THEJOURNAL OF BIOVXICAL CHF.MI~Y
Vol. 269, No. 5. Issue of February 4, pp. 3838-3845, 1994 Printed in U S A .
Q 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization and Functional Expression of Squid Retinal-binding Protein A NOVEL SPECIES OF HYDROPHOBIC LIGAND-BINDING PROTEIN* (Received for publication, July 12, 1993, and in revised form, October 11, 1993)
Koichi OzakiS, Akihisa TerakitaO, Mamiko Ozaki, ReikoHaran, and TomiyukiHaran From the Department of Biology, Faculty of Science, Osaka University, 'Ibyonaka, Osaka 560,Japan
Ikuko Hara-Nishimura, HitoshiMori, and Mikio Nishimura From the Department of Cell Biology, National Institute for Basic Biology, Okazaki 444, Japan
The primary structure of squid retinal-binding pro- In the eye, CRBP and CRABP bind cytoplasmic all-trans-retitein (RALBP) was determined by cDNA and proteinse- no1 and all-trans-rekinoic acid, respectively (7). Cellular retinalquencing. Squid RALBP contains 342 amino acid resi- binding protein (CRALBP) is also a cytoplasmic retinoid-bindduesinasingleN-terminal-blockedchainwitha ing protein (8).However, CRALBP binds 114s-retinal and11molecular weightof 39,111. The N,-blocking group was cis-retinol in the eye (7),and it is distinct from CRBP and identified as an acetyl moiety by mass spectrometry. TheCRABP in terms of primary structure (9). Interphotoreceptor amino acid sequence revealed that the protein is highly retinoid-binding protein (IRBP)is a high molecular weightprohydrophilic and acidic, but it has several hydrophobic tein located in theinterphotoreceptor matrix of the retina (10, regions that are located mainly in the middle part of the 111, and it transports retinoids between the retinal pigment polypeptide chain. It is also predicted that these hydroepithelial cells and thephotoreceptor cells (12,131. On the basis phobic regions form &sheet structures. Theprimary of the above studies, a possible pathway hasbeen proposed for structure of RALBP is,however,quitedistinctfrom those of other retinoid-binding proteins, showing that transport of retinoids to the photoreceptor cells (14-16). Briefly, all-trans-retinol bound to RBP is transported in plasma from squid RALBP is a novel hydrophobic ligand-binding prothe liver to the retinalpigment epithelial cells, where the retitein that functions in intracellular retinoid transport. Using the cloned cDNA, squid RALBP was expressedin nol is esterified to all-trans-retinyl ester andstored. When necvitro. By carrying out the translation at 20 "Cin reticu- essary, the retinyl ester is directly processed to 11-cis-retinol, followed by oxidation to retinal. Retinoids are carriedby CRBP locyte lysates, the protein having retinol binding activand CRALBP in the pigment epithelial cells, and they are ity was produced. transferred to extracellular IRBPfor transport to the photoreceptor cells. In thephotoreceptor cells, no retinoid-binding protein hasbeen identified, and themechanism for retinoid transIn vertebrates, five types of retinoid-binding protein have port in the photoreceptor cells remains to be clarified. been isolated, and both their primary structures and their funcThree typesof soluble retinal-binding protein (RALBP) have tions inretinoid transport havebeen studied indetail. Retinolbeen reported in the case of invertebrate retinas.Two such probinding protein (RBP)' belongs to a family of plasma proteins teins were found in thehoneybee retina, and they probably functhat bind and transport extracellular hydrophobic ligands (1, tion in theisomerization of retinal from all-trans to ll-cis-reti2). RBP transports all-trans-retinol in plasma from the liver to nal in the light (17,18). We found another RALBP in thesquid retinal pigment epithelial cells (1,3). Cellular retinol-binding retina (19). This protein is distributed throughout the outer and protein(CRBP) and cellularretinoicacid-binding protein inner segmentsof the photoreceptor cells (20). Our recent stud(CRABP) belong to a family of cytoplasmic proteins thatfuncies in vitro and invivo have revealed that squid RALBP transtion in the intracellular transport of hydrophobic ligands (4-6). ports 11-cis- and all-trans-retinalsbetween rhodopsin and retinochrome, andthat it regenerates photoproducts of both * This work was supported in part by Grants-in-Aid for Scientific pigments to maintain thehigh sensitivity of the photoreceptor Research onF'rioritykeas (62621001,6362001, and 0162001) (toK. 0.) cells (21,22).Although it has been suggestedthat squid RALBP from the Ministry of Education, Science andCulture, Japan. The costs of publication of this article were defrayed in part by the payment of might mediate the exchange of chromophores between retinopage charges. This article must therefore be hereby marked "aduertise- chrome and rhodopsin, details of the molecular mechanism for ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this such a n exchange still remain unknown. In the presentstudy, fact. we determined the primary structure of squid RALBP and ex$To whom correspondence shouldbeaddressed: Dept. ofBiology, Faculty of Science, Osaka University, lbyonaka, Osaka 560, Japan. pressed the protein in vitro. This study provides information that may helpefforts in to understand themolecular mechanism Fax: 81-6-845-7966. Tel.: 81-6-844-1151 (ext. 4318); E-mail: of the transport and the exchange of retinoid chromophores.
