Retinol-binding protein is synthesized in the mammalian eye

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 30, 1988

Pages 1078-1084

RETINOL-BINDING

PROTEIN IS SYNTHESIZED IN THE MAMMALIAN EYE

Robert L. Martone I, Eric. A. Schonl,2o DeWitt S. Goodman 3, Dianne ~& Soprano 3 and Joseph Herbert ~-

Departments of Neurology I, Genetics and Development 2 , and Medicine ~ Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032 Received October 20, 1988

As the chromophoric component of the visual pigment, retinol plays an essential role in vision. In the plasma, retinol is transported by retinol-binding protein (RBP) in complex with transthyretin (TTR, prealbumin). In previous work we demonstrated intraocular synthesis of TTR. To determine whether RBP is also synthesized in the eye, we performed Northern and Western blot analysis of rat eye, and detected both RBP mRNA and immunoreactive RBP. Regional Northern analysis of bovine eye localized RBP mRNA to ciliary body/iris and retina/RPE. Preliminary immunohistochemical studies revealed a widespread but heterogeneous distribution of RBP in rat eye. We postulate that ocular RBP and TTR are involved in the intraocular translocation of retinol. ©

1988 A c a d e m i c

Press,

Plasma

Inc.

retinol-binding

responsible complete

for

the

protein

plasma

nucleotide

transport

(5,6)

and

retinol

complex

with

another

thyroxine-binding transport

of

bloodstream

In

plasma

prealbumin, thyroid

and

the

the

plasma,

protein, by

been

and

of

The

reported.

holo-RBP

transthyretin

virtue

hormone)(10).

RBP

have

sequence

three

in the liver by the hepatocytes and, after binding retinol, (I).

RBP

(vitamin A)(I-4). The

(7,8)

structure holo-RBP

human

of acid

synthesized as

of

amino

dimensional secreted

(9)

(RBP) is a 21-kDa polypeptide which is

its

RBP

is is

forms a i:I molar (TTR, also known as

role

complex

in

the

circulates

plasma in

the

delivers retinol to vitamin A--requiring tissues

(3,4). TTR which

is are

sequestered

epithelium However, been

synthesized in the liver (13-18)

from

and

the

(11,12) and at two extrahepatic sites

the systemic circulation: retinal

pigment

the choroid plexus

epithelium

(RPE)(19-21)

the function of TTR within the central nervous system has not yet

established.

participate

By

analogy

with

its

function in the plasma, TTR may

in the transport of retinol or thyroxine

* To whom correspondence

should be addressed.

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

1078

(22) within the brain

Vol. 157, No. 3, 1988

and we

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

eye. Since retinoids are an essential component of the visual pigment, proposed that TTR may participate in the intraocular cycling of retinol

(19). If

ocular

plasma

TTR,

Although ~oes

TTR this

receptors

not

cross

~etinoid-binding ~hemically investigated

plays would

role in retinol transport similar to that of imply

the presence of RBP within the eye.

for RBP have been reported on the RPE the

proteins

distinct

a also

from

blood-retina

barrier

are

present

in

RBP

and

not

do

the

(23), plasma RBP

(23). eye

complex

While

(24-35), TTR.

the possibility of intraocular RBP synthesis.

several these are

We therefore Here we present

~vidence for de novo synthesis of RBP within the mammalian eye.

MATERIALS AND METHODS Except Io. Adult tudies.

where otherwise indicated, reagents were from Sigma Chemical male Sprague-Dawley albinos were used for all rat tissue

