cDNA clones encoding bovine interphotoreceptor retinoid binding protein

Vol.

131,

No. 3, 1985

September

30,

BIOCHEMICAL

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RESEARCH

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Pages 1086-1093

1985

cDNA CLONES ENCODING BOVINE INTERPHOTORECEPTOR RETINOID BINDING PROTEIN David J. Barrett,* Daniel D. Oprian,+

T. Michael Redmond,* Barbara Wiggert,’ Gerald J. Chader,” and John M. Nickerson”

*Laboratory

of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD +Department of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139

Received

August

8,

1985

We have isolated a cDNA clone (AIRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal Xgtll cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine resulting in the isolation of two larger cDNA retina AgtlO cDNA library, clones containing inserts of 2.5 kb (XIRBP-2) and 1.5 kb (XIRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of XIRBP-1 and the 2000 bp fragment of AIRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP. 0 1985 Academic Press,

Inc.

Interphotoreceptor interstitial

Retinoid-Binding

retinoid-binding M, in the

(146,000

cow)

interphotoreceptor epithelium neural into (9). retinoid

retina, the

IPS,

it it

neural

in a light-dependent

protein

Abbreviations: bp, base pairs.

a role

has been

by the

found

HPLC, high

0006-291X/85 $1.50 Copyright 0 I985 by Academic Press, Inc. All rights of reproduction in any form reserved.

and,

(l-3,10,11

).

ocular

disease retinal

liquid

1086

a large in the

of the

pigment

is

cells

known as

found

synthesized

most

soluble

protein

interestingly

states.

degenerations

chromatography;

by the

and secreted

(6-8)

70% of the readily

in hereditary

performance

The protein

retinoid

in specific

is

(I-4)

layers

photoreceptor

exogenous manner

tissue

(5).

about

also

glycoprotein

the

retina

constitutes carries

matrix

between

most probably

IRBP may play this

(IPS)

(IRBP),

or 7s receptor

extracellular

and the

IPS where

In the

protein

space

(PE)

Protein

binds

A decrease (lZ,l3).

Kb, kilobases;

in In

Vol.

131,

No. 3, 1985

an allied

hereditary

also

apparent

BIOCHEMICAL

disease,

in retinal

making

it

(14).

In an autoimmune

IRBP into

likely

the

Because

footpad of the

and well-defined its

molecular

three

cDNA clones peptide

MATERIALS

which

loss

causes unique

matrix

sequences

bovine for

protein,

As a first

injection

step,

in IRBP is

morphological disease of highly in the

we thought we now report establish

it

damage

process purified rat

as a retinoid-binding

IRBP and unequivocally this

in the

uveoretinitis

of IRBP both

COMMUNICATIONS

decrease

minimal

event

system,

experimental nature

biology. for

demonstrate

model

RESEARCH

a striking

is an early

disease

extracellular

study

encode

this

BIOPHYSICAL

choroideremia,

areas

that

AND

(15). protein

of importance

to

the

of

that

isolation they

protein.

AND METHODS

Purification of Bovine IRBP. A crude interphotoreceptor matrix wash was obtained by soaking frozen bovine retinas (Hormel) in Dulbecco’s phosphatebuffered saline (1 ml per retina) for approximately 2 hours with occasional gentle stirring at 4V. The mixture was centrifuged for 20 min at 5,000 g, and the resulting supernatant was centrifuged at 100,000 g for 30 min. IRBP was purified from the supernatant using concanavalin A-Sepharose affinity chromatography followed by ion-exchange and size exclusion HPLC (16). Purity of the final IRBP preparation was assessed by SDS-polyacrylamide gel electrophoresis followed by silver staining. Determination of IRBP Protein Sequence. Approximately 700 ug (5 nanomoles) of bovine IRBP was digested using trypsin-TPCK (Worthington) at an enzyme:substrate ratio of 1:50 for 22 l/2 hours at 37°C. This digest was fractionated by HPLC using a Bondapak Cl8 column, eluting with a gradient of 0.1% trifluoroacetic acid (TFA) in H2C to 0.1% TFA in 80% 2-propanol. Specific peaks were lyophilized and rechromatographed using the same system but eluting with a gradient starting with 25 mM ammonium acetate, pH 6.0, and ending with a solution of (1:1.5) 50mM ammonium acetate:2-propanol. Individual peaks were collected and lyophilized. Peaks that indicated high yield and purity were then subjected to microsequencing on an Applied Biosystems 470A gas-phase protein sequencer (17). The resultant phenylthiohydantoin amino acids were identified by HPLC (18). Preparation of Anti-IRBP. Antiserum to purified bovine IRBP was obtained by 4 intramuscular injections (6OOpg each) of a female Nubian goat. The antiserum was absorbed against h bacteriophage and bacterial antigens using a cyanogen bromide activated Sepharose 48 column (19) linked with lysate from heat shocked E. coli BNN97 (20,21 ). Immunoelectrophoresis of homogenized bovine retina and eye cup washings and pure bovine IRBP revealed a single arc verifying the monospecificity of the antibody. Agtll Library Preparation. Bovine retina RNA was prepared by the guanidine thiocyanate method (22,23). Poly(A)+ RNA was isolated bv oligo(dT)-cellulose chromatography (24). First strand synthesis was via AMV Second strand synthesis was by the large fragment of reverse transcriptase. DNA Polymerase I (25). After Sl nuclease digestion the cDNA was treated with EcoR I methylase, and EcoR I linkers were added. Following EcoR I digestion, the cDNA was ligated into the EcoR I site of Xgtll, and packaged. About 350,000 independent h phage plaques were amplified in Y1088 (21). Screening of the Bovine Retina cDNA Library. The amplified hgtll library was plated on 2x YT agar, expression was induced with isopropyl 1087