[email protected]. 8 Present address: Institute of Biology, Faculty of Education, Oita University, Oita 870-11, Japan. MATERIALS AND METHODS 1Present address:Dept. of Biology, Kinki University School of MediPreparation of Squid Eyes-Squid, Warodes pacificus, were colcine, Osaka-Sayama, Osaka 589,Japan. lected at night fromthe southwestern watersof the Japan Sea, and they The abbreviations used are: RBP, retinol-binding protein, CRABP, cellular retinoic acid-binding protein, CFtALBP, cellular retinal-binding were immediately decapitated in the dark.The eyeswere then excised and stored at -20 "C until use. protein;IRBP,interphotoreceptorretinoid-bindingprotein;FtALBP, Preparation of Squid RALBp"AI1 procedures werecarried out under liquid retinal-binding protein, kb, kilobase(s); HPLC, high performance dim red light according to the method of Ozaki et al. (19). The frozen chromatography; PAGE, polyacrylamide gel electrophoresis; bp, base eyes were cut in half for removalof the anteriorpart and the lens.The pairs(s).
3838
cDNA Cloning and Expression of Squid Retinal-binding eyecups were shaken in 25 m~ histidine/HCl buffer that contained 0.3 M NaCl (pH 6.2). in order to detach the outer segments of the visual cells. The suspended outer segments were then homogenized and centrifuged at 25,000 x g for 1h. The resultant clear supernatant contained RALBP. After concentration, the supernatant was applied to a polybuffer exchanger column (PBE94, Pharmacia; 10mm inner diameter x 200 mm) and eluted by 25 m~ histidine/HCI buffer (pH 6.2) with a linear gradient from 0.3 to 1.0 M NaCI. Fractions containing RALBP were pooled and concentrated by ultrafiltration. In thesecond purification step, high performance size-exclusionchromatography was camed out on a Hitachi model 635 liquid chromatography system equipped with a size-exclusion column (TSK G-3000, Toyo-Soda; 7.5 mm inner diameter x 600 mm). The column was preequilibrated with 0.2 M phosphate buffer (pH 6.5), and 0.1 ml of the concentrated sample was injected into the column. The sample was chromatographed with the same buffer a t a flow rate of 0.5 mumin a t room temperature, and the separated proteins were monitored by absorbance at 280 or 360 nm. Fractions containing RALBP were pooled and concentrated a s described above, and stored a t -20 "C until use. Preparation of RALBP-specific Antiserum--Rabbitswere immunized by the standard procedure for preparation of antisera. Each animal received purified RALBP emulsified with Freund's complete adjuvant. After 3 weeks, a second injection of antigen in Freund's incomplete adjuvant was given. Samples of blood were collected 1or 2 weeks after the booster injection. Preparation of Retinal Poly(A)'RNA-RNA was prepared a s described by Ullrich et al. (23) with slight modifications. The frozen eyes were cut in half for removal of the anterior part and lens, and the eyecups were homogenized in buffer (5 mueyecup) that contained 6 M guanidine thiocyanate, 5 m~ sodium citrate, 0.1 M p-mercaptcethanol, and 0.5% sodium lauryl sarcosinate using a Hiscotron homogenizer. After centrifugation a t 80,000 x g for 30 min, the supernatant was mixed with CsCl(0.4 g/mU and layered onto a cushion of 5.7 M CsCI, 0.1 M EDTA. The homogenate was then centrifuged a t 20 "C for 24 h a t 80,000 x g, and the resultant pellet of RNA was dissolved in water. The solution was treated with phenoVchloroform. RNA was recovered by ethanol precipitation and redissolved in water. Poly(A)+RNA was separated from total RNA by affinity chromatography on a column of oligo(dT)-cellulose,according to the method previously described (24). Construction and Screening of a cDNA Library-A cDNA library was constructed from poly(A)* RNAusing vector-primer DNA, according to the method previously described (25). To prepare the vector-primer, PstI-digested pTTQ18 ( h e r s h a m ) was tailed with TTP by use of terminal transferase (Takara Shuzo), and the T-tailed vector was redigested with XbaI. The T-tailed vector-primer was annealed with the denatured poly(A)* RNAand the first-strand cDNA was synthesized by RAV-2 reverse transcriptase (Takara Shuzo). The second-strand cDNA replacement was performed with RNase H and DNA polymerase I (Takara Shuzo). The double-stranded cDNA with plTQl8 was then blunt-ended and ligated to form circular plasmid. Escherichia coli cells (Epicuriancoli SCS1; Stratagene) were used for the hosts of the plasmids. The library was screened with RALBP-specific antiserum. Protein A-alkaline phosphatase conjugate was used as thesecond antibody and positive clones were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. Five positive clones were isolated from approximately 1 x lo6 independent recombinants. Three of them remained positive in the second screening. Two positive clones (pTTB1 and pTTB5) contained the identical cDNAinserts of 2.8 kb, a s revealed by analysis with various restriction endonucleases. The other clone (pTTB7) contained a shorter (2.0 kb), truncated insert. DNA Sequencing and Analysis-Both strands of the cDNA insert of pTTB5 were sequenced by the dideoxy chain terminationmethod using a DeazaGTP Sequencing kit (Nippon Gene). The sequencing strategy is shown in Fig. 1. For sequencing of the coding strand, the cDNA insert of p " B 5 was subcloned into the M13 mp19 vector and deletion mutants of the clone were constructed by treatment with exonuclease 111 and mung bean nuclease, using a Kilo-sequence Deletion kit (Takara Shuzo). In order to sequence the antisense strand, the cDNA was subcloned into the M13 mp18 vector (Toyobo) and the deletion mutants were constructed as described above. In addition, the cDNA insert was digested with AatI, AcyI, BamHI, HincII, HindIII, HinfI,andNaeI, and the resultant fragments were subcloned in the M13 mp18 vector for sequencing. Northern Analysis of Retinal Poly(A)+ RNA-Total RNA (22.3 pg) from the squid retina was subjected to electrophoresis on 1.5% agarose, 6% formaldehyde gels, as described by Maniatis et al. (24); capillarilly transferred to Hybond-N nylon membrane ( h e r s h a m ) in 20 x SSC (1
