From the Division of Gastroenterology, Department of
Medicine,
Clinical Nutrition Research Unit, and ¶ Mass
Spectrometry Research Center, Department of Biochemistry, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232
Received for publication, November 16, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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Selenoprotein P is a plasma protein that
has oxidant defense properties. It binds to heparin at pH 7.0, but most
of it becomes unbound as the pH is raised to 8.5. This unusual heparin
binding behavior was investigated by chemical modification of the basic amino acids of the protein. Diethylpyrocarbonate (DEPC) treatment of
the protein abolished its binding to heparin. DEPC and
[14C]DEPC modification, coupled with amino
acid sequencing and matrix-assisted laser desorption ionization-time of
flight mass spectrometry of peptides, identified several
peptides in which histidine and lysine residues had been modified by
DEPC. Two peptides from one region (residues 80-95) were identified by
both methods. Moreover, the two peptides that constituted this sequence
bound to heparin. Finally, when DEPC modification of the protein was
carried out in the presence of heparin, these two peptides did not
become modified by DEPC. Based on these results, the
heparin-binding region of the protein sequence was identified as
KHAHLKKQVSDHIAVY. Two other peptides (residues 178-189 and 194-234)
that contain histidine-rich sequences met some but not all of the
criteria of heparin-binding sites, and it is possible that they and the histidine-rich sequence between them bind to heparin under some conditions. The present results indicate that histidine is a
constituent of the heparin-binding site of selenoprotein P. The
presence of histidine, the pKa of which is 7.0, explains the release of selenoprotein P from heparin binding as pH
rises above 7.0. It can be speculated that this property would lead to
increased binding of selenoprotein P in tissue regions that have low pH.
Selenoprotein P is an unusual, extracellular glycoprotein that is
the major form of selenium in rat plasma (1, 2). Its mRNA has 10 UGAs in the open reading frame that specify selenocysteine incorporation (3). The protein is secreted by the liver, and its
mRNA is present in most other tissues, implying that they secrete
it as well (4). An immunohistochemical study has shown that
selenoprotein P is strongly associated with endothelial cells (5).
Thus, selenoprotein P is present in extracellular fluid and bound to cells.
There is increasing evidence that selenoprotein P has a role in defense
against oxidative injury. Administration of low doses of diquat to
selenium-deficient rats causes lipid peroxidation, massive liver
necrosis, and death within a few hours (6). These adverse events can be
prevented by injection of a physiological dose of selenium 12 h
before administration of the diquat. The selenium-dependent
protection correlates with the appearance of selenoprotein P in plasma
but not with the appearance of glutathione peroxidase in liver or in
plasma (7). A subsequent study using this model showed that the initial
lesion in the liver was injury of the centrilobular sinusoidal
endothelial cells and loss of them within 2 h (8). Endothelial
cell loss was followed within an hour by necrosis of the exposed
hepatocytes. The results of these studies are compatible with
protection by selenoprotein P against diquat-induced oxidative injury
of endothelial cells. Two reports have appeared recently suggesting
that selenoprotein P has enzymatic activities of an antioxidant nature
(9, 10).
Selenoprotein P is a heparin-binding protein (11). It binds to
heparin-Sepharose columns when applied at pH 7.0, but most of it is
eluted when the pH is raised to 8.5. This suggests that histidine
residues, which have pKa values of 6.5-7.0, are
important in the binding. Elution of bound selenoprotein P with a pH
gradient from 7.0 to 8.5 yielded several peaks, suggesting the presence
of isoforms of the protein that have differing affinities for heparin
(12). Two of the eluted peaks contained single proteins that shared the
same N terminus. One was the full-length form of selenoprotein P, and
the other was a short form that terminated at the second UGA (13).
Other isoforms appear to exist, and all of them bind to heparin
(12).
This report, which presents the first structural data on selenoprotein
P, describes experiments carried out to locate its heparin-binding
site(s). Site-directed mutagenesis is often used to assess the
significance of specific amino acid residues in binding of proteins to
heparin. However, expression of animal selenoproteins is generally not
possible in bacteria and is even difficult to achieve in animal cells.
Thus, site-directed mutagenesis could not be used to study heparin
binding by selenoprotein P. The older, but classic, techniques of
chemical modification, peptide mapping, and mass spectrometry were used
to identify histidine and lysine residues that are responsible for the
interaction of selenoprotein P with heparin.
Materials--
p-Hydroxyphenylglyoxal
(HPG)1 was from Pierce. DEPC,
[14C]DEPC, TNBS, chymotrypsin, trypsin, and
heparin-agarose were purchased from Sigma. FPLC and heparin HiTrap
columns were obtained from Amersham Pharmacia Biotech. All other
reagents were analytical grade or better.
For reverse phase HPLC, a Hewlett-Packard XDB-C18 column (2.1-mm inner
diameter × 15 cm) was used. For tryptophan emission experiments,
a SPEX-Fluorolog-1681 photon-counting fluorometer was used. UV-visible
spectroscopy was performed on a Hewlett Packard P8453 spectrophotometer
linked to a Hewlett Packard-Vectra XM microcomputer.
Purification of Selenoprotein P--
Selenoprotein P was
purified from rat plasma by a simplification of a previously described
method (1). A 10-ml immunoaffinity column, made with the monoclonal
antibody 8F11 (1), was equilibrated with phosphate-buffered saline
containing 0.5 mM EDTA and 0.02% NaN3. Rat
plasma, containing a small amount of 75Se-labeled
selenoprotein P, was applied to the column. Typically, 50 ml of plasma
was applied to the column and then washed with 150 ml of
phosphate-buffered saline. The column was washed with 15 ml of 1 M NaCl to elute nonspecifically adsorbed plasma proteins. Selenoprotein P was then eluted from the column with 0.1 M
glycine, pH 2.8, and collected in 1-ml fractions. The eluted protein
was neutralized with 50 µl of 1 M Tris-Cl, pH 9.0, so
that the final concentration of Tris-Cl was 50 mM.
