Deamidation and Isoaspartate Formation in Smeared Tau in Paired Helical Filaments
UNUSUAL PROPERTIES OF THE MICROTUBULE-BINDING DOMAIN OF TAU*

Atsushi WatanabeDagger §, Koji TakioDagger , and Yasuo Iharaparallel **

From the Dagger  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 parallel  Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Kawaguchi, Saitama 332-0012, Japan

    ABSTRACT
Top
Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    EXPERIMENTAL PROCEDURES

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 alpha -cyano-4-hydroxycinnamic acid as a matrix.

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 Nalpha 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).

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).

    RESULTS

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


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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.

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.


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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.

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).

                              
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Table I
Sequence and MS analyses of the peptides released from smeared CN5 (a) and soluble CN5 (c)
A few small peaks between peaks 8 and 9 in Fig. 3A, a and b represent CN2 peptides derived from contamination of larger CN2-CN5 fragment. Peaks a12-a14 and c11-c12 were found to contain the peptides having the same amino-terminal sequence, SPVV...(396-), 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.

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).


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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 alpha -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.

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.


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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).

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.


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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.

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.


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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

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 epsilon -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?

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.


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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.

    ACKNOWLEDGEMENTS

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.

    Note Added in Proof

A new mutation, S305N, causing FTDP-17 has recently been found (Iijima et al. NeuroReport, in press).

    FOOTNOTES

* 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.

    ABBREVIATIONS

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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