Vol.
185,
No.
3, 1992
June
30,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
Association
of the Carboxy-Terminus
Precursor Claudia
With Alzheimer
B. Caputo,
William
Irene
F. Brunner,
Pharmacology
Paired Helical
R. Evangelista
Department,
Received
May 11,
Sobel,
Clay W.Scott,
Group, ICI Americas,
DE 19897
ICI Pharmaceuticals
Macclesfield,
Filaments
and David P. Blowers*
ICI Pharmaceuticals Wilmington,
*Biotechnology
of @-Amyloid Protein
Peter T. Barth*
Department,
1034-1040
Cheshire,
Division,
Alderley
Park,
UK
1992
sumary:
We investigated whether a peptide fragment from the C-terminus of &amyloid protein precursor is associated with Alzheimer paired helical filaments (PAPS). Antiserum BR188, to the last 20 amino acids of the precursor, did not cross-react with tau protein, known to be in PHFs. It did react with all five pronase-treated PEF preparations assayed by ELISA and immunogold-labelled the same PHF fibrils that a PHF-specific tau antibody labelled. Neither antibody labelled $/A4 fibrils. These results suggest that a fragment from the C-terminus of $-amyloid precursor protein copurifies with pronase-treated PHFs and may play a role in their molecular pathogenesis. 0 1992 Academic Press, Inc.
Two types of amyloid brains
of Alzheimer
completely associated difficult
patients A highly
protein
tau is present
to solubilize
form amyloid
of their fibrils,
vitro with
derived
Copyright All rights
accumulate
The composition
of PHFs is not
phosphorylated
(5), another
in the
form of the microtubule-
in PHFs (3,4).
which has precluded
composition.
However PHPs are a complete
chemical
Since tau has not been reported
more fibrilogenic
protein
are comprised of B/A4, a peptide
from APP (6).
(7). Recent studies
suggest
PHFs.
antibodies
Abbreviations: protein. 0006-291X192
PHFs and f3-amyloid,
to
may also be
in PHFs.
f3-Amyloid fibrils acids
(1,2).
known.
identification present
fibrils,
Several
B/A4 forms amyloid that
PHFs, paired
the C-terminus to the C-terminus
helical
filaments;
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1034
fibrils
of up to 43 amino spontaneously
of APP may label
be
tangles
in
associated (8,9)
and
APP, #3-amyloid precursor
Vol.
185,
the
No.
3,
1992
dystrophic
lesions
BIOCHEMICAL
neurites
that
of senile
contain
observed
upon
addition
a synthetic
PHFs.
sequence
fibrils
in
induced
to
by tau
We investigated immunologically substantial
MATERIALS
A sequence
peptide vitro
(15)
on isolated amounts
of
of
loosely
from
resemble
C-terminus
PHFs that bound
the
were
COMMUNICATIONS
two Alzheimer
C-terminus
of
PHF material to
and remain the
the
solubilized
which
RESEARCH
(g-12),
corresponding
protein,
whether
BIOPHYSICAL
plaques
analysis
amyloid
form
AND
the
APP was (13,14).
C-terminus
of APP forms
PHFs morphologically, tightly
bound
of APP could treated
contaminating
with
In
to tau
are (16).
be detected pronase
to remove
protein.