Protein
3839
x SSC contains 150 m~ NaCl and 15 m~ sodium citrate), and fixed on the membrane by irradiation with UV light. The clonedcDNA for RALBP was labeled with [a-32PldCTPby use of a random primer labeling kit(Nippon Gene) for use as a probe for detection of the mRNA for squid RALBP. Hybridization was camed out at 42 "C for 18 h in 50% formamide, 5 x SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, and0.2% SDS.The membrane was then washed successively in 2 x SSC a t 25 "C, in 2 x SSC that contained 1% SDS at 65 "C, and in 0.1 x SSC a t 25 "C, and then itwas exposed to x-ray film (X-Omat AR; Kodak). AminoAcid Analysis and Sequencing-Twentynmol of purified RALBP were denatured in 6 M urea and cleaved with lysyl endopeptidase. Resultant peptides were fractionated by reverse-phase HPLC on a CIScolumn (4-mminner diameter x 300 mm; Nacarai Tesque).Amino acid compositions of the purified peptides were determined with an amino acid analyzer (A-5500; Inca) afterhydrolysis by HCI in the presence of thioglycolic acid. Amino acid sequences of peptides were determined by manual Edman degradation. Mass Spectrometry-The N-terminal blocking group was identified by analysis by fast atom bombardment mass spectrometry of the Nterminal lysyl peptide (K14) obtained by the method described above. The peptide (1.5 nmol;3 pl) was mixed with glycerol and then placed as a target in thexenon ion beam. Synthesis of lbtal Retinal Protein in Vitro-Total RNA (6 pg; 6 pl) from the squid retina was mixed with 12 pl of a rabbit reticulocyte lysate (Amersham) and [35Slmethionine(37 TBq/mmol; 10 p ~ 2 ;1.11).The mixture was incubated a t 30 "C for 1h, and the reaction was terminated by adding 8 pl of 10%SDS to the reaction mixture. RALBP was isolated from other retinal proteins synthesized in vitro, as follows. After the termination of synthesizing reaction by SDS, 30 pl of the reaction mixture was mixed with 8 pl of 20% Triton X-100 and 400 pl of STE buffer (100 m~ NaCI, 1m~ EDTA, 10 m~ TridHCI; pH 8.0), and the RALBPantibody conjugates were formed by adding 2 pl ofRALBP-specific antiserum, with subsequent incubation at 4 "C for 12 h. The conjugates were recovered by batch chromatography on Protein A-Sepharose (1.7
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FIG.1. Sequencing s t r a t e g y for the cDNA and Northern hybridization of the mRNA of squid RALBP.A, sequencing strategy for squid RALBP. An open box shows the open reading frame of RALBP cDNA. Arrows indicate the directions and lengths of fragments identified by sequencing. Stars represent the locations of possible polyadenylation signals (AATAAA). B, Northern hybridization of squid retinal RNA. Total RNA(22.3 pg) was fractionated by electrophoresis on a 1.5% agarose-formamide gel, blotted onto a nylon membrane, and hybridized with the 32P-labeledRALBP cDNA. Major (2.8 kb) and minor (1.7 kb) transcripts of squid RALBP gene are indicated by large and small arrowheads, respectively. The numbers on the left indicate the positions and the sizes (kilobases) of RNA size markers.
cDNA Cloning and Expression of Squid Retinal-binding
3840
Protein
GGACTTGGMCCGTTTGTTGAGTGTT TAGMTGTGGACGAMGCTACATCAMTCTGTGCTTTATATTTTTMCT~TCCTCTTTACTATTTTGACGATTCCTCATCTGMCTMGC
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ACGTGATCGTCTCTATMlLMMTTGGTGCCATMGTTTTCACGTTGMMGCTTCTGTTCTGTTTTTTTTTCCATTATATT~TTTCCTA 1710 A T G G A G T T T T G T T T T G A M T A G M C M G A G T T M C G G C A A M T G T A T T T T T A M G M T G A T A T M G C M T A T T A T C T T T A T M G T A T T T T 1800 CAGAMTTAGTACCGGTTTCCTTACATCATCATATTACTTACAMTACATCTCTATATTTTTTTTCATCTTTMCAMTTAG~CAGATATG lago T A C T T T C A T A T C A T G T A T T C T T C A T M C T T T T T A T T T A T C T G T ~ T T ~ T C A A M T M G T A T T T T G T T T M C ~ T A C1980 CM ATMTTlVLMTATTGCGTGATCTGMCAGCGCGCACTMGTGGGTATAGTAGAGCAGTACTCTTGGGAGCAMTAGGCTTTATGMTCCG 2070 GCACGCCGATTTCATTTTATGTGATGTTAGGCCAMTCGGCATTTATGGTGTTCCTTCTTTTCATGGTCGTCMCCTCCTTGAMCATTCTG 2160 A G T T G C A C C A M T A T A T A T M C A C T C T C M C A G A G C C T T T G G A A M C C A T A T T T C C A G A C T G A M T M C A G G C C T A T C T M C A T M C M C 2250 C M T C A T T T T C T T T A G M T C G T C T T T T A M T T C A T M M G G G A T T C G T T G A G M C C M T C G A C A T G T T G T T C M C T T C T C G C G T G A M C A G C 2340 TCGTGATMTTGCTTGMCGTGATGAGAGACCAGGCTTTATTTCTACGTGTGCATTTM~CACATTGGCTACCTGMTGTTGTATGTCAC2430 AGTGCCGTTC~TTATATMGTCATTGTGCCCGGCMCGTACGCTACAGCCACATTTAGTACATATTMCCACTAGTTGTTGATATAG
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CTMTATTTGGAGACGTCTGGAGTTATTCTTATCTTCMTCATTTGTTTGMGCTGTTTCGGTATCCCTTGTGATTCTTTGATGTATCCA 2610 CGAMCTGACATTTTTGMTAMTATAGTTTMTCAGAGCAT(PoIp(A))
2652
FIG.2. The nucleotide sequence of thecDNA (upper) and deduced amino acid sequence (lower) of quid RALBP. Underlined nucleotides in the 3’-untranslatedregion represent the possible polyadenylation signals. Polypeptides obtained by digestion with lysyl endopeptidase and subsequent separation by HPLC, as shown in Fig. 3, are underlined and numbered (Kl-Kl6).
