Proc. Natl. Acad. Sci. USAVol. 86, pp. 5646-5650, July 1989Neurobiology

Identification and localization of a T peptide to paired helicalfilaments of Alzheimer disease

(Alzheimer neurofibrillary tangles/amino acid sequence/protein phosphorylation/immunocytochemistry/microtubule-associated proteins)

KHALID IQBAL*t, INGE GRUNDKE IQBAL*, ALAN J. SMITHS, LALU GEORGE*, YUNN-CHYN TUNG*,AND TANWEER ZAIDI**New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314; and *Department of Biological Chemistry, School ofMedicine, University of California, Davis, CA 95616

Communicated by Dominick P. Purpura, April 14, 1989 (received for review March 3, 1989)

ABSTRACT Amino acid sequencing of a CNBr digest ofthe r protein isolated from bovine brain revealed an amino acidsequence of 17 residues, Pro-Gly-Leu-Lys-Glu-Ser-Pro-Leu-Gln-Ile-Gly-Ala-Ala-Pro-Gly-Leu-Lys, which we call peptide I,with heterogeneity at position 11 of glycine (peptide Ia) andproline (peptide Ib); peptide I showed no homology with thepreviously reported cDNA-derived mouse and human 7 se-quences. Antisera raised to synthetic peptides corresponding topeptides Ia and lb labeled all the bovine 7 polypeptidesrecognized by other monoclonal and polyclonal antibodies tobovine 7. Antisera to peptide lb did not label any mouse 7polypeptides; however, an anti-Ia antiserum labeled two of thefour mouse T polypeptides. Antisera to both peptides labeledpaired helical filaments (P1F) as neurofibrillary tangles,plaque neurites, and neuropil threads in Alz heimerdiseasebrain and PHF polypeptides on immunoblots. Immunotainingwith anti-Ia antisera of PHF in tissue sections and PHFpolypeptides, but not bovine x, on immunoblots was markedlyincreased when pretreated with alkaline phosphatase. Thesestudies suggest that (i) the amino acid sequences of someisoforms of 7 peptide might be different from that predictedfrom cDNAs, (ii) a 7 peptide that is absent in the predictedsequences is present in PHF in Alzheimer disease, and (iii) T inPHF is abnormally phosphorylated.

Microtubule-associated protein X is a family of closely relatedpolypeptides with a molecular weight range of 55,000-68,000on sodium dodecyl sulfate/polyacrylamide gel electrophore-sis (SDS/PAGE) (1, 2). These polypeptides also differ in theirisoelectric points but have similar peptide maps and aminoacid compositions (2). r stimulates microtubule assembly andcoassembles with tubulin into microtubules in vitro. T poly-peptides microinjected into cells increase tubulin polymer-ization and decrease the rate of microtubule depolymeriza-tion, suggesting their role in assembly of microtubules in vivo(3).Recent studies (4, 5) have shown that there are at least four

molecular species of bovine T, some of which, depending onthe degree of phosphorylation, have different electrophoreticmobilities on SDS/PAGE. T, in a phosphorylated form, hasbeen shown to be present in paired helical filaments (PHF),the aberrant fibrils making the intraneuronal neurofibrillarytangles in Alzheimer disease (6-8). In addition to the neu-ronal cell body, the PHF also accumulate in the neurites ofthe neuritic (senile) plaques and in the neuropil as "neuropilthreads" (9) in affected brains.The cDNA-derived sequences of mouse and human 7 have

been reported (10, 11). In addition a fragment of human T hasbeen shown to be intimately associated with PHF (12). In this

paper we describe the presence of a unique T peptide notpresent in either cDNA sequence and the localization of thisunique 7 peptide to PHF in Alzheimer disease.

Preliminary communications on the amino acid sequencingof the CNBr digest of bovine X have been reported (13, 14).