AND METHODS
Preparation of PHFs, Proteins and Peptides. PHFs were isolated from Alzheimer brains as the if11 fraction and treated with pronase as previously described (17). PHFs were prepared from five Alzheimer brains (FD52, FD69, FD72, FD76, and FD80;18). Bovine and porcine tau were isolated from microtubules as described previously (19). The human tau isoforms (20) with no inserts (~3) and with all inserts (~4~~) were expressed in E. coli and purified (21). APP,,,-,,, amino acids 647-695 of the intracellular domain of APP695 (6), was expressed in E. coli. Polymerase chain reaction was used to generate the corresponding DNA fragment from the APP cDNA clone pcEco1056. The vector pLB015 was ligated with the purified fragment and this plasmid was used to transform E. coli C600 hosts. Protein in the inclusion body fraction was solubilized in 6 M guanidine-HCl and dialyzed. The insoluble fraction was dissolved in 0.1% trifluoroacetic acid and reverse phase HPLC in acetonitrile was used to isolate the peptide to greater than 80% purity. Peptides were synthesized which correspond to amino acids 585-600 of APP695 (APP 585-600), 597-610 (AH’,,,-,,,), 611-624 WP,,l-,,), 649-668 (APP,,,-,,), 662-681 (APP,,,-,,), and 676-695 (C-APP). Their structures were confirmed by mass spectroscopy. The $/A4 peptide corresponding to amino acids 597-636 (APP,,,-,,,) was purchased from Bachem. Antibodies. BR188 was raised in rabbits against the C-terminal 20 residues of APP conjugated by glutaraldehyde to keyhole limpet hemocyanin. BR188 and monoclonal antibody 423 were kindly provided by Drs. Claude Wischik and Michal Novak. 423 recognizes a fragment of tau from PHFs but not soluble human tau (17). ELISA. Direct ELISAs were performed as described previously (18). Tau proteins were plated at 0.01 to 1.0 uM in 25 mM tris buffer, pH 7.1 or 50 mM carbonate buffer, pH 9.6. APP,,,-,, was plated at 0.0001 to 0.1 uM in 2% formic acid and synthetic peptides were plated at 0.01-10 uM in water. Control wells were plated with 0.7% bovine serum albumin. The values from wells treated with albumin instead of BR188 were subtracted from those for wells with primary antibody. Where indicated, BR188 was preincubated with 0.01 uM APP,,,-,, for one hour at 37OC before being added to the ELISA wells. Color reagent was ABTS. Statistical significance of mean ELISA values was assessed using the Student’s t test. Immunoelectron microscopy. PHFs in the if11 fraction were applied to carbon and formvar-coated grids, blocked with 0.1% gelatin in phosphate-buffered saline, floated on drops of diluted BR188 or antibody 423 diluted 1:lO for one hour at 30° C and then transferred to goat anti-rabbit immunoglobulin G or antimouse immunoglobulin G, respectively, conjugated with gold particles. In one experiment grids were exposed to BR188, followed by its secondary antibody conjugate and then to 423 and 1035
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
then to its secondary antibody conjugate. PHFs were negatively stained with 1% lithium phosphotungstic acid. Samples were observed on a Zeiss EM-1OA electron microscope at 25,000 and 40,000 X magnification. Immunoblot analysis was performed as described previously (18).
RESULTS Antiserum
BR188 reacted
and showed limited
reactivity
partially
with
overlaps
in ELISA with peptides with APP,,,-,,,
that
of C-APP (fig.
peptides APP,,,-,,,, APP,,,-,,,, BR188 also did not cross-react proteins
or bovine
tau by immunoblot fragment released with
antibody
or porcine analysis. from if11
reacted
a peptide 1).
and C-APP,
whose sequence
BR188 did not react
APP,,,-,,, APP,,,-,,,, or APP649-68v with up to 1 uM recominant human tau tau
ELISA or with either
by
BR188 also
failed
PHFs by formic
human or bovine
to cross-react
acid
treatment,
with
the tau
which reacts
with
with PHF preparations
PHF preparations
by ELISA.
in a dose-related
Antiserum
manner (fig.
BR188
2).
was not enhanced by pretreating PHFs with formic acid or 2), in contrast to the enhancement seen with several
1.6
1.2
0.4
0.0 .OOOl ,001
Peptide Figure
with
423 (17).
BR188 Immunoreactivity Immunoreactivity guanidine (fig.
APP,,,-,,
1. Immunoreactivity BR188 to peptides domain of APP695. squares, APP,,,-,,;
.oi
.,
Concentration
by ELISA of corresponding to Circles, APP,,,-,,; color development
1036
1
10
(PM)
a 1:lOO dilution of antiserum sequences from the intracellular triangles, C-APP (APPs76-95); time was 5 min.
