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INTRODUCTION |
The cellular prion protein (PrPC)1 and
Doppel (Dpl) are glycosylphosphatidylinositol-anchored proteins
with
-helical C-terminal domains. Whereas PrPC is
expressed in the central nervous system and many peripheral tissues,
the most notable site of Dpl expression is the testis (2, 3), although
transient expression in endothelial cells of the developing central
nervous system has also been described (4). Both proteins can engender
neurodegenerative diseases. PrPC serves as a precursor to a
malfolded isoform denoted PrPSc in prion infections
such as scrapie and is mutated in familial prion diseases such as
familial Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome (5). In the case of Dpl,
neuronal expression results in the death of cerebellar cells by
apoptosis (6, 7).
Although PrPC was first described in 1985 (8), discerning
its physiological function has proven to be challenging (9). The
absence of PrP in homozygous null mice was once thought to cause a
profound cerebellar ataxia (10), but it is now clear that this syndrome
is an artifact of the genetic engineering used to create the deleted
PrP gene (Prnp) alleles, resulting in ectopic expression of
Dpl (2, 6, 7, 11). Recent analyses have instead focused on a potential
role for PrPC in signal transduction (12), interactions
with other cell surface proteins (13), and perhaps elements of the
extracellular matrix (14-16). Several reports have established that
under conditions of oxidative or cellular stress, PrPC can
serve a protective role (17-19).
One particularly intriguing connection concerns the ability of
PrPC to bind copper ions. Many studies have documented the
selectivity, multivalence, and pH dependence of Cu(II) binding, as well
as binding constants potentially compatible with Cu(II) occupancy in vivo (20-23). There is a broad consensus that the
N-terminal region of PrP is the major contributor to copper binding
ability. This glycine-rich domain, absent from Dpl, contains five
"octarepeat" motifs. Although the first repeat is of the general
form PGGGGWGQ, the four following identical repeats PHGGGWGQ bear
histidine residues and bind copper in a cooperative fashion (Hill
coefficient, ~3.5) (21, 23, 24). One plausible scenario for
physiological activity, based upon stimulation of endocytosis by
extracellular copper (25, 26), is that PrPC plays a role in
copper uptake or scavenging, perhaps germane to the synaptic location
of this molecule. An alternative viewpoint is that PrPC
possesses a superoxide dismutase-like enzymatic activity (27). Irrespective of these two contrasting views of the physiological role
of PrPC, it is to be emphasized that genetic evidence
exists for the importance of the main copper-binding domain, insofar as
alterations in the number of octarepeats cause familial prion diseases
(28-31).
In addition to the octarepeat Cu(II) binding site of PrPC,
there has been interest as to the existence of additional
Cu(II)-binding sites C-terminal to these motifs. In the course of our
studies to inventory Cu(II)-binding sites in recombinant PrP, we
reasoned that Dpl, a PrP-like protein lacking octarepeats, would
comprise a useful control. Unexpectedly, as described below, Dpl was
found to exhibit selective Cu(II) binding with an affinity comparable with that of PrP.
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MATERIALS AND METHODS |
Dpl Peptide and Proteins--
Mouse Doppel 27-154
(MoDpl(27-154)) refolded by a copper catalysis method (3) and mouse
Doppel 26-157 (MoDpl(26-157)) refolded in the presence of glutathione
(32) were prepared as described previously. Intrinsic copper content
was determined by atomic absorption spectrophotometry. A MoDpl
helix-loop-helix peptide was synthesized by standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry. This peptide
corresponds to residues 101-145 of the mouse doppel and includes the
interrupted
-helix B (
B/B'), the
-helix C (
C), and the loop
between these two helical structures (32). The disulfide bond
was introduced by treating the peptide overnight in an aerated solution
of ammonium bicarbonate. Protein concentrations were calculated by
amino acid hydrolysis (33). Far-UV CD measurements, endoproteolysis,
and S-carbamidomethylation of cysteines to determine the
presence of integrity of disulfide bonds were carried out as described
previously (3, 33).
Fluorescence Spectroscopy and Titrations--
Steady-state
fluorescence spectra were recorded on a Photon Technology International
QM-1 spectrophotometer. Emission spectra were collected from 310 to 400 nm (
ex = 280 nm, 1 s/nm, band pass = 1 nm for
excitation and emission). For Cu(II) binding experiments, MoDpl(101-145), MoDpl(27-154) or MoDpl(26-157) were diluted to a
final concentration of 0.7 µM in 25 mM
N-ethylmorpholine, pH 7.4, 150 mM KCl (NEMO-KCl
buffer). The sample volumes were 1 ml, and the experiments were
performed at room temperature. The protein was titrated with a
concentrated Cu(II) stock such that sample dilution was never more than
1.7%. To detect metal binding selectivity, 1, 2, or 3 µM
CuCl2, ZnCl2, NiSO4,
MgCl2, CaCl2, or MnCl2 was mixed
with 0.7 µM MoDpl(27-154). The emission spectrum was
collected after each CuCl2 addition, and fluorescence
intensities from 310 to 380 nm were integrated.