For Northern analysis, rat eyes and livers were frozen rapidly under iquid nitrogen. Bovine eyes were obtained from a local slaughterhouse nd frozen under liquid nitrogen after being dissected into regions: ornea and lens were dissected free and the retinal tissue removed from he sclera in the plane of the choroid, posterior (retina/RPE region) and nterior (ciliary body/ iris region) to the ora serrata. Total RNA was extracted using the guanidinium thiocyanate method (36). ~rthern blotting analysis was performed as described previously (14) sing 20 ug total RNA per sample. Filters were hybridized with a nick ranslated probe ( 3 7 ) prepared from a partial-length cDNA (602 nt) ncoding rat RBP (38) or an almos~ full-length cDNA (500 nt) encoding /man TTR (13), and labeled with JzP-dNTP's (New England Nuclear) to a 9ecific activity of approx. 108 cpm/ug DNA. For Western blot analysis, whole eyes of saline-perfused rats were ~mogenized in phosphate-buffered saline (PBS), pH 7.2, and sodium dodecyl ~ifate (SDS) was added to a final concentration of 2%. After mixing, the 9mogenate was centrifuged and the supernatant diluted 5:1 in sample iffer (39) containing 5% 2-mercaptoethanol. After boiling for ten inutes, samples containing 50 ug of total protein or 0.3 ug of purified at RBP were electrophoresed through a 0.75mm SDS-15% polyacrylamide gel 39). The separated proteins were then transferred to nitrocellulose liters by electroblotting (40). Filters were saturated with 8% non fat fy milk in PBS containing 0.05% Tween 20, and then incubated with a :2,000 dilution of a monospecific rabbit antiserum to rat RBP in the same ~lution. After washing, filters were treated with a I:I,000 dilution of ~roxidase-conjugated goat anti-rabbit IgG and developed in 1 mg/ml aminobenzidine, 1 mg/ml imidazole, and 0.001% hydrogen peroxide. For immunohistochemistry, rats were sacrificed by paraformaldehyde ~rfusion and tissues prepared for cryostat sectioning. ~unohistochemistry was performed using a modification of the method of u et al. (41). Sections were preincubated for 30 mins with 1% hydrogen roxide and then incubated overnight with I:i,000 - 1:4,000 dilutions of bbit antiserum to rat RBP in 50 mM sodium phosphate pH 7.2 and 0.1% vine serum albumin. Sections were then washed and incubated 45 min. th 7.5 ug/ml biotinylated goat anti-rabbit IgG (Vector Laboratories). ter further washing, sections were treated with a i:i,000 dilution of idin-biotinylated peroxidase complex (Vectastain, Vector Laboratories) d developed for 6-10 min in 0.05% diaminobenzidine and 0.01% hydrogen roxide in phosphate buffer, pH 7.2.

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Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS Using

a rat RBP c D N A probe for N o r t h e r n analysis,

hybridizing

band

preparations with

the

nt)

of

rat

rehybridized

approximately

total rat eye and liver RNA

(930

nt) RBP c D N A

with

migrating

a

at

human

Northern

TTR

approximately

w i t h our p r e v i o u s findings

RBP

at

in

(at least 882

(5,6). The same filter, w h e n stripped and c D N A probe, y i e l d e d a single hybridizing 700 n u c l e o t i d e s

in the rat

(fig.

IB),

in agreement

(13).

blot a n a l y s i s of regions of b o v i n e eye s h o w e d the p r e s e n c e of

m R N A in h o m o g e n a t e s p r e p a r e d from retina/RPE and c i l i a r y body/iris; no

Western whole

rat

blot eye

demonstrated

Preliminary

the

p r e s e n c e of a m a j o r i m m u n o r e a c t i v e band

immunohistochemical

immunoreactivity inner

consistent of

primary

and

(fig. 3).

studies

revealed

for RBP in the retinal g a n g l i o n cells, outer

staining

the RPE

(fig. 2).

a n a l y s i s of p u r i f i e d RBP and total p r o t e i n isolated from

m i g r a t i n g at a p p r o x i m a t e l y 21 kDa in each lane

and

nucleotides

(fig. IA). T h i s is consistent

RBP m R N A was d e t e c t e d in h o m o g e n a t e s of lens or cornea

the

we d e t e c t e d a single

900-1,000

size d e d u c e d from the n u c l e o t i d e s e q u e n c e of h u m a n

and

band

migrating

plexiform of

the

layers

of

the

intense

strong staining of

retina,

and lesser but

inner segments of the r e t i n a l photoreceptors

(fig. 4). S t a i n i n g was c o m p l e t e l y a b o l i s h e d by omission of