MATERIALS AND METHODSIsolation of 7. 7 was isolated from bovine brain, obtained

from a local slaughter house, by the method ofGrundke-Iqbalet al. (15). Microtubule proteins obtained by three cycles ofassembly/disassembly (16) were heat-treated at pH 2.7, andr from the heat-stable microtubule-associated proteins werethen extracted in 2.5% perchloric acid (17, 18) and dialyzedagainst 2.5 mM Tris (pH 7.6). No protein bands other than 7bands were detected on Coomassie blue-stained SDS/PAGE.Amino Acid Sequencing. Amino acid sequence 1-17 of

bovine T peptide I (see structure I) was obtained by auto-mated Edman sequencing (13) of a CNBr digest after onecycle ofmanual Edman degradation and subsequent blockingwith o-phthalaldehyde. The CNBr digestion was performedon 100 Ag ofpurified T, and the sequence was recovered at the700-pmol level. Residues in parentheses at positions -1 and-2 (see structure I) were deduced from mixed sequenceinformation by comparison with the cDNA and were includedfor the purpose of designing a synthetic peptide for produc-tion of antibodies.SDS/PAGE. SDS/PAGE of bovine and mouse brain mi-

crotubules and Alzheimer PHF for subsequent immunoblots(Western blots) was carried out by using the Tris-glycinesystem (19) and a 5-15% polyacrylamide gradient.

Synthesis of Peptides. Two peptides, Ia and lb collectivelycalled peptide I, corresponding to amino acid residues of aCNBr fragment of X peptide differing only in one residue (13,14), were synthesized commercially (Biosearch). Cysteinewas added at the carboxyl termini in amide form to conjugateto keyhole limpet hemocyanin (KLH). The peptide Ia had thefollowing sequence: (Met)-(Ala)-Pro-Gly-Leu-Lys-Glu-Ser-Pro-Leu-Gln-Ile-Gly-Ala-Ala-Pro-Gly-Leu-Lys, withresidues numbered as in structure I. In peptide lb, residue 11was proline instead of glycine.

Production of Antisera to the KLH-Conjugated SyntheticPeptides Ia and lb. The amount of total protein in theKLH-conjugated peptides was determined by amino acidanalysis. Two New Zealand White rabbits, weighing about2-2.5 kg each, were immunized with each peptide as de-scribed (20). Initially each rabbit received an emulsion of 3

Abbreviations: KLH, keyhole limpet hemocyanin; PHF, pairedhelical filaments.tTo whom reprint requests should be addressed at: New York StateInstitute for Basic Research in Developmental Disabilities, 1050Forest Hill Road, Staten Island, NY 10314.

5646

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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mg of peptide-KLH conjugate in complete Freund's adju-vant. After 40 days, the rabbits received in intervals of 2weeks two boosters, each containing 0.13 mg of peptide-KLH conjugate emulsified in incomplete Freund's adjuvantadministered intramuscularly and 0.7 mg of unconjugatedpeptide in alumina Cy administered intravenously. Threeweeks later, a third booster, containing 1.0 mg of unconju-gated peptide in alumina Cy, was administered intrave-nously. Eight days after the final booster, the animals weresacrificed.

Isolation and in Vitro Dephosphorylation ofPHF. PHF wereisolated from frozen autopsied brains of patients with Alz-heimer disease (21). The PHF (1 mg/ml) were incubated at370C for 16 hr with alkaline phosphatase (0.1 mg/ml) (Sigmatype VII-S, from Sigma) in 50 mM MgCl2/10 mM phenyl-methylsulfonyl fluoride/0.02% sodium azide/2 ,ug of leupep-tin per ml/100 mM Tris, pH 8.8. After incubation the samplewas centrifuged through a cushion of0.5M sucrose at 200,000x g for 10 min in a Beckman TL-100 ultracentrifuge. ThePHF pellet was resuspended in water and used for immuno-absorption.

