Affinity gels for purification of retinoid-specific binding protein (RSBP)

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 503-508

Vol. 155, No.l, 1988 August 30, 1988

AFFINITY

GELS

FOR PURIFICATION

RETINOID-SPECIFIC

Hiroyuki

Kagechika,

BINDING

Yuichi

Hashimoto,

Koichi

Faculty

of P h a r m a c e u t i c a l 7-3-1

Hongo,

OF

PROTEIN

(RSBP)

Emiko

Kawachi

and

Shudo

Sciences,

Bunkyo-ku,

University

Tokyo

113,

of T o k y o

Japan

Received June 20, 1988

Retinoids are d e f i n e d as c o m p o u n d s which elicit specific biological effects such as control of cell growth and cell d i f f e r e n t i a t i o n b y b i n d i n g to a s p e c i f i c r e c e p t o r . R e c e n t l y , we d e m o n s t r a t e d the p r e s e n c e of a p r o t e i n (RSBP) w h i c h s a t i s f i e s the c r i t e r i a for t h e r e t i n o i d r e c e p t o r . F o r p u r i f i c a t i o n of RSBP, w e p r e p a r e d two t y p e s of a f f i n i t y g e l s w i t h r e t i n o i d a l l i g a n d s (GelAm and Gel-Ch) based on synthetic ret i n o b e n z o i c acids which p--ossess v e r y p o t e n t retinoidal activities. R S B P in t h e c r u d e fraction extracted from cultured cells could be purified about 300-fold by affinity column chromatography using these affinity gels. © 1988 Academic Press, Inc.

Retinoids controlling believed

that

(retinoidal though not

the

well

receptor

new

Among

molecular

drawing

and

retinoids

by

been

effects class

the s y n t h e s i z e d

a

the

retinoidal

acid

for

(RA),

synthetic

reported

the

relationships acids. S - ~

4-(5,6,7,8-tetrahydroacid

(Am80) ~

(E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-l-propenyi]benzoic

acid

(Ch55) ~

compounds to

are

retinoid

many

we

5,5,8,8-tetramethyl-2-naphthalenylcarbamoyl)benzoic and

is

effects

a

retinobenzoic

acids,

It

activities such

and

Recently,

retinoids,

retinobenzoic

agents

r e c e p t o r , I-4~

and s t r u c t u r e - a c t i v i t y

of

as

biological

specific

ligand

r e p o r t e d . I, z~

biological structural

specific to

classical

all-trans-retinoic

have

the

of

attention

d i f f e r e n t i a t i o n . ', 2'

binding

mechanisms A

much

cell

elicit

activities)

is

synthesis, a

been

growth

understood.

retinoids

of

have

cell

the

should

biological

more

assay

potent

has

been

potent

systems)

retinoidal

their biological

r e c e p t o r . 3, 4~

receptor show

quite

also eliciL

retinoid

retinoid (which

possess

One

of

the

CRABP. ~, 2~

to C R A B P

503

effects

than

with much

These

by binding

candidates

However,

bioactivities

bind

activities.

for

Am80 RA lower

such

and

in

a

Ch55

various

affinity

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 155, No. 1, 1988

than

RAg-~

estimated true

;

the

to be

retinoid

possess

affinities

1/80 and

recently,

to

amount

Ch55 w i t h

very high

protein

receptor,

is a p r o t e i n

and

reported

named

steroid cloned gate

genes

were

and to reveal essential In (Figs. partial

1

this

acid

we

based

on

the

weight

which

receptor acid

mechanism

describe new-type

of R S B P

of

possess-

10 l° M-I ). 14~ and E v a n s I ~

on the b a s i s

of

should

resemble

of the p r o d u c t s

of t h e i r

of c l o n e d

To

genes

of r e t i n o i d a l

the

and

(association

50 kDa. ~5-~7~

products

protein

fraction,

o f 95 kDa,

gene

no and

retinoid

binding

receptor

weights

Am80

m a n n e r . 14~

true

a n d Ch55

in

high

possess RA,

of C h a m b o n ~5. ~6~

to be a r o u n d these

to to

competitive for

Am80

a n d to c h a r a c t e r i z e

paper,

binds

are of the o r d e r

retinoic

reported between

reported

that

for RA,

The m o l e c u l a r

purification

Materials

The

of a p r o t e i n

in the n u c l e a r

the g r o u p s

the m o l e c u l a r

2)

is )

molecular

retinoids

the

to i s o l a t e

and

were

(which possesses

retinoid-specific

of r e t i n o i c

that

relation

CRABP

compounds

the p r e s e n c e

criteria

affinity

these

receptors.

