc Natl Acad Sci. 1998 PSGL-1
rat liver rat liver
Salivary Glands
William S.,et al. Arch. Biochem. Biophys.
EAY 1997 Rat Submandibular
rat Salivary Glands (TPST)
Kasinathan C,et al. Gen. Pharmacol. 1995 EAY
Rat Salivary Glands rat Salivary Glands (TPST)
Niehrs C,et al. EMBO J. 1990 (PKG or others)
bovine adrenal medulla bovine (TPST)
Lin W.H.,et al. Biochem. Pharmacol. 1990 EAY
human liver Human (TPST)
Niehrs C,et al. J Biol. Chem. 1990 CCK; Sgi
bovine adrenal medulla bovine (TPST)
Rens-Domiano S.,et al. J Biol. Chem. 1989 EAY
Mishiro E.,et al. J. Biochem.2006 PSGL-1
293T cell human TPST-1 & 2
Seibert C.,et al. Proc Natl Acad Sci. 2002 PSGL-1;CCR5
HEK293T cell human TPST-1 & 2
293T cell mouse TPST
Ouyang Y., et al. Proc Natl Acad Sci. 1998 293T cell
human TPST-HPC4 PSGL-1
CHO-K1 cell human TPST-HPC4
Liu M.C., et al. J. Biochem. 1987 3Y1
Kasinathan C.,et al. Int. J. Biol. Sci. 2007 Statherin; EAY
Human Saliva Human Saliva (TPST)
Kasinathan C.,et al. Alcohol. 1998 EAY
rat liver rat TPST
Ramaprasad P.,et al. Gen Pharmacol. 1998 EAY
Rat Liver Rat Liver (TPST)
Ouyang Y. et al.Proc Natl Acad Sci. 1998 PSGL-1
rat liver rat liver
Salivary Glands
William S.,et al. Arch. Biochem. Biophys.
EAY 1997 Rat Submandibular
rat Salivary Glands (TPST)
Kasinathan C,et al. Gen. Pharmacol. 1995 EAY
Rat Salivary Glands rat Salivary Glands (TPST)
Niehrs C,et al. EMBO J. 1990 (PKG or others)
bovine adrenal medulla bovine (TPST)
Lin W.H.,et al. Biochem. Pharmacol. 1990 EAY
human liver Human (TPST)
Niehrs C,et al. J Biol. Chem. 1990 CCK; Sgi
bovine adrenal medulla bovine (TPST)
Rens-Domiano S.,et al. J Biol. Chem. 1989 EAY
Mishiro E.,et al. J. Biochem.2006 PSGL-1
293T cell human TPST-1 & 2
Seibert C.,et al. Proc Natl Acad Sci. 2002 PSGL-1;CCR5
HEK293T cell human TPST-1 & 2
293T cell mouse TPST
Ouyang Y., et al. Proc Natl Acad Sci. 1998 293T cell
human TPST-HPC4 PSGL-1
CHO-K1 cell human TPST-HPC4
Liu M.C., et al. J. Biochem. 1987 3Y1
Figure 1a Schematic representation of protein modifications related to the regulation of biological processes.
a Adapted from Seo et al. 2004
Figure 2 a. Sulfate activation and tyrosine O-sulfation.
a Adapted from Moore et al. 2003
Figure 3a. Schematic representation of cell entry by HIV-1 following sulfation of CCR5 by a tyrosylprotein sulfotransferase.
a Adapted from Chapman et al. 2004.
Y276, Y278, Y279 Y276, Y278, Y279
Figure 4a GPIbα from amino acid 200-294
a Adapted from Murata et al. 1991
Figure 5 a. Tyrosine sulfation plays an important role in the immune response.
a Adapted from Kehoe et al. 2000
Figure 6. Graphical presentation of the far-Western immunoblot technique.
Figure 7. The catalyzed reaction of tyrosylprotein sulfotransferase.
Figure 8. Sequence alignment and transmembrane domain analysis of tyrosyl protein sulfotransferase. The sequence pairwise alignment is 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.
