From the Division of Biomolecular Characterization,
The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama
351-0198, Japan, ¶ Department of Neuropathology, Faculty of
Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and
Core Research for Evolutional Science and Technology
(CREST), Japan Science and Technology Corporation (JST), Kawaguchi,
Saitama 332-0012, Japan
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ABSTRACT |
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An extensive loss of a selected population of
neurons in Alzheimer's disease is closely related to the formation of
paired helical filaments (PHFs). The most striking characteristic of PHFs upon Western blotting is their smearing. According to a previously described protocol (Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993) Neuron
10, 1151-1160), smeared tau was purified, and its peptide map
was compared with that of soluble (normal) tau. A CNBr fragment from
soluble tau (CN5; residues 251-419 according to the 441-residue
isoform) containing the microtubule-binding domain migrated at 15 and
18 kDa on SDS-polyacrylamide gel electrophoresis, whereas that from
smeared tau exhibited two larger, unusually broad bands at ~30 and
~45 kDa, presumably representing dimers and trimers of CN5. In the
peptide map of smeared tau-derived CN5, distinct peaks eluting at
unusual locations were noted. Amino acid sequence and mass
spectrometric analyses revealed that these distinct peptides bear
isoaspartate at Asn-381 and Asp-387. Because no unusual peptides other
than aspartyl or isoaspartyl peptide were found in the digests of
smeared tau-derived CN5, it is likely that site-specific deamidation
and isoaspartate formation are involved in its dimerization and
trimerization and thus in PHF formation in vivo.
One of the neuropathological hallmarks of Alzheimer's disease
(AD)1 is the formation of
innumerable neurofibrillary tangles (NFTs) throughout the cortex. NFTs
were originally found by silver staining as intracytoplasmic fibrous
structures surrounding neuronal nuclei and running into the proximal
portion of apical dendrites, thus often providing an image of a flame
(flame-shaped tangle) (1). Smaller NFTs are also formed in dendrites
and senile plaque neurites. Dendritic NFTs largely represent curly
fibers (neuropil threads), and their remarkable abundance in the AD
brain has been uncovered by specific immunostaining (2, 3). The unit
fibrils of NFTs are called paired helical filaments (PHFs), which are
20 nm in diameter and constrict to 10 nm at a periodicity of 80 nm.
This unusual morphology allows us to readily distinguish PHFs from normal neuronal cytoskeletons including microtubules and neurofilaments under an electron microscope. Because NFTs, especially curly fibers, are always dense in areas of neuronal loss, they have been considered to be closely related to neuronal death in AD (4). The neuronal loss,
in turn, is closely correlated with the degree of dementia, the
essential symptom for making the diagnosis of AD (4).
The major components of PHFs are tau and ubiquitin (5-7), the former
of which constructs the framework of PHFs (8, 9). The striking
characteristics of tau in PHFs are insolubility and hyperphosphorylation. Thus far, the abnormal phosphorylation of PHF-tau
(hyperphosphorylated full-length tau) has been intensively focussed
upon, and the phosphorylation sites have been determined with
immunochemical and protein chemical procedures (10-12). However, it is
now known that the microtubule-binding domain makes up the framework of
PHFs (8), and that phosphorylation in the flanking regions is not
required for the assembly of tau into PHFs in vitro (9).
Thus, the role of hyperphosphorylation of tau in PHF formation remains
to be elucidated. Despite the presence of heparin or heparan sulfate
(13) or RNA (14), a stimulator for PHF assembly, the concentrations of
tau required are too high to apply to the in vivo situation,
and the formed filaments are easily disaggregated. Therefore, we
believe that there should be other factors to promote and stabilize tau
aggregation in vivo.
We have been concerned about the smearing on the blot of the
PHF-enriched fraction using PHFs or tau antibodies (15). The smear on
the blot ranged from the gel top to ~10 kDa, with the high molecular
mass region showing the most intense immunoreactivity. This smear is
not an artifact during electrophoresis but appears to represent a real
molecular form because: (i) it is fractionated according to molecular
size on gel filtration (16), (ii) a close inspection of the blot
reveals that the smear in the low molecular mass region consists of
very fine, closely spaced, discrete bands, and the minimal unit of
reactive bands may migrate at 10-20
kDa,2 and (iii) the smear
reacts strongly with tau antibodies to the carboxyl-terminal portion
but does not react at all or reacts only faintly with antibodies to the
amino-terminal portion.3
Thus, the smear may be composed of carboxyl-terminal fragments that
should have a strong tendency to aggregate into oligomers. This means
that the analysis of the smear should provide us with an important
insight into the assembly of tau into PHFs and their unusual
stabilization in vivo.
There are two kinds of smears: ubiquitin-positive smears and
ubiquitin-negative smears (16). This indicates that ubiquitination is
not responsible for the smearing phenomena. In the present work, the
ubiquitin-negative, smeared tau was subjected to protein chemical
analysis. We show here that the major difference between the soluble
and smeared forms of tau is the presence of structurally altered
asparaginyl and aspartyl residues in the microtubule-binding domain of
the latter. It is possible that isoaspartate formation at specific
sites in the microtubule-binding domain of tau induces a profound
conformational change by which the tau can self-assemble into PHFs and
provides unusual stability in vivo.
Materials--
All reagents for peptide synthesis, namely,
Fmoc-L-amino acids (SynProPep® Reagents SM8-Pack
PB-Mix), Fmoc-D-Asp (OtBu)-OH, and MAPS resins
(TAKO8-Cys-WTGS), were obtained from Shimadzu (Kyoto, Japan).
