BIOCHEMICAL
Vol. 169, No. 1, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 171-l 76
May 31, 1990
SYNTHETIC PEPTIDE ANTIGENS ELICIT MONOCLONAL AND POLYCLONAL ANTIBODIES TO CYTOCHROME P450 IA2 Michael
J. Myers, Gao Liu, Haruko Miller, Harry V. Gelboin. Richard C. Robinson and Fred K. .Friedman
Laboratory
Received
is, Nat ional Carcinogenes Bethesda, MD 20892
of Molecular
March
28,
Inst itute,
Cancer
1990
Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent antisera to both peptides detected both rat IA2 and IA1 on assay. However, immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IAl. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies. e1990 Academic Press,
1°C.
Cytochrome a wide
array
drugs
and
disparate 450
P-450
chemical
enzymes,
of
of
role
of
have
been
a given
P-450s
and
family
antibodies, single
many of
P-450 are
P450.
directed
against
several
P45O's.
specific Thus
the
forms
of
individual
exists
for
goal
epitopes
But,
among
P-450s
generated
towards
of
a given may
rather
171
be
are
to
P-
substrate
powerful
due
in
the the
tools
a high belong
to
degree to
of
the
crossreact
monoclonal
a given
that
radioimmunoassay, to
that
useful
individual
activation.
P450 family
than
and
understanding
to one P-450 these
large
enzymes
via
(3,4).
approach
P450 enzyme
of P-450
P-450s
Accordingly,
the
overlapping
multiplicity
inhibition antibodies
or
metabolize
prostaglandins,
multiplicity
unique
to
system
metabolize
and metabolic
P-450
(6-8).
a different a single
the
an important
antibodies
which
to
of
either role
characterize
oxidase steroids,
ability
detoxification
similarity
(S),
The
the is
and enzyme
related
function including
possess of
polyclonal to
sequence
mixed
a result
substrate
employed
immunopurification gene
which
in metabolism,
Monoclonal
other
of
is
Elucidation
metabolism
the
(1,2).
chemicals
many
specificities.
of
and xenobiotics,
carcinogens
number
protein
enzymes
of endobiotics
same with
and polyclonal rather
than
elicit
antibodies
epitope
for
a
common to
0006-291X/90 $1.50 Copyrighi 0 1990 by Academic Press, Inc. ALI rights of reproduction in any form reserved.
Vol.
169, No. 1, 1990
One
potential
generated
by
sequence
which large
synthesis. towards with
is rat
epitope
site of
these
used
as
the
IA2
the
P-450.
to the the
MATERIALS
of
the
can
of this small
our
anti-peptide ref
peptide
technique
peptide
obtained
lacking
details
antibodies,
synthesized
be
generally
report
as per
specific
The advantages that
and polyclonal
site
a chemically
are
(nomenclature
RESEARCH COMMUNICATIONS
of
structure
antigen This
antigen.
use
agent
advantages
monoclonal
P450
with
a given
amounts of
AND BIOPHYSICAL
immunizing
for
Both generating
the
is
as the
is unique the
relatively
solution
using
in restricting
molecule
BIOCHEMICAL
and in the by
when
chemical
the
entire
successful
antibodies
are
efforts which
react
9).