Vol.

131,

No. 3, 1985

BIOCHEMICAL

AND

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b-D-thiogalactopyranoside, and the fusion proteins were transferred to DA85 nitrocellulose filters (Schleicher and Schuell). These filters were washed in 50mM Tris pH 8.0, 150mM NaCl and 20% fetal calf serum, then incubated with the absorbed goat anti-bovine IRBP antiserum, washed and incubated with rabbit anti-goat IgG to which horseradish peroxidase had been conjugated (Miles) (20,21,26). Control filters containing purified bovine IRBP, E. coli BNN97 lysate, and wild type (no cDNA inserts) Agtll bacteriophage were incubated and developed in an identical fashion. The positive clones were plaque purified, and rescreened serially until all plaques were positive on a filter. While several different plaques were picked from the initial screening, only one original plaque was carried through the entire procedure. Subcloning into Ml 3 mp8 and -mpl8 and Nucleotide Sequence Analysis. The EcoR I fragments from the cDNA clones were isolated and ligated into the EcoR I site of bacteriophage Ml3 mp18 vector (27) and used to transform E. coli JMI Olr-. The 2 Kb EcoR I cDNA fragment from hIRBP-2 was sheared by sonication, .blunt-ended and ligated into the Sma I site of Ml3 mp8 (28) with minor modification (29) of the method of Deininger (30). Nucleotide sequencing was by the chain terminator method (31) using 35S-dATP and salt gradient gels (32). Hybridization to RNA Blots. Bovine retinas were dissected from fresh eyes and RNA was isolated by the guanidine thiocyanate/CsCl method (22,23 ). Poly(A)+ RNA was obtained by oligo(dT)-cellulose chromatography (24). Total The RNA RNA, from chicken lens and liver were also prepared as controls. samples were electrophoresed on a denaturing formamide-formaldehyde gel (33) The and transferred to a nylon membrane (Gene Screen, New England Nuclear). 250 bp fragment of clone XIRBP-1 and the 2000 bp fragment of clone AIRBP-2 were labeled with 32P-dCTP by nick translation (34) and used to probe the filters. Blots were washed 2 times in 2 x SSC at room temperature and 4 times at 52OC in 0.1 x SSC 0.1 I SDS for about 15 min per wash.

RESULTS Isolation clone

for

and Characterization

IRBP was obtained

4 x 106 phage using This

clone,

dization-positive

XIRBP-2

were

clones analyzed

to have a 2.5

and 500 bp, and clone fragments

screening

further.

obtained

of approximately

enzyme

bovine

retina

(23,36,37).

1088

of was

cDNA library Sixteen

Two clones (Fig.

EcoR I fragments

of a 1.5 kb cDNA insert

1500 bp and 50 bp.

1).

EcoR I digestion

enzyme digestion

containing

to consist

(Fig.

The 250 bp fragment

and purified.

Restriction

kb cDNA insert XIRBP-3

techniques

(20,21),

probe

by restriction

a hgtl0

cDNA

of approximately

library

as the

of 250 bp and 75 bp.

were

(26)

a 325 bp cDNA insert.

to screen

One putative

cDNA expression

and analyzed

revealed

plaque

screening

IRBP antibody

was purified

fragments

standard

retina

anti-bovine

and used as a probe

using

hIRBP-3)

bovine

2) which

cDNA yielded

isolated (35)

goat

AIRBP-1, (Fig.

by immunological

a hgtll

monospecific

digestion the

from

of IRBP cDNA Clones.

hybri-

(XIRBP-2

and

2) showed of 2000 with

bp

EcoR I

Vol.