"Cold" selenoprotein P (without 75Se-labeled plasma
added) could be purified using the same procedure. The unlabeled
protein was used for labeling with [14C]DEPC, proteolytic
digestion, separation by HPLC, and 14C determination by
liquid scintillation.
Chemical Modification of Selenoprotein P--
Modification by
DEPC was performed either on purified protein or on rat plasma. For
DEPC modification of selenoprotein P, a final concentration of 6.9 mM DEPC in 0.2 M sodium phosphate buffer, pH
7.0, was used unless otherwise indicated. For modification of lysine
residues with TNBS, the final concentration of TNBS was 0.2 mM in 0.2 M sodium carbonate buffer at pH 7.0 or pH 8.5. Modification of arginine residues was done with 1 mM HPG in 0.2 M sodium carbonate, pH 9.0.
Chemical Modification of Selenoprotein P Followed by
Heparin-Agarose Chromatography--
Four separate columns of
heparin-agarose (0.8 ml) were equilibrated with 50 mM
sodium bicarbonate, pH 7.0. Purified selenoprotein P (50 µg) was
treated for 2 h with either TNBS, DEPC, or HPG, or was not
treated. Modification of selenoprotein P with DEPC was done in 0.2 M sodium phosphate buffer, pH 7.0. Modification of
selenoprotein P with TNBS was done in 0.2 M sodium
bicarbonate, pH 8.5. Modification of selenoprotein P with HPG was done
in 0.2 M sodium bicarbonate, pH 9.0. After modification,
the samples were dialyzed against 4 liters of 50 mM sodium
bicarbonate, pH 7.0, for 4 h at 4 °C. After dialysis, each
sample was applied to a heparin-agarose column. The columns were each
washed with 11 ml of 50 mM sodium bicarbonate, pH 7.0. 75Se in the wash was determined by radioactive measurement
of 1-ml aliquots of wash in an LKB Compugamma 1282 Monitoring the Extent of Chemical Modification with UV-visible
Spectroscopy--
Carbethoxylation of histidine residues by DEPC
causes an increase in absorbance at 240 nm (14). Selenoprotein P (50 µg) in 1 ml of 0.1 M sodium phosphate buffer was used as
a reference blank, and DEPC was added to an identical sample to give a
final concentration of 6.9 mM. The extent of modification,
as measured by an increase in absorbance at 240 nm, was monitored over
10 min. The same experiment was carried out with heparin (5 mg/ml) in
the solution.
The extent of modification of lysine residues was monitored using the
lysine-specific reagent, TNBS. When TNBS reacts with the pH Dependence of DEPC modification of Selenoprotein
P--
Modification of histidine residues by DEPC is dependent upon
the pH, since the positively charged imidazolium ion will not react
with DEPC. For these studies, 50 µg of selenoprotein P in 1 ml of 0.2 M sodium phosphate buffer with pH values from 5.5 to 8.0 was modified with 6.9 mM DEPC, and the extent of
modification was followed at 240 nm.
Modification of Selenoprotein P in the Plasma and Reversal of
Modification by Hydroxylamine--
For heparin HiTrap chromatography,
0.1-ml aliquots of plasma labeled with 75Se diluted to 1 ml
with buffer A (100 mM ammonium acetate, 50 mM
Tris base, and 1 mM Na2EDTA, pH 7.0) were used.
One sample was then modified with 6.9 mM DEPC for 5 min in
the starting buffer. A second sample was modified with 6.9 mM DEPC for 5 min in the starting buffer and then treated
with Proteolytic Digestion of Selenoprotein P Followed by Heparin
HiTrap Chromatography--
400 µg of selenoprotein P in 10 mM Tris-Cl, pH 7.0, was mixed with 2 µg of chymotrypsin,
2 µg of trypsin, or neither and incubated for 3 h at 37 °C.
After incubation, the sample was loaded onto a 1-ml heparin HiTrap
column equilibrated with 10 mM Tris-Cl, pH 7.0 (buffer C).
The column was then washed with 10 ml of buffer C, after which a salt
gradient was applied. Buffer D was the same as buffer C except that it
contained 1 M NaCl. The flow rate was 0.25 ml/min, and the
gradient was set at 2.22% buffer D/ml.
Proteolytic Digestion of [14C]DEPC-modified
Selenoprotein P and HPLC Separation of Fragments--
A 200-µg
sample of selenoprotein P was dissolved in 1 ml of 0.1 M
sodium phosphate buffer, pH 7.0. [14C]DEPC was added to a
final concentration of 6.9 mM (specific activity 1.6 mCi/mmol). The sample was incubated for 5 min at room temperature and
then dialyzed against 4 liters of 10 mM NaHCO3 buffer for 4 h at 4 °C. This was followed by digestion with 10 µg of chymotrypsin for 5 h at room temperature. The sample was then dried in a SpeedVac to reduce the volume, redissolved in 100 µl
of column buffer, and injected onto the HPLC column. The HPLC column
was a 2.1-mm inner diameter × 15-cm narrow bore C18 column
(XDB-C18). Solvent E consisted of 1 mM sodium phosphate, pH
5.5, 0.1% trifluoroacetic acid. Solvent F consisted of 30% solvent E
and 70% acetonitrile. The initial conditions were 95% E, 5% F for 5 min followed by a linear gradient to 100% F in 70 min, followed by a
return to the initial conditions over 2 min. Selenoprotein P was also
modified in the presence of heparin. Conditions for modification,
digestion, and HPLC were the same as above except that the sample
contained 5 mg/ml heparin.