Fmoc-L-Asp-OtBu and Fmoc-L-Lys (Boc)-resin were obtained from Calbiochem-Novabiochem (La Jolla, CA), piperidine was
obtained from Wako Pure Chemical Industries (Osaka, Japan), and
anisole, 1,2-ethanedithiol, N-methylmorpholine, and
piperidine were obtained from Nacalai Tesque (Kyoto, Japan).
Purification of Soluble Tau, PHF-Tau, and Smeared
Tau--
Soluble (normal) tau was purified from normal control or AD
brains according to the protocol described previously (6). Because the
peptide maps of normal soluble tau and AD-soluble tau were
indistinguishable from each other (10), in the present study, we used
AD-soluble tau to compare with smeared tau. PHF-tau and smeared tau
were purified from Sarkosyl-insoluble pellets of AD brain homogenates,
as described previously (10, 16). The Sarkosyl-insoluble pellets were
solubilized with 6 M guanidine-HCl, and the supernatants
were carboxymethylated with iodoacetate after reduction with
dithiothreitol. After gel filtration on a TSK gel G-3000SWXL column
(7.8 × 300 mm × 2 (tandem); Tosoh, Tokyo, Japan), pooled
fractions for PHF-tau or smeared tau eluting earlier than PHF-tau were
further purified on an Aquapore RP300 column (2.1 × 30 mm;
Applied Biosystems; Foster City, CA) by reverse-phase high performance
liquid chromatography (RP-HPLC; model 1090 M; Hewlett-Packard,
Waldbronn, Germany) with a linear gradient of 20-40% acetonitrile in
0.1% trifluoroacetic acid (TFA) for 10 min at a flow rate of 0.2 ml/min (10, 16). Thus, the smear that has a molecular mass larger than
that of PHF-tau was subjected to protein chemical analysis. By using
this step, ubiquitin-negative, smeared tau was largely separated from
ubiquitin-positive, smeared tau that eluted later (16).
Cyanogen Bromide and Protease Digestion--
Purified soluble
tau, PHF-tau, and smeared tau were cleaved with CNBr, and the fragments
generated were separated by HPLC on a Superspher Select B column
(2.0 × 119 mm; Merck, Darmstadt, Germany), which was developed
with a linear gradient of 16-44% acetonitrile in 0.1% TFA. The
ubiquitin-negative CNBr fragment was further separated from the
contaminating ubiquitin-positive fragment that eluted later from CN5
(see Fig. 2). The absence of ubiquitin was confirmed by Western
blotting with an anti-ubiquitin antibody. The two CNBr fragments
bearing many phosphates, CN2 (residues 128-250) and CN5 (251-419),
were treated with alkaline phosphatase (Escherichia coli,
type IIIR; Sigma). Dephosphorylated CN2 and CN5 were digested with
Achromobacter lyticus protease I (API), as described
previously (10, 16). The digests were separated by RP-HPLC on a
Superspher Select B column with a linear gradient of 0-48%
acetonitrile in 0.1% TFA for 48 min at a flow rate of 0.2 ml/min. The
API peptide bearing the tau-1 portion (residues 191-225) from CN2 was
further digested for 15 h at 37 °C with chymotrypsin (Cooper
Biomedical) in 50 mM Tris-HCl (pH 8.0) at an
enzyme:substrate ratio of 1:50 (w/w). The digest was separated under
the same conditions as described above. All of the separated peptides
were subjected to protein sequencing and mass spectrometry (see below).
Dephosphorylated CN5 was electrotransferred onto a polyvinylidene
difluoride membrane (Immobilon; Nihon Millipore Ltd., Yonezawa, Japan)
after SDS-PAGE. After staining with Coomassie Brilliant Blue, each
broad band at ~45 or ~30 kDa was excised from the membrane and
blocked with 2% polyvinylpyrrolidone-40 in methanol for 30 min (17).
After rinsing the membrane several times with distilled water, each
band on the membrane was digested with API at a 1:50 enzyme:substrate
ratio at 37 °C overnight. The digest was similarly separated as
described above.
Amino Acid Sequence and Mass Spectrometric Analyses of
Peptides--
Fractionated peptides were sequenced on an Applied
Biosystems 477A Protein Sequencer equipped with an on-line 120A PTH
Analyzer or on an Applied Biosystems 473A Protein Sequencer, as
described previously (10). Mass spectrometric analysis was performed by matrix-assisted laser desorption ionization time of flight mass spectrometry on REFLEX (Bruker-Franzen Analytik, Bremen, Germany) equipped with a delayed extraction or Voyager-DERP (PerSeptive Biosystems, Framingham, MA). Samples were prepared by mixing
DL-Amino Acid Analysis--
The samples were dried
and hydrolyzed in a vapor of 6 N HCl containing 0.1% (w/v)
phenol at 108 °C for 6 h, conditions under which most of the
Asp residues are released from the peptide bond (18). After drying in a
vacuum centrifuge, the samples were dissolved in 10 µl of 100 mM borate buffer at pH 9.0 and incubated with 7.5 µl of
acetonitrile and 2.5 µl of 18 mM (+)-1-(9 fluorenyl)ethyl chloroformate (Eka Nobel AB) in acetone at room temperature for 25 min.
The reaction was terminated by the addition of 10 µl of 100 mM cysteic acid dissolved in 100 mM borate
buffer at pH 9.0. The sample solution was mixed with 14 µl of 9 N acetic acid and 56 µl of sodium acetate buffer (pH
3.7), and then a 20-µl aliquot was subjected to RP-HPLC (LC-10AVP;
Shimadzu) on a Superspher® 100 RP-18(e) column (4 µm; 4 mm × 24 cm; Merck) equilibrated with Buffer A (0.1 M sodium
acetate (pH 4.22):acetonitrile:tetrahydrofuran (19:3:3, v/v) and 0.1 mM EDTA). Elution was conducted with a stepwise increase in
the proportion of Buffer B (0.1 M sodium acetate (pH 4.75):acetonitrile:tetrahydrofuran (4:3:3, v/v) and 0.1 mM
EDTA) at a flow rate of 0.6 ml/min (19, 20). The processes of
derivatization to analysis were automated for reproducible
results.4 Independent
multiple samples were analyzed for each protein or fragment.