131,

BIOCHEMICAL

No. 3. 1985

AND

BIOPHYSICAL

RESEARCH

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WILD TYPE Agtll BACTERIOPHAGE PLAQUES (NO cDNA INSERTS)

**

,*

1_ * mi

I ,’ I 1”

_

*

_. _*

il*

1 3 10

0

‘-

0.1 0.3

POSITIVE PLAQUES FROM IIRBPl CLONE (CONTAINS IRBP cDNA

‘/”

ng OF PURIFIED

INSERT)

STANDARDS E. COLI LYSATE

IRBP

Figure 1. Screening of a Agtll bovine retina cDNA expression library with anti-(bovine IRBP) antibodies. Top. Wild type hgtll plaques which contain no cDNA inserts give no positive signals. [diddle. After serial screenings and plaque purification a clone containing a 325 bp cDNA insert was identified that Purified gave positive signals with anti-(bovine IRBP) antibodies. Bottom. bovine IRBP from 0 to 10 ng was mixed with 100 pg/ml bovine serum albumin and spotted onto nitrocellulose and tested with anti-bovine IRBP with the above filters. E. coli BNN97 lysate (2 ~1 at 80 mg/ml) was also spotted onto the filters.

Eco Rl AlRBPl (325 bpl

5’1

Taq 1 Taq 1 AIRBP3 (1.5 Kb)

1

Taq 1

1

Eco Rl Kpn

1

5.‘3?

Taq 1 AIRBP2 (2.5 Kb)

Kpn

Hind

I

I

III 1

13

Eco Rl

u

0

0.5

1.0

1.5

2.0

2.5

3.0

KILOEASES

Figure -digested

2.

Restriction with the the restriction

maps of three bovine IRBP cDNA clones. The three clones indicated restriction enzymes. In the case of Taq I the order of fragments was not unequivocally determined where identified by brackets. There are no Bgl I, Sma I, Sph I, Sal I, or BamH I sites in any of the clones. A limited nucleotide sequence analysis has verified the order of the EcoR I fragments and overlaps of the 3 clones. The 3’ end of URBP-2 does not correspond to the 3’ end of the mRNA, since it does not contain Poly(A) by sequence analysis. 1089

3.5

BIOCHEMICAL

Vol. 131, No. 3, 1985

IRPB

NUCLEOTIDE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND

AMINO

ACID

SEQUENCE

3. Comparison of the deduced amino acid sequence with an authentic amino acid sequence from an IRBP tryptic identity unequivocally establishes that these cDNA clones

from

Figure

the

peptide. encode

cDNA clones This bovine IRBP

and provides amino acid sequence of IRBP from an internal tryptic peptide. The underlined amino acid residues in the tryptic peptide sequence are the most but their identification was not absolutely likely amino acid residues, positive. The top line is the authentic amino acid sequence, the middle is the deduced amino acid sequence predicted from the mRNA. The bottom line is the nucleotide sequence of the cDNA, which contains the unique Kpn I site shown in Fig. 2.

Analysis clones

were

were

shown

revealed

we have

from

AIRBP-2.

species Fig.

the

match.

This

identity

unequivocally

authentic

size

of the mRNA for

for

the

sequence

from

deduced

a 3.

A

amino

acid

establishes

IRBP.

as chicken

The RNAs were

transferred

250 bp fragment

Both

probes 8

kb long

size

and lens

to a nylon AIRBP-1

to a unique

in bovine

retina

stringency

molecular

weight

synthesized

in the

photoreceptor

cell

bovine 4 kb.

IRBP,

mRNAs for

have large

of transducin

(38)

1090

membrane

and probed

2000 bp fragment

exceptionally

This

retina

RNAs by gel

large

and poly(A)+

to chicken

of washing.

to exceed

bovine total

or the

total

was no hybridization

of mRNA encoding

a-subunit

liver

from

hybridized

there

to high

predicted

the

IRBP, we separated

RNA as well

As expected,

are

cDNA clones

acid

in Figure

a perfect

polypeptide

examples

shown

with

translated

RNAs at moderate with

IRBP and is also

Some

(31).

was obtained

sequence

approximately 4).

sequence

amino

acid

the

electrophoresis. nick

deduced

igt

3

sequences

method

amino

and poly(A)+

with

acid

the

Nucleotide

terminator

corresponding

from

Analysis.