Protection of Heparin-binding Site of Selenoprotein P from DEPC
Modification--
Plasma (25 µl) containing selenoprotein P
radiolabeled in vivo was brought to a volume of 0.5 ml with
buffer A, pH 6.9. This sample was applied to a 1-ml heparin HiTrap
column and washed with 10 ml of buffer A, pH 6.9, and successively with
10-ml volumes of the same buffer containing 0.0069, 0.069, 0.69, and
6.9 mM DEPC. Then 10-ml of buffer A, pH 6.9, containing 1 M NaCl was passed over the column to elute the remaining
selenoprotein P. This experiment is shown in Fig. 7A.
Fractions 62-64 shown in Fig. 7A were combined and
extensively buffer exchanged with buffer A, pH 6.9. The
buffer-exchanged sample was applied to another heparin HiTrap column as
shown in Fig. 7B and washed with 10 ml of buffer A, pH 6.9. Then 10 ml of buffer B, pH 8.5, was applied, followed by 10 ml of
buffer B, pH 8.5, containing 1 M NaCl. 1-ml fractions were collected.
Peptide Sequencing and MALDI-TOF Mass Spectrometry to Identify
DEPC-modified Peptides--
Peptides isolated from proteolytic digests
by HPLC as described above were sequenced using an Applied Biosystems
492 Procise protein sequencer.
MALDI-TOF mass spectrometry analysis was done with a Vestec Voyager
Elite Time-of-Flight Mass Spectrometer using an acceleration potential
of 20 kV. The instrument was calibrated with insulin as an external
calibration standard.
2 µg (~40 pmol) of full-length selenoprotein P, prepared as peak 2 on a heparin HiTrap column (12), was dissolved in 20 µl of 100 mM ammonium bicarbonate. Modified trypsin (0.04 µg) was
added to give an enzyme/protein ratio of 1:50, and then the solution
was incubated at 37 °C for 18 h. 1 µl of the digested mixture
was analyzed using mass spectrometry.
2 µg (~40 pmol) of full-length selenoprotein P was dissolved as
above, and DEPC was added to it at a concentration of 6.9 mM. The reaction was incubated at room temperature for 5 min and then dialyzed against water for 6 h. It was then dried
using a SpeedVac and redissolved in 20 µl of 100 mM
ammonium bicarbonate. Digestion and mass spectrometric analysis was
performed as described for the unmodified selenoprotein P.
Tryptophan Fluorescence of Native and DEPC-modified
Protein--
In order to measure the structural integrity of the
DEPC-modified protein, tryptophan emission spectra were taken of the
modified and native proteins. Excitation was done at 295 nm, and the
tryptophan emission was recorded between 300 and 420 nm. Spectra were
recorded in 0.2 M sodium phosphate buffer, pH 7.0, at a
protein concentration of 25 µg/ml for both native and DEPC-modified
selenoprotein P.
The Effect of Chemical Modification of Basic Amino Acids on the
Affinity of Selenoprotein P for Heparin--
Heparin binding by
proteins is mediated by basic amino acids arranged to form a
heparin-binding site. In order to determine which basic amino acids are
present in the heparin-binding site(s) of selenoprotein P, the
75Se-labeled protein was treated with reagents that
selectively modify basic residues. Then the protein was passed over a
heparin-agarose column equilibrated with buffer of pH 7.0. It was
reasoned that modification of residues in the binding site would
interfere with heparin binding of the protein. The amount of
selenoprotein P bound to the column was determined by subtracting the
amount of 75Se that passed through the column from the
amount of 75Se applied to the column.
Selenoprotein P was modified with DEPC, TNBS, and HPG in separate
experiments. DEPC binds to unprotonated histidine residues, but it can
also modify tyrosine, lysine, and cysteine residues (18). TNBS modifies
primary amino groups in proteins, especially lysine residues. HPG is
highly specific for modifying arginine residues (19).
The results in Table I show that DEPC
treatment of selenoprotein P abolished its heparin-binding property at
pH 7.0. TNBS treatment blocked most heparin binding. However,
modification of selenoprotein P by HPG had only a small effect on the
affinity of the protein for heparin. These results implicate histidine and lysine residues in the binding of selenoprotein P to heparin.
Monitoring Extent of Modification of Histidine Residues with DEPC
Using UV Spectroscopy--
The modification of histidine and tyrosine
residues by DEPC can be followed spectrophotometrically. When histidine
becomes carbethoxylated, there is a corresponding increase in
absorbance at 240 nm (14). Modification of tyrosine side chains
produces a decrease in absorbance at 278 nm (20). In order to assess histidine and tyrosine modification, purified selenoprotein P was
treated with DEPC, and UV spectra were recorded. A progressive increase
in absorbance at 240 nm occurred with time, but no decrease took place
at 278 nm (spectra not shown). Therefore, modification of histidine
occurs, but no evidence was found for modification of tyrosine side chains.
When selenoprotein P was modified with DEPC, histidine binding reached
saturation in about 2 min at pH 7.0 (Fig.
1). When selenoprotein P was modified in
the presence of heparin, the increase in absorbance at 240 nm was not
as great as seen with the unliganded protein (Fig. 1). This indicates
that the presence of heparin prevents DEPC from modifying some of the
histidine residues, implicating histidine in the heparin binding.
DEPC reacts with unprotonated histidine residues. In order to
approximate the pKa of the modifiable histidine
residues, the modification of selenoprotein P by DEPC was carried out
at varying pH values. The modification of selenoprotein P in sodium phosphate buffer with pH values ranging from 5.5 to 8.0 is shown in
Fig. 2. A marked increase in reaction
rate occurred when the pH reached 6.8. The data from Fig. 2 indicate
that the pKa values of the modified histidine
residues are in the range of 6.4-6.8.
