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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5384-5388, June 1992 Biochemistry -dependent epitopes of neurofilament on tau and relationship with Alzheimer tau (-associated /Alzheimer paired helical filaments/protein engineering/Ser-pro-directed protein kns) B. LICHTENBERG-KRAAG*, E.-M. MANDELKOW*, J. BIERNAT*, B. STEINER*, C. SCHROTER*, N. GUSTKE*, H. E. MEYERt, AND E. MANDELKOW** *Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, D-2000 Hamburg 52, Federal Republic of Germany; and tInstitute for Physiological Chemistry, Ruhr-Universitat, Universitatsstrasse 150, D-4630 Bochum, Federal Republic of Germany Communicated by Hans J. Muller-Eberhard, January 29, 1992 (receivedfor review November 7, 1991)

ABSTRACT We have studied the phosphorylation of tau The data suggested that the neurofilament antibodies of protein from Alzheimer paired helical filaments, of tau from Sternberger et al. (3) might be suitable for analyzing the normal , and of recombinant tau isoforms. As a phosphorylation of tau. In this study we have monitored the tool we used monoclonal antibodies against neurofiament phosphorylation of tau in three ways: (i) sequencing of protein [Sternberger, N., Sternberger, L. & Ulrich, J. (1985) phosphopeptides; (ii) blotting with phosphorylation- Proc. Nad. Acad. Sci. USA 82, 4274-4276] that crossreact with dependent antibodies, which yields information about abnor- tau in a phosphorylation-dependent manner. This allowed us to mal states; and (iii) SDS/PAGE mobility shift induced by deduce the state ofphosphorylation in normal and pathological certain types ofphosphorylation (23, 24), which provided one tau, as well as epitopes. The epitope of antibody of the early clues that PHF tau was abnormally phosphory- SM133 is at the first Lys-Ser-Pro sequence motif (residues lated (11). These approaches were used to study how PHF tau 234-236) and requires an unphosphorylated Ser-235. Antibody differs from normal or recombinant tau. SMI31 binds between Ser-396 (in the second Lys-Ser-Pro motif) and Ser-404, both of which must be phosphorylated. SM134 MATERIALS AND METHODS has a conformational epitope that depends on the interaction between regions on either side of the microtubule-binding The preparation of tau from normal human or bovine brain region; it also requires phosphorylation. The phosphorylatable was done as described (24). PHF tau was prepared according detected by the SMI antibodies are part of Ser-Pro to ref. 18. Alzheimer brain tissue was obtained from the motifs and can be phosphorylated by a protein kinase activity Kathleen Price Bryan Brain Bank (Duke University). Phos- that can be used to induce a paired helical filament-like state in phorylation of tau was done with porcine brain extract. The human brain tau in vitro. The phosphates are incorporated in extract was prepared by homogenization in 10 mM Tris HCl, several stages that can be identified by antibody reactivity and pH 7.2/5 mM EGTA/2 mM MgCl2/2 mM dithiothreitol/ gel shift. This suggests a role for the phosphorylation sites in protease inhibitors (leupeptin, aprotinin, pepstatin A, a2- Alzheimer disease, as well as the involvement of a Ser-Pro- macroglobulin, phenylmethylsulfonyl fluoride) and centrifu- gation at 100,000 X g for 30 min at 40C. The supernatant was directed protein kinase. used after addition of2 mM ATP and 10 ,uM okadaic acid. For phosphorylation, 1 ul of extract was added to 40 tkl of tau Alzheimer disease brains contain paired helical filaments protein solution (0.5-1 mg/ml) at 370C. (PHFs) whose nature has been a matter of debate (reviewed Human tau cDNA clones were kindly provided by M. in refs. 1 and 2). Two main possibilities were considered, Goedert (19, 20). The cDNA inserts were subcloned into the neurofilament protein and , based on antibody expression vector pNG2, a