cDNA Cloning and Expression
of Squid Retinal-binding Protein
mg). After through washing of the RALBP-antibody-Protein A-Sepharose complex with STE buffer, proteins were solubilized in SDS-containing buffer for SDS-PAGE. In order to check the specificity of the antiserum and the possibility of the nonspecific binding of synthesized proteins to the antibody-ProteinA-Sepharose complex,a similar experiment was carried out by use of the antiserum previously incubated with purified RALBP. Synthesis of Cloned U B P in Vitro-In order to synthesize capped mRNA of RALBP in vitro, full-length cDNA was subcloned in the pBS vector (Stratagene) with the T7 RNApolymerasepromoter (pBSB1).The plasmid was linearized with SphI, treated successively with proteinase K andphenol/chloroform,and recovered by ethanol precipitation. RNA with a 5'-cap structure was synthesized from the linearized DNA byuse of an mCAP mRNA Capping kit (Stratagene).T7 RNA polymerase was used for RNA synthesis. After incubation a t 37 "Cfor 1h, template DNA was digested with deoxyribonuclease.The synthesized capped RNAwas recovered by ethanol precipitation and dissolved in water. The pBS vector,carrying 5'-end-truncated RALBP cDNA(pBSBlD1X was constructed as follows (Fig. 7A). The plasmid pBSBl was doubledigested with EcoRI and StyI, blunted with mung bean nuclease (Takara Shuzo), and ligated with T4 DNA ligase. As a result of this treatment, the complete 5'-untranslated region and the first 134 nucleotides of the translated region were deleted. After the truncation, it was anticipated that translation would be initiated at Met-50, resulting in the deletion of 49 N-terminal amino acids. Capped RNA from pBSlDl was also synthesized in the same manner as described above. For expression in vitro of full-length and truncated RALBP, a solution of capped RNA (3 pg; 3 pl) and [Wlmethionine (37 TBq/mmol; 10 p ~ 3 ;pl) were added to 19 pl of a rabbit reticulocyte lysate (Amersham). The mixture (25 pl) was then incubated at 20 "C for 2 h for synthesis of RALBP. For control experiments, 3 pl of water were added to the reaction mixture instead of the solution of capped RNA. In order to measure retinol binding activity of RALBP, 2 pl of r3H1retinol in a3% solution of bovine serum albumin plus 1 pl of unlabeled L-methionine ( 1 111~) were used instead of 3 pl of radiolabeled methionine. SDS-PAGE andRetinol-bindingAssay of Cloned U B P - T h e RALBP-antibody conjugates were formed by adding 1 pl ofRALBPspecific antiserum and25 p1 of phosphate-bufferedsaline (pH 7.3) to 25 pl of reaction mixture, with subsequent incubation a t 20 "C for 1h. The conjugates wererecovered by batch chromatography on Protein A-Sepharose (1.7 mg). After through washing of the RALBP-antibodyProtein A-Sepharose complex with phosphate-buffered saline, proteins were solubilizedin SDS-containingbuffer. The solubilized solution was
I
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3841
used directly for analysis by SDS-PAGE. SDS-PAGE was performed on slab gels by the method of Laemmli (26). The concentration of acrylamide was 5% in the stacking gel and 12% in the running gel. f i r electrophoresis, the proteins were stained with Coomassie Brilliant Blue R-250, incubated in enhancer solution (EN3HANCE; DuPont-New England Nuclear), and exposed to x-ray film (X-OmatAR; Kodak). For the retinol-binding assay, 10 pl of SDS-solubilizedsample were added to 10 pl of scintillation mixture (Clear Sol; Nacarai Tesque), and photons in the 3H-window were counted in a liquid scintillation counter (LS9000; Beckman) for 10 min. RESULTS
Cloning and Seqwncing of Squid U B P cDNA-Three clones recognized by polyclonal RALBP-specific antibodies were isolatedby screening 1x lo5 recombinant plasmids. Analysis with restriction endonucleases indicated that two of these clones (p'I"l'B1 and p'l"B5) contained a n identical cDNAinsert of about 2.8 kb, and another clone (p'lTB7) contained a 2.0-kb insert thatlacked the 5"region (0.8 kb)of the longer insert of p'ITB1 and P'ITB5. Therefore, we sequenced the cDNA insert of p'l"B5. Fig. lA shows the restriction map anda summary of the sequence analysis of squid RALBP cDNA. The results of sequencing are summarized in Fig. 2, together with the deduced amino acid sequence. The cDNA consists of 2768 bp; it contains a 5"untranslated region (116 bp), a long open reading frame that encodes 343 amino acids, a termination codon, and a 3"untranslated region (1620 bp). The long open reading frame begins withATG at nucleotides 1-3, and terminateswith an ochre codon (TAA) at nucleotides 1030-1032. A polyadenylation signal, AATAAA, can be found 24 bases upstreamfrom the 3'-end. Moreover, two additional AATAAA sequences are present at nucleotides 1474-1479 and 1521-1526. Northern analysis of the squid retinal mRNA (Fig. 1B) showed that the RALBP mRNA consists of at least two classes of transcripts, a major 2.8-kb and a minor 1.7-kb transcripts, suggesting that the AATAAA sequence at nucleotides 1474-1479 and/or nucleotides 1521-1526 is also used as a polyadenylation signal. The RALBP mRNA is characterized by a long 3"untrans-
