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Heparin-induced Conformational Change in Microtubule-associated Protein Tau as Detected by Chemical Cross-linking and Phosphopeptide Mapping

1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês

10.1074/jbc.274.12.8029

1083-351X

Hemant K. Paudel, Wei Li,

Connective tissue disorders research

In Alzheimer's disease, microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments. Sulfated glycosaminoglycans such as heparin and heparan sulfate were shown to accumulate in pretangle neurons, stimulate in vitro tau phosphorylation, and cause tau aggregation into paired helical filament-like filaments. The sulfated glycosaminoglycan-tau interaction was suggested to be the central event in the development of neuropathology in Alzheimer's disease brain (Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996) Nature383, 550–553). The biochemical mechanism by which sulfated glycosaminoglycans stimulate tau phosphorylation and cause tau aggregation remains unclear. In this study, disuccinimidyl suberate (DSS), a bifunctional chemical cross-linker, cross-linked tau dimers, tetramers, high molecular size aggregates, and two tau species of sizes 72 and 83 kDa in the presence of heparin. In the absence of heparin only dimeric tau was cross-linked by DSS. Fast protein liquid chromatography gel filtration revealed that 72- and 83-kDa species were formed by intramolecular cross-linking of tau by DSS. These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species. Heparin-induced structural changes in tau molecule depended on time of heparin exposure. Dimerization and tetramerization peaked at 48 h, whereas conformational change was completed within 30 min of heparin exposure. Heparin exposure beyond 48 h caused an abrupt aggregation of tau into high molecular size species. Heparin stimulated tau phosphorylation by neuronal cdc2-like kinase (NCLK) and cAMP-dependent protein kinase. Phosphopeptide mapping and phosphopeptide sequencing revealed that tau is phosphorylated by NCLK on Thr212 and Thr231 and by cAMP-dependent protein kinase on Ser262 only in the presence of heparin. Heparin stimulation of tau phosphorylation by NCLK showed dependence on time of heparin exposure and correlated with the heparin-induced conformational change of tau. Our data suggest that heparin-induced conformational change exposes new sites for phosphorylation within tau molecule. In Alzheimer's disease, microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments. Sulfated glycosaminoglycans such as heparin and heparan sulfate were shown to accumulate in pretangle neurons, stimulate in vitro tau phosphorylation, and cause tau aggregation into paired helical filament-like filaments. The sulfated glycosaminoglycan-tau interaction was suggested to be the central event in the development of neuropathology in Alzheimer's disease brain (Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996) Nature383, 550–553). The biochemical mechanism by which sulfated glycosaminoglycans stimulate tau phosphorylation and cause tau aggregation remains unclear. In this study, disuccinimidyl suberate (DSS), a bifunctional chemical cross-linker, cross-linked tau dimers, tetramers, high molecular size aggregates, and two tau species of sizes 72 and 83 kDa in the presence of heparin. In the absence of heparin only dimeric tau was cross-linked by DSS. Fast protein liquid chromatography gel filtration revealed that 72- and 83-kDa species were formed by intramolecular cross-linking of tau by DSS. These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species. Heparin-induced structural changes in tau molecule depended on time of heparin exposure. Dimerization and tetramerization peaked at 48 h, whereas conformational change was completed within 30 min of heparin exposure. Heparin exposure beyond 48 h caused an abrupt aggregation of tau into high molecular size species. Heparin stimulated tau phosphorylation by neuronal cdc2-like kinase (NCLK) and cAMP-dependent protein kinase. Phosphopeptide mapping and phosphopeptide sequencing revealed that tau is phosphorylated by NCLK on Thr212 and Thr231 and by cAMP-dependent protein kinase on Ser262 only in the presence of heparin. Heparin stimulation of tau phosphorylation by NCLK showed dependence on time of heparin exposure and