Peptide Synthesis--
Each synthetic peptide consisting of
residues 386-395 (TDHGAEIVYK) with the second position being replaced
by L-Asp, L-isoAsp, or D-Asp was
synthesized by the solid-phase method with
N Antibodies and Immunochemical Methods--
The peptide
T(isoD)HGAEIVY was synthesized on a branched lysine carrier core. An
antiserum against the peptide was raised using the multiple antigen
peptide method (22). A New Zealand White rabbit was immunized with 1 mg
of multiple antigen peptide and boosted four to five times with 0.5 mg
at 2-week intervals, starting 4 weeks after the first immunization.
Titers of the antisera were assessed by enzyme-linked immunosorbent
assay and dot blotting.
The antibodies used were 5E2 (epitope, residues 214-233, according to
the longest human tau isoform) (23, 24), tau-C4 (residues 354-369)
(6), tau-C6 (residues 420-430) (6), DF2 (a monoclonal antibody that
recognizes ubiquitin) (7), and anti-human tau (residues 68-73 and
103-130) (25).
Western blotting was performed using the avidin-biotin method
(Vectastain ABC kit; Vector Laboratories, Burlingame, CA), as described
previously (10, 16).
Smeared Tau Consisted Mainly of Amino-terminally Processed
Tau--
The levels of soluble tau were significantly decreased in the
AD brain as compared with the normal brain (Fig.
1A). On the other hand, tau
was undetectable in the Sarkosyl-insoluble pellets of normal brain
homogenates by the immunochemical procedure used (Fig. 1A),
whereas the Sarkosyl-insoluble pellets of AD brain homogenates
contained a large amount of tau; the tau in these pellets exhibited
slowly migrating triplets (PHF-tau) and a smear on the blot (Fig.
1A). Whereas PHF-tau was immunoreactive with all of the
antibodies (their epitopes spanned the whole tau molecule), the smear
reacted to lesser extents with antibodies to the amino-terminal half of
tau (Fig. 1A; see Fig. 5 for the locations of their
epitopes).3
The Sarkosyl pellets from AD brain were solubilized with 6 M guanidine hydrochloride, and the solubilized sample was
subjected to gel filtration (10, 16). The fractions eluting earlier than PHF-tau and those containing PHF-tau were pooled separately, and
each pool was subjected to RP-HPLC (Fig. 1B). PHF-tau was recovered as a distinct peak (Fig. 1B, a), whereas the pool
rich in high-mass smear provided very broad, rather indistinct peaks (Fig. 1B, b). An early-eluting peak represents
ubiquitin-negative smeared tau, whereas a late-eluting peak represents
ubiquitin-positive smeared tau (Ref. 16; see also Fig.
2C). Thus, the smear we have
characterized here represents only a part of smeared tau: the smear
with molecular masses larger than that of PHF-tau.
Purified soluble (normal) tau and smeared tau were cleaved with CNBr,
and the generated fragments were separated on a RP column (Fig.
2A). Two large CNBr fragments, CN2 (residues 128-250) and CN5 (residues 251-419), emerged (see Fig. 5 for the sequence of tau)
(6). CN5 contains the microtubule-binding domain (residues 256-367),
whereas CN2 contains the tau-1 portion (residues 191-225). The CN5
from smeared tau eluted slightly later and showed some tailing as
compared with that from soluble tau (Fig. 2A). These two
major fractions containing CN2 and CN5 were treated with alkaline phosphatase and subjected to SDS-PAGE and Western blotting (Fig. 2,
B and C).
The CN2 fragments from soluble tau and smeared tau were examined for
their reactivities with anti-human tau, 5E2, tau-C4, or DF2 (Fig.
2B). The CN2 fragments from soluble and smeared tau on the
blot showed a discrete band and a somewhat indistinct band with a
minimally retarded mobility, respectively (Fig. 2B). CN5 from soluble tau (lane a in the tau-C4 blot) consisted of
two discrete bands at ~18 and ~15 kDa (Fig. 2C). These
are most likely derived from four-repeat and three-repeat tau,
respectively. In contrast, CN5 from smeared tau showed two very broad,
major bands at ~45 and ~30 kDa, in addition to the two
aforementioned bands that were only faintly labeled. These broad bands
were unusually stable and never converted to the bands at ~18 kDa or
~15 kDa by pretreatment with concentrated formic acid (26) or neat
trifluoroacetic acid (27) (data not shown). As expected from the
purification procedure, the two broad bands showed no immunoreactivity
with DF2 (Fig. 2C). Thus, it is quite reasonable to
speculate that the smearing on the blot of PHF-enriched fractions is
largely due to alterations in the CN5 region containing the
microtubule-binding domain.
The presence of these larger molecular forms after CNBr cleavage may
raise the possibility that some unknown modifications cause incomplete
CNBr digestion of tau. In fact, a smear of broad bands above ~40 kDa
is detected in lane b in the 5E2 blot or the tau-C6 blot,
respectively (Fig. 2C). However, it is most likely that
these bands represent CN2 or CN8 (residues 420-441) attached with CN5
due to incomplete CNBr cleavage, respectively. The presence of the
larger CN2-CN5 fragment is clearly indicated in lane a of
the 5E2 blot (Fig. 2C). This view is also supported by the observation that the API digest of the larger CN5 from smeared tau
contains essentially the same peptides found in the digest of the CN5
from soluble tau (see below and Table
I).