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80
100
T i me (min) FIG. 3.Separationof peptides derivedfrom squid RALBP by digestion withlysyl endopeptidase.As described in the text, RALBP that had been purified from the squid retina was digested with the enzyme, and the digest was fractionated by reversed-phase HPLC. Material in the peptide peaks indicated (Kl-Kl6) was collectedand subjected to amino acid analysis. Sequence analysis was also carried out on peptides K4 and K9. K14 was subjected to mass spectrometory (Fig. 4).
cDNA Cloning and Expression of Squid Retinal-binding Protein
2616.3
F P6i 401
C
I
I,
I
, I
B
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0
2 o M p o o 2 u K ) a B a o 2 8 0 0 3 M x ) s w 3 u a
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FIG.4. Analysis by mass spectrometoryof the N-terminalihgment derived from squid RALBP. The N-terminal fragment (K14, isolated as indicated in Fig. 3)of squid RALBP was subjected to mass spectrometory. The ion peak showing the exact mass ([M + HI+) was observed at m / z 2617.3,indicating that the molecular weight of the fragment was 2616.3.This value coincides exactlywith the theoretical molecular weightof the fragment aRer loss of 1methionine residue and acetylation.
C
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: C C
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66
6
lated region which accounts for about 60% of the total length. Although the role of 5'- and 3'-untranslated regions of the A s of mRNA has not been clarified, it should be noted that two other retinal proteins in the squid, rhodopsin and retinochrome, also have an expanded 3'-untranslated sequence like RALBP (25, 27). Molecular Structure of Squid RALBP-From the nucleotide sequence ofFWLBPcDNA,we deduced the amino acid sequence of the encoded polypeptide(Fig. 2). In order to confirm the deduced sequence, we determined the amino acid composition of 16 peptides (Kl-Kl6) derived from purified RALBP by digestion with lysyl endopeptidase (Fig. 2, underlined; Fig. 3). Partial amino acid sequences of K4 and K!3 peptides were also determined by Edman degradation. Of the 16 peptides, 15 peptides (Kl-K13, K15, K16) had amino acid compositions identical to those of corresponding peptides in thededuced sequence (Table I). Amino acid sequences of K4 and K9 were also consistent with those of the deduced sequence. K10 was expected to be the C-terminal peptide, since it was the only fragment without a lysine residue. In fact, the amino acid compositionof K10 corresponded to that expected fromthe deduced sequence of the C-terminal peptide. The amino acid composition of the K14 peptide corresponded t o that of the N-terminal fragment of the deduced sequence except that 1of 2 methionine residues was missing. Sincethis result suggested that thefirst methionine of RALBP might have been removed by post-translational modification, we tried to determine the N-terminal sequence of purified RALBP. However, since no amino acid could be detected by Edman degradation, it appeared that RALBP has an N-terminal blocking group. Therefore, we carried out mass spectrometric analysis of the N-terminal lysyl peptide (K14).As shown in Fig. 4, the ion peak showing the exact mass ([M+ HI') was observed at mlz 2617.3, indicating that themolecular weight of the original peptide was 2616.3. This value coincides with the theoretical weight of the K14 peptide, if 1methionine residue is removed fromthe peptide and the peptide is acetylated. From these results,we concluded that RALBP was subjected to posttranslational modifications in which the N-terminal methionine is removed and the second residue, serine, is acetylated. From the above results, the molecular weight ofRALBP, including the N-terminal blocking group, was calculated to be 39,111. This value is somewhat smaller than that of purified RALBP, as estimated by SDS-PAGE (48,000; Fig.6).' However, In a previous paper (19),we estimated the molecular weight of RALBP to be 51,000.However, in the case of RALBP, estimation of molecular weight by SDS-PAGE is very significantly affected by the
cDNA Cloning and Expression of Squid Retinal-binding
Protein
3843
+3.0
Hydropathy index
0 -3.0 1.5
Potentials of a-helix (----p-sheet (-
)
1.0
I 0.5
+3.0 0 -3.0 1.5
1 .o 0.5 150
250
200
300
350
Residue number FIG.5. Hydropathicity profile and predictions of secondary structure of the deduced amino acid sequence of squid RALBP. Hydrophobicity values were calculatedby the method of Kyte and Doolittle (28)for a window size of 5 amino acid residues. The averagehelical (broken line) and p-sheet (solid line)potentials were calculated by the method of Chou and Fasman(29)(averages of 4 amino acid residues). Filled areas indicate thehydrophobic regions expanded over 5-10 amino acid residues.