Coordination, Modification, and Digestion of MoDpl(101-145)
Peptide--
Prior to MALDI-MS analysis,
MoDpl helix-loop-helix (90 µM) was incubated with 10-fold
molar excess CuCl2, ZnCl2, NiSO4,
MgCl2, CaCl2, MnCl2, or
FeSO4 in 25 mM NEMO-KCl buffer, pH 7.4, at room temperature for 1 h. Sequencing grade chymotrypsin (Roche
Molecular Biochemicals) was used to digest the intact peptide and
copper-peptide complex for 2 h at room temperature (the ratio of
enzyme:protein was 1:20). In some cases, before chymotrypsin digestion,
the samples were incubated with 5-fold molar excess diethlpyrocarbonate
(DEPC) at room temperature for 30 min. The samples were then analyzed by using MALDI-MS as described previously (1).
Equilibrium Dialysis--
MoDpl(101-145) and control protein
(HuPrP(23-98): (21)) were prepared in NEMO-KCl buffer, pH 7.4. Chelate
solution (Gly:Cu:Gly) was prepared by mixing CuSO4 solution
with a 2-fold molar excess of glycine and then adjusting the pH to 7.4. The equilibrium dialysis experiments were carried out in multiple
5-cell equilibrium dialyzers (Spectrum Industries). One ml of protein
plus chelated Cu(II) mix (retentate) and 1 ml of buffer (dialysate)
were placed on opposite sides separated by a 1-kDa cut-off cellulose
ester membrane (Spectrum Industries) in a dialysis chamber. For a given
experiment, the concentration of protein was held constant, and the
concentration of chelated Cu(II) was varied over a Cu(II):protein ratio
from 0.5:1 to 40:1. The system was rotated to come to equilibrium for 108 h at 4 °C (verified by analysis of "blank reactions"
not containing protein), and the concentration of all forms of copper
in each half-chamber was measured by atomic absorption spectroscopy.
2.5 µM protein was used in all of the equilibrium
dialysis experiments.
Copper Determinations--
Copper was determined by means of
graphite furnace atomic adsorption spectrophotometry. Protein samples
for analysis were diluted with 0.5% nitric acid (nitric acid Ultrex
II; J. T. Baker) to concentrations within the linear range of graphite
furnace copper measurements. The dilution range varied from 10 to 520 times. The diluted samples were transferred to Eppendorf tubes and
placed within the autosampler of the atomic adsorption
spectrophotometer (Varian Graphite Furnace GTA 100/Varian Spectra 880),
and absorbance was measured using furnace conditions developed in the
trace element laboratory. Calibration was against working standard
solutions prepared by dilution with 0.5% nitric acid from a 1000 mg/liter copper standard (J. T. Baker). Concentrations of stock
solutions used for fluorescence quenching studies were verified by
atomic absorption spectroscopy.
Mice--
Tg(Dpl) and non-Tg littermate mice (Tg(Dpl) line
10329) have been described previously (7). Mice were between 15 and 20 weeks of age at the time of analysis.
Antibodies--
The anti-Dpl rabbit polyclonal antibody E6977
raised against recombinant mouse doppel (7) was used as
described below. The anti-nitrotyrosine antibody was from Cayman
Chemical (a gift from N. Cashman), whereas antibodies against
2,4-dinitrophenylhydrazine were obtained from Intergen.
Western Blotting--
10% (w/v) homogenates of half brains were
prepared in 0.32 M sucrose supplemented with one
complete-mini protease inhibitor tablet (Roche Diagnostics) on ice.
Homogenates were aliquoted and stored at
80 °C until needed. The
total protein concentration of each sample was determined using
Bradford assay reagents (Bio-Rad) as per the manufacturer's
instructions. For the immunoblot analysis of Doppel expression and
nitrosylation, samples of 50 µg of total brain protein were prepared
and boiled in loading buffer for 9 min and then centrifuged at
20,800 × g for 5 min prior to fractionation by SDS-polyacrylamide
gel electrophoresis. The samples were loaded onto a 10-20% Tricine
gradient gel (Novex) and electrophoresed at 100 V for ~100 min. The
samples were then transferred onto 0.2 µm nitrocellulose (Schleicher
& Schuell) for ~80 min at 25 V (Novex semi-dry apparatus). The blots
were blocked with 5% (w/v) milk powder in Tris-buffered saline (pH
7.4), 0.02% Tween 20 (Buffer A) for 1 h with gentle agitation at
room temperature and then probed with the appropriate primary antibody
(anti-Dpl E6977 used 1:4000, anti-nitrotyrosine used 1:5000) in 0.5%
milk powder in Tris-buffered saline (pH 7.4), 0.02% Tween 20 overnight
at 4 °C. The appropriate horseradish peroxidase-conjugated secondary
antibodies were used. The blots were visualized using ECL detection
reagents (Amersham Biosciences) and exposed on X-Omat AR film (Eastman Kodak Co.).
Detection of Nitrotyrosine Residues--
A positive control for
the nitrosylation blot was made just prior to SDS-PAGE. Peroxynitrite
was added to 50 µg of a wild type mouse brain homogenate in sodium
phosphate buffer while vortexing to create a final concentration of 15 mM peroxynitrite. After 2 min, 2× gel loading buffer was
added to quench the reaction, and the sample was kept on ice. The
positive control as well as the sample homogenates of 50 µg of total
brain protein were subjected to Western blot analysis with a monoclonal
anti-nitrotyrosine primary antibody in Buffer A.
