(Received for publication, October 10, 1995; and in revised form, November 13, 1995)
From the
Photoaffinity labeling with 2-azidoadenosine
3`,5`-[5`-P]bisphosphate was used to identify
and characterize adenosine 3`,5`-bisphosphate-binding proteins in human
liver cytosol and recombinant sulfotransferase proteins. The
sulfotransferases investigated in these studies were the human phenol
sulfotransferases, HAST1, -3, and -4, dehydroepiandrosterone
sulfotransferase, and estrogen sulfotransferase. The cDNAs for these
enzymes have been previously cloned and expressed in COS-7 cells or Escherichia coli. Photoaffinity labeling of all proteins was
highly dependent on UV irradiation, was protected by co-incubation with
unlabeled adenosine 3`,5`-bisphosphate and phosphoadenosine
phosphosulfate, and reached saturation at concentrations above 10
µM. To verify that the 31-35-kDa photolabeled
proteins were indeed sulfotransferases, specific antibodies known to
recognize human sulfotransferases were used for Western blot analyses
of photolabeled proteins. It was shown unequivocally that the proteins
in the 31-35-kDa region recognized by the antibodies also
photoincorporated 2-azidoadenosine
3`,5`-[5`-
P]bisphosphate. This is the first
application of photoaffinity labeling with 2-azidoadenosine
3`,5`-[5`-
P]bisphosphate for the
characterization of recombinant human sulfotransferases. Photoaffinity
labeling will be also useful in the purification and functional
identification of other adenosine 3`,5`-bisphosphate-binding proteins
and to determine amino acid sequences at or near their active sites.
Sulfation is an important pathway in the biotransformation of
many drugs, xenobiotics, neurotransmitters, bile acids, and hormones.
The sulfate donor for these reactions is 3`-phosphoadenosine
5`-phosphosulfate (PAPS). ()Adenosine 3`,5`-bisphosphate
(PAP) is a product of the reaction catalyzed by all sulfotransferases
(STs) and competitively inhibits PAPS binding(1, 2) .
Direct photoaffinity labeling with radioactively labeled PAPS has
been used to identify PAPS-binding proteins. Lee et al.(3) used direct labeling with 3`-phosphoadenosine
5`[P]phosphosulfate to identify an M
= 34,000 protein involved in PAPS
translocation in Golgi membrane preparations of bovine adrenal medulla (3) . Otterness et al. (4) utilized a similar
approach, using [
S]PAPS for direct photoaffinity
labeling of human liver STs. It was shown that UV irradiation of
[
S]PAPS in the presence of partially purified
human liver thermostable phenol sulfotransferase (PST) resulted in the
labeling of 35-kDa proteins with properties identical to those of
thermostable PST. These results indicated that
[
P]PAPS and [
S]PAPS can
be used as direct photoaffinity ligands for the study of ST and other
PAPS-dependent proteins; however, the reaction with the enzyme depends
on high energy UV light activation of pyrimidine residues, and it is
not always possible to distinguish covalent binding from nonspecific
enzyme inactivation.
Another strategy for affinity labeling and
probing nucleotide binding sites in protein molecules is to use
photoreactive substrate analogs or inhibitors containing an azido
group, such as 2-azidoadenosine and
8-azidoadenosine(5, 6) . A new photoreactive
pyrimidine analog, 2-azidoadenosine
3`,5`-[5`P]bisphosphate
(2-azido-[
P]PAP), was synthesized by Sylvers et al. (7) and applied to the photoaffinity labeling
of Escherichia coli ribosomes.
In this paper, we report
photoaffinity labeling studies of human and rat liver cytosolic ST and
several recombinant human STs with
2-azido-[P]PAP synthesized and characterized
according to Sylvers et al.(7) . Studies were
performed that demonstrated specific photoinsertion of
2-azido-[
P]PAP into these enzymes and also
indicated the potential usefulness of this approach in the purification
and molecular characterization of purified native and cloned PAPS- and
PAP-binding proteins, including the STs. Moreover, photolabeling with
2-azido-[
P]PAP will be also useful in studies
investigating the structure of the ST active sites.
PAP, PAPS, 4-nitrophenol, and other reagents were purchased from Sigma. 2,6-Dichloro-4-nitrophenol (DCNP) was obtained from K and K Laboratories (Plainview, NY).