the

for

r e s p e c t i v e l y . 9~ the

line H L - 6 0

predominantly

an a p p a r e n t

a cloning

assumption

Ch55

all

in a m u t u a l l y

the

O n the o t h e r hand,

the

and

to

which

affinity

binding

toward

cell

C R A B P 9. 12, ~

exists

with

ing v e r y h i g h constants

of

was

RSBP

bind

we d e m o n s t r a t e d

leukemia

satisfies

and

(RSBP). 14~

should

retinoids,

detectable

This

Am80

of that of RA,

a c t i v i t y 4~

promyelocytic

response

of

1/530

receptor

retinoidal Very

human

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

investia n d RSBP,

action,

it is

RSBP. synthesis

of

retinobenzoic

b y use of the p r e p a r e d

affinity acids,

gels

and

affinity

the

gels.

and M e t h o d s

S y n t h e s i s of the A f f i n i t y L i g a n d 3 (Fig. I): 5,6,7,8-Te-t-rahydro-5,5,8,8-tetramethyl-2-naphthoic a c i d (I, 1.512 g) w a s c o n v e r t e d to the a c i d c h l o r i d e b y thionyl c h l o r i n e treatment, and was condensed with methyl p-aminosalicylate (1.08 g) in the p r e s e n c e of a c a t a l y t i c a m o u n t of 4 - d i m e t h y l a m i n o p y r i d i n e in a m i x t u r e of d r y b e n z e n e (30 ml) a n d p y r i d i n e (3 ml) to g i v e the a m i d e c o m p o u n d 2 in the y i e l d of 64.6% (11.6 g, mp. 2 2 7 . 5 - 2 2 8 . 0 ~ ). The compou-nd 2 (0.5 g) w a s t r e a t e d w i t h s o d i u m h y d r i d e (NaH) in d r y d i m e t h y l f o r m a m i d e ( 5 ml), a n d t h e n m i x e d w i t h BrCHzCHzCHzNHCOOCH2Ph (0.44 g). The m i x t u r e w a s s t i r r e d at r o o m temperature for 15 hr. The protected affinity ligand thus o b t a i n e d (yield: 50.8%) w a s d e p r o t e c t e d w i t h p o t a s s i u m h y d r o x i d e ( h y d r o l y s i s of the e s t e r group, yield: 93.5%) a n d t h e n w i t h trifluoroacetic acid (CF3COOH; debenzyloxycarbonylation) to give a f f i n i t y l i g a n d 3 in the y i e l d of 68.1%. S y n t h e s i s of the--Affinity L i g a n d 7 (Fi~. i): 5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthylmethylk e t o n e (4, 36.8 g) was h e a t e d w i t h m - c h l o r o p e r b e n z o i c a c i d (mCPBA, 45 g) in--chloroform (200 ml) for s e v e r a l h o u r s to g i v e c o m p o u n d 4 in the y i e l d of 87.6% (34.5 g). C o m p o u n d 5 w a s h e a t e d at 130140 ~ with aluminum chloride (AICI~, 37--g) to g i v e acetylrearranged product, 5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetra-

504

Vol. 155, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

O ~COOCH3

2)

~

c°°cH3

2

H2N" Y -OH I) NaH 2) 3) BrCH2CH2CH2NHCOOCH2Ph KOH )

O /~COOH ~'V~OCH2CH2CH2NH2

4) CF3COOH

m

ococ

i

2

1) Nail 2) BrCH2CH2CH2NHCOOCH2Ph 3) OHC ~

c

COOCH3

s

0

CH2CH2CH2NH2 |

>

~

4) CF3COOH

C

O

O

H

0 7

Fig. i.