AND METHODS
Peptides were synthesized on a BioSearch 9600 automated Peotide Svnthesis synthesizer using standard FMOC chemistry. The protected amino acids purchased from BioSearch. Peptide purity was assessed by amino acid composition and by analytical HPLC. The latter was performed on a Spectra-Physics system with Model 8000 pump Model 4270 integrator. The program consisted of a Brownlee C4 cartridge (10 x 25 mm) column and a linear gradient of 0 to 60% acetonitrile, in 0.1% trifluoracetic acid. Antibodv Generation The peptides were coupled to either ovalbumin or bovine serum albumin using I-ethyl-3(3-dimethylaminopropyl)-carbodiimide chemistry (10). Antisera to the ovalbumin-peptides were generated using Freunds Adjuvant and purified by ammonium sulfate precipitation and hydroxyapatite chromatography (11). Monoclonal antibodies were generated as previously described (8). Microsomes Control and 3-methylcholanthrene-induced rat (MC) microsomes were prepared from either untreated male Sprague-Dawley rats or rats treated for three days with 3-MC (40 mg/kg/d). Human livers were obtained from The University of Miami Kidney Transplant Unit. Liver microsomes were isolated by differential centrifugation (12) and were suspended in buffer containing 0.25 M sucrose and stored at -80°C. Protein content was measured by the Pierce BCA protein assay system (Pierce). Immunoblots and Enzvme Immunoassav (EIA) Microsomal proteins and immunopurified P-450s (6) were subjected to sodium dodecyl sulfate gel electrophoresis, transferred to nitrocellulose paper for Western blot analysis (13) and developed as previously described (14). The primary antibodies were used at a final titer of 1:50 (anti-Dl) or 1:lOO (anti-D2). EIA was performed as previously described (15) with the following modifications: the antigens were allowed to bind to the wells in phosphate-buffered saline; the primary antibody was allowed to react overnight and the second alkaline phosphatase conjugated antibody was used at a 1:600 dilution. Control antibodies did not bind in either EIA or Western blot assays. RESULTS AND DISCUSSION
the
Two 17-mer structurally
termed sequence
these
the
peptides
comprised
D2 corresponds sequence
peptide similar
sequences present P450 IA1 form were Dl
and
of residues
to the
carboxyl
D2.
Dl
208-224 terminal
in cytochrome P450 IA2 selected and synthesized.
corresponds with
the
sequence
MKPRTCEHVQAWPRFSK. 172
to sequence
an
internal
but
not in We have
amino
FPRKSEEMLNLVKSSKD,
commencing
at residue
acid and
497 with
Vol.
169, No. 1, 1990
0 0 n Cl
.3
anti-Dl, anti-Dl, anti-O*. anti-02.
P450 P450 P450 P450
BIOCHEMICAL
IA2 IA1 IA2 IA1
.8
II l ’ II I r’ /
.*
2
0 0 A 0
?
/
: {
AND BIOPHYSICALRESEARCH
8
96
4
.4
anti-Dl. anti-Dl, anti-D2, anti-D*,
COMMUNICATIONS
4
MC-mics C-mics MC-mics C-mics
I I I /
f
.l .2
0
0 10
0
1
10
Protein
100
02
(ng)
Protein
1000 (ng)
Fisure 1: Determination of anti-peptide antisera specificity using purified IA2 and IAl. Antisera generated against either peptide Dl (anti-01) or peptide D2 (anti-D2) were allowed to react with varying amounts of purified P450 IA2 and P450 IAl. Both antisera were used at a 1:lOO dilution. Fioure 2: Determination of anti-peptide microsomes obtained from either untreated animals (MC). Conditions are as detailed
Antisera
were
generated
immunizing
agent.
Anti-peptide
immunoblot
assay,
using
which
immunized
were
titers.
The
and
Negligible
binding limit
of 10 ng.
in
IA2
binding
to with
while
and
IA2,
it
anti-D2,
antisera
solely
protein.
This
(500 from
was necessary to
from
IA2 at
MC microsomes As microsomes
2).
to P450
was especially
In
ng)
(data
control
not
or
if to
important
these
the this with 173
of
to
the
affinity
(Fig. and
1). 1). P-450
specific of
MC microsomes
these
antiserum as compared
antiserum
did
2).
antisera
Both
not
100 ng of protein),
(using
1:8000
were
Anti-D2
anti-01
1:2000
1:00
antisera
microsomes.
microsomes
or untreated
ascertain IA2,
(Fig.
examined
of
(Fig. for
the
of
dilutions
one 7-fold
respectively
a dilution
binding
control
demonstrating
P450 IA1 was observed
contrast,
a dilution at
IAl,
that
in of
using
at
We further
level
(Fig.