To clarify nomenclature, HAST1 corresponds to p-PST or thermostable PST, HAST3 is m-PST or
thermolabile PST, and HAST4 is a new form of PST differing from HAST1
by 12 amino acids. HAST4, unlike HAST1 and HAST3, is incapable of
sulfating dopamine, and its K for 4-nitrophenol
sulfation (74 µM) (
)is markedly different from
those of HAST1 (0.6 µM) and HAST3 (2200
µM)(12) .
Figure 1:
Photoaffinity labeling of
human and rat liver cytosol and endoplasmic reticulum with
2-azido-[P]PAP. Autoradiograph of proteins (50
µg) from human and rat liver cytosol and endoplasmic reticulum were
photolabeled with 40 µM 2-azido-[
P]PAP as described under
``Materials and Methods.'' Lanes 1-4 are human
liver ER. lane 1, no UV irradiation; lane 2, with UV
irradiation; lane 3, with 200 µM unlabeled PAP; lane 4, with 0.05% Triton X-100. Lanes 5-7 are
human liver cytosol. lane 5, no UV irradiation; lane
6, with UV irradiation; lane 7, with 200 µM unlabeled PAP. Lanes 8-11 are rat liver endoplasmic
reticulum, and lanes 12-14 are rat liver cytosol treated
in the same manner.
Several protein bands in rat liver
microsomes photoincorporated 2-azido-[P]PAP (Fig. 1, lane 9), but the photolabeling was inhibited
by unlabeled PAP only in the approximately 40-kDa band, and even here
protection was not complete (Fig. 1, lane 10). The only
significant effect of detergent treatment of microsomes was to
completely abolish photolabeling of a protein band of approximately 28
kDa, outside the mass range of the STs (Fig. 1, lane
11).
With cytosolic preparations from rat liver, on the other hand, proteins having molecular masses of 30-38 kDa, corresponding to the reported molecular masses of rat liver STs, were specifically photolabeled (Fig. 1, lane 13). Except for a band at approximately 40 kDa, the labeling was effectively inhibited by preincubation with 200 µM cold PAP (Fig. 1, lane 14). The pattern of rat liver cytosolic STs is different from the human ST pattern and is consistent with the presence of different forms of ST.
Figure 2:
Concentration dependence of
photoincorporation of 2-azido-[P]PAP into HAST3.
Autoradiograph is shown of HAST3 protein (50 µg) photolabeled with
increasing concentrations of 2-azido-[
P]PAP as
described under ``Materials and Methods.'' Concentrations of
2-azido-[
P]PAP are as follows: lane 1,
0.1 µM; lane 2, 0.25 µM; lane
3, 1.0 µM; lane 4, 2.5 µM; lane 5, 10 µM; lane 6, 25
µM; lane 7, 50 µM; lane 8,
control with no UV irradiation.
Figure 3:
Effect of inhibitors on photoincorporation
of 2-azido-[P]PAP into HAST3. Autoradiograph is
shown of HAST3 protein (50 µg) photolabeled with 10 µM 2-azido-[
P]PAP as described under
``Materials and Methods'' in the presence of increasing
concentrations of various inhibitors. Lane 1, no inhibitor; lanes 2-4, 10, 20, and 50 µM PAP; lanes
5-7, 10, 20, and 50 µM PAPS; lanes
8-10, 1, 10, and 100 µM 4-nitrophenol; lanes 11-13, 1, 10, and 100 µM DCNP.
Figure 4:
Photoaffinity labeling of human liver
cytosol and COS-7 cell-expressed human recombinant sulfotransferases.
Autoradiograph is shown of human liver cytosol, control,
non-transfected COS-7 cells, and COS-7 cell-expressed HAST1, -3, and -4
proteins (50 µg) photolabeled with 10 µM 2-azido-[P]PAP as described under
``Materials and Methods.'' Lanes 1-3, human
liver cytosol without and with UV irradiation and with 50 µM unlabeled PAP, respectively. Lanes 4-6,
non-transfected COS-7 cells without and with UV irradiation and with 50
µM unlabeled PAP, respectively. Lanes 7-9,
HAST1 without and with UV irradiation and with 50 µM unlabeled PAP, respectively. Lanes 10 and 11,
HAST3 with UV irradiation and with 50 µM unlabeled PAP. Lanes 12 and 13, HAST4 with UV irradiation and with
50 µM unlabeled PAP.