Detection of Reactive Carbonyl Groups--
The amount of
carbonyl groups introduced into the total protein of the brain
homogenates via oxidative modification was detected using the OxyBlot
protein oxidation detection kit (Intergen). Samples of 15 µg of total
brain proteins in 0.32 M sucrose and 2% 2-mercaptoethanol
were denatured and then derivatized with 2,4-dinitrophenylhydrazine,
electrophoresed, and electroblotted as per the manufacturer's
instructions. The blots were blocked with 1% (w/v) bovine serum
albumin in phosphate-buffered saline (pH 7.4), 0.05% Tween 20 for
1 h at room temperature and then probed with a rabbit anti-DNP
primary antibody, used at a concentration of 1:150 in blocking solution
overnight at 4 °C. Following incubation with the goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody, blots were
visualized using ECL or ECL-Plus detection reagents as described above
or subjected to protein quantification using ImageQuant software
(Molecular Dynamics, Inc.), respectively.
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RESULTS |
Recombinant Dpl Proteins and a Synthetic Dpl Peptide--
To
exclude trivial or idiosyncratic effects pertaining to expression and
purification of Dpl proteins from Escherichia coli, we used
two recombinant polypeptides prepared by different methods (3, 32) (in
the absence of the N-terminal sequences of mature Dpl isolated from
mammalian cells, the lengths of these two proteins, Dpl(27-154)
versus Dpl(26-157), merely reflect ambiguities in assigning
N- and C-terminal signal peptides by different algorithms). For
Dpl(27-154) prepared by oxidative refolding in catalytic
concentrations of Cu(II), we used atomic adsorption spectroscopy to
quantify residual metal content (<3 × 10
4 mol of
copper/mol of dialyzed protein). Extrapolating from studies attributing
special significance to the helix B/B'-loop-helix C region of Dpl (34),
we hypothesized that this area of Dpl may comprise a site with
biological activity and synthesized a corresponding peptide,
Dpl(101-145) (Fig. 1A). This
peptide encompasses one of the two disulfide bonds present in
full-length Dpl, the "inner" Cys109-Cys143
linkage located at an equivalent position to the single disulfide bond
present in PrP. Measured in NEMO-KCl buffer at pH 7.4, Dpl(101-145) exhibited a CD spectrum with minima at 208 and
222 nm, indicative of a high
-helical content (Fig. 1B).
MALDI-MS analysis of Dpl(101-145) peptide showed a single charged
peptide signal at m/z 5162.9 (Fig. 2A), in excellent agreement
with the calculated molecular mass of 5162.9 Da as
[M+H]+. S-Carbamidomethylation (3, 33) was
used to verify the presence of appropriate disulfide linkages in the
recombinant polypeptides (not shown) and Dpl(101-145) peptide (see
Fig. 6A) used for the following analyses.

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Fig. 1.
Properties of the Dpl(101-145) peptide.
A, schematic showing predicted chymotrypsin cleavage sites
and the disulfide linkage. B, CD spectrum of the
Dpl(101-145) peptide (48 µM) in 25 mM
N-ethylmorpholine, 30 mM KCl (NEMO-KCl) buffer,
pH 7.4, measured at room temperature. The spectrum indicates a high
-helical content.
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Fig. 2.
MALDI-MS analysis of metal binding to
MoDpl(101-145). A-H, spectra obtained with or without the
indicated divalent cations, as described under "Materials and
Methods."
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MALDI-MS Analysis of Dpl(101-145)--
Because MALDI-MS provided
some of the first evidence in favor of copper binding by PrP (20), this
technique was applied to Dpl(101-145). After incubation with 10×
molar excess of CuCl2 in NEMO-KCl buffer, pH 7.4, at room
temperature for 1 h, Cu(II)-peptide complexes were detected. In
addition to intact protein at m/z 5162.9, additional peaks at m/z 5225.4 and 5286.7 were
observed in the presence of copper (Fig. 2B). Averaged over
three experiments, the starting material and derivatives were
established as 5162.63 ± 1.42, 5225.90 ± 0.78, and
5287.23 ± 1.09 Da, yielding individual mass increments of 63.3 and 61.3 Da, respectively, and an averaged figure of 62.3 Da. These
data are consistent with up to 2 mol of copper (mass 63.5 Da) added per
mole of peptide under the conditions of this assay, although intrinsic
errors for the mass determinations do not permit conclusions as to how
many protons are expelled from the protein commensurate with copper
binding. To detect whether other divalent ions bound to Dpl,
MoDpl(101-145) was incubated with 10× molar excess of Zn(II), Ni(II),
Mg(II), Ca(II), Mn(II), or Fe(II) in NEMO-KCl buffer, pH 7.4, at room
temperature for 1 h prior to MALDI-MS analysis. However, in
agreement with fluorescence quenching studies presented in Fig.
3, no signals corresponding to
metal-peptide complexes were detected in these other analyses (Fig. 2,
C-H).

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Fig. 3.
Effect of Cu(II) on Dpl proteins and
Dpl(101-145) peptide measured by fluorescence spectrometry.