According to recent in vitro experiments, three-repeat and
four-repeat tau make up paired helical filaments and straight
filaments, respectively (13). Because it is known that the
microtubule-binding domain constructs the framework of these filaments,
it can be speculated that the products at ~45 and ~30 kDa may be
composed of homodimers of four-repeat and three-repeat tau-derived CN5, respectively. However, this is not the case; the peptide mapping of the
two bands at ~45 and ~30 kDa clearly showed that they both contained three-repeat and four-repeat domain-derived peptides (data
not shown). Furthermore, these two HPLC profiles were very similar
(data not shown), strongly suggesting that the ~45-and ~30-kDa
proteins represent multimers of the minimal unit.
Deamidation and Isoaspartate Formation in CN2 and CN5 from Smeared
Tau--
Each of the CNBr fragments (CN2 and CN5) purified from
soluble and smeared tau was digested with API, and the generated
peptides were separated by RP-HPLC (Figs.
3A and 4A). There
were distinct differences between the soluble and smeared tau peptide
maps. Peak 8 in the CN5 smear was markedly reduced as compared with that in the normal CN5. This is presumably because the peptide it
contained, IGSTENLK, is mostly phosphorylated in the CN5 smear (see
Table I, footnote c), and the phosphorylated form elutes earlier than peak 8. Peaks a10 and a13 in the map of CN5 from smeared
tau are not found in the map of that from soluble tau (Fig.
3A). Because these peaks were also present in the map of PHF-tau (Fig. 3A, b, peaks b10 and b13), their presence
should also characterize PHF-tau. Therefore, these two peaks were
subjected to mass spectrometric and protein sequence analyses (Fig.
3B).
In peak a10, a signal of (M+H)+ monoisotopic ion was
detected at m/z 1132.5 (Fig. 3B). This
corresponds to the calculated molecular mass (1132.6 atomic mass units)
of TDHGAEIVYK (residues 386-395), but the sequencer provided no
significant signal after the first cycle. In peak a13, a signal of
(M+H)+ monoisotopic ion was detected at m/z
979.3. This seemed to be slightly larger than the calculated (978.5 atomic mass units) or observed molecular mass (978.3 atomic mass units)
of LTFRENAK (residues 376-383) in peak a12. Sequencing of the peptide
in this peak provided no significant signal after the fifth cycle.
Normally, TDHGAEIVYK elutes at the position of peak c11, and LTFRENAK
elutes at the position of peak c12 (see Fig. 3A, c). These
findings strongly suggest that Asn-381 and Asp-387 are converted to
isoAsp, resulting in anomalous elution and resistance to Edman degradation.
To further confirm the abovementioned observations, each synthetic
TDHGAEIVYK peptide with the second residue replaced by L-Asp, L-isoAsp, or D-Asp was
subjected to HPLC (Fig. 3C). The isoAsp peptide eluted at a
position exactly corresponding to that of peak a10 in Fig.
3A. With the solvent system consisting of 0.1% TFA in water
and acetonitrile, the isoaspartyl peptide (peak a10) usually elutes
before the aspartyl peptide (peak a11) (28), and the asparaginyl
peptide (peak a12) typically elutes close to (either before or after)
the corresponding isoaspartyl form (peak a13) (28).
It is also noted that the height of one peak from smeared tau (peak a15
in Fig. 3A) was remarkably decreased as compared with that
of the corresponding peak from soluble tau (peak c15 in Fig. 3A). These two peaks (peaks a15 and c15) gave the same
sequence, SPVVSGDTSPRHLSNVSSTGSID-Hse> or -Hse (residues 396-419;
Hse>, homoserine lactone; Hse, homoserine), by both mass spectrometry and protein sequencing (Table I). A few small peaks close to peak a15
were found to contain peptides having the same amino-terminal sequence,
SPVV ... (396-) (see Table I). One of the reasons for low recovery
of the peptide could be incomplete removal of the phosphate (10, 12).
The presence of multiple peaks in the peptide map of CN5 from smeared
tau could be due to various numbers of phosphates remaining, different
terminal residues (homoserine, homoserine lactone, or carboxyl-terminal
truncation), and deamidation and/or isomerization of Asn-410 and/or
Asp-402 and Asp-418 in the peptide. All the remaining peaks were
subjected to mass spectrometry and protein sequencing, which revealed
no significant differences between soluble and smeared CN5 (see Table
I).
With regard to CN2, one peak in the peptide map of smeared tau was
missing when compared with that of soluble tau (peak c1 in Fig.
4A). Peak c1 gave signals
corresponding to GQANATRIPAK (residues 164-174) by mass spectrometry
and protein sequencing. Peak a2 or c2 eluting immediately after peak c1
contained the deamidated form of GQANATRIPAK. Thus, Asn-167 is largely
deamidated in smeared tau, whereas this residue is only partially
deamidated in soluble tau (10). The poor recovery of PTH-Asp in the
third cycle of peptides in peak a3 suggests that Asp-193 is partially converted to an isoaspartyl residue. To investigate this further, peak
a3 from smeared tau and peak c3 from soluble tau were digested with
chymotrypsin, and the generated peptides were separated (Fig. 4A,
b and d). Peaks b1 and b2 from smeared tau and peak d2
from soluble tau gave the same signals corresponding to the calculated molecular mass (741.3 atomic mass units) of SGDRSGY (191-197) by mass
spectrometry (Fig. 4B), whereas peak b1, but not peak b2 or
d2, provided no significant signal after the second cycle (data not
shown). These findings strongly suggest that Asp-193 of the b1 peptide
is isomerized; thus, Asp-193 in smeared tau is partially converted to
isoAsp.