Figure 5:
Photoaffinity labeling of human liver
cytosol and E. coli-expressed human recombinant
sulfotransferases. Autoradiograph is shown of human liver cytosol and E. coli expressed DHEA-ST and EST proteins (50 µg)
photolabeled with 10 µM 2-azido-[P]PAP as described under
``Materials and Methods.'' Lanes 1-3, human
liver cytosol without and with UV irradiation and with 50 µM unlabeled PAP, respectively. Lanes 4 and 5,
DHEA-ST with UV irradiation and with 50 µM unlabeled PAP. Lanes 6 and 7, EST with UV irradiation and with 50
µM unlabeled PAP.
Figure 6:
Western blot analysis and photolabeling of
human liver cytosol and COS-7 cell-expressed human recombinant
sulfotransferases. Western blot (A) developed with a specific
anti-human PST antibody and autoradiograph (B) of human liver
cytosol, COS-7 cell-expressed HAST1, -3, and -4 and control,
non-transfected COS-7 cell proteins (50 µg) photolabeled with 10
µM 2-azido-[P]PAP as described
under ``Materials and Methods'' are shown. In both A and B, samples are as follows: lanes 1-3,
human liver cytosol without and with UV irradiation and with 50
µM unlabeled PAP, respectively; lanes 4-6,
HAST1 without and with UV irradiation and with 50 µM unlabeled PAP, respectively; lanes 7 and 8,
HAST3 with UV irradiation and with 50 µM unlabeled PAP. Lanes 9 and 10, HAST4 with UV irradiation and with 50
µM unlabeled PAP. Lanes 11-13,
non-transfected COS-7 cells without and with UV irradiation and with 50
µM unlabeled PAP,
respectively.
Figure 7:
Western blot analysis and photolabeling of
human liver cytosol and E. coli-expressed human recombinant
sulfotransferases. Western blot (A) and autoradiograph (B) of human liver cytosol and E. coli-expressed EST
and DHEA-ST proteins (50 µg) photolabeled with 10 µM 2-azido-[P]PAP are shown as described under
``Materials and Methods.'' On the Western blot, human
cytosolic ST was detected with anti-human PST antibody and E.
coli-expressed EST and DHEA-ST with specific anti-human EST and
DHEA-ST antibodies. In both A and B, samples are as
follows: lanes 1-3, human liver cytosol without and with
UV irradiation and with 50 µM unlabeled PAP, respectively; lanes 4 and 5, EST with UV irradiation and with 50
µM unlabeled PAP; lanes 6 and 7, DHEA-ST
with UV irradiation and with 50 µM unlabeled
PAP.
Direct photoaffinity labeling of various proteins with
adenosine derivatives, such as ATP, ADP, and S-adenosyl-L-methionine, has been described
previously(22, 23) . Additionally, reports in the
literature have suggested that direct affinity labeling with adenosine
3`-[P]phosphate 5`-phosphosulfate can be used to
label PAPS-binding proteins(3) . The mechanism of
photoactivation of purines and purine nucleosides postulates the
involvement of the C-8 position of the purine ring system(23) .
Another possibility is that UV irradiation results in the formation of
free radicals of aromatic amino acids, which scavenge the nucleotide.
In this work, we have developed an approach to ST labeling using PAP
containing a 2-azido function as a photoreactive group. This mechanism,
which involves covalent binding of the azido groups into specific amino
acids, uses different functional groups than the procedure described
above and, thus, targets different areas of the protein. Therefore, it
can be anticipated that different amino acids could become
radiolabeled. Moreover, labeling with probes that contain
P allows for a much higher level of sensitivity in
detecting the photolabeled proteins due to the higher energy of the
decay products. This feature is especially important when using
radiolabeling for the purification and characterization of the
recombinant proteins.
A first series of studies was used to
determine whether the probe, 2-azido-[P]PAP,
could be used as an effective photoaffinity ligand for specific
labeling of PAPS-binding proteins in the mixture of proteins from crude
human cytosolic preparations. Additionally, photoaffinity labeling of
recombinant STs from COS-7 cell and E. coli expression systems
was examined. We were able to demonstrate that, under standard
conditions, the 2-azido-[
P]PAP probe efficiently
bound to cytosolic STs as well as to several recombinant proteins with
a high specificity (Fig. 1, Fig. 4, and Fig. 5).
As the next step, optimal photolysis conditions were determined
using COS-7 cell-expressed human liver HAST3. We demonstrated that
incubation of HAST3 with 2-azido-[P]PAP followed
by UV irradiation resulted in the photolabeling of a single 34-kDa
protein (Fig. 2). The binding of
2-azido-[
P]PAP was concentration dependent with
half-maximal binding at approximately 7.5 µM.