Vol.
AND BIOPHYSICAL
BIOCHEMICAL
185, No. 3, 1992
RESEARCH COMMUNICATIONS
1.6 0.8
1A
B
1.2 0.8 : 4
0.6
0.4
0.0
100 300
30
0
2
Antibody
1000
Dilution
0
0.0
3
Factor
-
--
Antibody
Pretreatment
+
With APPw-95
Figure
2. Immunoreactivity as determined by direct diluted 1:250 with 25 guanidine-HCl (triangles), albumin (circles) was time was 30 minutes.
Figure
3. Immunoreactivity of a 1:lOO dilution of antiserum BR188 with as the plated antigen or (B) pronase-treated (A) 0.001 PM APP,,,wg5 PHFs diluted 1:250 in tris buffer. Antiserum was incubated for one hour alone (striped bars) or in the presence of APP,,,-,s (solid and hatched bars) prior to assay by ELISA. Preincubation of BR188 with or without the peptide is also indicated by + or -, respectively. Color development time was ten minutes.
anti-tau
antibodies
preparations values
using
showed
of
0.718
The
This
assay
the
(Fig.
same PHF preparations immunoreactivities,
for
PHFs in
difference
immunoreactivity
was substantially
antiserum BR188 with pronase-treated PHFs ELISA. Prior to plating, if11 PHFs were ml4 tris buffer, pH 7.1 (diamonds), 2 M or 2% formic acid (squares). Bovine serum plated in control wells. Color development Means k S.E.M.s are shown.
similar
k 0.057
in guanidine.
of
of
reduced
acid
of
and 0.883
f 0.047
peptide
BR188
BR188 with
five
PI-IF for
PHFs
significant.
APP,,,-,,
by preincubating
All
mean absorbance
statistically
BR188 with
Preincubation
3).
with
formic
was not
(18).
tau
with
and with
APP647-95
PHFs
prior
had no effect
to
on
immunoreactivity. Immunogold
Labelling
antibody
dilutions
antibody
with
of
1:lO
than
in
treated antibodies fibrils
or
C-APP
1:30.
the
of of
both (fig.
which
(fig.
peptide
The
BR188 4B).
contaminate
4A)
BR188 all
seen
and 423, No labelling these
for
each
fibril
4C,
of
on the
the dilutions
pattern, 17).
was decorated
was observed preparations.
PHFs at
by BR188 at
in a periodic 423 (fig.
1037
if11
Preabsorption
labelling
was distributed
pattern
labelled
and 1:lO.
abolished
label
continuous
with
PHFs by BR188.
1:30
rather
When PHFs were with
typical
both B/A4
Figure
4. Electron micrographs of PHFs immunogold-labeled with antiserum BR188 and 423 are shown. Pronase-treated PHPs in an if11 preparation were applied to grids and treated with BR188 (A) or 423 (C) or BR188 followed by 423 (B). Secondary antibodies were conjugated to 10 nm gold particles in panels A and C. In panel B the secondary antibody for BR188 was conjugated to 15 nm gold particles and for 423 was conjugated to 5 nm particles. Magnification, ~23,000.
DISCUSSION The the
present
C-terminus
antiserum
used,
study
demonstrates
of APP is BR188,
present
appears
immunologically
that
in pronase-treated to be specific 1038
a fragment
PHF preparations. for
the
C-terminus
from The
of APP as
Vol.
185,
it
No.
3, 1992
BJOCHEMICAL
does not cross-react
Specificity
with
other
BIOPHYSICAL
regions
RESEARCH
of APP or with
was also shown by loss of the observed
BR188 preabsorbed Electron decorated
with
a C-terminal
microscopy
presentations
Alternatively
is presented
immunoreactivity
along the fibril,
by BR188 or may reflect
a C-APP like
proportion
tau proteins.
that not all of the fibril This pattern may reflect
dilution.
recognized
COMMUNICATIONS
with
APP peptide.
revealed
at any antibody
in the way the antigen serum.