To measure total

analysis.

chain

amino bovine

cDNA fragments

authentic

isolated

RNA Blot

sequence

and the

of purified

of this

for

Several

dideoxynucleotide

Authentic

3.

peptide

sequence

Ml3

sequence

in Fig.

comparison

into

by the

nucleotide

tryptic

that

subcloned

determined

of the is

of IRBP cDNA sequences.

result

lens is

estimated other

RNA (See or liver

consistent by its

retinal

3’ untranslated

and rhodopsin

RNA

proteins regions,

(35)

which

two have

Vol.

131,

No. 3, 1985

BIOCHEMICAL

AND

BOVINE RETINA m-

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

CHICKEN

Figure 4. Northern Blot Analysis. Bovine retina total RNA and Poly(A)+ RNAs (two independent preparations) and chicken lens and liver total RNAs were blotted and probed with the 2.0 kb XIRBP-2 cDNA insert. Only electrophoresed, an 8 kb RNA species was identified in retina RNA specific for IRBP.

untranslated also

regions

synthesized

region

of

present size. of this

size.

normal

homologous

bovine

be able

kb,

expression

will

for

kb

its

is

apparently

remarkable

with

untranslated

at 2.6

kb (40)

of IRBP at the

Cultured

demonstrates

to produce

human probes,

LDL receptor

states.

conditions,

3’ untranslated

of up to 4

accounting

6-transducin,

mRNA

regions and inter-

(41).

to study

which

Whereas

has a typical

region IRBP,

are the

and pathological

culture

(39)

untranslated

up to 3.9

can be induced

defined

respectively.

kb,

few mRNAs have been identified

human retinoblastoma

its

I.4

photoreceptor,

Two examples

We now will

tance,

.2 and

A large

a very

2 receptor,

both

1

in the mRNA encoding Only

lukin

in the bp.

142

of

Y-73 cells,

a distinct

IRBP by

butyrate

and in conjunction be crucial

in the 1091

derived

genetic (42). with study

gene level

in

from

a

mode of inheriThis our

system

under

cDNA clones

of IRBP control

or and

Vol.

131,

No. 3, 1985

BIOCHEMICAL

synthesis.

The analysis

polypeptide

is also

have been sequenced. the monkey protein bovine this

protein corresponds

large

a-transducin, aspect

conserved

a terminal

region

the two other

of gene expression

RESEARCH

COMMUNICATIONS

of the gene, mRNA and the amino acids

of IRBP

in the cow and monkey, but

5 amino acid sequence not found in the

DNA sequence analysis

in this

divergence of the bovine

of the IRBP mRNA (8

untranslated

structures

They are strongly

processing

size

BIOPHYSICAL

Only the N-terminal

of interest.

to evolutionary

post-translational exceptional

of the primary

contains

(16).

AND

kb)

will

of the sequences IRBP polypeptide.

is of particular

along with

similar

regions

proteins

studied

to date,

in photoreceptor

region

determine

if

or to co-or The

interest.

in rhodopsin may indicate

The and a unique

cells.

ACKNOWLEDGEMENTS We wish to thank Dr. J. Nathans for kindly providing his bovine AgtlO retina cDNA library. We also thank Drs. D. Borst, J. Dean, D. Lenar, and E. Ginns for helpful discussions. We are indebted to Drs. P.-M. Yuan and R.W. Blather of Applied Biosystems for their help in the isolation and sequencing of peptides. REFERENCES Wiggert, B., Bergsma, D.R., Lewis, M. and Chader, G.J. (1977) Neurochem. 29: 947-954. Lai, Y., Wiggert, B., Liu, Y. and Chader, G. (1982) Nature 2. 1.

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Liou, G.J., Bridges, C.D., Fong, S., Alvarez, R.A. and Gonzalez-Fernandez, F. (1982) Vision Res. 22: 1457-1467. Adler, A.J. and Martin, K.J., (1982) Biochem. Biophys. Res. Commun. 103: 1601-1608.

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Bunt-Milam, A.H. and Saari, J.C. (1983) J. Cell. Biol. 97: 703-712. Hollyfield, J.G., Fliesler, S.J., Reyborn, M.E., Fong, S., Landers, R.A. and Bridges, C.D. (1985) Invest. Ophthalmol. Vis. Sci. 26: 58-67. P.J. and Chader, G.J. (1984) Biochem. Wiggert, B, Lee, L., O'Brien, Biophys. Res. Commun. 118: 789-796. R-A., Gonzalez-Fernandez, Fong, S., Liou, G.I., Landers, R.A., Alvarez, F ., Glazebrook, P.A., Lam, O.M. and Bridges, C.D. (1984) J. Neurochem. -42: 1667-1675.