The proportion of isoaspartyl residues in each corresponding peptide
was roughly estimated. With regard to Asp-193 and Asp-387, they were
calculated from the HPLC profiles (Figs. 3A and
4A) on the assumption that each peptide was recovered at the
same yield from the RP column. With regard to Asn-381, the proportion of isoAsp was estimated from the yields in Edman degradation before the
blocked cycle. The proportions of isoAsp for these peptides from
smeared tau were about ~24%, ~30%, and ~50% for Asp-193, Asn-381, and Asp-387, respectively.
The sequence of tau and the locations of the major
deamidation/isomerization sites and the antibody epitopes are shown in Fig. 5.
Racemization of Smeared Tau and Soluble Tau--
The proportions
of D-Asp (D-Asp:L-Asp + D-Asp) were 12.2 ± 0.6% (n = 4),
9.9 ± 1.1% (n = 3), 6.0 ± 0.5%
(n = 4), and 5.9 ± 0.5% (n = 3)
for smeared tau, PHF-tau, AD-soluble tau, and normal soluble tau,
respectively. Thus, in terms of racemized Asp content, there was no
difference between AD-soluble tau and normal soluble tau. The
proportions of D-Asp in CN5 were 8.4 ± 1.2%
(n = 4), 7.0 ± 0.7% (n = 3),
4.1 ± 0.6% (n = 4), and 4.4 ± 0.5%
(n = 3) for smeared tau, PHF-tau, AD-soluble tau, and
normal soluble tau, respectively. Under the same conditions, the
proportions of D-Asp were 2.4 ± 0.2%
(n = 2) for lysozyme and 2.0 ± 0.2%
(n = 2) for bovine serum albumin. These values can be
considered to represent hydrolysis-induced racemization. Similarly, the
proportions of D-Asp in CN2 were 18.8 ± 2.2%
(n = 3), 18.9 ± 2.5% (n = 3),
9.8 ± 2.2% (n = 4), and 11.1 ± 1.4%
(n = 3) for smeared tau, PHF-tau, AD-soluble tau, and
normal soluble tau, respectively. This strongly suggests that the
extent of racemization in the CNBr fragments is not related to their
oligomerization. The relative levels of D-Asp determined in
this study in PHF-tau, AD-soluble tau, and soluble tau are
significantly higher than those reported previously (29-31).
Immunocytochemical Analysis of AD Brain with an isoAsp-387-specific
Antiserum--
An antiserum was raised against T(isoD387)HGAEIVYK
using the multiple antigen peptide method (22). This antibody intensely stained innumerable neurofibrillary tangles and curly fibers in a
formalin-fixed paraffin-embedded AD section (Fig.
6A). To assess its
specificity, the antiserum was preabsorbed with each synthetic peptide
with the second residue replaced by L-Asp (Fig.
6B), L-isoAsp (Fig. 6C), or
D-Asp (Fig. 6D). Although preabsorption with the L-Asp peptide minimally decreased the intensity of
immunoreactivity, most of the specific staining remained (Fig.
6B). In contrast, preabsorption with the
L-isoAsp or D-Asp peptide largely eliminated (Fig. 6C) or greatly reduced (Fig. 6D) the
specific immunoreactivity, respectively. Because both
L-isoAsp and D-Asp are recognized by protein
L-isoaspartate (D-aspartate)
O-methyltransferase (EC 2.1.1.77), the similar extents of
absorption may be explained by their conformational similarities (32,
33). Thus, we have shown that tau in neurofibrillary tangles and curly
fibers in vivo is isomerized at Asp-387 residues. This
indicates that isoaspartyl formation at Asp-387 is not an artifact that
may have occurred postmortem and/or during the preparation of smeared
tau.
The most significant finding in the present work is that the CN5
obtained from the smeared tau migrated on SDS-PAGE as very broad,
smear-like bands at ~45 and ~30 kDa, whereas the CN5 from soluble
tau showed discrete bands at 18 and 15 kDa, as expected. The larger,
smear-like CN5 from smeared tau generated peaks distinct from those of
soluble CN5 by API digestion (Fig. 3A). The distinct peaks
are found to be due to deamidation and/or isomerization of Asn-381 and
Asp-387, and all of the remaining peaks contained the expected peptides
and no other unusual peptides suggesting the involvement of cross-links
(Table I). In particular, we can exclude the presence of Lys-involved
cross-links in the CN5 smear, because API cannot cleave Lys-X when the
Isoaspartate is mainly formed by spontaneous intramolecular deamidation
at Asn-X (36, 37). This reaction occurs most often at
Asn-Gly and Asn-Ser when they are located in flexible peptides or
domains of proteins (38-40). Besides these sites, deamidation also
occurs in many other sequences including Asn-Asp (41, 42), Asn-Glu
(43), Asn-His (44), Asn-Ala (45), Asn-Ile (46), and Asn-Met (45, 47).
In CN5, there are 10 potential deamidation sites (see Fig.
5); seven Asn residues are followed by
bulky hydrophobic residues including Val, Leu, and Ile, and two are
followed by Lys, one of which is partially deamidated (Asn-279), and
one (Asn-381), the major deamidation/isomerization site identified
here, is followed by a small residue, Ala. Isoaspartate can also arise
from direct isomerization of Asp (36, 37). There are 10 Asp sites for potential isomerization in CN5 (see Fig. 5); 5 are followed by bulky
residues including Leu, Phe, and Met, 2 are followed by Asn, 2 are
followed by basic residues, one of which is the major isomerization
site (Asp-387), and the remaining site is followed by a hydroxyl
residue. Thus, deamidation and isomerization sites in CN5 generally
follow the principle obtained using various synthetic peptides (38): a
smaller-sized following residue leads to an increased rate of
succinimide formation (Fig. 7) (38).