AND
surface was variations
with only certain
the low potency
of the
peptide may account for only a small leaving large stretches of the fibril
of the PAP structure, While a C-APP-containing fragment may not comprise devoid of the peptide. a major portion of the PHF structure, it also does not seem to be an adventitious
contaminant,
resistent
as the association
fragment
with PHI% is
to pronase.
Although
B/A4 contaminates
be associated
with
tangles
due to a B/A4-containing synthetic
that
labelled
(22),
BR188 reactivity of APP.
and B/A4 fibrils
were labelled
with
PAP preparations
fragment
$/A4 peptides
The fibrils also
of this
and has been reported does not appear
an antibody
were never labelled
specific
with
to detect
other
with
BR188.
PEFs, as they were
for PHF-tau.
BR188 is also probably not reacting with an APP fragment that includes the N-terminal region of the intracellular domain of APP. were unable
to be
BR188 does not cross-react
with BR188 were clearly
to
intracellular
APP regions
We
in PBF preparations
with
four antisera (W298, W299, W300, BR215) to peptide APPs49-6( (Caputo, Sygowski , and Brunner, unpublished observation). These antisera detect as little as 0.4 nH APPsrsess by ELISA. Furthermore BR188 did not cross-react
with APP,,,-,,.
The presence two major amyloid Alzheimer’s protein,
of C-APP in PHFs provides fibrils
disease. APP, may give
which together
and the lesions
The proteolytic rise
a biochemical
link
which they comprise
processing
between the in
of the same precursor
to both B/A4 and C-APP-containing
PHF fibrils,
form both plaques and tangles.
ACKNOWLEDGHENTS The authors thank Dr. Michel Goedert for the tau expression plasmids, Dr. John Hardy for the APP cDNA clone, and Dr. Craig Thornber for the synthetic peptides used in this study.
REFERENCES 1. 2.
Glenner, G.G. (1980) New Eng. J. Bed. 302, 1283-1292. Kirschner, D.A., Abraham, C., and Selkoe, D.J. (1986) Acad. Sci. USA 83, 503-507. 1039
Proc. Nat.
Vol.
185, No. 3, 1992
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Wood, J.G., Mirra, S.S., Pollock, N.J., and Binder, L.I. (1986) Proc. Nat. Acad. Sci. USA 83, 4040-4043. Grundke-Iqbal, I., Iqbal, K., Tung, Y.-C., Quinlan, M., Wisniewski, H.M. and Binder, L.I. (1986) Proc. Nat. Acad. Sci. USA 83, 4913-4917. Selkoe, D.J., Ihara, Y. and Salazar, F.J. (1982) Science 215, 1243-5. Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Hasters, C.L., Grzeschik, K.-H., Hulthaup, G., Beyreuther, K., and Muller-Hill, B. (1987) Nature 325, 733-736. Gorevic, P.D., Castano, E.M., Sarma, R., and Frangione, B. (1987) Biochem. Biophys. Res. Comm. 147, 854-862. Dickson, D.W., Fishman, A., Mattiace, L.A. and Yen, S.-H. (1991) J. Neuropathol. Exp. Neurol. 50, 317. Yamaguchi, H., Ishiguro, K., Shoji, M., Yamazaki, T., Nakazato, Y., Ihara, Y. and Hirai, S. (1990) Brain Res. 537, 318-322. Ishii, T., Kametani, F., Haga, S. and Sato, M. (1989) Neuropathol. Appl. Neurobiol. 15, 135-147. Arai, Il., Lee, V.M.-Y., Otvos, L., Greenberg, B.D., Lowery, D.E., Sharma, S.K., Schmidt, N.L. and Trojanowski, J.Q. (1990) Proc. Nat. Acad. Sci. USA 87, 2249-2253. Shoji, M., Hirai, S., Yamaguchi, H., Harigaya, Y. and Kawarabayashi, T. (1990) Brain Res. 512, 164-168. Wischik, C.M., Novak, H., Jakes, R., Edwards, P. and Harrington, C.R. (1991) Kumamoto Med. J. 42, S15-S16. Caputo, C.B., Wischik, C., Sobel, I.R.E., Kirschner, D.A., Fraser, P.E. and Brunner, W.F. (1991) Sot. Neurosci. Abstr. 