antiserum

(not shown). A m o r e extensive m o r p h o l o g i c a l analysis is

in progress. 1 A L

2

3

4

5

B E

L

E

-28 S -18S

-28S

g

-18S RBP - TTR

Q

Q

~iii•• iji!i~i~!i

Figure I. Northern blot analysis of RBP (A) and TTR (B) mRNA in rat eye. Twenty ug each of total RNA prepared from rat liver (L) and eye (E) were hybridized with ~ RBP cDNA (A) or human TTR cDNA (B) labelled by nick translation with . Hybridizing bands of expected size for RBP (approximately 1,000 nt) and for TTR (approximately 700 nt) are present in both tissues. Fiqure 2. Northern blot analysis of RBP mRNA in bovine eye. Twenty ug of total RNA prepared from bovine liver (lane i), lens (lane 2), cornea (lane 3), retina/RPE (lane 4) and ciliary body/iris (lan~5) were hybridized with rat RBP cDNA labelled by nick translation with ~ P . Hybridizing bands of expected size for RBP (approximately 1,000 nt) are present in ciliary body/iris and retina/RPE. Lanes negative for RBP had intact, non-degraded 18S and 28S ribosomal RNA bands indistinguishable from the RBP-positive lanes (not shown).

1080

Vol. 157, No. 3, 1988

R

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

E

,ILM

GC

IPL

INL OPL ONL ,ELM

RPE

®

®

Fiqure ~. Western blot analysis for RBP protein in rat eye. Three hundred ng of purified rat RBP (R) and 50 ug of total protein prepared from rat eye (E) were electophoresed through a 0.1% SDS-15% polyacrylamide gel and immunoblotted as described in Materials and Methods. A major immunoreactive band migrating at approximately 21 kDa is present in both lanes. Molecular weight markers (BioRad) are indicated in kDa. Fiqure 4. Immunohistochemical localization of RBP in rat retina using a i:i,000 dilution of a monospecific antiserum to rat RBP. The immunoperoxidase procedure is described in Materials and Methods. Intense immunostaining is present within the retinal ganglion cells (GC), and moderately intense staining is present in the inner plexiform layer (IPL), the outer plexiform layer (OPL), the inner segments of the photoreceptors (IS) and the retinal pigment epithelium (RPE). Other abbreviations: ILM, inner limiting membrane; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segments of photoreceptors; ELM, external limiting membrane; P, photoreceptor layer. DISCUSSION As

the c h r o m o p h o r i c group of the visual pigment,

~sential

role

lantities le

in

of retinol

photoreceptors

.:ter

a

strong

~gments .ther

vision

and is

(42) .

The

from the plasma

where

bleach,

recycled

RPE

takes

the r e t i n o i d s p l a y an up

(42). Retinol

and

stores

large

is t h e n t r a n s p o r t e d to

it is o x i d i z e d and i n c o r p o r a t e d into rhodopsin. retinal may be reduced by the p h o t o r e c e p t o r outer

to

the

RPE.

It is then i s o m e r i z e d in the RPE and

t r a n s p o r t e d b a c k to the p h o t o r e c e p t o r layer or is stored in the

~E. Several ,teract

retinoid-binding

with

ocular

retinol

.~tinoid-binding

proteins

~.tinol-binding

protein

"otein

(CRABP) (31,32)

'oRAIBP) (28,30,31) . :tinoid-binding ~le Le

in

Two

protein

proteins, and

present

all c h e m i c a l l y d i s t i n c t from RBP,

its in

derivatives. the

(CRBP) (26,31) , and

RPE

cellular

cellular

r e t i n a are c e l l u l a r

retinoic

acid-binding

retinaldehyde-binding

interphotoreceptor (IRBP) (29-31)

and

A m o n g the cytosolic

and

m a t r i x proteins, purpurin

protein

interstitial

(34,35), may play a

the s h u t t l i n g of retinol b e t w e e n the retina and the RPE. However,

precise

m e c h a n i s m s u n d e r l y i n g retinol c y c l i n g w i t h i n the eye have not

~t b e e n established.