Similar results have also been obtained with isomerization of the Asp peptide (38). These observations suggest that the affected
microtubule-binding domain of tau may have been unbound from
tubulin.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-cyano-4-hydroxycinnamic acid as a matrix.
Fmoc-amino acids on a PSSM-8 peptide
synthesizer (Shimadzu) using the standard protocol (21). After repeated
washing with methanol and butyl methyl ether, the synthesized peptides
were released from the resin, and side chain-protecting groups were
removed by treatment with a cleavage mixture (94% TFA (v/v):5%
anisole (v/v):1% 1,2-ethanedithiol (v/v)) at room temperature for 2 h. All of the peptides were purified by RP-HPLC on an Aquapore RP300 column and a Superspher Select B column (2.0 × 119 mm; Merck).
RESULTS
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Fig. 1.
Western blots of soluble and
Sarkosyl-insoluble fractions from normal and AD brains with tau
antibodies (A) and purification of PHF-tau and smeared tau
by RP-HPLC (B). A, the same amounts of
Tris-saline-soluble fraction (S) and Sarkosyl-insoluble
fraction (P) from normal and AD brains were run on a 10%
polyacrylamide gel and transferred onto a polyvinylidene difluoride
membrane, followed by immunostaining with tau-C4 (a) or
anti-human tau (b) using the avidin-biotin method. The
levels of soluble tau were decreased in AD brains, whereas the levels
of Sarkosyl-insoluble tau were remarkably increased in AD brains.
tau-C4, but not anti-human tau, reveals an extensive smear in the
Sarkosyl-insoluble pellets from AD brain homogenates. The bands below
44 kDa in a and b probably represent the
degradation products of tau. B, each of the two pools for
PHF-tau and smeared tau obtained from gel filtration in 6 M
guanidine hydrochloride was subjected to RP-HPLC. The pool for PHF-tau
provided a distinct peak (1 in a), whereas the
pool for smeared tau provided very broad, rather indistinct peaks. The
early-eluting peak (2 in b) represents
ubiquitin-negative smeared tau, whereas the late-eluting peak
(3 in b) represents ubiquitin-positive smeared
tau.
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Fig. 2.
HPLC profile (A) and Western
blots (B and C) of CNBr fragments from soluble
tau and smeared tau. A, purified soluble tau and smeared tau
were cleaved with CNBr, and the fragments generated (CN1-CN8) (6) were
separated on a Superspher Select B column that was developed with a
linear gradient of 16-44% acetonitrile in 0.1% TFA. The CN5 from
smeared tau (b) eluted slightly late when compared with that
from soluble tau (a). The asterisk indicates CN8
(residues 420-441). The CN8 from smeared tau eluted earlier, due to
one phosphate presumably at Ser-422 (12), as judged by both mass
spectrometry and protein sequencing (data not shown). B, the
same amounts of CN2 from soluble tau (a) and smeared tau
(b) were run on a 17.5% polyacrylamide gel and transferred
onto a polyvinylidene difluoride membrane, followed by immunostaining
with anti-human tau, 5E2, tau-C4, or DF2, according to the
avidin-biotin method. The CN2 from smeared tau showed a somewhat
indistinct band at ~14 kDa with minimally retarded mobility.
C, the same amounts of CN5 from soluble tau (a)
and smeared tau (b) were analyzed similarly for their
immunoreactivities with 5E2, tau-C4, tau-C6, or DF2. The CN5 from
soluble tau (lane a in the tau-C4 blot) consisted of two
major bands at ~18 and ~15 kDa, probably representing the
four-repeat and three-repeat domains, respectively. In contrast, the
CN5 from smeared tau showed two major smeared bands at ~45 and ~30
kDa, in addition to the two rather discrete bands seen in soluble CN5.
The bands at ~35 and ~32 kDa (lanes a in the 5E2 blot or
tau-C4 blot) presumably represent incompletely digested fragments
containing both CN2 and CN5 (residues 128-419). Similarly, a smear of
broad bands at above 40 kDa in lane b in the 5E2 blot or the
tau-C6 blot is most likely to be smeared CN5 attached with CN2 or CN8,
respectively.
Sequence and MS analyses of the peptides released from smeared CN5 (a)
and soluble CN5 (c)
), but corresponding mass numbers were not
found. As can be seen below, dephosphorylation of smear CN5 is
incomplete. This may be due to oligomerization of smear CN5, a
condition that may prevent free access by phosphatase. The unidentified
peptides by sequencing were NVK (255-257), SK (258-259), Cm-CGSK
(291-294), VTSK (318-321), SEK (341-343), and AK (384-385). These
may have been lost in the breakthrough
fractions.
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Fig. 3.