Reactivity of Peptide Antisera with i and with AlzheimerNeurofibrillary Tangles. Immunoreaction of each antiserumwas tested on Western blots of the SDS/PAGE of isolatedPHF, bovine T, and mouse brain microtubules as described(15).The reactivity of antisera was also examined on paraffin

sections of hippocampus from Alzheimer diseased brain asdescribed (15).Immunoabsorption. For immunoabsorption, the antisera

were incubated at 1/10th of their final dilution with variousamounts of each antigen at 22°C for 1 hr and at 4°C for 18 hr(20).

Antibodies to r. Antibodies to whole bovine T used in thisstudy were a monoclonal antibody r-1 (22) and a rabbitantiserum 92e (17).

RESULTSAmino Acid Sequencing ofBovine r. Amino acid sequencing

of the CNBr digest of bovine r after phenylisothiocyanatetreatment revealed several peptides, one ofwhich had prolineas the second residue (see refs. 13 and 14). By takingadvantage of this observation, a CNBr digest of T wassubjected to one manual cycle of Edman degradation, fol-lowed by blockage with o-phthalaldehyde of all amino terminiexcept the one starting with proline. Amino acid sequencingof the o-phthalaldehyde-treated CNBr digest of bovine Trevealed the presence of a previously unknown peptide:

(Met)-(Ala)-Pro-Gly-Leu-Lys-Glu-Ser-Pro-Leu-Gln-Ile-Gljy-17

Ala-Ala-Pro-Gly-Leu-Lys.I

This fragment from bovine T (13, 14), which we call peptideI, has no homology with the cDNA-predicted sequences ofmouse (10) and human (11) T. Two other peptides identifiedfrom the CNBr digest of bovine r are highly homologous tothe cDNA-predicted sequences (13). A computer search fromdata bank failed to reveal any homologies between peptide Iand any other known amino acid sequence.

Production of Antisera to r Peptide I and Their Reactionwith r and PHF Polypeptides. Each of the four rabbitsimmunized had produced antibodies to the r peptides. Onimmunoblots of bovine microtubules, all four peptide anti-sera labeled the r polypeptides identically to polyclonal andmonoclonal antibodies against the total bovine T (Fig. 1; seealso Fig. 3). However, on blots of mouse microtubules, only

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from mouse (lane M-MT), human (lanes H-MT), and bovine (lanesB-MT) brain. Immunolabeling with antiserum 102c to peptide Ia wasat 1:1000 antiserum dilution (lanes a.Ia) and monoclonal antibody T-1(ref. 22) to T was at 1:50,000 ascites dilution (lanes tau-1). Boundantibodies were visualized by using peroxidase-antiperoxidase andavidin-biotin complex techniques, respectively. Electrotransferswere from SDS/polyacrylamide gels (5-15% acrylamide, 8 x 6 x0.75 cm). * - indicates blots pretreated with alkaline phosphatase at86 ,Lg/ml in 0.1 M Tris, pH 8.0/1 mM phenylmethylsulfonyl fluoridefor 3 hr at 37°C prior to immunostaining. Molecular size standards areindicated in kDa: myosin (200), phosphorylase b (92.5), bovine serumalbumin (68), ovalbumin (43), and a-chymotrypsinogen (25.7). Theother three antisera to peptide I-i.e., 101c to peptide Ia and 103c and104c to peptide lb (not shown in this figure)-also reacted withbovine and human rand r in PHF, but the pretreatment with alkalinephosphatase only increased the reactivity with peptide Ia antisera.

antiserum 102c against peptide Ia reacted with two of theslowest moving T species (Figs. 2 and 3). The three other rpeptide antisera did not react with any of the mouse rpolypeptides. No significant change in immunostaining ofmurine or bovine T polypeptides occurred when the blotswere treated with alkaline phosphatase prior to application ofthe antisera (Figs. 1 and 2). Reaction of the peptide antiserawith PHF polypeptides on immunoblots was weak. How-ever, on blots pretreated with alkaline phosphatase, thestaining of the PHF polypeptides corresponding to the slow-est moving T polypeptides was increased with the antisera topeptide Ia (Figs. 1 and 2) but not with the antisera to peptideIb (figure not shown). No enhancement of the immunostain-ing was observed when the blot was treated with alkalinephosphatase in the presence of 25 mM pyrophosphate, aninhibitor of alkaline phosphatase (figure not shown). The rpolypeptides in PHF immunolabeled with the anti-peptide Iantisera migrated slower than the polypeptides recognizedwith the T-1 antibody on SDS/PAGE (Figs. 1 and 2).