17, 1446. Caputo, C.B., Fraser, P.E., Sobel, I.R.E., and Kirschner, D.A. (1992) Arch. Biochem. Biophys. 292, 199-205. Caputo, C.B., Sygowski, L.A., Scott, C.W and Sobel, I.R.E. (1992) Neurobiol. Aging, in press. Wischik, C.M., Novak, H., Thogersen, H.C., Edwards, P.C., Runswick, M.J., Jakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A. (1988) Proc. Nat. Acad. Sci. USA 85, 4506-4510. Caputo, C.B., Wischik, C., Novak, M., Scott, C.W., Brunner, W.F., Montejo de Garcini, E., Lo, M.M.S., Norris, T.E. and Salama, A.I. (1992) Neurobiol. Aging 13, 267-274. Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.-C., Zaidi, N.S. and Wisniewski, A.M. (1986) J. Biol. Chem. 261, 6084-6089. Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, D. and Crowther, R.A. (1989) Neuron 3, 519-526. Scott, C.W, Blowers, D.P., Barth, P.T., Lo, M.M.S., Salama, A.I. and Caputo, C.B. (1991) J. Neurosci. Res. 30, 154-162. Masters, C.L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R.N., and Beyreuther, K. (1985) EMBOJ. 4, 2757-2763.
1040
185,
No.
3, 1992
June
30,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
Association
of the Carboxy-Terminus
Precursor Claudia
With Alzheimer
B. Caputo,
William
Irene
F. Brunner,
Pharmacology
Paired Helical
R. Evangelista
Department,
Received
May 11,
Sobel,
Clay W.Scott,
Group, ICI Americas,
DE 19897
ICI Pharmaceuticals
Macclesfield,
Filaments
and David P. Blowers*
ICI Pharmaceuticals Wilmington,
*Biotechnology
of @-Amyloid Protein
Peter T. Barth*
Department,
1034-1040
Cheshire,
Division,
Alderley
Park,
UK
1992
sumary:
We investigated whether a peptide fragment from the C-terminus of &amyloid protein precursor is associated with Alzheimer paired helical filaments (PAPS). Antiserum BR188, to the last 20 amino acids of the precursor, did not cross-react with tau protein, known to be in PHFs. It did react with all five pronase-treated PEF preparations assayed by ELISA and immunogold-labelled the same PHF fibrils that a PHF-specific tau antibody labelled. Neither antibody labelled $/A4 fibrils. These results suggest that a fragment from the C-terminus of $-amyloid precursor protein copurifies with pronase-treated PHFs and may play a role in their molecular pathogenesis. 0 1992 Academic Press, Inc.
Two types of amyloid brains
of Alzheimer
completely associated difficult
patients A highly
protein
tau is present
to solubilize
form amyloid
of their fibrils,
vitro with
derived
Copyright All rights
accumulate
The composition
of PHFs is not
phosphorylated
(5), another
in the
form of the microtubule-
in PHFs (3,4).
which has precluded
composition.
However PHPs are a complete
chemical
Since tau has not been reported
more fibrilogenic
protein
are comprised of B/A4, a peptide
from APP (6).
(7). Recent studies
suggest
PHFs.
antibodies
Abbreviations: protein. 0006-291X192
PHFs and f3-amyloid,
to
may also be
in PHFs.
f3-Amyloid fibrils acids
(1,2).
known.
identification present
fibrils,
Several
B/A4 forms amyloid that
PHFs, paired
the C-terminus to the C-terminus
helical
filaments;
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1034
fibrils
of up to 43 amino spontaneously
of APP may label
be
tangles
in
associated (8,9)
and
APP, #3-amyloid precursor
Vol.