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Vol. 157, No. 3, 1988

RBP

is

the

solubilizes on

target

hydrophobic

tissues, in

stabilizing into

p r i n c i p l e plasma t r a n s p o r t p r o t e i n for retinol

this

circulates

up

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

the

prevents

serum

in

the R B P - r e t i n o l

the

basolateral

RPE

vitamin,

a

its i:i

o x i d a t i o n and excretion. molar

interaction

(1-4). RBP

directs its t r a n s p o r t to receptors

complex

with

(I0). In the eye,

Holo-RBP

TTR,

thereby

retinol is taken

cells after d e l i v e r y to p u t a t i v e RBP receptors on their

surface

(23),

but n e i t h e r plasma TTR

(43) nor RBP

(23) cross

the b l o o d - r e t i n a barrier. Our

p r e v i o u s finding of an i n d e p e n d e n t source of ocular TTR in the RPE

(19-21)

led

study, rat

we

eye,

to

postulate

the

intraocular s y n t h e s i s of RBP. In this

and of RBP m R N A in bovine ciliary body and retina. These findings

establish RBP

us

report the p r e s e n c e of RBP and RBP m R N A in h o m o g e n a t e s of whole

de

novo s y n t h e s i s of RBP w i t h i n the m a m m a l i a n eye. Since plasma

reportedly

does

not

cross the b l o o d - r e t i n a b a r r i e r

(23), we suspect

that the m a j o r fraction - if not all - of ocular RBP is p r o d u c e d locally. Our

p r e l i m i n a r y i m m u n o h i s t o c h e m i c a l studies i n d i c a t e d a w i d e s p r e a d but

heterogeneous observed less

distribution

within

intense

the

the g a n g l i o n cells,

ciliary

widespread

and the RPE. R e g i o n a l N o r t h e r n

iris

and

distribution TTR

synthesized other

and

a n a l y s i s of bovine eye

that s y n t h e s i s was r e s t r i c t e d to cells w i t h i n the neural retina, body,

However,

inner and o u t e r p l e x i f o r m layers,

but c o n s i s t e n t s t a i n i n g was p r e s e n t in the inner segments of

photoreceptors

suggested

of RBP w i t h i n the rat eye. S t r o n g staining was

mRNA

RPE. of

In our p r e v i o u s studies we also d e t e c t e d a

TTR

i m m u n o r e a c t i v i t y in the rat eye

was l o c a l i z e d u n i q u e l y to the RPE,

(19-21).

s u g g e s t i n g that TTR

in the RPE was s e c r e t e d and t r a n s p o r t e d to the n e u r o r e t i n a and

ocular

structures.

In

situ h y b r i d i z a t i o n studies will demonstrate

w h e t h e r RBP is also s y n t h e s i z e d in the RPE. While

the function of ocular RBP is unknown,

involved

in

possible

that,

thereby and

protein

(25)

retinol

the

eye

translocation as

in

the

of

the d e l i v e r y of retinol. reported

in

both

a

high

serum

retinol.

plasma,

If

so,

it is

TTR c o m p l e x e s with RBP,

It is n o t e w o r t h y that Wiggert

molecular

weight

p r o t e i n capable of

and in extracts of RPE. The size of that

(75 kDa) was c o n s i s t e n t w i t h that of the R B P - T T R complex.

Abnormalities pathogenesis both

intraocular in

modulating

Chader

binding

the

we b e l i e v e that it may be

of

of the

retinoid pigmentary

metabolism

have

retinopathies

been

suspected

in

the

(44). Our d e m o n s t r a t i o n of

TTR and RBP s y n t h e s i s w i t h i n the m a m m a l i a n eye may be of relevance to

these disorders.

ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by the Charles A. Dana and A a r o n and Irene Diamond Foundations, the M u s c u l a r Dystrophy Association, NIH grants NSI1766, DK05968 and NINCDS Clinical I n v e s t i g a t o r D e v e l o p m e n t A w a r d NS01155.

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