Identification of Selenoprotein P Peptides and Residues Labeled
with [14C]DEPC--
Peptides that become modified by
DEPC treatment of the native protein are predicted to be
solvent-exposed and thus might be involved in binding of selenoprotein
P to heparin. To determine which peptides of the native protein can be
labeled by DEPC, [14C]DEPC was employed. After labeling,
the purified selenoprotein P sample was dialyzed to remove excess
[14C]DEPC. Then it was digested with chymotrypsin.
Peptides were separated by reverse phase HPLC (chromatogram not shown).
Peaks that contained 14C were submitted for amino acid
sequence analysis. The peptides identified are shown in Table
II. Two of them are KHAHL and
KKQVSDHIAVY, which correspond to residues 80-84 and 85-95,
respectively. Although it has been reported that loss of label is
usually observed during the Edman degradation procedure (21), Lemaire
and colleagues (22) observed an additional peak that did not correspond
to any standard PTH-derivative when sequencing a peptide containing a
single histidine residue that had been labeled with
[14C]DEPC. Such a peak appeared along with a
PTH-derivatized histidine residue during its turn in the Edman cycle.
Similarly, we were able to identify a secondary peak with a retention
time slightly less than PTH-proline that was always associated with
DEPC-labeled histidine. A potential interpretation of this result is
that some of the carbethoxylated histidine residues survived the
sequence analysis and appeared as a new peak in the sequencing
chromatogram. These sequencing results (not shown) indicate that
His-81, His-83, and His-91 become carbethoxylated upon treatment of the
protein with DEPC (Table II).
We also found evidence for lysine modification by DEPC. Again, the
sequence analysis provided the evidence. The PTH-derivatized lysine
residues in the peptides in Table II did not elute in the position for
PTH-lysine but were shifted so that the retention of the modified
lysine was slightly greater than that of PTH-valine. A previous study
that used DEPC to modify chick liver glutathione S-transferase demonstrated that DEPC modified lysine
residues and that the N-carbethoxylated PTH-lysine eluted in
a similar position to that reported here (23). Thus, Lys-80, Lys-85,
Lys-86, Lys-112, and Lys-261 became modified with DEPC based on the
sequencing chromatograms (not shown).
Identification of DEPC-labeled Peptides and Residues by MALDI-TOF
Mass Spectrometry--
A second approach that used MALDI-TOF mass
spectrometry for detecting DEPC-labeled peptides was employed. It has
been reported that peptides containing carbethoxylated histidine
residues can be detected by using MALDI-TOF mass spectrometry (24-27).
Fig. 3A shows the mass
spectrum of a total tryptic digest of the native full-length isoform of
selenoprotein P. 12 peaks with the predicted masses of tryptic peptides
could be identified in the spectrum. After treatment with DEPC and
subsequent digestion, shifts of 72 mass units, corresponding to the
addition of a C(O)OCH2CH3 group to an imidazole
nitrogen atom or to an
Fig. 4 presents the amino acid sequence
of selenoprotein P as derived from its cDNA (3). The
solid lines under the
sequence coincide with the peptides identified by mass
spectrometry of the tryptic digest (Fig. 3A). The
broken lines with arrowheads above the sequence indicate the peptides that
were detected to be shifted in mass by DEPC treatment of the protein
(Fig. 3B). Residues in boldface type
indicate the DEPC-modified peptides identified by sequencing (Table
II).
These results demonstrate that at least five stretches of the
selenoprotein P sequence are accessible to modification by DEPC in the
native protein. These stretches are residues 81-120, 178-193, 233-238, 256-262, and 292-297. These results do not indicate that no
other parts of the molecule interact with heparin. The largest peptide
detected (residues 190-232 in Fig. 3A,
HGHEHLGSSKPSENQQPGALDVETSLPPSGLHHHHHHHK) represents the sequence
between the second and third stretches identified as potential
heparin-binding sites. Because of its richness in histidines, this
peptide might be expected to bind to heparin. After DEPC modification
of the protein, this peptide could not be detected in the trypsin
digest (no peak in Fig. 3B). Peptides on either side of it
were detected, so it must have been present in the digest. This result
implies that the peptide had been modified by DEPC. It seems likely
that the modification impaired the ability of the peptide to be ionized
and therefore detected. Thus, this stretch of the sequence would appear
to be a candidate for a heparin-binding site along with the adjacent
histidine-rich sequences.
Identification of Proteolytic Fragments of Selenoprotein P That
Bind to Heparin--
In order to determine directly the regions of
selenoprotein P that can interact with heparin, the protein was
digested with chymotrypsin or trypsin, and the resulting fragments were
loaded onto a heparin HiTrap column, which was washed with 10 mM Tris-Cl, pH 7.0. This approach has been used to identify
the heparin-binding sites of several proteins (28, 29). Fragments that
bound to the column were eluted with a NaCl gradient and detected by
their absorbance at 214 nm. The results of the heparin HiTrap
chromatography of the chymotrypsin and trypsin digests are shown in
Fig. 5, A and B,
respectively. In the chymotrypsin digest, two prominent peaks eluted
from the heparin column at salt concentrations of 220 (peak I) and 270 mM NaCl (peak II), respectively. These peaks were collected
and subjected to electrospray ionization mass spectrometry that was
coupled to a reverse phase C18 column (liquid chromatography-mass spectrometry). The peptides were identified by the mass of the chymotryptic fragments. The results are summarized in Table
IV. The results of the peptide mass
mapping show that the peptides KHAHL (elutes in peaks I and II) and
KKQVSDHIAVY (elutes in peak II) bound to heparin.