correlated with the heparin-induced conformational change of tau. Our data suggest that heparin-induced conformational change exposes new sites for phosphorylation within tau molecule. paired helical filament Alzheimer's disease N,N-dimethyl formamide disuccinimidyl suberate fast protein liquid chromatography monoclonal antibody neuronal cdc2-like protein kinase cAMP-dependent protein kinase polyacrylamide gel electrophoresis high performance liquid chromatography Paired helical filaments (PHFs),1 the major fibrous component of the neurofibrillary tangles associated with Alzheimer's disease (AD), are composed mainly of microtubule-associated protein tau (1, 2; for review, see Ref. 3Goedert M. Trends Neurosci. 1993; 16: 460-465Abstract Full Text PDF PubMed Scopus (549) Google Scholar). PHF-tau (tau isolated from PHFs) has retarded mobility on an SDS-gel, is highly insoluble, is abnormally phosphorylated (i.e. contains more phosphate than normal tau), and is functionally inactive (1Lee V.M.-Y. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1253) Google Scholar, 2Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar, 3Goedert M. Trends Neurosci. 1993; 16: 460-465Abstract Full Text PDF PubMed Scopus (549) Google Scholar). After dephosphorylation, PHF-tau migrates as normal tau on an SDS-gel and regains the ability to bind to, and regulate, microtubule dynamics (4Alonso A.D.C. Zaidi T. Grundke-Iqbal I. Iqbal K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5562-5566Crossref PubMed Scopus (605) Google Scholar, 5Wang J.-Z. Gong C.-X. Zaidi T. Grundke-Iqbal I. Iqbal K. J. Biol. Chem. 1995; 270: 4854-4860Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Abnormal phosphorylation may prevent tau from performing microtubule-related functions, resulting in cytoskeleton instability, loss of axonal transport, and PHF formation (3Goedert M. Trends Neurosci. 1993; 16: 460-465Abstract Full Text PDF PubMed Scopus (549) Google Scholar). Tau is a natural phosphoprotein, and sites that are phosphorylated in normal adult brain (6Watanabe A. Hasegawa M. Suzuki M. Takio K. Morishima-kawashima M. Titani K. Arai T. Kosik K.S. Ihara K.Y. J. Biol. Chem. 1993; 268: 25712-25717Abstract Full Text PDF PubMed Google Scholar) are also phosphorylated in PHF-tau (2Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). Fetal tau (6Watanabe A. Hasegawa M. Suzuki M. Takio K. Morishima-kawashima M. Titani K. Arai T. Kosik K.S. Ihara K.Y. J. Biol. Chem. 1993; 268: 25712-25717Abstract Full Text PDF PubMed Google Scholar), much like PHF-tau (2Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar), is also hyperphosphorylated. Strikingly, both normal adult tau and fetal tau do not form PHFs. Furthermore, PHF-like filaments can be reconstituted from tau molecules that do not contain any phosphate (7Schweers O. Mandelkow E.-M. Biernat J. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8463-8467Crossref PubMed Scopus (351) Google Scholar, 8Crowther R.A. Olesen O.F. Jakes R. Goedert M. FEBS Lett. 1992; 309: 199-202Crossref PubMed Scopus (140) Google Scholar, 9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar). These observations have raised the possibility that abnormal phosphorylation alone may not be sufficient, and another factor(s) may be involved in converting tau to PHFs. There are 19 phosphorylation sites within PHF-tau (2Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). Aberrant activation of tau-specific kinase (s) has been suggested to lead to the abnormal phosphorylation of tau in AD brain (3Goedert M. Trends Neurosci. 1993; 16: 460-465Abstract Full Text PDF PubMed Scopus (549) Google Scholar), because adult tau is phosphorylated only on four sites (6Watanabe A. Hasegawa M. Suzuki M. Takio K. Morishima-kawashima M. Titani K. Arai T. Kosik K.S. Ihara K.Y. J. Biol. Chem. 1993; 268: 25712-25717Abstract Full Text PDF PubMed Google Scholar). Therefore, considerable effort is being made by many investigators to identify kinases that phosphorylate tau. 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Biernat J. Schmitt-Ulms G. Meyer H.E. Mandelkow E.-M. Mandelkow E. J. Biol. Chem. 1995; 270: 7679-7688Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Surprisingly, none of these kinases has been shown to be activated in AD brain. Furthermore, the above-mentioned kinases normally phosphorylate a diverse group of proteins in neurons, but in AD brain only phosphorylation of tau is significantly up-regulated (3Goedert M. Trends Neurosci. 