Synthesis of affinity ligands

3 and 7

methyl-2-naphthylmethylketone (6) in the y i e l d of 77.7% (48.2 g). The h y d r o x y l g r o u p of 6 (2.532 g) was a l k y l a t e d w i t h B r C H z C H z C H 2 N H C O O C H z P h by the m e t h o d d e s c r i b e d a b o v e to g i v e the or__tho-alkoxy k e t o n e in the y i e l d of 83.6% (3.70 g). The aldol c o n d e n s a t i o n of this ketone(2.0 g) w i t h terephthalaldehydic acid methyl ester (0.75 g) in a m i x t u r e of 2 N s o d i u m h y d r o x i d e (5 ml) and e t h a n o l (30 ml) g a v e p r o t e c t e d a f f i n i t y l i g a n d 7, w h i c h was d e p r o t e c t e d by r e f l u x i n g in C F 3 C O O H to g i v e a f f i n i t y i T g a n d 7. P r e p a r a t i o n of the A f f i n i t y Gels A m and Ch (F~g~2): The N - h y d r o x y s u c c i n i m i d e e s t e r of a g a r o s e (Affi-Gel i0) was obtained from Bio-Rad Laboratories. The gel (2 ml in i s o p r o p a n o l ) was w a s h e d t h r e e times w i t h 2 ml of d i o x a n e a n d was r e s u s p e n d e d in 2 ml of d i o x a n e . To this suspension, ligand 3 or l i g a n d 7, p r e p a r e d as d e s c r i b e d above, and t r i e t h y l a m i n e ~ 1 2 . 5 ~ i) w e r e a d d e d at 25 °C and r e a c t e d for 2 hr. T h e n the u n r e a c t e d Nhydroxysuccinimide e s t e r w a s b l o c k e d by t r e a t m e n t w i t h 0.1 ml of 1 M e t h a n o l a m i n e for 1 hr. The m i x t u r e was d i l u t e d by a d d i t i o n of dioxane (6 ml), and c e n t r i f u g e d (2000 rpm x 4 min), and then decanted. The gel was w a s h e d s u c c e s s i v e l y w i t h dioxane, e t h a n o l and p h o s p h a t e b u f f e r e d s a l i n e (PBS; 136.9 m M NaCI, 2.68 m M KCI, 8.10 m M NazHPO4, 1.47 m M KHzPO4, 0.9 m M CaCI2, 0.49 m M MgCI2), and stocked in PBS containing 0.02% sodium azide at 4 °C . The coupling efficiencies w e r e e s t i m a t e d from U V a b s o r p t i o n m e a s u r e ments. E x t r a c t i o n of R S B P from C u l t u r e d H L - 6 0 Cells: HL-60 cells were cultured, collected and washed as described p r e v i o u s l y . ~4) The cell p e l l e t was homogenized and f r a c t i o n a t e d as d e s c r i b e d p r e v i o u s l y TM e x c e p t that the h o m o g e n i z 505

Vol, 155, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COOH

f

Gel-Am

(CH2)3NHCO(CH2)2CONH(CH2)2NHCOCH20 <

~

COOH

0 Gel-Ch (A = agarose) Fig.

2.