Fluorescence quenching by addition of copper was determined for
Dpl(27-154) (A and B), Dpl(26-157)
(C and D), and MoDpl(101-145) peptide
(E and F). A, C, and
E, fluorescence emission spectra at different Cu(II)
concentrations using protein at 0.7 µM. B,
D, and F, the change in fluorescence intensity
integrated over a wavelength range of 310-380 nm plotted against
copper concentration (abscissa); thus, 1.0 on the
ordinate indicates 100% (maximal) observed fluorescent
change. The binding curves represent the best fits of Equations 1 and 2
(main text) to the data with the polypeptide concentrations set at the
measured values of 0.7 µM. The data were also fitted with
the concentration of polypeptide fixed at 1.4 µM
(i.e. the total concentration of two binding sites) for
comparison of residuals and chi-squared values (curves not shown: see
main text). The residual plots (lower panels of
B, D, and F) indicate deviations
between data points and the model with the polypeptide concentration
set at 0.7 µM (filled circles, 1:1 binding
model) or 1.4 µM (open circles, 2:1 binding
model). The chi-squared values, whose magnitudes are inversely
proportional to the closeness of model and the data points, are:
Dpl(26-154), 1:1 binding, 0.028; 2:1 binding, 0.078; Dpl(27-157), 1:1
binding, 0.014; 2:1 binding, 0.058; Dpl(101-145), 1:1 binding, 0.006;
and 2:1 binding, 0.022. G, the fluorescence intensity change
integrated over the wavelength range of 310-380 nm of 0.7 µM Dpl(27-154) in 3 µM of various metal
ions.
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Copper-Protein Interactions Assessed by Fluorescence
Quenching--
Fluorescence quenching analysis for recombinant mouse
Doppel (recDpl) was undertaken exploiting the intrinsic properties of four tryptophan residues at positions 35, 85, 136, and 151, and five
tyrosine residues at positions 78, 79, 84, 91, and 92 (present within
both Dpl(27-154) and Dpl(26-157)). To confirm that these residues
undergo a change in environment, we measured the change of fluorescence
intensity of Trp and Tyr residues upon Cu(II) addition. The diminutions
in intrinsic fluorescence (integrated from 310 to 380 nm) induced by
Cu(II) addition were 26% for Dpl(27-154) and 30% for Dpl(26-157).
However, the change in the intensity was not accompanied by a
significant shift in the wavelength position of the maximum
(
max), suggesting that the solvent accessibility of Trp
residues are not affected significantly by copper binding. Also, CD
spectra were not notably altered by copper addition (not shown),
suggesting that copper binding is not accompanied by a significant
change in the secondary structure of recDpl. MoDpl(101-145) contains a
single tryptophan residue at position 136 (Fig. 1A), facilitating similar fluorescence spectroscopic studies to those performed for the aforementioned full-length recDpl molecules.
Binding affinities and stoichiometries for copper binding to the
various proteins and peptides were quantified by fitting of the
conventional model expressed in Equations 1 and 2 to the fluorescence data in Fig. 3,
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(Eq. 1)
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where F is the measured fluorescence, normalized by
taking the difference in the first and last data point as 1, F0 is the fitted fluorescence before addition of
copper, and
F is the fitted change in fluorescence. The
concentration of the Dpl-copper complex, [bound] is described in
Equation 2,
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(Eq. 2)
|
where Kd is the dissociation constant of the
copper-binding site(s), [Cu]tot is total copper
concentration, and [P] is protein concentration. In accord with data
presented in Figs. 2 and 5, binding was considered in terms of
"one-site" and "two-site" models. The protein concentration
terms in Equations 1 and 2 were first set to the measured value of 0.7 µM (i.e. one copper-binding site/molecule),
and the best fits of the model to the data are shown in the lower
panels of Fig. 3 (B, D, and F).
The affinities of Dpl(27-154) and Dpl(27-157) for copper were essentially identical. For Dpl(27-154), a unique binding site would
have a Kd value of 0.16 ± 0.08 µM (Fig. 3B), and for Dpl(26-157), the
analogous value is Kd = 0.20 ± 0.05 µM (Fig. 3D). Remarkably, copper was found to
interact with the 44-mer, Dpl(101-145) with nearly the same
characteristics as seen for the larger fragments; for a unique site,
the Kd for a copper-peptide complex is estimated to
be 0.36 ± 0.04 µM (Fig. 3F).
Residual plots and chi-squared values for the best fits to the data of
Equation 1 were also ascertained for when [P] was set at 1.4 µM, which would hold if two copper ions bound to two
similar sites in one protein or peptide molecule. Smaller deviations
between model and data for the 1:1 model than for the 2:1 model were
apparent in the residual plots (Fig. 3, B, D, and
F, lower panels). Chi-squared values, which
increase with differences between the model and data, were smaller in
all cases for the 1:1 binding model than for the 2:1 model (Fig. 3,
legend). Modeling the stoichiometry at levels of 3:1 and beyond by
increasing [P] during fitting led to progressively poorer agreement
with the data (not shown). Taken together, these results do not support
the hypothesis of two similar copper sites/polypeptide, in agreement
with the absence of palindromic or repeated amino acid sequences in
Dpl. These data are compatible with an interpretation that each mole of
protein binds 1 mole of copper at a single predominant site under the
particular conditions of the assay. However, the presence of a weaker
binding site or of a second binding site associated with a
metal-peptide complex with different fluorescent properties cannot be
excluded based solely upon these fluorescence titrations.