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Pfeffer, B., Wiggert, B., Lee, L., Zonnenberg, B., Newsome, D. and Chader, G. (1983) J. Cell. Physiol. 117: 333-341. Saari, J.C., Bredberg, L. and Garwin, G.G. (1982) J. Biol. Chem. 259: 13329-l 3333. Adler, A.J. and Evans, C.D. (1983) Biochem. Biophys. Acta 761: 217-222. Bergsma, D., Wiggert, B., Funahashi, M., Kuwabara, T. and Chader, G. (1977) Nature 265: 66-67. Bridges, C., Alvarez, R. and Fong, S.-L. (1982) Invest. Ophthalmol. Vis. Sci. 24 (Suppl): 141a. RodriEes, M.M., Ballintine, E.J., Wiggert, B.N., Lee, L., Fletcher, R.T. and Chader, G.J. (1984) Ophthalmology 91: 873-883. Cery, I., Wiggert, B., Redmond, M., Kuwabara, T., Crawford, M.A., Vistica, B.P. and Chader, G.J. (1985) Invest. Ophthalmol. Vis. Sci. 26 (Suppl): 77a. 1092

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878-884. Young, R.A. and Davis, R.W. (1983) Proc. Natl. Acad. Sci. 80: 1194-1198. Young, R.A. and Davis, R.W. (1983) Science. 222: 778-782. IJllrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J. and Goodman, H.M. (1977) Science 196: 1313-1319. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York. Acad. Sci (USA) 69: 1408-1412. Aviv, H. and Leder, P. (1972) Proc. Natl. J. Biol. Chem. 253: Wickens, M.P., Buell, G.N. and Schimke, R.T. (1970) 2483-2495. DeWet, J.R., Fukushima, H., Dewjii, N.N., Wilcox, E., O'Brien, J.S. and Helinski, D.R. (1984) DNA 3: 437-447. Norrander, J., Kempe, T. and Messing, J. (1983) Gene 26: 101-106. Messing, J. and Vieira, J. (1982) Gene 19: 269-276. Nickerson, J-M., Wawrousek, E.F., Hajikins, J.W., Wakil, A.S., Wistow, C.J., Thomas, G., Norman, B.L. and Piatigorsky, J. (1985) J. Biol. Chem. (in press). Deininger, P.L. (1983) Anal. Biochem. 129, 216-223. S. and Coulson, A.R. (197'7) Proc. Natl. Acad. Sci. Sanger r (USA) ;4F';46N:_C:;:;' . Biggin, il.D., Gibson, T.J. and Hong, F.F. (1983) Proc. Natl. Acad. Sci. (USA) 80: 3963-3965. Lehrach, H., Diamond, D., Wozney, J.M. and Boedtker (1977) Biochemistry 16:

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Maniatis, T., Jeffrey, A. and Kleid, D.G. (1975) Proc. Natl. Acad. Sci. (USA) 72: 1184-1188. Nathans, J. and Hogness, D.S. (1983) Cell 34: 807-814. Benton, W.D. and Davis, R.W. (1977) Science 196: 180-182. Woo, S.L.C., DugaiczyK, A., Tsai, M.-J., Lai, E.C., Catterall, J.F. and O'Malley, B.W. (1978) Proc. Natl. Acad. Sci. 75: 3688-3692. Tanabe, T., Nukada, T., Nishikawa, Y., Sugimoto, K.., Suzuki, H., Takahashi, H., Noda, M., Haga, T., Lchiyama, A., Kangawa, K., Minamino, N ., Matsuo, H. and Numa, S. (1985) Nature 315: 242-245. Yatsunami, K., Pandya, B.V., Oprian, D., Khorana, H.G. (1985) Proc. Natl. Acad. ,Sci. USA 82: 1936-1940. Yamamoto, T., Davis, C.G., Brown, M-S., Schneider, W.J., Casey, M.L., Goldstein, J.L., and Russell, D.W. (1984) Cell 39: 27-38. Miller, J., Maler, T.R., Leonard, W.J., Greene, W.C., Shevach, E.M., and Germain, R.N. (1985) J. Immunol. 134: 4212-4217. Kyritsis, A., Wiggert, B., Lee, L., Chader, G. (1985) Invest. Ophthalmol. Vis. Sci. 26 (Suppl): 17a.

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