1993; 16: 460-465Abstract Full Text PDF PubMed Scopus (549) Google Scholar). Therefore tau, in AD brain, may be phosphorylated either by a kinase (s) that is yet to be identified or by a known kinases in the presence of a tau-specific substrate modulator, which renders tau more susceptible to phosphorylation. Sulfated glycosaminoglycans (such as heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate) are sulfated copolymers of glucosamine and uronic acid residues (23Roden L. Lennarz W.J. The Biochemistry of Glycoproteins and Proteoglycans. Plenum Publishing Corp., New York1980: 267-371Crossref Google Scholar). Several studies have indicated the presence of glycosaminoglycans in senile plaques and neurofibrillary tangle (9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar, 24Perry G.S. Siedlak L. Richey L. Kawai M. Cras P. Kalaria R.N. Galloway P.G. Scardina J.M. Cordell B. Greenberg B.D. Ledbetter S.R. Gambetti P. J. Neurosci. 1991; 11: 3679-3683Crossref PubMed Google Scholar, 25Su J.H. Cummings B.J. Cotman C.W. Neuroscience. 1992; 51: 801-813Crossref PubMed Scopus (152) Google Scholar, 26DeWitt D. Silver J. Canning D.R. Perry G. Exp. Neurol. 1993; 121: 149-152Crossref PubMed Scopus (156) Google Scholar, 27Snow A.D. Nochlin D. Sekiguchi R. Carlson S.S. Exp. Neurol. 1996; 138: 305-307Crossref PubMed Scopus (84) Google Scholar). Recently, heparan sulfate was shown to accumulate in pretangle neurons (9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar), to stimulate in vitro tau phosphorylation by various kinases (28Brandt R. Lee G. Teplow D.B. Shalloway D. Abdel-Ghany M. J. Biol. Chem. 1994; 269: 11776-11782Abstract Full Text PDF PubMed Google Scholar, 29Hasegawa M. Jakes R.M. Crowther R.A. Lee V.M.-Y. Ihara Y. Goedert M. FEBS Lett. 1996; 384: 25-30Crossref PubMed Scopus (149) Google Scholar, 30Mawal-Dewan M. Sen P.C. Abdel-Ghany M. Shalloway D. Racker E. J. Biol. Chem. 1992; 267: 19705-19709Abstract Full Text PDF PubMed Google Scholar, 31Yang S.D. Yu J.S. Shiah S.G. Huang J.J. J. Neurochem. 1994; 63: 1416-1425Crossref PubMed Scopus (56) Google Scholar, 32Hasegawa M. Crowther R.A. Jakes R. Goedert M. J. Biol. Chem. 1997; 272: 33118-33124Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), to prevent tau from binding to microtubules, and to cause tau to aggregate into PHF-like filaments (9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar). An increase in the sulfated glycosaminoglycans within the nerve cells was suggested to trigger the hyperphosphorylation of tau, destabilization of microtubules, and assembly of PHFs (9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar). The interaction of sulfated glycosaminoglycan and tau was suggested to be the central event in the development of neuropathology in AD (9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar, 33Beyreuther K. Masters C.L. Nature. 1996; 383: 476-477Crossref PubMed Scopus (29) Google Scholar). However, the biochemical mechanism by which glycosaminoglycans enhance tau phosphorylation and cause tau to aggregate into PHFs remains unclear. In this study, we have investigated the effect of heparin on the structure and phosphorylation of tau by chemical cross-linking and phosphopeptide mapping. Herein we report that heparin, in addition to causing aggregation of tau, also changes tau's conformation, exposing new sites within the tau molecule for kinase phosphorylation. Tau protein used in this study was purified from extracts of Escherichia coli overexpressing the longest isoform of human tau (htau 40) as described (19Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), except effluent from Q-Sepharose column was chromatographed through an S-Sepharose column. Neuronal cdc2-like protein kinase (NCLK) was purified from fresh bovine brain extract as described previously (34Lew J. Beaudette K. Litwin C.M.E. Wang J.H. J. Biol. Chem. 1992; 267: 13383-13390Abstract Full Text PDF PubMed Google Scholar). cAMP-dependent protein kinase (A kinase), catalytic subunit of A kinase (C subunit), trypsin, and thermolysin (protease type X), and Kemptide (LRRASLG) were from Sigma. Preparations of polyclonal antibody against bovine brain tau and synthetic