since the molecular weight of RALBP expressed in vitro from 1 2 3 4 5 both the total retinal mRNA (Fig. 6) and the cloned RALBP x103 cDNA(Fig. 7B)was also estimated to be 48,000by SDS-PAGE, it is most likely that SDS-PAGE provided a n overestimate of 94 the molecular weight of RALBP. Possibly, this overestimation might be due to the highly acidic nature (pK = 4.55,calculated 67 from the sequence) of RALBP. Considering both the nucleotide sequence and the amino acid 43 sequence, we searched for the sequences homologous to squid RALBP in theGenBank, EMBL, PIR, and SWISS-PROT data libraries. However, no sequences with significant homology to 30 squid RALBP were found. We next examined more closely the sequence homology between squid RALBP and other known retinoid-binding proteins (RBP, CRBP, CRABP, CRALBP, and IRBP), but found no homology. These data strongly suggest 20 that squid RALBP is a novel retinoid-binding protein, which does not belong to anyof the families of the hydrophobic ligandbinding proteins reported to date. 14 The analysis of hydropathicity of the amino acid sequence with Kyte and Doolittle (28)showed that squid RALBP is, on FIG.6. In vitro translationof squid retinal proteins. Total RNA the average,hydrophilic but possesses several hydrophobic rewas extracted from squid retina, andused for translation in the rabbit gions, each of which is composed of 5-10 amino acid residues reticulocyte lysate expression system as described under "Materials and (Fig. 5; residues 10-15, 89-95, 110-119, 130-136, 137-146, Methods." Lanes 1 3 , fluorograms showing the results of SDS-PAGE 148-152,159-164,195-200,220-226,277-282,and 294-2991, analysis of the proteins synthesizedin vitro in thepresence of ["Slmeand might be able to have hydrophobic interaction with reti- thionine. From the total productof retinal RNA (lane 3). RALBP (M,= 48,000)was specifically immunoprecipitated with RALBP-specific annoid. Predictions of secondary structure with Chou and Fas- tiserum and ProteinA-Sepharose (lane 1 ), but it was not precipitated man (29)indicated that most of these hydrophobic regions are when the antiserum was pretreated with purifiedRALBP squid (lane2 ) . I
concentration of polyacrylamide and the nature of molecular weight marker proteins. Under the conditions for SDS-PAGE used in the present study, the molecular weight of RALBP purified from squid retina was calculated to be 48.000.
Lane 4 , Coomassie Brilliant Blue R-250(CBB R-250)stain of RALBP that was extradedfrom squid retina and purified as described under "Materials andMethods." Lane 5, Coomassie Brilliant Blue R-250stain of molecular weight marker proteins. Molecular weight of these proteins are shown on the right side.Arrowheads indicate the bandof RALBP.
cDNA Cloning and Expression of Squid Retinal-binding
3844
A StyI
Protein
pBSB1 EcoRI T7 Promotor 135
1
---TAATACGACTCACTATAGGGCGaattcgagc----atgagtctc----ccatgG~TTACTTGGACATG~----
M
S
---
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pBSB1Dl T7 Promotor ( 135 1 ---TAATACGACTCACTATAGGGCGGGGTTACTTGGACATGW----
M
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(50)
B mRNA
94
-
pBSB1
pBSBlDl
-
-
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+
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-
-
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pBSBlD1
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........ ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... .....
+
mRNA
FIG. 7.Expression in oitro of full-lengthand truncated RALBP.Capped RNAs were synthesizedin vitro from intact and truncated RALBP cDNA, and they were used for translation in the rabbit reticulocyte lysate expression system as described under "Materials and Methods." A, construction of 5"truncated squid RALBP cDNA.The pBS vector with wild-type squid RALBP cDNAas insert (pBSB1) was double-digested with EcoRI and StyI, blunted with mung bean nuclease, and ligated (pBSBlD1).As a result of this treatment, the complete 5'-untranslated region and the first 134 nucleotides of the translatedregion were deleted (nucleotides indicated by lower-caseletters).ARer such truncation, translation should be initiated a t Met-50, with resultant deletion of 49 N-terminal amino acids. Underlining indicates the promotor sequence of T7 RNA polymerase, and the EcoRI and StyI recognition sites, respectively. E, fluorogram showing the results of SDS-PAGE analysis of full-length and truncated RALBP. RALBP was expressed in vitro in thepresence of [35Slmethionine,recovered by batch chromatography withRALBP-specific antibodies and Protein A-Sepharose, and subjected to SDS-PAGE. For the control, expression was camed out in theabsence of RALBP RNA. The numbers and arrows on both sides indicate thepositions and the molecular weights of protein markers. C,retinol-binding assay of full-length and truncated RALBP. RALBP was expressed in vitro in the presence of E3H1retinol, recovered as indicated above, and solubilized with SDS-containing buffer. Radioactivity (cpm) of the solubilized samples was measured in a liquid scintillation counter as described under "Materials and Methods." Control experiments were carried out in theabsence of RALBP RNA. Averaged results of five independent experiments are given with standard errors.