185,
the
No.
3,
1992
dystrophic
lesions
BIOCHEMICAL
neurites
that
of senile
contain
observed
upon
addition
a synthetic
PHFs.
sequence
fibrils
in
induced
to
by tau
We investigated immunologically substantial
MATERIALS
A sequence
peptide vitro
(15)
on isolated amounts
of
of
loosely
from
resemble
C-terminus
PHFs that bound
the
were
COMMUNICATIONS
two Alzheimer
C-terminus
of
PHF material to
and remain the
the
solubilized
which
RESEARCH
(g-12),
corresponding
protein,
whether
BIOPHYSICAL
plaques
analysis
amyloid
form
AND
the
APP was (13,14).
C-terminus
of APP forms
PHFs morphologically, tightly
bound
of APP could treated
contaminating
with
In
to tau
are (16).
be detected pronase
to remove
protein.
AND METHODS
Preparation of PHFs, Proteins and Peptides. PHFs were isolated from Alzheimer brains as the if11 fraction and treated with pronase as previously described (17). PHFs were prepared from five Alzheimer brains (FD52, FD69, FD72, FD76, and FD80;18). Bovine and porcine tau were isolated from microtubules as described previously (19). The human tau isoforms (20) with no inserts (~3) and with all inserts (~4~~) were expressed in E. coli and purified (21). APP,,,-,,, amino acids 647-695 of the intracellular domain of APP695 (6), was expressed in E. coli. Polymerase chain reaction was used to generate the corresponding DNA fragment from the APP cDNA clone pcEco1056. The vector pLB015 was ligated with the purified fragment and this plasmid was used to transform E. coli C600 hosts. Protein in the inclusion body fraction was solubilized in 6 M guanidine-HCl and dialyzed. The insoluble fraction was dissolved in 0.1% trifluoroacetic acid and reverse phase HPLC in acetonitrile was used to isolate the peptide to greater than 80% purity. Peptides were synthesized which correspond to amino acids 585-600 of APP695 (APP 585-600), 597-610 (AH’,,,-,,,), 611-624 WP,,l-,,), 649-668 (APP,,,-,,), 662-681 (APP,,,-,,), and 676-695 (C-APP). Their structures were confirmed by mass spectroscopy. The $/A4 peptide corresponding to amino acids 597-636 (APP,,,-,,,) was purchased from Bachem. Antibodies. BR188 was raised in rabbits against the C-terminal 20 residues of APP conjugated by glutaraldehyde to keyhole limpet hemocyanin. BR188 and monoclonal antibody 423 were kindly provided by Drs. Claude Wischik and Michal Novak. 423 recognizes a fragment of tau from PHFs but not soluble human tau (17). ELISA. Direct ELISAs were performed as described previously (18). Tau proteins were plated at 0.01 to 1.0 uM in 25 mM tris buffer, pH 7.1 or 50 mM carbonate buffer, pH 9.6. APP,,,-,, was plated at 0.0001 to 0.1 uM in 2% formic acid and synthetic peptides were plated at 0.01-10 uM in water. Control wells were plated with 0.7% bovine serum albumin. The values from wells treated with albumin instead of BR188 were subtracted from those for wells with primary antibody. Where indicated, BR188 was preincubated with 0.01 uM APP,,,-,, for one hour at 37OC before being added to the ELISA wells. Color reagent was ABTS. Statistical significance of mean ELISA values was assessed using the Student’s t test. Immunoelectron microscopy. PHFs in the if11 fraction were applied to carbon and formvar-coated grids, blocked with 0.1% gelatin in phosphate-buffered saline, floated on drops of diluted BR188 or antibody 423 diluted 1:lO for one hour at 30° C and then transferred to goat anti-rabbit immunoglobulin G or antimouse immunoglobulin G, respectively, conjugated with gold particles. In one experiment grids were exposed to BR188, followed by its secondary antibody conjugate and then to 423 and 1035
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
then to its secondary antibody conjugate. PHFs were negatively stained with 1% lithium phosphotungstic acid. Samples were observed on a Zeiss EM-1OA electron microscope at 25,000 and 40,000 X magnification. Immunoblot analysis was performed as described previously (18).