The protein was also digested with trypsin, and the resulting
chromatogram is shown in Fig. 5B. There are two very
prominent peaks in this chromatogram, labeled III and
IV. The peptide in peak III was determined to be
TTEPSEEHNHHK. This peptide had been shown to be labeled with DEPC in
the previous MALDI-TOF experiment (Fig. 3). Peak IV contained two
peptides having the sequences HAHLK and HAHLKK. These two peptides
overlap with the chymotryptic fragments in Fig. 5A. This
suggests that His-81, His-83, Lys-85, and Lys-86 are capable of being
involved in binding selenoprotein P to heparin. Lys-80 might also be
important for the binding interaction, because trypsin removes this
residue from the peptide KHAHL (Fig. 5A, peak
I). The elution of the undigested protein is shown in Fig.
5C as a comparison. Several other peptides were detected as
binding to heparin (Table IV), but the only one of them that had been
shown to be labeled by DEPC was HKGQHR. The histidine and lysine
residues in the unlabeled peptides are probably not on the surface of
the protein, and those peptides are thus not likely to be significant
to the heparin-binding properties of the protein.
Heparin Protects Peptides KHAHL and KKQVSDHIAVY from Modification
with [14C]DEPC--
The results from peptide mapping
with DEPC indicated that several peptides containing lysine and
histidine residues were modified with DEPC (Table II and Fig. 4). Among
these were the peptides KHAHL and KKQVSDHIAVY. Table IV summarizes the
assessment of the heparin-binding properties of these peptides and
indicates that these same two peptides, KHAHL and KKQVSDHIAVY, had the
strongest affinity of the peptides for heparin.
Selenoprotein P was labeled with [14C]DEPC in the
presence and in the absence of heparin. The protein was labeled as
described under "Experimental Procedures" and digested with
chymotrypsin. Peptides were separated using HPLC. The 14C
content of each chromatographic fraction was determined by liquid scintillation. The resulting chromatogram is shown as Fig.
6. Radioactive peptides were submitted
for sequencing. The peaks that correspond to the peptides KHAHL and
KKQVSDHIAVY did not appear in the presence of 5 mg/ml heparin as is
shown in Fig. 6. This result demonstrates that heparin protects these
two peptides from modification and further implicates them in the
binding of selenoprotein P to heparin.
Protection of Heparin-binding Site of Selenoprotein P from DEPC
Modification by Binding to Heparin HiTrap Column--
Radiolabeled
selenoprotein P was bound to a heparin HiTrap column at pH 6.9, and then DEPC at concentrations from 0.0069 to 6.9 mM was
applied to the column (Fig.
7A). When the DEPC was washed
off the column, no selenoprotein P eluted with it. Selenoprotein P
eluted when 1 M NaCl was added to the buffer.
The eluted radiolabeled selenoprotein P was buffer-exchanged and
applied to a second heparin HiTrap column at pH 6.9. It bound to the
column and behaved like native selenoprotein P, being partially eluted
when buffer of pH 8.5 was applied and completely eluted by 1 M NaCl (Fig. 7B). This demonstrates that binding
to heparin protects the heparin-binding site(s) of selenoprotein P from
DEPC modification.
Reversal of Modification by Hydroxylamine--
The results of the
above experiments demonstrated that the peptides KHAHL and KKQVSDHIAVY
can bind to heparin and that the lysine and histidine residues of these
peptides become modified with DEPC. The reversibility of DEPC
modification was tested by treating the modified protein with
hydroxylamine. DEPC bound to histidine in a 1:1 ratio can be removed
from it by treatment with hydroxylamine. Therefore, restoration of the
activity of an enzyme by hydroxylamine after its inactivation by DEPC
has been used as evidence that the enzyme activity is dependent
on histidine (16, 18). For this experiment, selenoprotein P was
modified directly in the plasma with 6.9 mM DEPC, and the
plasma sample was injected onto a heparin HiTrap column. Untreated
plasma was used as the control (Fig.
8A).
As is shown in Fig. 8B, after treatment with DEPC, the
selenoprotein P in the plasma elutes in the flow-through volume. This occurs after a 5-min treatment with DEPC. Thus, the protein can be
modified rapidly, abolishing its ability to bind to heparin. The
heparin binding capability of selenoprotein P can be restored after
DEPC modification by treating the sample with 200 mM
hydroxylamine at pH 7.0 for 30 min (Fig. 8C). This
reversibility of DEPC modification by hydroxylamine demonstrates
several points. First, at this ratio of protein/DEPC, modification of
histidine residues with two DEPC molecules apparently does not occur,
because a doubly modified histidine residue is irreversibly modified
(18). Second, the reversibility and restoration of heparin binding
implies that the protein structure is not altered upon modification.
Although reversibility of DEPC modification has been used as a test to distinguish between the importance to function of modified lysine and
histidine residues (17), it has been shown that hydroxylamine treatment
can also reverse modification of lysine residues (30). This should be
especially true in the case of highly reactive lysine residues. This
last point will be addressed under "Discussion."
Structural Integrity of Native and Modified Selenoprotein P as
Judged by Fluorescence Spectroscopy--
Since modification of
selenoprotein P with DEPC caused loss of its heparin binding, a method
was needed to assess the structural integrity of selenoprotein P upon
chemical modification. It seemed possible that modification of
selenoprotein P with DEPC could cause the protein to unfold, thereby
abolishing binding to heparin. Selenoprotein P contains two tryptophan
residues in the N terminus of the protein, near many of the potentially
modifiable histidine residues. A wavelength of 295 nm was chosen for
excitation in order to eliminate emission from tyrosine residues. The
emission spectra of native and DEPC-modified selenoprotein P were
essentially identical (not shown). Both native and modified
selenoprotein P had a The major conclusion of this study is that a motif located in
residues 80-95 of selenoprotein P mediates binding of the protein to
heparin. Several lines of evidence have been presented to support this
conclusion. First, when selenoprotein P was modified with DEPC, the
protein lost its ability to bind to heparin. DEPC forms adducts with
unprotonated histidine and lysine residues, and these are the basic
amino acids in the putative binding sequence. Such adduct formation
would be expected to block heparin binding by preventing protonation of
histidine and lysine side chains. Moreover, histidine and lysine
residues of the putative binding sequence were shown to become modified
when selenoprotein P was treated with DEPC. This was shown by peptide
sequence analysis using Edman chemistry (Table II) and by labeled
peptide identification using MALDI-TOF mass spectrometry (Fig. 3).