peptide substrate of NCLK (KTPKKAKKPKTPKKAKKL) were described previously (19Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The concentration of tau was estimated spectrophotometrically (35Cleveland D.W. Hwo S.Y. Kirschner M.W. J. Mol. Biol. 1977; 116: 227-247Crossref PubMed Scopus (635) Google Scholar). Amounts of A kinase, C subunit, and Kemptide were based on their dry weights. Concentration of synthetic peptide NCLK substrate was determined by amino acid analysis. The amount of NCLK was estimated by enzymatic activity (34Lew J. Beaudette K. Litwin C.M.E. Wang J.H. J. Biol. Chem. 1992; 267: 13383-13390Abstract Full Text PDF PubMed Google Scholar). Unless otherwise stated, NCLK activity was measured as described previously (19Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) in an assay mixture containing 25 mm Hepes (pH 7.2), 0.1 mm EDTA, 0.2 mm dithiothreitol, 60 mm NaCl, 0.5 mm [32P]ATP, 10 mmMgCl2, 50 μm peptide substrate, or 0.5 mg/ml tau and 400 units/ml NCLK. The assay was initiated by the addition of 5 μl of kinase to a 20-μl mixture containing the rest of the assay mixture components. After 20 min at 30 °C, aliquots were withdrawn and analyzed for the amount of radioactivity incorporated into the substrate by phosphocellulose strip assay. Activity of C subunit was determined as above, except Kemptide was used as the peptide substrate, and the concentration of C subunit was 10 μg/ml. A kinase was assayed in a manner similar to that described above for C subunit, except the assay mixture also contained 10 μm cAMP. Chemical cross-linking of tau by disuccinimidyl suberate (DSS), a homobifunctional chemical cross-linker with an 11.4-Å spacer arm, was performed essentially as described previously (36Paudel H.K. J. Biol. Chem. 1997; 272: 28328-28334Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) in a mixture containing 1.5 mg/ml tau, 0.1 mm EDTA, 0.2 mm dithiothreitol, 60 mm NaCl, 1 mm DSS (Pierce) and 2%N,N-dimethyl formamide (DMF). The reaction was initiated by the addition of 1 μl of DSS stock solution in DMF to 49 μl of mixture containing the rest of the cross-linking mixture components. After various time points at room temperature, aliquots were removed, mixed with an equal volume of SDS-PAGE sample buffer (0.1 mTris-HCl, pH 6.8, 25% glycerol, 0.2% bromphenol blue, 10% β-mercaptoethanol, and 2% SDS), boiled, and electrophoresed on a 7.5% Laemmli SDS-gel. The amounts of cross-linked bands were quantitated by scanning the gels using a Molecular Dynamics SI personal densitometer. Band intensities were determined by dividing the optical density of each cross-linked band with the band intensity of tau control (treated with the solvent). The amount of tau that was not recovered in the gel was expressed as the higher molecular size species that did not enter the gel after DSS cross-linking. Apparent molecular weights of various cross-linked species were determined essentially as described previously (36Paudel H.K. J. Biol. Chem. 1997; 272: 28328-28334Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Tau (0.5 mg), phosphorylated for 6 h by NCLK or A kinase, was digested with trypsin and subjected to HPLC C18 reverse phase chromatography essentially as described previously (19Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To purify phosphopeptide 1, peak 1 fractions depicted in Fig. 5 A were combined, concentrated to ∼0.5 ml, and chromatographed through a Sephadex G-25 column (0.5 × 25 cm) preequilibrated and eluted with 0.1% trifluoroacetic acid. The effluent fractions (0.5 ml each) were collected. Only one radioactive peak eluted from the column. Fractions containing radioactivity were combined, concentrated, and injected into an HPLC column as described above. The column was eluted with an acetonitrile gradient of 0–30% in 50 min. Phosphopeptide 3b was purified from peak 3 (see Fig.5 B) fractions and is shown in Fig. 6 B. To purify phosphopeptide 4, peak 4 fractions (see Fig. 5 B) were vacuum dried and redissolved in 0.2 ml of 50 mmNH4HCO3 (pH 8.0) containing 25 μg/ml thermolysin. The sample was then incubated at 37 °C for 3 h and then injected into an HPLC column. The peptide was then eluted from the column by a linear gradient of acetonitrile (0–40%) in 50 min. To purify phosphopeptide e, peak e fractions (see Fig. 7 B) were combined, vacuum