RESULTS Antiserum
BR188 reacted
and showed limited
reactivity
partially
with
overlaps
in ELISA with peptides with APP,,,-,,,
that
of C-APP (fig.
peptides APP,,,-,,,, APP,,,-,,,, BR188 also did not cross-react proteins
or bovine
tau by immunoblot fragment released with
antibody
or porcine analysis. from if11
reacted
a peptide 1).
and C-APP,
whose sequence
BR188 did not react
APP,,,-,,, APP,,,-,,,, or APP649-68v with up to 1 uM recominant human tau tau
ELISA or with either
by
BR188 also
failed
PHFs by formic
human or bovine
to cross-react
acid
treatment,
with
the tau
which reacts
with
with PHF preparations
PHF preparations
by ELISA.
in a dose-related
Antiserum
manner (fig.
BR188
2).
was not enhanced by pretreating PHFs with formic acid or 2), in contrast to the enhancement seen with several
1.6
1.2
0.4
0.0 .OOOl ,001
Peptide Figure
with
423 (17).
BR188 Immunoreactivity Immunoreactivity guanidine (fig.
APP,,,-,,
1. Immunoreactivity BR188 to peptides domain of APP695. squares, APP,,,-,,;
.oi
.,
Concentration
by ELISA of corresponding to Circles, APP,,,-,,; color development
1036
1
10
(PM)
a 1:lOO dilution of antiserum sequences from the intracellular triangles, C-APP (APPs76-95); time was 5 min.
Vol.
AND BIOPHYSICAL
BIOCHEMICAL
185, No. 3, 1992
RESEARCH COMMUNICATIONS
1.6 0.8
1A
B
1.2 0.8 : 4
0.6
0.4
0.0
100 300
30
0
2
Antibody
1000
Dilution
0
0.0
3
Factor
-
--
Antibody
Pretreatment
+
With APPw-95
Figure
2. Immunoreactivity as determined by direct diluted 1:250 with 25 guanidine-HCl (triangles), albumin (circles) was time was 30 minutes.
Figure
3. Immunoreactivity of a 1:lOO dilution of antiserum BR188 with as the plated antigen or (B) pronase-treated (A) 0.001 PM APP,,,wg5 PHFs diluted 1:250 in tris buffer. Antiserum was incubated for one hour alone (striped bars) or in the presence of APP,,,-,s (solid and hatched bars) prior to assay by ELISA. Preincubation of BR188 with or without the peptide is also indicated by + or -, respectively. Color development time was ten minutes.
anti-tau
antibodies
preparations values
using
showed
of
0.718
The
This
assay
the
(Fig.
same PHF preparations immunoreactivities,
for
PHFs in
difference
immunoreactivity
was substantially
antiserum BR188 with pronase-treated PHFs ELISA. Prior to plating, if11 PHFs were ml4 tris buffer, pH 7.1 (diamonds), 2 M or 2% formic acid (squares). Bovine serum plated in control wells. Color development Means k S.E.M.s are shown.
similar
k 0.057
in guanidine.
of
of
reduced
acid
of
and 0.883
f 0.047
peptide
BR188
BR188 with
five
PI-IF for
PHFs
significant.
APP,,,-,,
by preincubating
All
mean absorbance
statistically
BR188 with
Preincubation
3).
with
formic
was not
(18).
tau
with
and with
APP647-95
PHFs
prior
had no effect
to
on
immunoreactivity. Immunogold
Labelling
antibody
dilutions
antibody
with
of
1:lO
than
in
treated antibodies fibrils
or
C-APP
1:30.
the
of of
both (fig.
which
(fig.
peptide
The
BR188 4B).
contaminate
4A)
BR188 all
seen
and 423, No labelling these
for
each
fibril
4C,
of
on the
the dilutions
pattern, 17).
was decorated
was observed preparations.