derivative of pET-3b (25). Re- reactivities. Sternberger et al. (3, 4) generated neurofilament combinant plasmids were used to transform Escherichia coli antibodies that crossreacted with PHFs and distinguished BL21(DE3) cells (25). The protein was isolated on the basis different states of phosphorylation. This was used to show of its heat stability and by Mono S (Pharmacia) FPLC (24). that neurofilaments were phosphorylated (4-7), in agreement PCR amplifications were carried out with Taq polymerase with other antibody work (e.g., refs. 8 and 9), and suggested (Perkin-Elmer/Cetus). Residue numbering in this paper re- that PHFs consisted of phosphorylated neurofilaments. fers to the sequence of htau40 (20). However, antibodies against tau protein also reacted with Monoclonal antibodies SMI31 (IgG), SM133 (IgM), SM134 PHFs in a phosphorylation-dependent manner, suggesting (IgG), SM135 (IgG), and SMI310 (IgG) were obtained from that the PHFs consisted of tau protein in an abnormal state Sternberger Monoclonals (Baltimore) and used for blotting of phosphorylation (10-13). according to the manufacturer's instructions (dilution, 1:300 Later it was realized that neurofilament antibodies may to 1:1000). After SDS/PAGE (gradient of 4-20%o or 7-15% crossreact with tau (12, 14, 15). The major phosphorylation acrylamide), proteins were transferred to Immobilon mem- sites of neurofilament heavy chain occurred in Lys-Ser-Pro- branes (Millipore) for immunoblot analysis. Val (KSPV) motifs (9, 15), one ofwhich exists in tau as well. Antibodies against a phosphorylated synthetic peptide re- acted with A68 protein from Alzheimer tangles (16). This RESULTS confirmed that A68 was a variant of tau and that PHFs Neurofilament Antibodies SM133 and SML34/SMI31 Rec- consisted of tau, in agreement with results of molecular ogize PHF tau in a Complementary Fashion. To analyze cloning showing that normal and PHF tau occurred in several Alzheimer-specific abnormalities we have to combine an isoforms arising from alternative splicing (17-22). agent causing the abnormality (a kinase activity), a substrate (tau protein), and a detector (monoclonal antibodies, gel The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: PHF, paired helical filament. in accordance with 18 U.S.C. §1734 solely to indicate this fact. lTo whom reprint requests should be addressed. 5384 Downloaded by guest on September 24, 2021 Biochemistry: Lichtenberg-Kraag et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5385

shift, etc.). Detectors used in this study were the neurofila- 0 30t 6090' 2h 3h 6h 8h10h14h16h 20h24 ment antibodies SMI31, SMI34, SMI35, or SMI310 (with a PAGE phosphorylated epitopes) and SM133 [unphosphorylated epitopes (3)]. The first step was to ascertain the antibody reactivities with tau from different sources. Coomassie blue of normal human tau in the "native" state of phosphorylation (as isolated) shows several bands because b AR there are several isoforms and phosphorylation states (Fig. la, lane 1). The bands of PHF tau are higher than those of normal tau, as if PHF tau were more highly phosphorylated (Fig. la, lanes 1 and 3). SMI33 recognizes normal human brain tau (Fig. lb, lane 1) 1 2 3 4 5 6 7 8 9 10 11 1213 but not PHF tau, unless it is dephosphorylated (lane 4). This suggests that the epitope of SM133 is blocked by some c phosphorylation in the PHF-like state. Both SMI31 and SM134 are complementary to SM133, recognizing tau in the PHF-like state (Fig. 1 c and d, lanes 3), but not when it is dephosphorylated (lanes 4), and not the normal tau control (lanes 1). Alzheimer-Like Phosphorylation of tau Can Be Induced in Vito. In contrast to the kinases we tested previously (27), the 0 5 10 1 I kinase activity used here can phosphorylate normal brain tau time [h] (bovine, porcine, or human) or recombinant tau so that it acquires PHF-like immunoreactivities (Fig. 1). The phos- FIG. 2. Time course (0-24 hr) ofphosphorylation of recombinant phorylation can be seen by the shift in apparent size (Fig. 2a) human tau (htau34). (a) SDS/PAGE (Coomassie blue stain) showing and by autoradiography (Fig. 2 b). The gel shift reveals three an increase of apparent molecular mass in three main stages: stage main stages of phosphorylation (Fig. 2a): Starting from the 1, 2-3 hr; stage 2, 10 hr; and stage 3, 24 hr. (b) Autoradiogram (AR) unphosphorylated protein (58 kDa for the recombinant of a. (c) Time course of intensity of bands in a. *, unshifted protein htau34), the first main stage is completed around 2-3 hr, when (0); o, stage 1; o, stage 2; *, stage 3. most of the protein is shifted up to 62 kDa. The second stage is reached after about 10 hr (64 kDa), and the third after about phorylated epitope that is specific for PHFs. No reactivity is 24 hr (66 kDa). The stages are well separated in time, observed up to the first stage, but the reactivity appears suggesting that the phosphorylation proceeds sequentially during the second stage and remains throughout the third. (Fig. 2c). There are 1.5-2 Pi incorporated per stage, so that Similar time courses are found with antibodies SM134, after 24 hr there are 5-6 Pi incorporated. Similar time courses SM135, and SMI310 (Fig. 3 c, g, and h). For comparison we are obtained with other tau isoforms (data not shown). Next we asked how the antibodies react with different 0'3076090* 2 h 3 h 6h 8n10h 14h 16h1 8h20h24h phosphorylation states. SM133, whose epitope is masked by a PHF-specific phosphorylation (Fig. lb, lane 3), reacts with PAGE a VW W*. the unphosphorylated protein (Fig. 3d, time 0); the reactivity ht23 -0 -"a remains through the first stage but is lost during the second stage. Thus, the third or fourth Pi incorporated destroys the SM131 b -w._wwwW" epitope ofSMI33. Antibody SM131 has the opposite behavior (Fig. 3b, and compare Fig. 1c, lane 3): it requires a phos- SM 134 C Uwwwwww" ht PHF-t SM 131

- + + - - + +-_ a c SM 133 W 4 = la .I }t W. .44 e T- 1 s s0 ., 1 2 3 4 1 2 34 ff1*

AT-8 t 8. _ t -

SM 1 33 SM1 34 b d 1 2 3 4 5 6 7 8 9 1011 12 1314 SM135 ht34 9 1 2 3 4 1 2 3 4 FIG. 1. SDS/PAGE and immunoblots of normal human (ht) tau and PHF tau (PHF-t). (a) Lane 1, SDS/PAGE of human brain tau, SM1310 five to six bands of 55-65 kDa; lane 2, after phosphorylation, all bands shifted up; lane 3, blot of PHF tau with antibody 5E2, which tau of phosphorylation (26); lane 4, PHF recognizes independently 0 30 6090 2h 3h 6h 8h10h 14h 16h 20h24h tau after dephosphorylation (alkaline phosphatase), bands shifted down. (b) Blot of a with SM133, which recognizes normal human tau FIG. 3. Time course of phosphorylation and immunoblots. (a) (lane 1), and PHF tau after dephosphorylation (lane 4). (c and d) SDS/PAGE of recombinant htau23. (b-f) Blots of htau23 with SMI31 and SM134 recognize normal human tau after phosphorylation SMI31, SM134, SM133, TAU1, and AT8; (g and h) blots of htau34 (lanes 2) and PHF tau (lanes 3). with SM135 and SMI310. Downloaded by guest on September 24, 2021 5386 Biochemistry: Lichtenberg-Kraag et al. Proc. NatL Acad Sci. USA 89 (1992) also include the blots with AT8 (Fig. 3f), an Alzheimer tangle antibody (28), and TAU1, an antibody against delphosphory- SMI--34 lated tau (10); both have epitopes in the region S199-S202 (29). The time course of the AT8 reaction resemlbles that of SMI31, SM134, SMI3S, and SM1310, while TAU1 is similar Pi + + I to SM133, even though the epitopes differ (see below). The b SMlI--33 d SMI--31 striking feature is that in each case it is the stage 2 phos- phorylation that determines the antibody respons;e, and that there is a precise relationship between gel shift, lphosphory- lation, and immunoreactivity. Xr..1L t A 4 -:) Antibody Epitopes. The major phosphorylati(Dn sites of FIG. 5. (a) SDS/PAGE oftau constructs