dried, dissolved in 500 μl of 50 mmNH4HCO3 containing 25 μg/ml thermolysin, and incubated at 37 °C for 3 h. After incubation, the sample was loaded onto a ∼1-ml DEAE-Sephacel (Sigma) column pre-equilibrated in 25 mm Hepes (pH 7.0). The column was washed with 10 ml of equilibration buffer and eluted with 0.25 m NaCl in equilibration buffer. Effluent fractions (0.2 ml each) were collected. Fractions containing radioactivity were combined and concentrated to ∼0.2 ml, and the phosphopeptide was purified by HPLC as above using acetonitrile gradient 0–40% in 50 min. Phosphopeptides were sequenced using a gas phase amino acid sequencer (19Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) at the Department of Biochemistry and Microbiology, University of Victoria.Figure 6HPLC peptide maps of peak 3 fractions from Fig. 5, A and B. Peak 3 fractions from Fig. 5, A and B were pooled separately and fractionated through a Sephadex G 25 column. Effluent fractions containing radioactivity were pooled, lyophilized, redissolved in 0.2 ml of 0.1% trifluoroacetic acid, and rechromatographed by HPLC. All the chromatographic conditions were same as in Fig. 5, except the acetonitrile gradient was 0–45% in 50 min. Effluent-containing peptide peaks were manually collected, and 10 μl was counted in a scintillation counter. A, HPLC profile of peak 3from Fig. 5 A; B, HPLC profile of peak 3 from Fig. 5 B. Note that phosphopeptide 3b(arrow) is present only in B. This peptide was subjected to amino acid sequencing.View Large Image Figure ViewerDownload (PPT)Figure 7HPLC tryptic phosphopeptide maps of tau phosphorylated by A kinase in the absence (A) and presence (B) of heparin. Tau species (0.5 mg each) phosphorylated by A kinase in the absence and presence of heparin were trypsinized and subjected to phosphopeptide mapping as in Fig. 5.Insets, HPLC profiles.View Large Image Figure ViewerDownload (PPT) When tau was incubated with DSS and the product was analyzed by SDS-PAGE, there was a time-dependent formation of a heavier species with a concomitant decrease in the tau band intensity (Fig.1 A, lanes 2–4). The molecular size of the heavier band on an SDS-gel was estimated to be ∼153 kDa. In a previous study we have shown that the tau isoform used in this study that migrates as a 65-kDa band on the SDS-gel is a mixture of tau monomers and dimers when purified from bacterial lysate. These dimers, when cross-linked by DSS, migrate with a size of ∼151 kDa on an SDS-gel (36Paudel H.K. J. Biol. Chem. 1997; 272: 28328-28334Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, the cross-linked band in Fig.1 A, lanes 3 and 4, is the dimeric tau. Cross-linking of tau under identical conditions in the presence of heparin showed four major differences when compared with tau cross-linked alone (Fig. 1). First, the intensity of the dimeric band was higher in tau cross-linked in the presence of heparin (Fig. 1, compare lanes 3, 4 and 6–8). Second, a band with size of ∼246 kDa was formed in the presence, but not in the absence, of heparin. Because the molecular size of tau is ∼65 kDa (Fig.1 A), this 246-kDa band is ∼3.8 times heavier than tau and must therefore be tetrameric tau. Third, a protein band that migrated as a streak on the top portion of the gel was formed only when heparin was present in the cross-linking mixture (Fig. 1, lanes 7and 8). This band must be a heavy molecular size tau aggregate cross-linked by DSS. Fourth, in the presence of heparin, two species of molecular sizes 72 and 83 kDa were also cross-linked by DSS (Fig. 1, lanes 6–8). When immunoblotted using an anti-heparin monoclonal antibody (mAb; Chemicon), none of the above cross-linked bands displayed immunoreactivity (data not shown), indicating that the DSS cross-linked bands were not formed by tau-heparin cross-linking. Densitometric quantitation of various bands in Fig. 1 A indicated that at the 15-min time point 3.5% dimeric tau was cross-linked by DSS in the absence of heparin, whereas in the presence of heparin 4.1, 6.2, 10.5, 7.5, and 3.0% tau was cross-linked into 72-kDa, 83-kDa, dimer, tetramer, and higher molecular size species, respectively. The cross-linking of dimeric, tetrameric, and higher aggregates by DSS in Fig. 1 suggested that heparin promotes dimerization and causes the formation of tetrameric and higher molecular sized