HPLC profile and mass spectrometric analysis
of CN5-derived peptides. A, CN5 from smeared tau
(a), PHF-tau (b), or soluble tau (c)
was digested with API, and the peptides generated were separated by
RP-HPLC. Peaks a10 and a13 in the map of CN5 from smeared tau are not
detected in the map from soluble tau. Additionally, peak a15 in the map
of smeared tau decreases markedly when compared with c15 in the map of
soluble tau. A decreased height of peaks a8 and b8 is ascribed to
phosphorylation of IGSTENLK (residues 260-267) on Ser-262 in smeared
tau and PHF-tau, respectively. The phosphorylated peptides eluted
earlier. B, mass spectrometric analysis was performed by
matrix-assisted laser desorption ionization time of flight mass
spectrometry with -cyano-4-hydroxycinnamic acid as a matrix. In peak
a10, a signal of (M+H)+ monoisotopic ion was detected at
m/z 1132.5. This value corresponds to the observed (a11;
1132.8 atomic mass units) or calculated molecular mass (1132.6 atomic
mass units) of TDHGAEIVYK (residues 386-395). In peak a13, a signal of
(M+H)+ monoisotopic ion was detected at m/z
979.3. This value is slightly larger than the observed (a12; 978.3 atomic mass units) or calculated molecular mass (978.5 atomic mass
units) of LTFRENAK (residues 376-383). C, elution profiles
of TDHGAEIVYK from smeared tau (a) and corresponding
L-Asp (b), L-isoAsp (c),
or D-Asp peptide (d). Each synthetic
L-Asp, L-isoAsp, or D-Asp peptide
was subjected to HPLC, and its elution position was compared with that
of the peptide from smeared tau. The L-isoAsp and
L-Asp peptides eluted at positions corresponding to those
of the peptides in peaks a10 and a11, respectively. The elution
conditions were the same as those described in A.
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Fig. 4.
HPLC profile and mass spectrometric analysis
of CN2-derived peptides. A, API peptides of CN2 from smeared
tau (a) and soluble tau (c). Peak a1 is missing
in the map of CN2 from smeared tau. Peak c1 gave signals corresponding
to GQANATRIPAK (residues 164-174) by mass spectrometry and protein
sequencing. Peaks a2 or c2 eluting immediately after peak c1 contained
the deamidated form of GQANATRIPAK. Thus, Asn-167 is largely deamidated
in smeared tau, whereas this residue is only partially deamidated in
soluble tau. Broader peak a3 may be due to incomplete removal of
phosphate in addition to isomerization. Peaks a3 and c3 (tau-1 portion:
191-225) were subjected to chymotrypsin digestion, and the peptides
generated were separated, as in b and d. Peaks b1, b2, and d2 were
subjected to sequencing and mass spectrometry. B, peaks b1,
b2, and d2 gave signals at m/z 741.8, 741.7, or 741.6, corresponding to the calculated molecular mass (741.3 atomic mass
units) of SGDRSGY (residues 191-197).
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Fig. 5.
Amino acid sequence of the longest isoform of
human tau. The locations of the antibody epitopes are indicated by
dashed lines. The amino-terminal inserts and second repeat
are in brackets. Asterisks indicate the sites of
missense mutation for frontotemporal dementia (50-52). The major
deamidation/isoaspartate formation sites are circled.
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Fig. 6.
Immunostaining of serial AD sections with an
antiserum specific for tau isoAsp-387, with or without
preabsorption. An antiserum was raised against isoAsp-387 of tau,
T(isoD)HGAEIVYK (residues 386-395), using the multiple antigen peptide
method (22). A, the antiserum (1:250) immunostained
innumerable neurofibrillary tangles and curly fibers in layer two of
the entorhinal cortex from an AD brain. B, after
preabsorption with the L-Asp peptide (~2.5 µg/ml), the
specific immunoreactivities, although slightly decreased, remained.
C, preabsorption with the L-isoAsp peptide
(~2.5 µg/ml) completely eliminated the immunoreactivity, as
expected. D, preabsorption with the D-Asp
peptide (~2.5 µg/ml) also greatly reduced the immunoreactivity.
Because both L-isoAsp and D-Asp are recognized
by L-isoaspartate (D-aspartate)
O-methyltransferase (EC 2.1.1.77), the absorption effect can
be explained by their conformational similarity. Asterisks
in A-D indicate the same blood vessel. Bar, 50 µm.
DISCUSSION
-amino group of Lys is modified (16). Thus, the only unique
characteristic of the SDS-stable CN5 dimers and trimers appears to be
the presence of isomerized Asn-381 and Asp-387. It is most likely that
the bands at ~45 and ~30 kDa represent dimers and trimers of CN5
because: (i) each of the two larger CNBr fragments contained both
three- and four-repeat tau-derived peptides (Table I), and (ii) the HPLC profiles of the 45-and 30-kDa proteins were very similar (data not
shown). This strongly suggests that the structural alterations revealed
here should underlie the oligomerization of CN5 and the unusual
stability of these oligomers in SDS. These SDS-stable dimers and
trimers are probably related to the tau fragments in the
protease-resistant PHF core, which were originally found by Wischik and
his colleagues (34, 35). They found a discrete band at ~12 kDa and
broad bands with higher molecular masses, each of which appeared to be
composed of both three- and four-repeat tau-derived fragments. Thus,
the high molecular mass bands were interpreted to represent dimers and
trimers of the 12-kDa protein. This so-called "minimal protease
resistant tau unit" encompasses approximately three repeats and
terminates mostly at Glu-391 (34, 35) and thus indeed contains Asn-381
and Asp-387. However, the presence of deamidation/isomerization at
Asn-381 and Asp-387 was not previously noted (35). On the other hand,
CN2 from smeared tau migrated as a monomer at ~14 kDa on SDS-PAGE,
although it produced a somewhat indistinct band, and Asp-193 was found
to be partially isomerized. Taken together, it is reasonable to
speculate that the smearing characteristic of the tau in PHFs is
derived from altered CN5 that is primarily composed of the
microtubule-binding domain and that the altered CN5 has a strong
tendency to oligomerize, resulting in very broad bands of CN5 dimers
and trimers. If so, the question is why are these unusually broad,
smear-like bands generated?
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Fig. 7.