Localization of Peptide I to PHF and the Effect of Dephos-phorylation on Immunostaining. On tissue sections of Alz-heimer hippocampus, all four antisera strongly labeled intra-cellular neurofibrillary tangles, neuropil threads, and occa-sional plaque neurites, which are locations where PHFaccumulate in the brain of patients with Alzheimer disease(Fig. 4). Staining of the extracellular tangles or "tomb-stones," the putative end stages of the tangles (23) or of

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PHF M-MT PHF

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M-MT). Immunostaining was with monoclonal antibody -1 (ref. 22)to T(lane tau-1) and antiserum 102c to peptide Ta (lanes a.Ta) with(* )

and without pretreatment of the blot with alkaline phosphatase (seeFig. 1 for experimental details). Molecular size standards are indicatedin kDa as in Fig. 1. The other three antisera to peptide I-i.e., 101c topeptide Ta and 103c and 104c to peptide Tb (not shown in thisfigure)-also reacted with in PHF but did not label any ofthe mouseT polypeptides even when pretreated with alkaline phosphatase.

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plaque core amyloid, was not observed with any of theantisera. In the case of antisera lOic and 102c to peptide Ia,pretreatment of the sections with alkaline phosphatase priorto immunostaining considerably increased both staining in-tensity and the number of immunostained tangles and neu-ropil threads. This change was most dramatic in plaques,most of which became immunopositive only after dephos-phorylation. No change in the staining pattern was observedwhen the tissue was incubated with alkaline phosphatase in

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FIG. 3. Comparison of immunolabeling of T in bovine and mousemicrotubules (lanes B-MT and M-MT, respectively) by antiserum92e (ref. 17) to bovine T (lanes a.tau) and antisera 102c and 104c topeptides Ta and Tb (lanes a.Ta and a.Tb, respectively). See Fig. 1 forexperimental details. Molecular size standards are indicated in kDaas in Fig. 1.

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FIG. 4. Immunocytochemical staining of paraffin sections ofhippocampus from an Alzheimer disease case with antisera topeptide Ta and the effect of dephosphorylation. Antisera at 1:1000dilution were incubated with the tissue sections, and the boundantibodies were visualized by the peroxidase-antiperoxidase tech-nique. (a and c) Immunostaining of a tissue section that had beenincubated with 130 kg alkaline phosphatase per ml for 2.5 hr at 320Cprior to labeling with anti-peptide Ia antiserum 102c. Neurofibrillarytangles (arrow in c), plaque neurites (arrowheads in c) and fineneuropil threads (double arrows in c) are labeled by the antibody. (b)Significantly fewer numbers of tangles and plaques in the same areaas in a are stained by antiserum 102c when the preincubation withalkaline phosphatase was in the presence of 25 mM pyrophosphate,an inhibitor of this enzyme. Antiserum 104c to peptide Tb stainedalmost as many tangles and plaques as did antiserum 102c in a (notshown in this figure); plaque neurites, but not plaque amyloid cores,were stained with the peptide antisera. (a and b, x 100; c, x500.)

the presence of 2 mM each of EDTA and EGTA or 25 mMpyrophosphate, which are phosphatase inhibitors. In the caseof antisera 103c and 104c to peptide Tb, pretreatment of thesections with alkaline phosphatase only minimally changedthe staining pattern and intensity.The immunostaining of PHF was eliminated when the

peptide I antisera were preabsorbed with 10-50 ng of uncon-jugated peptide per ml of diluted antisera (Table 1). In thecase of the antisera to peptide Ia, the immunostaining wasremoved equally well by absorption with peptide Ia or Tb. Inthe case of the antisera to peptide Tb, the staining wasobliterated with peptide Tb but not with peptide Ia with levelsas high as 500 ng. Absorption ofantisera 102c (peptide Ta) and

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5648 Neurobiology: Iqbal et al.