tau species. These observations are consistent with previous reports and indicate that heparin causes tau aggregation (9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar, 32Hasegawa M. Crowther R.A. Jakes R. Goedert M. J. Biol. Chem. 1997; 272: 33118-33124Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). To interpret the cross-linking of 72- and 83-kDa bands by DSS in the presence of heparin (Fig. 1), we wished to know whether these two cross-linked bands were formed by cross-linking of tau intermolecularly or intramolecularly. Because DSS cross-linked dimer and tetramer migrated with molecular sizes of 153 and 246 kDa, respectively, on an SDS-gel (Fig. 1 A), the sizes 72 and 83 kDa are too small to be tau dimer, trimer, or tetramer that would have been formed if tau were cross-linked intermolecularly. To substantiate the idea that 72- and 83-kDa bands are formed by intramolecular cross-linking, we cross-linked tau (0.75 mg) with DSS in the presence of heparin as described under "Materials and Methods." The cross-linked tau was then fractionated through a fast protein liquid chromatography (FPLC) Superose 12 gel filtration column (Fig.2 A), and various column fractions were immunoblotted using anti-tau antibody (Fig.2 C). Tau tetramers were recovered within fractions 32–36 with peak fraction 34. Tau dimers were present within fractions 36–40 with peak fraction 38. The 72- and 83-kDa species were present within fractions 40–44 with peak fraction 42. Importantly, tau chromatographed through the same column under identical conditions eluted with peak fraction 42 (Fig. 2 B). Thus, the sizes of 72- and 83-kDa bands correspond to monomeric tau. As shown in Fig. 1, 72- and 83-kDa species are cross-linked by DSS only in the presence of heparin. These observations indicated that heparin causes a conformational change that exposes groups reactive to DSS within tau molecule leading to intramolecular covalent cross-linking of tau by DSS. These cross-linked tau species migrate slightly slower than tau on SDS-gels. In a previous study (9Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (870) Google Scholar), heparin-induced aggregation of tau was reported to be dependent on time of incubation, and PHF-like filaments were formed after incubating tau with heparin for >48 h. We therefore preincubated tau with heparin for various time points. Aliquots were removed from the preincubation mixture, treated with DSS, and subjected to SDS-PAGE. The intensities of various bands on the gel were quantitated. The amount of tau that was not recovered on the gel was regarded as the high molecular size aggregate, which, after cross-linking, did not enter the SDS-gel. As shown on Fig. 3, A andB, the intensities of the dimeric and tetrameric bands increased with increasing time of preincubation, peaked at 48 h (Fig. 3 A, lane 11) and diminished significantly at 72 and 96 h. The formation of the higher aggregate tau, which was slow until 48 h, sharply increased with concomitant decrease in the intensities of all other cross-linked and monomeric tau bands when incubated beyond 48 h (Fig. 3 B). Like the dimeric band, 72- and 83-kDa bands were visible within a few min of preincubation with heparin (Fig. 3 A, lane 3) and displayed a biphasic effect with respect to preincubation time (Fig. 3 B). However, these two bands peaked at the 30-min time point (Fig. 2 A, lanes 6 andB), remained constant for several hr, and then declined very slowly until 48 h (lane 11). At 72 and 96 h, both bands progressively faded. In conclusion, the dimerization and tetramerization of tau increased with the increase in heparin incubation time until 48 h. The formation of higher molecular size aggregate was slow during the initial period of heparin incubation until 48 h. Incubation with heparin beyond 48 h converted almost all tau species into the higher molecular size aggregate. Heparin-induced conformational change of tau, as detected by DSS cross-linking of 72- and 83-kDa species, completed within ∼30 min of heparin exposure. In addition to causing aggregation, heparin is known to stimulate tau phosphorylation by various kinases (28Brandt R. Lee G. Teplow D.B. Shalloway D. Abdel-Ghany M. J. Biol. Chem. 1994; 269: 11776-11782Abstract Full Text PDF PubMed Google Scholar, 29Hasegawa M. Jakes R.M. Crowther R.A. Lee V.M.-Y.