PHFs at
by BR188 at
in a periodic 423 (fig.
1037
if11
Preabsorption
labelling
was distributed
pattern
labelled
and 1:lO.
abolished
label
continuous
with
PHFs by BR188.
1:30
rather
When PHFs were with
typical
both B/A4
Figure
4. Electron micrographs of PHFs immunogold-labeled with antiserum BR188 and 423 are shown. Pronase-treated PHPs in an if11 preparation were applied to grids and treated with BR188 (A) or 423 (C) or BR188 followed by 423 (B). Secondary antibodies were conjugated to 10 nm gold particles in panels A and C. In panel B the secondary antibody for BR188 was conjugated to 15 nm gold particles and for 423 was conjugated to 5 nm particles. Magnification, ~23,000.
DISCUSSION The the
present
C-terminus
antiserum
used,
study
demonstrates
of APP is BR188,
present
appears
immunologically
that
in pronase-treated to be specific 1038
a fragment
PHF preparations. for
the
C-terminus
from The
of APP as
Vol.
185,
it
No.
3, 1992
BJOCHEMICAL
does not cross-react
Specificity
with
other
BIOPHYSICAL
regions
RESEARCH
of APP or with
was also shown by loss of the observed
BR188 preabsorbed Electron decorated
with
a C-terminal
microscopy
presentations
Alternatively
is presented
immunoreactivity
along the fibril,
by BR188 or may reflect
a C-APP like
proportion
tau proteins.
that not all of the fibril This pattern may reflect
dilution.
recognized
COMMUNICATIONS
with
APP peptide.
revealed
at any antibody
in the way the antigen serum.
AND
surface was variations
with only certain
the low potency
of the
peptide may account for only a small leaving large stretches of the fibril
of the PAP structure, While a C-APP-containing fragment may not comprise devoid of the peptide. a major portion of the PHF structure, it also does not seem to be an adventitious
contaminant,
resistent
as the association
fragment
with PHI% is
to pronase.
Although
B/A4 contaminates
be associated
with
tangles
due to a B/A4-containing synthetic
that
labelled
(22),
BR188 reactivity of APP.
and B/A4 fibrils
were labelled
with
PAP preparations
fragment
$/A4 peptides
The fibrils also
of this
and has been reported does not appear
an antibody
were never labelled
specific
with
to detect
other
with
BR188.
PEFs, as they were
for PHF-tau.
BR188 is also probably not reacting with an APP fragment that includes the N-terminal region of the intracellular domain of APP. were unable
to be
BR188 does not cross-react
with BR188 were clearly
to
intracellular
APP regions
We
in PBF preparations
with
four antisera (W298, W299, W300, BR215) to peptide APPs49-6( (Caputo, Sygowski , and Brunner, unpublished observation). These antisera detect as little as 0.4 nH APPsrsess by ELISA. Furthermore BR188 did not cross-react
with APP,,,-,,.
The presence two major amyloid Alzheimer’s protein,
of C-APP in PHFs provides fibrils
disease. APP, may give
which together
and the lesions
The proteolytic rise
a biochemical
link
which they comprise
processing
between the in
of the same precursor
to both B/A4 and C-APP-containing
PHF fibrils,
form both plaques and tangles.
ACKNOWLEDGHENTS The authors thank Dr. Michel Goedert for the tau expression plasmids, Dr. John Hardy for the APP cDNA clone, and Dr. Craig Thornber for the synthetic peptides used in this study.
REFERENCES 1. 2.
Glenner, G.G. (1980) New Eng. J. Bed. 302, 1283-1292. Kirschner, D.A., Abraham, C., and Selkoe, D.J. (1986) Acad. Sci. USA 83, 503-507. 1039
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