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

REFERENCES :: 3. 4. 5. 6.

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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8. 9.

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Park, S.S., Fujino, T., West, Il., Guengerich, F.P., Gelboin, H.V. Cancer Research 42, 1998-1808. Nebert, D. W., Nelson, D. R., Adesnik, M., Coon, M. J., Estabrook, Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, Kemper, B., Levin, W., Phillips, I. R., Sate, R. and Waterman, M. R. DNA 8, 1-13. Frey, A. B., Waxman, D. J., Kreibich, G. (1985) J. Biol. Chem. 260, 15265. Stanker, L.H., Vanderlaan, M., Juarez-Salinas, H. (1985) J. Immunol.

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Lu, A.Y.H., Towbin, H.,

Levin, W. (1972) Biochem. Biophys. Res. Commun. 46, 1334-1339. Staehelin, T., Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A.

76, 4350-4354. 14. 15. 16.

Robinson, R. C., Shorr, R. G. L., Varrichio, A., Park, S. S., Gelboin, H., Friedman, V ., Miller, F. K. (1989) Pharmacol. 39, 137-144. Myers, M. J., Schook, L. B. (1987) Int. J. Immunopharm. 9, 817-825. Edwards, R. J., Singleton, A. M., Sesardic, D, Boobis, A. R., Davies, S. (1988) Biochem. Pharmacol. 37, 3735-3741.

176

H. D.

Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2.

Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and pol...
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