P450
antisera
while
higher
MC microsomes purified
anti-D1
detected
fold
to
suggested
P-450s
animals
equivalent
findings
P450 IA2,
similar
IAl.
to to
All
having
our
as the
a dot-blot
peptides.
compared
both
in
albumin.
antisera
detected
as
results
relative
microsomes
distinguish
IA2 of
These
a three
control
detected
structurally
respect
demonstrated to
to
serum
detail
liver
to ovalbumin
screened
bovine
report two
antisera
to
the
to
this
of the
signal
or D2 coupled initially
and generated
in
to each
detection
IA2 was about antisera
positive
and anti-D2
higher
Ill was
coupled
presented
anti-Cl1
5-fold
The lower
peptides
antisera
Both
either
antisera
tested
results
representative
using
antisera specificity using rat (C) or 3-methylcholanthrene-induced above.
1:2000
respectively,
shown). animals EIA
data
P-450
and
respect
are known reflected some other, to anti-D2
to contain binding
P450 of
both
crossreactive antisera,
as this
Vol.
169, No. 1, 1990
antisera
reacted
between
control
analysis
was
criterion
for
almost and P-450
The
latter, or
both
exceeds
that
of
also in
(lane
with present
control
two
truncated
form
of either
unrelated
protein this
positive
chemical
agent.
to determine (lanes
1-3,
(lane
5).
orthologs
Human Mic 123
is
Figs. This of rat
control
The
p450
band
in
this
blot
additional
(lane
6), of
of this
related
to
shows
that
(lane level
P450 IA2
However, (Figs.
both
3 & 4).
by
that
this
is
unlikely
antisera band
following
under
an
with with
3-
respect
exposure
is currently
Human
Mic
to
this
investigation
form.
a protein antisera
p450
in human liver to
that
microsomes
of
cross-react
rat
P450
with
the
IA2
human
1234567
) w&l. _-
*_-
microsomes of human and rat liver Fiqure 3: Western Blot analysis antisera. Lanes 1, 2 and 3 contained human liver microsomes three separate individuals. Lanes 4 & 5 contained immunopurified
blot
analysis
using
6 and 7 contained untreated rat liver
anti-D2
antisera.
1
38245
1
2
3
4
-
u
5
6
05
Dl
IA2 (100 ng each), respectively while lanes 3-MC induced rat liver microsomes (MC) or Arrow indicates position of P450 IA2.
Rat
Human -)I
u
33u3
04
5pg
using anti(100 pg) from
rat
each of microsomes
Conditions
IA1 and either (CO).
are
as
Fiqure 5: Western Blot analysis of human and rat liver microsomes using anti02 monoclonal antibodies. Lanes 1, 2 contained human liver microsomes (100 ug, samples 1 & 3 from above). Lane 4 contained control rat liver microsomes (25 pg): Lanes 5 & 6 contained immunopurified rat IA1 and IA2 (lob ng each), respectively while lane 7 contained 5 uq 3-MC induced rat liver microsomes.Arrow indicates position of P450 IA2. '-
174
a
decreased
induction regulated
is
that
the
following
than
IA2.
&Q&
Western above.
not
greatly
the
7).
4).
was much lower
as it
corresponded
both
blot
data IA1
54 kD protein
IA2
P-450
migration
detected
is inversely
with
of
Furthermore,
band
either
immunoreacted
also
IAl,
IA1 and lower
level
suggests
obtained
protein
fold,
the
the this
related
by Western
P450 IA1 (lane
microsomes
sequences.
P450
_.
4:
an
(5-7
microsomes control
samples
that
which
IA2 peptides
both
of
-4
Fiqure detailed
distinguish Western
IA2 of
overloaded
in
IA2 or the
is%,0 4567
03
not as
P450 amount
antisera
detection P450
3 & 4) whose P450
in
not
possess
also result
6)
6) but
The identity it
was
Both
regulation
antisera
it
fold.
P450
protein
if
Both
(lane
different
suggests
the
when
a 54 kD protein
would
methylcholanthrene to
did
mobility
an equal
5-10
microsomes.
against
of
but
Accordingly,
purified
not
in MC microsomes
directed
levels
5) but
MC-microsomes
band in MC microsomes Although
IA2
1 & 2).
and D2 detected
lanes
IA2 by about
reacted
purified
electrophoretic
was observed
the
antisera that
Di
3 & 4,
in
with (Figs.
utilizing
however,
shown),
exclusively
RESEARCH COMMUNICATIONS
identification.
to
(Figs.