Pathways for the spontaneous degradation of
asparaginyl and aspartyl residues. L-Asparaginyl and
L-aspartyl residues can be converted spontaneously via a
succinimidyl intermediate to form L-isoaspartyl,
D-aspartyl, and D-isoaspartyl residue (modified
from Ref. 33).
Asparaginyl peptide can be nonenzymatically converted to isoaspartyl peptide (70-85%) and aspartyl peptide (15-30%) via cyclic imide formation (Fig. 7) (36, 37). The degree of microheterogeneity generated by deamidation and through the succinimide pathway is remarkable; partial degradation of a single Asn residue can produce seven variants at the site, i.e. L-Asn, L-imide, L-isoAsp, L-Asp, D-imide, D-isoAsp, and D-Asp, of which at least three (L-Asn, L-isoAsp, and L-Asp) are the major contributors (see Fig. 7) (48, 49). Similarly, partial degradation of a single Asp residue can also provide six variants at that site, i.e. L-Asp, L-imide, L-isoAsp, D-imide, D-isoAsp, and D-Asp, of which at least two (L-Asp and L-isoAsp) are the major contributors. Thus, for CN5 derived from three-repeat smeared tau, 6 variants are generated because it bears Asn-381 and Asp-387 that can be converted; thus, 12 variants are generated for three- and four-repeat CN5. Each of these variants, if present in CN5, may provide similar but distinct mobility; thus, dimerization results in an even broader, smear-like band at ~30 kDa. In fact, the CN5 monomer derived from smeared tau shows rather broad background staining as compared with CN5 from soluble tau (Fig. 2C). If such CN5 is dimerized on the assumption that each variant can randomly interact with another, 36 variants are generated. Thus, these variants produced at Asn-381 and Asp-387 should greatly contribute to the smearing of the altered CN5 on the blot. Similarly, the alteration of Asp-193 in CN2 can produce several variants. This may explain the indistinct band at ~12 kDa and a minimally retarded mobility, although oligomerization is not a characteristic of CN2 (Fig. 2B).
It is of note that D-Asp content in CN5 is significantly lower than that in CN2. This difference may have been caused by a strong interaction of the microtubule-binding domain with tubulin. This kind of molecular interaction may possibly suppress deamidation and isoaspartate formation. The above result also indicates that the alteration of CN5 from smeared tau cannot be ascribed to the greater extent of racemization/isomerization. This points to the possibility that, distinct from other CNBr fragments, CN5 is quite unusual in that: (i) each of the abovementioned modifications at the specific sites induces various extents of conformational change that may be resistant to SDS, resulting in various mobilities on SDS-PAGE, and (ii) altered CN5 has a strong tendency to form oligomers that are unusually stable in SDS. Thus, the oligomerization of CN5 from smeared tau is most likely due to the properties of CN5 itself and deamidation and racemization/isomerization at specific Asn and Asp residues.
Finally, it is of particular importance to note that all of the tau mutations in the exon identified thus far in the families affected by frontotemporal dementia are localized to CN5, namely, Gly-272 to Val, Pro-301 to Leu, Val-337 to Met, Arg-406 to Trp (50, 51), and Asn-279 to Lys (52) (see Fig. 5). These mutations should significantly affect the properties of tau to promote tubulin assembly into microtubules. It is also possible that the mutations enhance the oligomerization of the microtubule-binding domain, when altered, and provide the binding domain with a greater extent of protease resistance. The generated carboxyl-terminal portion in neurons may have a longer half-life, resulting in more susceptibility to deamidation/isomerization and thus oligomerization.
It should be noted that the smear we have characterized here represents
only a part of smeared tau (the smear with molecular masses larger than
PHF-tau). Presumably, the lower molecular mass smear could be composed
of smaller units derived from CN5. These are the smears that can be
solubilized with guanidine-HCl, and the guanidine HCl-insoluble smeared
tau is left in the residue. This species of smeared tau can be
extracted with concentrated formic acid, but it is highly resistant to
API (data not shown). Thus, the smeared tau that we have characterized
here is a less processed, less modified one, and it is quite possible
that in the greater insoluble smeared tau, there is a greater extent of deamidation and/or isomerization and possibly as-yet-unidentified cross-links.
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ACKNOWLEDGEMENTS |
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We thank Drs. C. L. Masters and D. J. Selkoe for providing the AD brains used for this study, Dr. T. Masaki for the gift of API, M. Chijimatsu for instructions regarding DL-amino acid analysis, Dr. N. Dohmae for instructions regarding the amino acid sequencer, Dr. T. Nonaka for instructions regarding the peptide synthesizer, J. Saishoji for immunocytochemistry, and Dr. M. Morishima-Kawashima for valuable comments on this study.
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Note Added in Proof |
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A new mutation, S305N, causing FTDP-17 has recently been found (Iijima et al. NeuroReport, in press).
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FOOTNOTES |
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* 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.
§ A recipient of a postdoctoral fellowship from Special Postdoctoral Researchers Program and President's Special Research Grant of RIKEN.
** To whom correspondence should be addressed. Tel.: 81-3-3812-2111, ext. 3541; Fax: 81-3-5800-6852; E-mail: yihara{at}m.u-tokyo.ac.jp.
2 Y. Ihara, unpublished data.
3 M. Morishima-Kawashima and Y. Ihara, unpublished data.
4 M. Chijimatsu, and K. Takio, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: AD, Alzheimer's disease; API, Achromobacter lyticus protease I; CNBr, cyanogen bromide; NFT, neurofibrillary tangle; PHF, paired helical filament; RP, reverse phase; HPLC, high performance liquid chromatography; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid.
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REFERENCES |
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