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Table 1. Absorption of tangle-staining antibodiesAntigen, 1g/ml of diluted antiserum

Antigen used Anti-peptide Ia Anti-peptide lb Anti-Tfor absorption 101c 102c 103c 104c 92ePeptide Ia 0.05 0.05 >>0.5 >0.5 >5Peptide Tb 0.05 0.05 0.05 0.01 >>5Bovine MT 50 50 20 50 6Mouse MT >>100 >100 >>100 >>100 6PHF ND 2* ND 2* 1

Immunostaining ofAlzheimer neurofibrillary tangles by antisera topeptides Ia, Tb, and.' was affected.by preabsorption with syntheticT peptides Ta, Tb, microtubules (MT), and PHF. >>, No change intangles staining seen when preabsorbed with the amount of antigenshown; >, definite reduction in staining intensity seen when preab-sorbed with the amount of antigen shown; *, treated with alkalinephosphatase; ND, not determined.

104c (peptide Tb) with alkaline phosphatase-treated PHF,likewise, removed the tangle staining. When bovine micro-tubules were used as a source of T peptide for absorption, theimmunostaining of PHF by all four antisera was eliminatedwith 50 ug of antigen per ml of diluted antisera, whereasabsorption with 100 ,ug of mouse microtubules per nml onlyslightly reduced the immunostaining by antiserum 102c (pep-tide Ta) and had no effect in the case of the other threeantisera. In contrast, the staining of tangles and neurites byan antiserum to isolated bovine T (serum 92e) was completelyeliminated with as little as 6 ,ug of bovine or mouse micro-tubules or 1 ,g of PHF per ml of diluted antiserum.

DISCUSSIONAlthough T in brain microtubules was discovered as early as1975 (24), the biochemistry of this family of polypeptides isnow only beginning to be understood. The association of apolypeptide, now known to be T, with Alzheimer PHF wasobserved in 1974 (25). However, it was not definitely iden-tified until several years later (6-8, 12, 15, 20, 21, 26-28).Discovery of T in PHF in Alzheimer disease not only hasrevived and tremendously increased interest in this otherwiselittle-studied protein but also has provided some indication asto the possible function of T in the brain (6, 29). The presentstudy (i) identifies a previously unknown T peptide and (ii)shows immunochemically the presence of this peptide innormal human T and in PHF in Alzheimer disease.A comparison of the amino acid sequence of peptide I with

the previously reported cDNA-predicted sequences of mu-rine (10) and human (11) T shows no homology. Two otherCNBr peptides of bovine T have been found to be highlyhomologous to the cDNA-derived sequences (13). The ab-sence ofpeptide I in the cDNA-predicted sequences suggeststhat the latter represent only certain isoforms of T. Thedifferent molecular species of T might be due to differentialsplicing of the mRNA as proposed in the earlier reports (10,11).Immunostaining of bovine T polypeptides with antibodies

to the synthetic peptides corresponding to Ta and Tb showsthe presence of these peptides in the parent protein. Inter-estingly, all molecular species ofbovine Trare immunostainedby both antibodies to peptides Ta and Tb, suggesting that thesepeptides are present in most if not all molecular species of T

from bovine brain. Likewise, all ofthese bands are stained byboth monoclonal and polyclonal antibodies to T. In contrast,only one of the two peptide Ta antibodies recognizes thelarger two of the four molecular species of murine T seen onCoomassie blue-stained gels. However, all of those bandswere labeled prominently by the monoclonal and polyclonalantibodies to bovine T. Neither of the two antibodies topeptide Tb label any murine Xpolypeptides. These immuno-