To evaluate metal binding selectivity of the Dpl protein, we calculated
the fluorescence intensity change of 0.7 µM
Dpl(27-154) in the presence of 3 µM divalent
cations, e.g. Cu(II), Zn(II), Ni(II), Mg(II), Ca(II), and
Mn(II). Whereas no significant changes of Trp and Tyr fluorescence
signals were observed for other cations (1.18 ± 0.40% for
Zn(II), 2.85 ± 0.40% for Ni(II), 2.60 ± 0.20% for Mg(II),
2.60 ± 0.18% for Ca(II), and 1.91 ± 0.10% for Mn(II)) (Fig. 3G), Cu(II) induced a 25.85 ± 0.39% quench of
fluorescence intensity (Fig. 3G, first column).
Although we cannot exclude that Dpl complexes (which do not result in
fluorescent changes) with metals other than copper exist, we note that
similar selectivity was found in MALDI-MS analyses (Fig. 2). Also,
these data are strikingly reminiscent of prior studies of selective
interactions between copper and hamster PrP (22).
Copper Binding to Dpl(101-145) Assessed by Equilibrium
Dialysis--
In the equilibrium dialysis experiments, Cu(II) was
presented to 2.5 µM MoDpl(101-145) as a glycine chelate.
Binding data are shown in Fig. 4.
Graphing [bound] versus [Cu]tot yielded a plateau at about 2.5 µM, the same concentration as the
protein used in the experiments. Fitting Equation 2 to the data gives Kd = 0.4 ± 0.2 µM, the same
within experimental error as that determined by fluorescence titration,
and [protein] = 2.49 ± 0.13 µM, very similar to
the concentration of the protein used in the experiment, indicating a
1:1 correspondence between protein monomers and copper-binding sites.
Equilibrium dialysis therefore confirms both that there is one
copper-binding site/monomer and that fluorescence provides a reliable
signal for the rapid measurement of copper binding to Dpl.

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Fig. 4.
Copper binding to Dpl (101-145) peptide
assessed by equilibrium dialysis. A plot of the concentration of
the MoDpl-copper complex ([bound]) versus the total
concentration ([Cu]tot), where and [bound] = [Cu]retentate [Cu]dialysate. Copper was
presented to peptide in the form of a copper:glycine2
chelate, and the peptide concentrations were 2.5 µM
throughout. The solid line represents the best of Equation 2
to the data with Kd = 0.4 ± 0.2 µM and [protein] = 2.49 ± 0.13 µM.
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DEPC Footprinting of Dpl(27-154)--
In a further
technique to interrogate copper binding by Dpl(27-154), we exploited
the ability of copper to protect certain reactive amino acid residues
(predominantly histidine) from chemical modification with DEPC (1).
Upon incubation of native protein (15 µM) (Fig.
5A) with DEPC, up to nine DEPC
adducts were detected per mole of Dpl(27-154) (Fig. 5B),
assuming an increment of 72.06 Da/mono-carbethoxylated adduct. In the
case of protein preincubated with a molar excess of copper, there was a
shift of n
1 in the spectrum of adducts (Fig.
5C), indicating that one DEPC-reactive residue was protected
from modification by the presence of copper.

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Fig. 5.
DEPC footprinting of native Dpl(27-154).
A, unreacted protein. B, mass spectrum of
DEPC-modified Dpl(27-154) protein. C, mass spectrum
of DEPC-modified protein preincubated with Cu(II).
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Copper Binding in the Region of Dpl(122-136)--
Chymotrypsin
digestion was used to map the location of Cu(II)-binding site(s)
detected by the prior MALDI-MS analysis of Dpl(101-145). In the
absence of Cu(II) (Fig. 6A), a
prominent peak at m/z 1910.05 correspond to a
partially digested chymotryptic peptide containing amino acid sequences
122-136 (C3 + C4 + C5 + C6, calculated 1910.03 Da). An additional peak
at m/z 1032.70 corresponds to 137-145 (C7 + C8,
calculated 1032.58 Da; not shown), whereas the peak at 3286.31 corresponds to disulfide-linked (101-121)-S-S-(137-145) dipeptide. In
the presence of Cu(II), two new peaks were observed at
m/z 1973.50 and 2036.07 Da, indicating that up to
two copper ions can bind to the area between amino acids 122 and 136. This chymotryptic peptide has the sequence SREKQDSKLHQRVLW. Mass
increments upon addition of the first and second Cu(II) ions were 62.69 and 62.57 Da, respectively (Fig. 6B), yielding a net
increment of 125.26 Da, in close agreement with the figure of 124.60 Da
obtained for intact Dpl(101-145) peptide.

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Fig. 6.
Copper binding and DEPC footprinting of
Dpl(101-145)-derived chymotryptic peptides. A, chymotryptic
peptides. B, chymotryptic peptides observed with samples
subsequent to preincubation with Cu(II). Note the "additional"
peaks adjacent to the peak at m/z 1910.81, compatible with one or two copper adducts (1Cu and
2Cu, respectively) and invariance of the disulfide
cross-linked chymotryptic fragments. C, chymotryptic digest
of DEPC-reacted peptide. The prominent peak at
m/z 1910.08 in A and B is
diminished in amplitude, and two new peaks of mono-carbethoxylated
peptide (1m and 2m) are apparent at
m/z 1982.09 and 2055.08. D,
Cu(II)-protected DEPC reacted peptide analyzed with chymotrypsin. The
intensity of the 1m peak is diminished concomitant with the
reappearance of the 122-136 peptide fragment and 1- and
2-Cu-derived complexes.