associated with a higher probability than other parts of the RALBP polypeptide of adopting the 0-sheet structure (Fig. 5). Expression of Squid RAL,BP in Vitro-%tal mRNA extracted from the squid retina was translated in vitro using a rabbit reticulocyte lysate system. As shown in Fig. 6,RALBP was one of the major components of translated products (lane 3),and it was precipitated by forming complex with RALBP-specific antiserum and Protein A-Sepharose (lane 1 ). Since no labeled protein was recovered when such immunoprecipitation was carried out usingthe antiserum thatwas pretreated with the purified squid RALBP (lane 2), it was confirmed that RALBP, synthesized in vitro,was specifically bound to the RALBP-specific antibodies and was recovered by immunoprecipitation. This experiment also showed that themolecular weight of RALBPsynthesized in vitro coincides with that of the matureRALBP ex-
tracted from the squid retina (48,000;lane 4). This result suggests that RALBP is not derived from a larger precursor. Expression and functional analysis of the cloned wild-type and mutant RALBP may be useful for studies of the relationship between the molecular structure and the function of the protein. We first tried to express RALBP in E. coli. Although we could generate a fair amount of the RALBP peptide in such a system, all the desired product was denatured, with resultant aggregation and loss of retinoid binding activity (data not shown). Since it is likely that RALBP is thermally unstableat 37 "C,we next in vitroexpression system, since used a reticulocyte lysate as an it can function a t a lower temperature (20 "C).Using this system, we achieved expression of a wild-type and anN terminally truncated RALBP, and we compared retinol binding activity of mutant RALBP with that of wild-type RALBP. As shown in Fig.
cDNA Cloning and Expression of Squid Retinal-binding Protein
3845
showed that the radioactivity in the truncated RALBP stayed at background level, strongly suggesting that the binding of from the squid retina (Fig. 6).By contrast, corresponding to the retinol to the expressed full-length RALBP doesnot result from deletion of 49 N-terminal amino acids, the molecular weight of nonspecific binding of retinol to RALBP polypeptides. In addithe truncatedRALBP was reduced to 42,000. Onthese two pro- tion, the above results also suggest that themissing N-terminal teins, we measured retinol binding activity using 3H-labeled region of the protein forms the retinoid-binding site directly, or retinol. As shown in Fig. 7C,radioactivity of the sample that more likely, that the region is essential for formation of the contained the full-length RALBP (pBSB1,mRNA+)was signifi- correct configuration of W B P required to bind retinoid. cantly higherthan thatof the sample of the control experiment We also tried to express RALBP in E. coli, but RALBP pep(pBSB1, mRNA-).Therefore, this result indicated that thefull- tides synthesized in E. coli had completely lost the ability to length RALBP, synthesized in uitro, could bind L3H1retinol bind retinol, although much more RALBP peptide could be added to the reaction mixture. By contrast, radioactivity of the obtained from E. coli than from the in uitro system. A possible sample that contained the truncated RALBP was as low as that reason for our inability to obtain active RALBP in the E. coli of the control experiment. This result suggests that theN-ter- expression system is that squid RALBP is less stable to heat minal region of the protein is essential for forming a functional than other vertebrateretinoid-binding proteins. We tried growRALBP. Moreover, this result also excludes the possibility that ing E. coli at a lower temperature (20 "C) for expression of retinol binds nonspecifically to the synthesized protein, and con- RALBP, but thebacteria did not synthesize any RALBP at all at firms the previous result thatfull-length RALBP, expressed in the lower temperature. Using the reticulocyte lysate system, this system, has activity to bind retinol. we succeeded in expressing active RALBP at 20 "C. However, since the amount of RALBP expressed in this system was still DISCUSSION very low, the development of a more efficient system is now In the present study, we determined the primary structure of necessary if we are to synthesize sufficient RALBP for further squid RALBP, and it was clear from this structure that the molecular analysis. protein is a novel hydrophobicligand-binding protein. Although AcknowZedgments-We are gratehl to Dr. Hirozo Oh-oka and Prof. we previously reported that IRBP-specific antibodies reacted Hiroshi Matsubara, Osaka University, for the analysis of amino acid with squid RALBP (30),we could not find any sequences that composition and for peptide sequencing. We also thank Prof. Takekiyo were present in common in W B P and IRBP. This result sug- Matsuo, Osaka University, for his cooperative examination in the mass gests that the antibodies might possibly have recognized the spectrometry. higher structure of the two proteins. Using mass spectrometry, REFERENCES we characterized the N terminus of squid RALBP.We found that thefirst residue, methionine, corresponding to the initia1. Kanai, M., Raz, A,, and Goodman, Dew. S . (1968) J . Clin. Invest. 