Second, the peptides that make up the putative binding sequence bound
to heparin (Fig. 5). Third, the presence of heparin protected the
histidine and lysine residues in the putative binding sequence from
modification by DEPC treatment of selenoprotein P (Fig. 6). In a
complementary experiment, DEPC modification of selenoprotein P bound to
a heparin HiTrap column did not abolish its subsequent ability to
bind to another heparin HiTrap column (Fig. 7).
The conclusion that can be drawn from these results is that the peptide
KHAHLKKQVSDHIAVY is responsible for at least part of the selenoprotein
P-heparin interaction at pH 7.0. Thus, lysines 80, 85, and 86 and
histidines 81, 83, and 91 appear to mediate interaction between heparin
and selenoprotein P.
Several consensus sequences have been determined for proteins that bind
to heparin (31). One such sequence is
XBBXBX, where X is a
hydrophobic residue and B is a basic residue. The peptide LKHAHL, which
corresponds to residues 79-84 of selenoprotein P, matches this
consensus sequence and has been experimentally determined to bind to
heparin (see Fig. 5 and Table IV). Although chymotrypsin cleaves the
peptide bond between Leu-79 and Lys-80, the resulting peptide, KHAHL,
still binds to heparin.
The heparin-binding experiment shown in Fig. 5 raises the possibility
of a second heparin-binding site on selenoprotein P. Another group has
reported a study of the interaction of human selenoprotein P with
heparin using surface plasmon resonance (32). They detected high
affinity binding and low affinity binding. Interestingly, they stated
that their results indicated that heparin had two binding sites for
selenoprotein P. However, we feel their results are also consistent
with the presence of two heparin-binding sites on the protein.
When the protein was digested with trypsin, one of the peptides that
bound to heparin had the sequence TTEPSEEHNHHK. This same peptide was
modified by DEPC in the MALDI-TOF experiment (Fig. 3). Therefore, this
peptide could be modified by DEPC, and it was able to bind to heparin.
It is possible that this peptide is involved in the selenoprotein
P-heparin interaction, although its affinity for heparin is much weaker
(elutes at 130 mM NaCl) than those of peptides KHAHL and
KKQVSDHIAVY (Fig. 5).
Selenoprotein P has two histidine-rich regions (Fig. 4), and the
peptide TTEPSEEHNHHK is in the first histidine-rich region. The second
histidine-rich region contains a peptide with seven histidines in a
row. When digested with trypsin, this peptide has the sequence
HGHEHLGSSKPSENQQPGALDVETSLPPSGLHHHHHHHK. This peptide appears as a peak
with a mass of 4295.58 in the upper panel of Fig.
3. We were unable to detect this peptide as binding to heparin or being
modified with DEPC. However, its disappearance from the mass spectrum
after DEPC treatment of the protein suggests that it was modified.
Further experiments are needed to clarify the role(s) of this region of
selenoprotein P.
Two isoforms of selenoprotein P have been characterized, and two more
have been postulated (12, 13). All isoforms share the same N-terminal
sequence through amino acid residue 244, which is the residue
just upstream from the second selenocysteine residue. The
heparin-binding site should therefore be present in all isoforms. Indeed, all isoforms bind to heparin (12).
Chemical modification has been used to study the interactions between
heparin and many proteins (33-36). It was used in this case because
selenoprotein P cannot be expressed in bacterial systems, and therefore
site-directed mutagenesis could not be employed. The majority of
proteins that bind to heparin do so through the basic amino acid
residues lysine and arginine (31). Chemical modification of arginine
and/or lysine residues severely inhibits or completely abolishes the
heparin binding property of those proteins. As far as we are aware,
there is only one other example of a heparin-binding protein that is
suggested to use histidine as a basic residue in the
heparin-protein interaction (37). Interestingly, both
selenoprotein P and that protein, histidine-rich glycoprotein, are
found in the plasma and elute from heparin in a
pH-dependent fashion.
A study that employed a peptide library of random 7-mers demonstrated
that peptides that bound to heparin were rich in lysine and arginine
residues. However, their histidine content was not distinguishable from
their content of the majority of the nonbasic amino acids (38). This
reinforces the perception that histidine is rarely involved in heparin binding.
Rat selenoprotein P cDNA codes for 28 histidine residues (out of
366 residues), and 16 of them are concentrated in two stretches of
sequence. The first stretch is 14 residues in length (amino acid
residues 185-198) with eight histidines and two lysines; the second
stretch is 10 residues in length (amino acid residues 225-234) with
eight histidines and two lysines. The other 12 histidine residues are
not clustered in such a manner.
The lysine and histidine residues in the major heparin-binding peptides
(residues 80-96) could be unambiguously identified as being labeled
with DEPC. The sequencing results indicated that in the case of
histidine there was an additional peak in the sequencing chromatogram
that was present when histidine was being sequenced in the Edman cycle.
This agrees with a previous report that identified N-carbethoxylated histidine residues by Edman degradation
(22). We could also identify N-carbethoxylated lysine
residues because of the shift of the N-carbethoxylated
PTH-lysine derivative in the sequencing chromatogram. This also agrees
with a previous report that identifies N-carbethoxylated
lysine residues (23). Therefore, several lysine residues of
selenoprotein P are also important for the heparin-protein interaction.