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Histidine-dependent Copper Binding in
Dpl(101-145)--
DEPC footprinting was also used to assess the
histidine-dependent copper coordination within the
Dpl(101-145) peptide. Dpl(101-145) (90 µM) in NEMO-KCl
buffer, pH 7.4, was incubated with 5-fold excess DEPC and then digested
with chymotrypsin. Compared with the signal in Fig. 6A, the
signal at m/z 1910.09 corresponding to unmodified
residues 122-136 (C3 + C4 + C5 + C6, calculated 1910.03 Da) was
reduced in amplitude (Fig. 6C). Two new peaks were found at
m/z 1982.09 and 2055.08 and indicated one
mono- and two mono-carbethoxylations of fragment 122-136 (calculated 1982.09 and 2054.14 Da, respectively). A different mass spectrum was
obtained when Dpl(101-145) peptide was preincubated with 10-fold excess Cu(II) prior to reaction with DEPC (Fig. 6D). Here
the major peaks were at m/z 1910.34, 1973.28, 2036.21, 1982.38, and 2046.31. Diminution of the mono-carbethoxylated
peak at 1982.09 and an increase in the peak corresponding to unmodified
122-136 C1 + C2 + C3 peptide (compare with Fig. 6D)
indicated that a site of potential DEPC modification was blocked by
copper binding. As per Fig. 6B, peaks at
m/z 1972.53 and 2036.03 are indicative of the
122-136 peptide with one or two bound copper ions. Additionally, a
peak at 2046.31 is likely equivalent to one copper plus one mono-carbethoxylation (calculated 2045.59 Da) (Fig. 6D).
These data can be reconciled with hypotheses that (i) a second
copper-binding site within Dpl(101-145) is independent of the
His131 residue or (ii) binding of a second copper ion
involves the second nitrogen in the imidazole ring, with this nitrogen
being unmodified in the mono-carbethoxylated adduct. Also, in contrast
to Fig. 6B, instead of a peak at m/z
3286.84 Da corresponding to fragment (101-121)-S-S-(137-145), a new
peak at m/z 3358.53 Da corresponding to one
mono-carbethoxylation (calculated 3358.71) indicates one DEPC
modification within this disulfide-bridged fragment (Fig. 6C). No change was observed in this peak (3358.83 Da) in
copper and DEPC-treated versus DEPC-treated samples (Fig.
6D), indicating that Cu(II) did not interact with this fragment.
Fine Mapping DEPC Modification of His131 within
Dpl(122-136)--
The post-source decay technique of tandem analysis
was used to verify DEPC modification and copper protection of
His131. Partial post-source decay spectra of fragment
122-136 and its counterpart in DEPC- and copper-DEPC-treated samples
are presented in Fig. 7. Fig.
7A shows the scheme of nomenclature for peptide post-source
decay fragment ions of chymotryptic peptide 122-136 derived from
intact Dpl(101-145). In unmodified samples, a peak at
m/z 109.9 corresponds to an immonium histidine
ion (His131) (calculated 110.0 Da) (Fig. 7B).
After DEPC treatment, a new peak at m/z 181.7 indicated one mono-carbethoxylation of histidine (calculated 182.0 Da)
(Fig. 7C). Incubation of the sample with Cu(II) prior to
DEPC treatment resulted in the disappearance of one
mono-carbethoxylation of histidine and indicates that Cu(II) coordination to this histidine protected the imidazole ring from DEPC
modification (Fig. 7D). It is also of note that peaks at m/z 128.7, corresponding to an immonium arginine
ion (Arg123 or Arg133) (calculated 129.1 Da),
and at m/z 226.9, corresponding to a b2-NH3 ion (SR-NH3, calculated
227.1 Da) were always present (Fig. 7, B-D), indicating
that neither fragment ion reacted with DEPC or Cu(II). Another peak for
the y9 SKLHQRVLW ion at m/z 1166.7 (calculated 1166.7 Da) in the unmodified sample (Fig. 7B)
was replaced by a signal at m/z 1239.6, corresponding to one mono-carbethoxylation of the peptide ion
(calculated 1238.8 Da) in the DEPC-treated sample (Fig. 7C).
Incubation of the sample with Cu(II) before DEPC treatment led to the
disappearance of the mono-carbethoxylation signal and replacement by a
signal at 1230.9 (calculated 1230.2 Da), indicating the binding of one
copper ion. In sum, the results demonstrate that Cu(II) coordination to
a y9 ion protected the peptide ion from DEPC modification
and, in turn, the involvement of His131 in coordinating
copper ions in the context of the peptide. Given the similarities
between the peptide and protein in terms of their sequences, copper
affinities, and fluorescence changes in response to copper binding,
these results suggest that His131 will coordinate copper in
the context of the full-length protein as well.

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Fig. 7.