47, 2 0 2 5 2043 tion codon, had been removed and the second residue, serine, 2. Zimmennann, J. G. (1988) Zkn& Biochem. Sei. 13,64-66 had been acetylated in squid RALBP. Unlike IRBP, RALBP did 3. Heller, J. (1975) J. Biol. Chem. 260, 3613-3619 4. Bashor, M. M., TOR, D. O., and Chytil, F. (1973) Proc. Natl. Acad. Sci. U. S. A. not include a signal sequence. This resultis consistent with the 70,3483-3487 fact that W B P is not a secretory protein but functions in the 5. Ong. D.E., and Chytil, F. (1975) J. Biol. Chem. 260,611345117 photoreceptor cells to transport intracellular retinoid between 6. Takahashi, K, Odani, S., and Ono, T. (1982) Biochem. Biophys. Res. Commun. 106,1099-1105 rhodopsin and retinochrome molecules (22). 7. Saari, J. C., Bredberg, L., and Gamin, G. G. (19821 J . Bioi C h m . 257, Squid RALBP can bind retinol as well as retinal (19). This 13329-13333 property suggests that not formation of a Schiffs base but, 8. Futterman,S., Saari,J. C., and Blair, S . (1977) J. Biol. Chem. 262,32674271 9. Crabb, J. W.,Johnson, C. M., Carr,S. A,, Ames, L. G., and Saari, J. C. (1988) rather, hydrophobic interactions are essential for binding of J. Biol. Chem. 263, 18678-18687 retinoid to RALBP. In the present study, we found that squid 10. Adler, A. J., and Martin, K. J. (1982)Biochem. Biophys. Res. Commun. 108, 1601-1608 RALBP contains several hydrophobic regions which might Lai, Y.L., Wiggert, B., Liu, Y.P., and Chader,G. J. (1982)Nature 298,848-849 adopt a P-sheet structure. The studies on crystalline structure 11. 12. Okajima, T.-I. L., Pepperberg, D.R., Ripps, H., Wiggert, B., and Chader, G. J. of RBP had revealed that theprotein contains a p-barrel struc(1990) Proc. Natl. Acad. Sei. U. S . A. 87,69074911 ture in which hydrophobicligands are trapped (31).The hydro- 13. Lin, Z.-S., Fong, S.-L., and Bridges, C. D. B. (1989) Vision Res. 29,1699-1709 C. D.B., Alvarez, R.A,, Fong, S.-L.,Gonzalez-Fernandez, F., Lam,D. phobic regions in RALBP might form a similar tertiary struc- 14. Bridges, M. K., and Liou, G. I. (1984) Vision Res. 24,1581-1594 ture for binding of retinoid. 15. Rando, R. R.,Bernstein, P. S., and Barry, R. J. (1991) in Progress in Retinal Research 10 (Osborne, N., andChader, J., eds) pp.161-178,Pergamon By using a rabbit reticulocyte lysate system, and carrying Press, Oxford out translation at a relatively low temperature, we succeeded 16. Ozaki, K (1991) Biophysics 31, 129-134 in expressing RALBP that retained the ability to bind retinol. 17. Pepe, I. M.. and Cugnoli, C. (1980) Vision Res. 20.97-102 18. Pepe, I. M., and Cugnoli, C. (1992) J. Photochem. Phoiobiol. B Biol. 13, 5-17 Although the result on retinol binding activity of expressed 19. Ozaki, K., Terakita,A,, Hara, R.,and Hara,T. (1987) Viion Res. 27,1057-1070 RALBP is statistically significant and reproducible, wetook 20. Hara, R.. Hara,T., Ozaki, K, Terakita, A., Eguchi, G . , Kodama, R., and Takeuchi, T. (1987) inRetinul Proteins (Ovchinnikov,Yu.A., ed) pp. 447456, VNU concern about high background radioactivity observed in the Science Press, Utrecht control experiments. Since nonspecific binding of retinol to Pro- 21. Terakita, A,, Hara, R., and Hara, T.(1989) Vision Res. 29, 639-652 tein A-Sepharose or other contaminants seemed to increase the 22. Hara, T.,and Hara, R.(1991)in Progress in Retinal Research 10 (Osborne,N., and Chader, J., eds) pp. 179-206, Pergamon Press, Oxford background radioactivity, we added either much excess of bo- 23. Ullrich, A., Shine, J., Chirgwin, J., l'idet, R., Tischer, E., Rutter, W.J., and vine serum albumin or a diluted solution of detergent to the Goodman, H. M. (1977) Science IS& 1313-1319 T.,Fritsch, E. F.,andSambrook, J. (1982) Molecular Cloning: A 24. Maniatis, reaction mixture in order to block nonspecific binding. This Laboratory M a n w l . Cold Spring Harbor Laboratory, Cold Spring Harbor, treatment greatly suppressed not only background activity but NY also retinol-binding to RALBP. This result suggests that inter- 25. Hara-Nishimura,I., Mataumoto, T.,Mori, H., Nishimura, M., Hara, R., and Hara, T. (1990) FEES Lett. 271, 106-110 action between expressed RALBP and retinol is very weak. 26. Laemmli, U. K. (1970) Nature 227, 680-685 Such weak interaction is, however, observed in the native squid 27. Hara-Nishimura, I., Kondo, M., Nishimura, M.. Hara, R., and Hara, T. (1993) FEBS Lett. 3 1 7 , 5 1 1 RALBP, too, and is favorable for exchanging retinal with rho28. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Eiol. 167, 105-132 dopsin and retinochrome in thephotoreceptor cells (21,22). We 29. Chou, P. Y.,and Fasman, G. D. 11978)A&. Enzymol. 4 7 , 4 5 1 4 8 thus made an attempt to confirm retinol binding activity of 30. Fong, S.-L., Lee.P. G., Ozaki, K , Hara, R.,Hara, T., and Bridges, C. D.B. (1988) Vision Res. 28, 563473 expressed RALBP byexamining retinol-binding to an N termi- 31. Newcomer, M. E., Jones, T. A,, Aqvist, J., Sundelin, J., Eriksson, U., Rask, L., nally truncated RALBP similarly expressed in. uitro. The result and Peterson, P. A. (1984)EMBO J. 3, 1451-1454
7E,full-length RALBP had an apparent molecular weight of 48,000 on SDS-PAGE,a value similar to that of RALBPisolated