labeling findings are to be expected from the predicted aminoacid sequence of murine X (10) in that the latter does notcontain peptide T. These studies raise an intriguing possibilitythat the two murine rcDNA clones reported by Lee et al. (10)correspond to the two smaller molecular species of Xthat lackpeptide I and, hence, are negative on immunolabeling bypeptide I antibodies. Peptide Ta or a part of its sequenceappears to be present in the two larger molecular species ofmurine X that are labeled with the peptide Ta antibodies andthat probably do not correspond to the two reported XcDNAs. Peptide Tb, which differs from peptide Ta by a singleamino acid at residue 11 and whose antibodies do not labelany murine Xrspecies, might represent a species differencebetween calf and mouse.The PHF polypeptides have been identified immunochem-

ically to be abnormally phosphorylated Xpolypeptides (6).The present study also suggests that X in PHF is phospho-rylated because the immunostaining of these polypeptides inPHF with peptide Ta antisera is markedly increased ontreatment with alkaline phosphatase. This phosphorylation ofXpolypeptides in PHF appears to be different from phos-phorylation of bovine and murine Xin that the reactivity ofonly PHF polypeptides on Western blots with peptide Taantibodies, is increased when pretreated with the phospha-tase. This increase in the accessibility of the antigenic deter-minant(s) to peptide Ta antibodies with alkaline phosphataseappears to be due to the phosphatase action and not to anyproteolysis, since this effect is specifically inhibited in thepresence of phosphatase inhibitors.The epitopes recognized by the antisera to peptides Ta and

Tb appear to be different from that seen by the monoclonalantibody -1 to bovineT. Unlike -1, which labels all of thefour murine T polypeptides, the antisera to peptide I reactwith, at most, two of the polypeptides. Furthermore, unliker-1, the antibodies to peptide Ta recognize only the slowmoving T polypeptides in PHF. The phosphatase-sensitivereactivity of antisera to peptide Ta thus provides evidence,independent from that reported previously with monoclonalantibody T-1 (6, 8), for the abnormal phosphorylation of T inPHF. Phosphorylation of T in neuronal cell bodies anddendrites in normal rat brain perfused with fixatives contain-ing phosphate buffer has also been observed (30). However,the phosphorylated Tpeptide accumulates in filaments only inAlzheimer brain and not in normal brain. The number of thephosphate groups added and/or the site(s) of phosphoryla-tion of Tin Alzheimer disease might be different from that inthe normal brain. T promotes microtubule assembly (24), andthe phosphorylation of T depresses this assembly (31). Fur-thermore, the amount of cytosolic Tin Alzheimer brain invitro is about half of that in normal brain (32). Therefore, theaccumulation of Tin the form ofPHF in the affected neuronsmight deplete the necessary amounts of functional Tin theaffected neuron to maintain microtubules and thereby theaxoplasmic flow and neurotransmission. The inability toachieve in vitro assembly of microtubules from Alzheimerdisease brain (29) is consistent with this possibility.

Note Added in Proof. The amino acid sequences of peptides Ta and lbare highly homologous to amino acid residues 28-46 of the cDNA-predicted sequence of bovine T (33). The immunostaining ofPHF ontissue sections and of PHF polypeptides on Western blots withantisera to peptides Ta and Tb confirm our previous findings (6, 15)showing the. presence of T polypeptides in their entirety or nearentirety in PHF. Our findings predict that the cDNA-derived se-quences of human r (11, 34) reported to date do not represent allisoforms of T seen in PHF.

We thank Dr. L. I. Binder (University of Alabama, Birmingham)for a generous supply of monoclonal antibody v-1 to T polypeptidesand for critical reading of the manuscript, the Biomedical Photog-raphy Unit (Institute for Basic Research, Staten Island, NY) for the

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preparation of the figures, and Marie Cappola and Concetta Vene-ziano for typing the manuscript. This work was supported in part byNew York State Office of Mental Retardation and DevelopmentalDisabilities, National Institutes of Health Grants AG 05892, NS18105, and AG/NS 04220, and a grant from the Alzheimer's DiseaseResearch Program of the American Health Assistance Foundation(Rockville, MD).

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