It is interesting to note the different reactivities of the lysine
residues of selenoprotein P toward DEPC and TNBS. As observed by
kinetics of modification (not shown), modification of lysine residues
by TNBS is slow. In contrast, when the protein is modified with DEPC
for 5 min, its heparin-binding ability is abolished. The results in
Tables II and III indicate that lysine residues at positions 80, 85, and 86 become modified with DEPC. In order for lysine to be modified
with either DEPC or TNBS, lysine must be in its neutral, unprotonated
form. The fact that DEPC is able to modify lysine residues at pH 7.0 indicates that these lysine residues are highly reactive and most
likely have a lower pKa than is usual for lysine
residues (normally near 10). The slow reaction rate of the lysine
residues of selenoprotein P with TNBS is probably due to the presence
of the negatively charged sulfate group of TNBS, which could mimic the
sulfate groups of heparin. As the Similarly, the data in Fig. 2 demonstrate that the
pKa values of the modifiable histidine residues are
in the range of 6.4-6.8. Therefore, at pH 7.0, some of the histidine
residues would be in the unprotonated form, which would not be optimal for their binding to the negatively charged sulfate groups of heparin.
These histidine residues must have altered pKa values in the selenoprotein P-heparin complex to be responsible for
binding. Alternatively, histidine residues with higher
pKa values are responsible for heparin binding.
The reactivity of lysine residues in selenoprotein P is further
demonstrated by the restoration of heparin binding by
hydroxylamine treatment. Lys-80, Lys-85, and Lys-86 are
clearly important for the selenoprotein P-heparin interaction, as
demonstrated by the heparin-binding experiment in Fig. 5. After
modification with DEPC and treatment with 200 mM
hydroxylamine, the selenoprotein P-heparin interaction is restored
(Fig. 8). This indicates that hydroxylamine reverses the histidine
modification by DEPC and strongly suggests that it reverses the lysine
modification as well. DEPC modification of highly reactive lysine
residues in proteins can be reversed by hydroxylamine as reported by
Pasta et al. (30), although an earlier report had concluded
that reversibility was specific for histidine (16). Thus, reversal of
DEPC modification of both histidine and lysine residues in the
heparin-binding site of selenoprotein P seems likely because both
histidine and lysine residues clearly contribute to the protein-heparin
interaction. When it becomes possible to produce sufficient
quantities of mutant selenoprotein P by site-directed mutagenesis, more
details about the contribution of each of these residues can be learned.
It is clear that the native structure of selenoprotein P is required
for the most efficient heparin binding. The peptides KHAHL and
KKQVSDHIAVY bind to heparin less tightly than does the intact protein,
as judged by the salt concentrations (220 and 270 mM NaCl,
respectively) needed to elute them from heparin (Fig. 5). Denaturation
of the protein by the addition of the reducing agent 1,4-dithiothreitol
also resulted in impaired heparin binding (not shown). This indicates
that the native tertiary structure of the protein is needed to align
these histidine and lysine residues of selenoprotein P to maximize
their interaction with heparin.
Heparin binding by proteins and peptides is defined as "tight" when
a salt concentration of 1 M or greater is needed to break it (39). Displacement by 0.3-0.4 M salt is defined as
"weak" binding. Selenoprotein P eluted from the heparin column at
0.3 M salt (Fig. 5C). Thus, it would be classed
as having weak binding by established criteria. However, those criteria
were developed for heparin-binding sites that were made up of lysine
and arginine, and it is not certain whether they should be used when
histidine-mediated heparin binding is being studied. It is postulated
here that this "weak" heparin binding of selenoprotein P has
physiological significance.
Extracellular superoxide dismutase is an appropriate protein to compare
with selenoprotein P. It is an extracellular oxidant defense enzyme
that also binds to heparin (40-42). Its heparin binding is postulated
to be important in its function of regulating superoxide concentration
near cell membranes. Extracellular superoxide dismutase is
heterogeneous with respect to heparin binding, a property that it
shares with selenoprotein P. The enzyme elutes from heparin in three
fractions, designated as A, B, and C. (41). Fraction A does not bind to
heparin; fraction B elutes at a salt concentration of 0.2 M; and fraction C elutes at a salt concentration of 0.5 M. Selenoprotein P binds more tightly than does fraction B
but not as tightly as does fraction C.
An additional comparison can be made between selenoprotein P and
histidine-rich glycoprotein (37). Heparin binding by these two proteins
is pH-sensitive. Histidine-rich glycoprotein elutes from heparin
between 0.3 and 0.5 M NaCl. It has been proposed that
histidine-rich glycoprotein acts as a pH sensor, interacting with
negatively charged glycosaminoglycans only when it acquires a net
positive charge. Based on this proposal, the histidine residues of a
protein should be able to provide a mechanism for regulating binding of
the protein to cell surface proteoglycans. Local pH can drop by as much
as 1 pH unit in acidosis or anoxia (43), leading to an increase in
protonation of histidine residues and facilitating binding.
The heparin binding behavior of extracellular superoxide dismutase is
modulated by deletion of heparin-binding sites on one (fraction B) or
both (fraction A) of its subunits (44). Heparin binding by
selenoprotein P is modulated also. However, physical modification of
selenoprotein P does not appear to be involved. Instead, heparin
binding by selenoprotein P is modulated by pH.
It seems possible that the function of selenoprotein P depends on its
binding to cell surface and matrix heparan sulfate proteoglycans. If it
does serve as an oxidant defense as we have postulated (7), localization to areas of low pH, such as sites of inflammation, would
be logical so that it could protect the host cells in those areas.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-counter.
-amino
group of lysine, an increase in absorbance at 420 nm occurs (15).
Purified selenoprotein P was modified in 0.1 M sodium
phosphate buffer at pH 7 or at pH 8.5. The absorbance at 420 nm was
followed for 10 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Binding of chemically modified selenoprotein P to a heparin-agarose
column
View larger version (10K):
[in a new window]
Fig. 1.