Mapping of DEPC modification and
Cu(II)-binding site in Dpl by post-source decay analysis. A,
scheme of nomenclature of chymotryptic peptide Dpl(122-136) for
post-source decay peptide fragment ions. B-D, tandem
post-source decay mass spectra of chymotryptic peptide Dpl(122-136)
(B), chymotryptic carbethoxylated peptide Dpl(122-136)
(C), and chymotryptic carbethoxylated copper-peptide
Dpl(122-136) complex (D). m,
monocarbethoxylation.
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Dpl Expression and Oxidative Damage--
The proposal that Dpl
expressed in the central nervous system is a source of oxidative damage
(35) has a potential parallel in the notion that Dpl is metallated
in vivo and thus may be a source of free radicals. Prior
analyses in this area examined the Dpl-expressing Rcm0 line
of Prnp0/0 mice (35). To address Dpl as a source
of oxidative damage in vivo, we exploited Tg(Dpl) mice
created with the hamster PrP gene promoter, exhibiting potent central
nervous system expression of Dpl and ataxia at about 1 year of age (7).
Brain homogenates of non-Tg and Tg(Dpl)10329 mice were subjected to
polyacrylamide gel electrophoresis and probed with antibodies to known
markers of oxidative stress. As seen in Fig.
8A, Tg(Dpl) mice express Dpl
in the central nervous system, as demonstrated by the strong heterodisperse signals at ~34 kDa, when compared with non-Tg
controls. When samples of the same homogenates were analyzed for
nitrotyrosine modification of proteins using an anti-nitrotyrosine
antibody, low signal intensities were obtained, and no obvious
differences in the levels of nitrotyrosine formation between Tg(Dpl)
and non-Tg mice (Fig. 8B) were found. Furthermore,
modification of carbonyl groups, another direct indication of oxidative
stress, also appeared to be unrelated to increased doppel expression
(Fig. 8C). Quantification of this blot for reactive carbonyl
groups is presented in Fig. 8D, revealing no significant
differences between the Tg(Dpl) and non-Tg sample groups
(p = 0.97).

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Fig. 8.
Protein nitrosylation and carbonylation in
Tg(Dpl) mice. A, analysis with Dpl antibody. Non-Tg Dpl
(lanes 1 and 2) and Tg(Dpl) (lanes 3 and 4) mouse brain homogenates immunoblotted with the
anti-Dpl E6977 antibody demonstrate the relative levels of Dpl
expression in the samples used. Cross-reactive bands between 78 and 210 kDa present in all samples served to demonstrate uniform sample
loading. B, analysis of nitrotyrosinylation. Lane
1, peroxynitrate-treated wild type positive control
(+Ctl) brain homogenate. Non-Tg (lanes 2 and
3) and Tg(Dpl) (lanes 4 and 5) brain
homogenates were probed with an anti-nitrotyrosine antibody. These
analyses demonstrated similar, very low levels of immunoreactivity
(indicative of nitrotyrosine modification) in non-Tg and Tg(Dpl) mice.
C, analysis of protein carbonyl groups. 15-µg samples of
non-Tg (lanes 1 and 2) and Tg(Dpl) (lanes
3 and 4) total brain protein was derivatized with
2,4-dinitrophenyhadrazine and immunoblotted to detect relative levels
of reactive carbonyl groups. Underivatized control samples yielded no
signal (not shown). D, quantitative representation of
carbonyl immunoreactivity via phosphorimaging analysis. A paired
t test yielded a p value of 0.97. There is no
significant difference between the non-Tg and Tg samples.
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DISCUSSION |
Metal Binding to Dpl in Vitro--
Many studies have documented
binding of one copper ion/histidine containing octarepeat within PrP,
when measured at neutral pH (Ref. 36 and references therein). In
addition to these four sites, there is growing agreement upon the
existence of a fifth copper site within PrP located slightly C-terminal
to the octarepeats and likely involving histidines 95 and 111 (mouse
PrP numbering scheme) (1, 37-39). Although sequence alignments reveal
that none of these residues have an equivalent in Dpl, our in
vitro studies have revealed, nonetheless, that Dpl also possesses
copper binding properties.
It is unlikely that the copper binding detected in our studies has a
trivial origin. Although contaminants from E. coli capable of binding copper can be hypothesized, they would have to be present in
substantial quantities to account for the observed stoichiometry of
1
mol of copper/mol of polypeptide. We also note that similar fluorescence quenching properties were produced by two forms of recDpl
prepared by different chromatographic procedures. Furthermore, a
Dpl(101-145) peptide synthesized in vitro also had the
ability to bind copper (Fig. 2), as did a Dpl(122-136) proteolytic
derivative of this peptide (Fig. 6). Another notable feature of the
interaction with copper is the degree of specificity. Assessed by
fluorescence quenching, the Dpl substrates exhibited no discernable
affinity for other divalent cations, a pattern of selectivity
reminiscent of PrP itself (1, 20, 22). Although it is possible that peptide complexes formed by metals other than copper have fluorescent properties similar to that of Dpl apo-protein and thus appear not to
interact in this assay, the similar pattern of metal specificity derived from MALDI-MS analysis (Fig. 2) tends to argue against this
interpretation and in favor of an intrinsic metal selectivity.