K10 (lanes 1 and 2), K17 neurofilaments are motifs of the type KSP, where S is the (lanes 3 and 4), and K19 (lanes 5 and 6) before and after (+) phosphate acceptor (8, 15). tau has two such moitifs around phosphorylation. All constructs except K19 show a shift upon S235 and S396. They lie on either side ofthe repeatt region and phosphorylation. (b) Blot of a with SM133, which recognizes only are conserved in all tau isoforms. By analog)V one may K17 in the unphosphorylated form (lane 3). (c) SM134 recognizes K10 suspect that these sites are involved in the reacti4on with the and K17 in the phosphorylated form (only top bands, lanes 2 and 4), SMI neurofilament antibodies. We tested this by sequencing but not K19 (lanes 5 and 6). (d) SMI31 recognizes only the top band of tryptic peptides, by point of the serin ies, and by of the phosphorylated K10 (lane 2). making truncated tau constructs (Fig. 4). We first consider constructs K10, K17, and K1L9 and their site. Finally, antibody SM134 labels K10 and K17 in their response to phosphorylation (Fig. 5a). K10 and I(17 show a phosphorylated form but not K19 (Fig. 5c). The lack of K19 gel shift, but not K19. K10 shows three shifted bands reactivity argues that the epitope is not in the repeat region. indicating three phosphorylation sites in the C-tierminal re: The reactivity of K10 and K17 argues that the epitope is both gion. K17 shows only one shifted band, so that tIhere is only before and after the repeats, which seems mutually exclusive. one shift-inducing site in the region before the re-peats. Fig. Our interpretation is that SMI34 has a conformational epitope 5 b-d show the blots with SM133, SMI31, and SMI34; the data that depends on phosphorylated tails on either side of the on SM135 and SM1310 (not shown) are similar tto those on repeats, and that at least one tail must be present. SMI31. Antibody SM133 reacts only with K17 in 1the dephos- To find the phosphorylation sites directly, tryptic peptides phorylated state but not with K10 and K19 (Fig. 5b, lane 3). of htau34 were identified by HPLC and protein sequencing, This suggests that the epitope is in a region before 1the repeats, and phosphorylated residues were determined (30). There are between S198 and Q244, outside the sequences 4covered by two major phosphorylated tryptic peptides in these regions: the other constructs, and compatible with an epiitope at the peptide 1 (T231-K240, TPPKS(pPSSAK) contains the first first KSP site. Antibody SMI31 reacts with phosiphorylated KSP motif, phosphorylated at S235; peptide 2 (T386-R406, K10 but not K17 or K19 (Fig. Sd), indicating that the epitope TDHGAEIVYKS(p)PVVSGDTS(p)PR) contains the second is in the region T373-L441, compatible with the siecond KSP KSP site, phosphorylated at S3% and S404. We then made point mutants of the phosphorylatable residues 235 and 3% N K44 A103 C |- -J LI htau23 (Fig. 6) and analyzed them by gel shift and antibody reactivity Q244 E372 (Fig. 7). The parent protein htau40 and its KAP mutants have I 1 1.4A->s K19 nearly identical apparent molecular masses, and they all shift by the same amount after phosphorylation (Fig. 7a, lanes K1O 1-8). The reactivity of SM133 is strongly reduced when S235 (M) S198 is mutated to alanine (Fig. 7b, lanes 3 and 7) and obliterated K17 after phosphorylation (Fig. 7b, lanes 2, 4, 6, and 8). This P47 K2 means that the epitope of SM133 is around the first KSP site, K2 but phosphorylation at other sites has an influence as well P154 R221 (perhaps via a conformational change). The mutation at S3% K3M (second KSP site) has no influence on the SMI33 staining L344 K44 A103 A426 (Fig. 7b, lane 5 and 6). ______K4 D387 L....4*.'I I Fe **Le, , K5 . I .. L 1;I Q244 V30 -, K6 - ~~~K~f4 V337 ...... - L; i. K7 r. V337 Q244 IA LJ . I A ...... z K13 V306 L344 0244 gw ..n H388 K14 L344 H388 . . E 1FA. :. .?.. .v .-:-..I-- K15 . ---. .