Modification of histidine residues by DEPC in
the presence and absence of 5 mg/ml heparin. 50 µg of
selenoprotein P in 1 ml of 0.1 M sodium phosphate buffer,
pH 7.0, was modified with 6.9 mM DEPC, and
A240 was monitored. No heparin was present in
the experiment represented by the solid line, and
heparin was present in the one represented by the broken
line.
View larger version (17K):
[in a new window]
Fig. 2.
pH dependence of the reaction with
DEPC and selenoprotein P. The details of the reaction are
given under "Results."
Peptides Modified with [14C]DEPC
-amino group, were detected in eight peptides
(Fig. 3B and Table III).
View larger version (16K):
[in a new window]
Fig. 3.
MALDI-TOF mass spectrometry of selenoprotein
P digested with trypsin. A, a digest of unmodified
protein; B, a digest of protein that had been modified with
DEPC. Peptides that could be identified by mass in both
panels, indicating that they had been modified by DEPC, are
labeled as a-h. Their masses and sequence numbers are given
in Table III. Unmodified peptides are identified by their sequence
numbers in A.
Peptides labeled with DEPC and detected by MALDI-TOF mass spectrometry
View larger version (50K):
[in a new window]
Fig. 4.
Amino acid sequence of rat selenoprotein P
with indication of DEPC modification. Solid
lines under sequences indicate
detection in a tryptic digest by mass spectrometry (Fig.
3A). Broken lines with
arrowheads above sequences indicate
that the sequence mass shifted after DEPC treatment of the protein
(Fig. 3B). Sequences in boldface
letters indicate DEPC-modified peptides that were detected
by amino acid sequencing (Table II).
View larger version (20K):
[in a new window]
Fig. 5.
A, heparin HiTrap chromatography of
chymotrypsin digest. Selenoprotein P peptides resulting from digestion
with chymotrypsin were loaded onto the column and washed with 10 mM Tris-Cl, pH 7.0. Bound peptides were eluted with a NaCl
gradient and detected at 214 nm. Two prominent peaks, labeled as I and
II, were visible in the chromatogram. The peaks were collected and
submitted for mass mapping analysis (Table IV). B, heparin
chromatography of trypsin fragments. Peaks III and IV are prominent.
The peptides in IV, HAHLK and HAHLKK, overlap with the chymotryptic
digest in A. C, elution of the undigested protein
(peak VI) as a comparison. This shows that the intact protein elutes at
a higher salt concentration than the peptide fragments.
Mass mapping analysis of peptides bound to heparin
View larger version (22K):
[in a new window]
Fig. 6.
Heparin protection of peptides KHAHL and
KKQVSDHIAVY from modification with [14C]DEPC.
Purified protein was modified in the presence and absence of 5 mg/ml
heparin in 0.1 M sodium phosphate buffer, pH 7.0. The
digests (heparin present (solid line) and heparin
absent (broken line)) were subjected to HPLC.
Peaks in the chromatogram that were protected from modification by
heparin correspond to KHAHL and KKQVSDHIAVY.
View larger version (16K):
[in a new window]
Fig. 7.
Effect of DEPC treatment on selenoprotein P
bound to a heparin HiTrap column. Plasma containing
75Se-labeled selenoprotein P was applied to the column as
shown in A. Increasing concentrations of DEPC were applied
before the 75Se-labeled selenoprotein P was eluted with 1 M NaCl. After removal of the NaCl, the sample was passed
over another heparin HiTrap column as shown in B. The pH was
raised to 8.5, and then 1 M NaCl was applied. Details of
the experiment are given under "Experimental Procedures."
View larger version (13K):
[in a new window]
Fig. 8.
Heparin HiTrap chromatography of
75Se-labeled plasma after DEPC treatment. The protein
is eluted in a pH-dependent fashion (pH 7.0-8.5) from
fraction 20-60, after which the remaining bound protein is eluted with
2 M NaCl. Elution of the untreated protein is shown in
A. Treatment of the protein in the plasma with 6.9 mM DEPC resulted in the protein eluting in the void volume
(B). Treatment with 200 mM hydroxylamine
following modification with DEPC resulted in the restoration of heparin
binding (C).
max near 345 nm. If selenoprotein
P were to be unfolded upon modification with DEPC, the tryptophan
emission would be expected to be shifted to longer wavelengths. That
was not observed. At the very least, it can be concluded that the local
environment of the tryptophan residues did not change upon modification
of selenoprotein P with DEPC. Further evidence that modification of
selenoprotein P with DEPC did not cause structural damage to the
protein is provided by reversibility of DEPC modification by hydroxylamine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of lysine would
approach the sulfate group of TNBS for nucleophilic attack, the
pKa of the
-amino group would increase due to the
presence of the negatively charged sulfate group, making this reaction
slow. The pKa values of these lysine residues are
probably higher in the selenoprotein P-heparin complex than in the
protein alone. This probably contributes to the binding of the protein
with heparin.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Eric F. Howard of the Protein Chemistry Core for the peptide sequencing, to Amy K. Motley and Ansley Tharp for the purification of selenoprotein P, and to Dr. Amy Harms of the University of Wisconsin Mass Spectrometry Facility for the mass spectrometry on the samples in Fig. 5.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants ES 02497, DK 26657, and ES 07028.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706
** To whom correspondence should be addressed: C2104, Medical Center North, Vanderbilt Medical Center, Nashville, TN 37232-2279. Tel.: 615-343-7740; Fax: 615-343-6229; E-mail: raymond.burk@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010405200
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ABBREVIATIONS |
---|
The abbreviations used are: HPG, p-hydroxyphenylglyoxal; DEPC, diethylpyrocarbonate; TNBS, trinitrobenzene sulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PTH, phenylthiohydantoin; FPLC, fast protein liquid chromatography; HPLC, high pressure liquid chromatography.
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REFERENCES |
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