Our experiments demonstrate that Cu(II) binding to recDpl can be
largely attributed the
B/B'-loop-
C domain; using fluorescence quenching, Kd values for full-length protein and
Dpl(101-145) differ only by a factor of two. Binding assessed directly
by equilibrium dialysis and indirectly by fluorescence quenching
analysis can be accounted for by a single-site model, with the
additional site detected by MALDI-MS analysis, perhaps reflecting
contribution of a low affinity site only filled by high concentrations
of Cu(II). In the NMR structure for Dpl (32), the
B/B'-loop-
C
domain lies at the opposite end of the molecule from the free C
terminus (the site of the glycosylphosphatidylinositol anchor addition) and may therefore be displayed toward the extracellular environment in vivo. The notion of copper-binding sites within the
-helical domain of a cellular prion protein has a precedent from
studies of PrP (40). Of potentially greater importance, this region of
the Dpl molecule encompassing a kinked helix B (
B/B') contributes to
a triangular hydrophobic pocket with no exact equivalent in PrP (32).
It will be of interest to determine the details of how amino acid
residues in this vicinity contribute to copper binding. The observation
that a cluster of fCJD mutations in the helix B-loop-helix C region of
PrP recapitulate conserved residues in Dpl provides further impetus for
deciphering the biological properties of this region of the protein
(34).
Regarding the issue of binding affinity, some of our analyses use
copper in the form of CuCl2. Such solutions contain oxy and
hydroxy polymers of copper, which may be kinetically inert (41).
Accordingly, some studies of PrP have instead used copper presented in
the form of glycine- or histidine-chelated complexes. These experiments
still yield binding at micromolar concentrations of copper, but
back-calculations based upon the dissociation constants of the
metal-amino acid complexes can be used to derive a concentration for
free ionic metal and protein-metal binding constants estimated in the
range of 10
14 M (38). Our equilibrium
dialysis binding analysis of Dpl(101-145) was performed with
glycine-chelated copper and a Kd value for a high
affinity site of 0.4 ± 0.2 µM. Irrespective of the validity of back-calculating ionic copper concentrations from experiments using copper chelates (where ternary complexes of protein/copper/amino acid complexes might also have to be considered (42)), our numerical data (i) are nonetheless quite similar to
estimates of binding constants derived from "uncorrected" binding data for PrP obtained under similar conditions (21) and (ii) illustrate
that Dpl, like PrP, can extract copper from an amino acid chelate, a
form that might better approximate presentation in biological fluids
than in solutions of copper salts. "Exchangeable" copper in plasma
bound to amino acids has been estimated at 3.6 µM,
whereas the total copper levels in plasma and seminal fluid are
measured at 15.5 ± 9 and 5.9 ± 3.7 µM,
respectively (43, 44) and are thus about 1 order of magnitude above the
Kd values presented herein. These data strongly
suggest that metallated forms of Dpl could exist in vivo,
which is of potential relevance to physiological function(s) in
spermatogenesis and also to the pathological effect of central nervous
system expression.
Central Nervous System Expression of Dpl: Oxidative Damage and
Neurotoxicity--
Based upon prior connections between
copper-polypeptide complexes and the generation of reactive free
radical species, we investigated indicators of oxidative damage in
Tg(Dpl) mice (35). Ectopic expression of Dpl in the brain is known to
be toxic, and genetically engineered mice with this property succumb to
an ataxic syndrome characterized by apoptotic death of cerebellar cells (2, 6, 7). Our studies exploited the Tg(Dpl)10329 line of mice (7). In
contrast to another study (35), we found no evidence of enhanced
oxidative damage to brain proteins (Fig. 8). This discrepancy cannot be
attributed to the penetrance of the disease phenotype in the
Tg(Dpl)10329 mice studied here. Tg(Dpl)10329 mice develop an ataxic
syndrome at 375 ± 8 days, as compared with Rcm0 mice,
which develop ataxia much later in life at 611 ± 12 days of age.
Furthermore, the Tg(Dpl)10329 mice at age of sacrifice in the analyses
of oxidative markers described here were approximately twice as old as
Rcm0 mice analyzed for markers of oxidative damage (35). It
is possible that the discrepancy between our findings and those of Wong
et al. (35) reflects a contribution of modifier loci
deriving from different genetic backgrounds. Nonetheless, because the
Tg(Dpl)10329 mice described here have levels of protein carbonyl and
nitrotyrosine formation no different from those seen in non-Tg mice
(yet are certainly prone to loss of cerebellar cells), it seems quite
unlikely that the neurotoxic properties of Dpl depend upon causing
oxidative damage to proteins.
Competition between Dpl and PrPC: a Role for
Cu?--
Despite our failure to implicate oxidative damage in the
neurotoxic action of Dpl, it remains plausible that an ability to bind
copper ions is necessary for neurotoxicity proceeding by a different,
but as yet undefined, mechanism. The studies presented here were not
designed to elucidate what this mechanism might be. Nonetheless, by
revealing that the ability to bind copper is a property of both Dpl and
PrPC, they may speak to "competition" between the
PrPC and Dpl proteins in modulating this pathologic process
(7). Specifically, because expression of PrPC can nullify
the neurotoxicity of Dpl to cerebellar cells, the question now arises
as to whether the competing actions of these two
glycosylphosphatidylinositol-linked proteins somehow derive from a
shared predilection for this particular transition metal. Experiments
to recapitulate Dpl-mediated neurotoxicity in a tractable system may
allow us to appraise this possibility.