Figure 9. Prokaryotic expression and purification of human hTPST2. The prokaryotic expression of human TPST2 was optimized to reach the maximal soluble amount and purified to near homogeneity. Lane 1 was purified from HisTrap chromatography followed the procedure as described in the “Experimental procedures.” Lane M was protein marker consisted of β-galactossidase (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (44 kDa), lactate dehydrogenase (35 kDa), and restriction endonuclease Bsp98I (25 kDa).
116 66 45 35 25
kD M 1
hTPST2
Figure 10. The protein of LC-MS-MS fingerprinting analysis was identified for human TPST2. The excised spot from SDS-PAGE was identified for human TPST2 by LC-MS-MS. The sequence (red) by mass fingerprinting was mapped to the protein sequence with high confidence. (The result was particularly descrided in appendix)
Time (mins)
0 20 40 60 80 100 120 140
Sulfation number (pmole)
0 1 2 3 4 5 6 7
Figure 11. Reaction of polyEAY sulfation as a function of time. This experiment was performed as described under the “Experimental procedures.” Each assay
consisted of 50 mM MES at pH 6.0, 25 mM MnCl2 , 50 mM NaF, 0.5 μM [35S]PAPS (15Ci/mmole), 40 μM TSH-R signaling
polyEAY, 0.5 % Triton-X100 and 4 μg human TPST2 in a final volume of 10 μl.
Data shown here is the result of a typical experiment performed in triplicate.
Figure 12. Effect of Temperature on hTPST2 activity. This experiment was performed as described under the “Experimental procedures.” Using different temperature in the same mixture resulted in changes of the catalytic activity of human TPST2. Data shown here is the result of a typical experiment performed in triplicate.
Specific activity (pmole/min/mg)
Temperature (oC)
20 25 30 35 40 45 50 55
0 1 2 3 4 5 6
Figure 13. Different pH affects the catalytic activity of human TPST2. The experiment was performed as described under the “Experimental procedures.” The result indicated pH affects of tyrosine O-sulfation in human TPST2 enzyme assay.
The data was performed in triplicate.
5.5 6.0 pH 6.5 7.0
Sepcific activity (pmole/min/mg)
-1 0 1 2 3 4 5 6
Figure 14. Effect of MnCl2 on hTPST2 activity. This experiment was performed as described under the “Experimental procedures.”.Data shown here was the result of a typical experiment performed in triplicate. This experiment was repeated three times with similar results. Point A indicated the total reaction without polyEAY and MnCl2, and point B meant the total reaction without human TPST2 and MnC2.
MnCl2concentration
A B 0 5 10 15 20 25 30 35 40
Specific activity (pmole/min/mg)
0 1 2 3 4 5 6 7
EAY (microM)
0 10 20 30 40 50
Specific Activity (pmol/min/mg)
0 1 2 3 4 5
Figure 15. Kinetics of human TPST2 for polyEAY as substrate. Each assay consisted of 50 mM MES at pH 6.0, 25 mM MnCl2 , 50 mM NaF, 0.5 μM [35S]PAPS (15Ci/mmole), various concentrations of polyEAY, and 4 μg human TPST2 in a final volume of 10 μl. Data shown here was the result of a typical experiment performed in triplicate. The data indicated the Km was 10.5 ± 2.1 μM and Vmax was 4.8 ± 0.3 pmole/min/mg. This experiment was repeated three times with similar results.
M 1 2
PSGL-1 6
kDa
PSGL-1
M 1 2
PSGL-1 6
kDa
PSGL-1
Figure 16. The SDS-PAGE of PSGL-1 peptide purification. Lane M was protein marker consisted of β-galactossidase (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (44 kDa), lactate dehydrogenase (35 kDa), and restriction endonuclease Bsp98I (25 kDa). Lane 1 was the elution of GST-fused PSGL-1. Lane 2 was PSGL-1 peptide purified from GSTTrap chromatography after the thrombin digestion.
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