Additionally, the labeling was inhibited in a concentration-dependent
fashion by cold PAP and PAPS with 50% inhibition at 6.0 µM and 5.8 µM, respectively. This protection is an
obligatory feature for demonstrating the true specificity of the
photoaffinity labeling process. Moreover, labeling of HAST3 after
preincubation with DCNP, a known inhibitor of the
enzyme(24, 25) , resulted in a concentration-dependent
inhibition of photoincorporation. The inhibition of labeling of ST by
DCNP was in agreement with previously published studies on the effect
of DCNP on direct photoaffinity labeling with
[
S]PAPS as the affinity probe(4) . The
observation that DCNP, a cosubstrate directed inhibitor which
interferes with catalysis of the HAST3 reaction, also strongly
inhibited the covalent binding of 2-azido-[
P]PAP
could be explained by the possibility that PAP and/or PAPS bind to both
the cosubstrate and the PAP/PAPS binding sites. As has been
demonstrated previously(4) , 4-nitrophenol, a model sulfate
acceptor for the phenol-specific STs, was neither required for nor
enhanced the labeling of human HAST3 with
2-azido-[
P]PAP. Indeed, at high concentrations
of 4-nitrophenol, photolabeling was significantly inhibited. The
significance of this observation is not clear at the present.
After
determination of the optimal photolysis conditions, we designed
experiments to determine whether 2-azido-[P]PAP
could also be used to photolabel other human STs expressed in different
expression systems. Fig. 4and Fig. 5show the
photoaffinity labeling of COS-7 and E. coli-expressed human
STs. In both series of experiments, crude liver cytosol was
photolabeled for comparison. These studies demonstrated that three
human STs, HAST1, -3, and -4, were each expressed as one single protein
with apparent molecular masses of 32, 34, and 32 kDa, respectively. It
should be emphasized that, in our experience, only a fully expressed
enzyme that possesses catalytic activity can be photolabeled.
Therefore, the ability to photoincorporate the probe can be used as a
criterion of successful expression. The above considerations can be
also applied for the characterization of the bacteria-expressed
proteins. Fig. 5shows the photolabeling of two human
cDNA-expressed steroid STs, DHEA-ST and EST, which were expressed as
single proteins in an E. coli expression system. The labeling
was UV-dependent, competitively inhibited by unlabeled PAP, and the
labeled proteins were identified as STs by Western blot analysis using
specific anti-DHEA-ST and EST antibodies.
Although progress in molecular biology has provided a better understanding of the overall genetic organization and expression of the STs, the amount of information available on the relationship between the structure and function of these enzymes is limited. STs require the activated sulfate donor PAPS as a cofactor. A putative nucleotide binding motif in the STs was noted by Hashimoto et al. in 1992(26) . The consensus sequence they cited, GXXGXXK, was significantly similar to a previously described motif termed the P-loop, found in ATP- and GTP-binding proteins(27) , and the authors suggested that this consensus sequence might constitute the PAPS binding site. It was recently confirmed by Komatsu et al.(28) that the P-loop, highly conserved in all STs, is required, at least in part, for binding of the activated sulfate donor. In different studies, Falany et al.(29) , using site-directed mutagenesis techniques, investigated the suggestions from previous experiments (16) that a cysteine residue might be located near the PAPS binding site of the STs. Bacterial expression of human p-PST with the cysteine at position 70 converted to serine indicated that the cysteine was not essential for activity or substrate binding. However, the mutant enzyme is significantly more sensitive to thermal inactivation.
Recently, Zheng et al.(30) investigated the PAPS binding site using ATP dialdehyde as an active site-directed affinity label for the PAPS binding site of rat aryl ST IV. It was demonstrated that the affinity label was bound to a hexapeptide at both lysine 65 and cysteine 66. These affinity-labeled amino acids are located within a region in the sequence of AST IV that shows considerable homology with various STs possessing diverse specificities for acceptor substrates.
In this
paper, we have demonstrated that 2-azido-[P]PAP
can be used as a photoaffinity probe to covalently and specifically tag
PAP-binding proteins in cytosolic and membrane fractions of rat and
human liver and recombinant ST proteins. Investigations of the actual
site of this linkage of protein and radioactive probe are in progress
in our laboratory.