AND BIOPHYSICAL
MC microsomes
performed
Antisera analysis
BIOCHEMICAL
Vol.
169, No.. 1, 1990
Based used
for
d14,
was
BIOCHEMICAL
on the
the
results
generation
found
monoclonal
give
antibody
addition,
obtained
bound
Figs.
IA2
antibody
on
were
immunized
not
bind
the
polyclonal
the
utility
with
OVA-D2
One monoclonal,
a western
IA1 with
and
did
RESEARCH COMMUNICATIONS
antibodies.
binding
P450
3 & 4) as did
mice
monoclonal
positive
the monoclonal
1 &. 3 from
above,
of anti-D2
to
AND BIOPHYSICAL
blot
near
to human IA2 antisera,
(Fig.
equal
5).
The
intensity.
(lanes
nor
and
termed In
1 & 2, samples
did
it
bind
to
the
54 kD protein. These the
results
generation
peptide both
of
present
anti-P450 in both
to
P450
reactivity
IA2.
towards
antibody.
The
under
further
antibody for
However,
while
IA1 was
observed
this
P-450.
This
specificity
cannot
addition,
this
will
are not
available
synthesized
on the
The
criteria
binding
to
IAl,
situations
antisera Thus,
and of
although
to
the IA2) these
our
known for
the
P45Os
specific
structures
of
generation (IA2).
polyclonal
and monoclonal
cytochrome
P450.
This
for
P-450s.
a significantly
demonstrate two of
antibodies
for
and
when
which the
is
desired
molecule.
In
to P-450s
which
is
monoclonal
enzyme
antibodies
P450 IA2 However,
lesser
of
IA2
another
by 5-10
microsomal
the
utility
highly
peptides
that can be
of
extent, fold.
with
high
blots and
In
synthetic
cytochrome
be useful
to
Western
relative
will
relative
then
addition
P450
IA1
revealed only
in
binding
protein.
of
related
antibodies
technique
useful
but
monoclonal
DNA sequence.
IA1 was in excess
studies
the
cross-
present
sequence
entire
a
with
IA2,
first
peptide
of
sequences
to
at the
in generating
purified
to
and
unclear
describes
and screening, are
was observed
antisera
the
use react
peptide unique
especially
towards
the that
were is
using
be useful
a known
using
both
is
described
utilized
synthesized
approach
antisera EIA
in which
by both
of
study
also
be obtained also
basis
of
with
approach
antibodies
sequences
report
immunization
anti-peptide
by the
from
for
report
cross-reactivity
a chemically
antibody
method
this
these
This
against
a given
A recent
In contrast,
for
anti-peptide
IA1 and IA2 to elicit
investigation.
directed
unique
(16).
reason
of the
antibodies. P450
IA1 and IA2
P450
unique
demonstrate
high for
specificity
the
peptides P450
(IA1
proteins
specificity production for
designed
single
for of forms
one both of
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7.
Nebert, Lu, A. Gelboin, Friedman, Interact. Gonzales, Cheng, (1984) Friedman, Biochem.
D. W. & Gonzales, F. J. (1987) Annu. Rev. Biochem. 56, 945-993. Y. H. & West, S. B. (1980) Pharmac. Rev. 31, 277-295. H .V. & Friedman, K. F. (1985) Biochem. Pharm. 34, 2225-2234. H. V. (1985) Rev. Drug Metab. Drug F. K., Park, S. S. & Gelboin, 5, 159-192. F.J. (1989) Pharmacol. Rev. 40, 243-288. Gelboin, H.V., Song, B.J., Park, S.S. and Friedman, F.K. K.C., J. Biol. Chem. 259, 12279-12284. F.K., Robinson, R-C., Park, S.S. and Gelboin, H.V. (1985) Biophys. Res. Commun. 129, 926-933.
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BIOCHEMICAL
AND BIOPHYSICAL
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