. i ..i FIG. 4. Constructs of tau. Residue numbering is that of htau40 [not shown (see ref. 20); 441 residues (res.) four C-terminal repeats, Q244-E372, and two N-terminal inserts, E45-T102]; htau23 (352 res.) has three repeats and no inserts. K19 (99 res.) has only three repeats. K10 (168 res.), K17 (145 res.), K2 (204 res.), and K3M (335 .JLA res.) have leaders or tails as shown. K4 (270 res.), K5 (310 res.), K6 (322 res.), and K7 (321 res.) have two repeats. K13 (291 res.), K14 FIG. 6. Point mutants of htau40, htau34, and htau23, with Ser- (279 res.), and K15 (278 res.) have one repeat. 235, -396, and/or -404 replaced by alanine. Downloaded by guest on September 24, 2021 Biochemistry: Lichtenberg-Kraag et al. Proc. NatL. Acad. Sci. USA 89 (1992) 5387 ht40 235 396 2 35196 SMI1-31 pathological tau in a phosphorylation-dependent manner c (29). A combination ofpeptide sequencing, protein engineering, and immunoblotting was used to localize the epitopes. The epitope of SM133 is at the first KSP motif and requires an rI unphosphorylated S235 (Fig. 8a). SM133 recognizes normal SM 1-33 SMI-34 human tau, indicating that S235 is normally not b d phosphory- _s M-Ow0 lated, but does not react with PHF tau, except when it is .q.w .. w dephosphorylated (Fig. lb, lane 3 and 4). This means that phosphorylation of S235 is a factor contributing to the 1 2 4 5 6 7 pathological state. 3 8 1 2 3 4 5 6 7 8 SMI31 is an antibody against a major phosphorylated FIG. 7. SDS/PAGE and immunoblots of htau4O and point mu- epitope in neurofilament heavy chain. Since the main sites in tants ofFig. 6. (a) Lanes 1-8, SDS gel ofhtau4O and its mutants A235, the heavy chain are the repeated four-residue motifs KSPV A396, and A235/396 in the unphosphorylated and phosphorylated (9, 15), and since the four-residue motif KSPV occurs once (+, note shift) forms. (b) Blot of a with SM133. The antibody in tau, one would expect that S396 is crucial for the antibody response is strongly reduced when S235 is mutated, both in the reaction. However, the point mutation of S396 to alanine dephosphorylated and phosphorylated state (lanes 3 and 4, 7 and 8). does not eliminate the reactivity, suggesting that phosphor- When S396 is mutated to alanine the behavior is similar to that ofthe ylation sites other than S396 contribute to the epitope. The parent molecule (lanes 1 and 2, 5 and 6). (c and d) SMI31 and SMI34 recognize htau4O and all mutants in the phosphorylated form (lanes same is true for S404; but when both S396 and S404 are 2, 4, 6, and 8). changed to alanine the reactivity of SMI31 is lost. The combination of these two serines is therefore part of the As mentioned above, the epitope of SMI31 depends on the SMI31 epitope and phosphorylated in PHF tau, consistent phosphorylation ofsites behind the repeat region. When S3% with studies based on tau peptides (16). The epitope appears is mutated to alanine the antibody still reacts in a phos- to have a conformational aspect, because mutants containing phorylation-dependent manner, indicating that this is only one microtubule-binding repeat are not recognized not responsible for the epitope by itself (Fig. 7c, lane 6). (K13-K15). Mutating S404 to alanine yields the same result. However, if The search for the SMI34 epitope leads to an apparent both serines are mutated, the antibody no longer reacts upon contradiction. Elimination of the region before or after the phosphorylation (data not shown), showing that the epitope repeats (as in K10 or K17) leaves the epitope intact, suggest- includes these two phosphorylated serines. ing an epitope in the repeat region (Fig. 5). But this region (in SMI31 also has a the form of K19) shows no reaction either. We hypothesize conformational component: Constructs with only one repeat that SMI34 is a conformational antibody whose epitope is (K13-K15) are not recognized (data not shown). formed by two or more noncontiguous parts of the chain, SM134 shows the most complex behavior because it de- possibly in or near the repeat region (Fig. 8a). The "kinked" pends on phosphorylation sites before and after the repeat conformation is conducive to SM134 binding; it requires (i) region. The antibody recognizes all KAP mutants, so that phosphorylation, (ii) the presence of a tail before or after the S235 and S396 cannot play a major role. However, SM134 repeats, or both (Fig. 8 b, d, and e). Without these conditions, recognizes phosphorylated K17 and K10 but not K19 (Fig. the conformation becomes nonreactive with SM134 (Fig. 8 c Sc), suggesting that the regions before or after the repeats must cooperate with the repeats to generate the epitope. One a1-/ ()- SM133 possibility is that the epitope is noncontiguous; another is I N that it depends on the number and conformation of the lS235 repeats. To check these possibilities we made constructs with different combinations of two repeats (K5, K6, K7) and SM3 MC constructs with one repeat only (K13, K14, K15; Fig. 4). All C ofthese showed a shift upon phosphorylation, and all ofthem SM134 's _ SM131 were recognized by SM134. This means that the epitope of SMI34 does not depend on the number of repeats. However, the nature of the region just before the repeats seems to be b - N e important for the conformation and is sensitive to charges. Tau (+P) <3 K10 (+P) This can be deduced from constructs such as K2 or K3M, c'Iz- where charged sequences have been brought close to the - N f