Structures

of affinity

gels,

Gel-~ra and G e l - C h

ing b u f f e r w a s c h a n g e d to PBS c o n t a i n i n g 20 m M Tris (pH 8.0), 0.6 M KCI, 1 m M d i t h i o t h r e i t o l (DTT), 20 m M m o l y b d a t e (Na2MoO4) and a p r o t e a s e i n h i b i t o r c o c k t a i l (PIC: 1 m M p h e n y l m e t h y l m e t h a n e s u l f o n y l f l u o r i d e , 0.1 m g / m l b a c i t r a c i n , 0.1 m M leupeptin, i~ g/ml p e p s t a t i n , 0.i m g / m l a p r o t i n i n a n d 1.5 n ~ EDTA). The a m o u n t of p r o t e i n was e s t i m a t e d b y the C o o m a s s i e b l u e m e t h o d as d e s c r i b e d by B r a d f o r d . TM The a m o u n t of R S B P w a s e s t i m a t e d b y f i l t e r b i n d i n g a s s a y of the s a m p l e s i n c u b a t e d w i t h t r i t i u m - l a b e l e d r e t i n o i d s [ 3 H - A m 8 0 (65 C i / ~ n o l e ) , ~ H - C h 5 5 (20 C i / m m o l e ) or ~ H - R A (50 C i / m m o l e ) ] in the p r e s e n c e o r a b s e n c e of e x c e s s a m o u n t s of c o l d r e t i n o i d s (Am80, Ch55 o r RA) at 4 ~ as d e s c r i b e d p r e v i o u s l y . L4~ Z H - A m 8 0 and 3 H - C h 55 w e r e prepared by Amersham. 3H-RA was purchased from NEN Research Products. P a r t i a l P u r i f i c a t i o n of R S B P U s i n g A f f i n i t y Gel A m o r Ch A f f i n i t y gel A m or C h (2 ml) w a s p a c k e d in a g l a s s t u b e (0.7 x i0 cm) a n d e q u i l i b r a t e d w i t h w a s h i n g b u f f e r (20 m M T r i s pH 8, 0.3 M NaCI, 1 m M DTT). A crude soluble protein fraction c o n t a i n i n g R S B P e x t r a c t e d f r o m c u l t u r e d H L - 6 0 c e l l s as d e s c r i b e d a b o v e w a s a p p l i e d on the p a c k e d a f f i n i t y gel c o l u m n w i t h a f l o w rate o f 9 ml/h. T h e n the c o l u m n w a s w a s h e d w i t h 50 ml of the washing buffer (9 ml/h). R S B P a b s o r b e d o n the gel w a s e l u t e d with elution buffer (20 m M Tris pH8, 1 m M DTT) a l o n e or the elution buffer containing 20 m M A m 8 0 (g m l / h ) . The e l u e n t w a s applied on a Mono Q HR5/5 column (obtained from Pharmacia) and eluted with a linear gradient of 0 - 0 . 5 M N a C I in the e l u t i o n b u f f e r as d e s c r i b e d p r e v i o u s l y . '4~ R S B P was e l u t e d w i t h 0 . 2 0 - 0 . 2 5 M NaCI in the e l u t i o n b u f f e r . The a m o u n t s of p r o t e i n a n d R S B P were estimated b y the C o o m a s s i e blue method as d e s c r i b e d by B r a d f o r d TM a n d b y f i l t e r b i n d i n g assay, ~4' r e s p e c t i v e l y .

Results

and Discussion It

receptor action.

is

essential

to

elucidate

isolate

the

to

for the

purify

RSBP.

and

molecular

As w e h a d e s t a b l i s h e d

fies the c r i t e r i a started

to

characterize mechanism

the p r e s e n c e

retinoid For

506

receptor this

the of

retinoid

retinoidal

of RSBP,

which

in H L - 6 0

cells, ~4~

we

designed

the

purpose,

we

satis-

VOI. 155, No. 1, 1988

affinity

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

gels with

retinoidal

ligands

(Gel-Am

a n d Gel-Ch)

as

shown

in Fig.2. Fig. obtain

1

to a g a r o s e

10,

gels,

attached

absorb

was

the

360

ability

prepared

than

7.4,

pmole

recovered

of

of anion

ionic to

between

interaction.

of

2

of

was in

shown). 14~ Ch55 than

on

at

300-fold

a and

the

exchange 72

hr

for

affinity

column

purified

more

retinoid-binding protein

fraction

and

needed

not the

Q

HR5/5

Methods.

fraction

or absence

reaction the

is

and

4000-fold

activity (Table

I).

of

incubated

Q

with

the

about

8%

the

buffer low; of of

150 RSBP the

probably

enough

RSBP

to e l u t e

RSBP-Am80

complex

as

described

showed complex

with

only

one

(data

not

3H-Am80

amount

4 ~

a

it

took

plateau.

chromatography, total from

recovery the

o r 3H-

of Ch55.

, and

reach

A quantitative

the

inter-

salt was

pmole

(Pharmacia)

Mono

of

eluted with elution

Eluted

to

the

I.

also

at

passed

conditions),

RSBP-Am80

slow

RSBP

that

(25-30%,

30 ml w a s

binding

that

recovery

dilute

of an e x c e s s

507

60

the

chromatogram

of

was

very

specific

(Gel-Am) than

The time

the the

In a d d i t i o n ,

low

elute

column

in

overwhelming

with

to

7.2,

hydrophobic

excellent

than

high

corresponding

efficiency

in T a b l e

the

rather

efficiency

a

RSBP,

to

very

(less

the

to RSBP.

Though

quite

sufficient

suggests

to

eluted

as s h o w n

retention

The pooled

was

fraction

is

o n the gels w a s

Mono

in the p r e s e n c e

ligand

of was

when

at pH 6.8,

binding

on 2 ml o f the gels).

Materials peak

the

be

was

20 m M A m 8 0

absorbed

possess

to b e l o w

This

there

gels.

under

absorbed

the

the e l u t i o n

was

the

activity

containing

the

major

of

about

separated

could

buffer

ml

The R S B P

the R S B P

ligands

described

adjusted

in

ligands

though

denaturation

purified

buffer

RSBP

alone,

retinoid-binding

was

the

efficiently

to b i n d

that

the r e t i n o i d a l

elution on

because

absorption

Gel-Ch)

ligands

diminished

suggests

absorbed

the

of

conditions

absorption.

retinoidal

also

ionic

absorbed

UV

R S B P was d i m i n i s h e d ;

f o r m is e s s e n t i a l

action

buffer)

the

quantitatively

which

and

RSBP very

the pH was

without

strength

The

(Gel-Am

under

When

the

RSBP,

(elution

to

attached

e s t e r of a g a r o s e

the

O n e ml of the g e l s w a s

to a b s o r b

column

ionization

low

employed

were

the salt c o n c e n t r a t i o n

RSBP

almost

the

gels

From

5-i0~ mole

absorbed

and

and Methods.

carboxylate

ml

that

gels

They

of the gel

through

routes

ligands

N-hydroxysuccinimide

t h a n 0.3 M for N a C I ) .

Materials

These

Laboratories).

estimated

properties. higher

(more

synthetic

1 ml of the gel.

Both

pH w a s

was

the

3 a n d 7.

with

Bio-Rad

it to

similar

shows

ligands

by coupling

(Affi-Gel the

briefly

the a f f i n i t y

crude

consideration

The more By

RSBP

was

of

the

soluble suggests

Vol. 155, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I. Purification of RSBP in Crude Soluble Protein Fraction Extracted from HL-60 Cells Loaded Sample Protein RSBP (mg) (pmole)

Columns

Eluted Fraction Protein RSBP Yield (mg) (pmole) (%)

Purification (-fold)

Gel-Am a)

106.0

21.20

0.112

6.36

30

284

Gel-Ch ~

99.0

17.82

0.078

4.63

26

329

Gel-Am ~ / Mono Q

102.0

14.79

0.002

1.26

8.5

4345

Gel-Ch~) /

104.0

15.20

0.001

1.18

7.8

8108

Mono Q

a) RSBP absorbed on the gels was eluted with elution buffer alone. b) RSBP absorbed on the gel was eluted with elution buffer containing 20 mM Am80, and then separated on a Mono Q column.

that

another

sufficient In

step

of

to

for the p u r i f i c a t i o n conclusion,

affinity

were

designed

tive

for the p u r i f i c a t i o n

affinity

10-

gels

a n d prepared. and

50-fold of R S B P gels

for

The gels

of RSBP.

a M o n o Q column,

purification

would

be

to h o m o g e n e i t y . the

were

purification

confirmed

of

RSBP

to be e f f e c -

By the u s e of the p r e p a r e d RSBP

could

be p u r i f i e d

4000-

to

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