tides obtained from the in situ digestion of the RPE65 blotted on nitrocellulose ... The open reading frame encodes a novel protein of 533 amino acid residues ...
Vol. 268, No. 21, Issue of July 25, pp. 15751-15751,1993 Printed in U.S. A.
CHEMISTRY THEJOURNAL OF BIOLOGICAL
Molecular Cloning and Expressionof RPE65, a Novel Retinal Pigment Epithelium-specific Microsomal Protein That Is Post-transcriptionally Regulated in Vitro* (Received for publication, December 18, 1992, and in revised form, March 2, 1993)
Christian P. Hamel, Ekaterini Tsilou, Bruce A. Pfeffer, JohnJ. Hooks$, Barbara Detricks, and T. Michael Redmondli From the Laboratory of Retinal Cell and Molecular Biology and the $Laboratoryof Immunology and Virology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892 and the §Department of Pathology, the George Washington University Medical Center, Washington, D. C. 20037
Studiesreported previously fromthislaboratory have shown that microsomal membranes of the vertebrateretinal pigment epithelium (RPE)contain an RPE-specific 65-kDa protein, RPE65, which bears the determinant recognized by the strictly tissue-specific monoclonal antibodyRPE9, and which is developmentally regulated (Hamel, C. P., Tsilou, E., Harris, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Neurosci. Res. 34, 414-425). Microsequencing of 17 tryptic and chymotryptic peptides obtained from thein situdigestion of the RPE65 blotted on nitrocellulose yielded primary sequences that were used to generate oligonucleotide probes. An 84-nucleotide guessmer was used to isolate twoclones from a bovine RPE XZap I1 cDNA library. Rapid amplification of cDNA ends was used to complete the 5’ and 3’ ends, resulting in a 3,115-base pair composite cDNA. The open reading frameencodes a novel protein of 533 amino acid residues witha computed molecular weight of 60,940. This protein does not match any other sequence in the databases. The 231 amino acids obtained from peptide sequencing match 43% of the amino acid sequence deduced fromthe cDNA. The protein hasa calculated PI of 6.41 and isnot predicted to have any transmembranesegments. The open reading frameexpressed in Escherichia coli has an apparent molecular weight identical to that of the native protein and is recognized by the monoclonal antibody RPES, further corroborating its validity. Northern blot analysis detected a major mRNA species of 3.15 kilobases for RPE65, as well as shorter species, only in RPE andnot in other tissues (including other ocular tissues). Cultured RPE cells (7 weeks in primary culture) contained RPE65 mRNA in amounts equivalent to fresh RPE. Such cells, however, contained no immunodetectable RPE65. The possible structure of this RPE-specific protein and hypotheses for the absence of translation in vitroare discussed.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reportedin thispaperhas been submitted accession number(s) to the GenBankTM/EMBLDataBankwith L11356. ll To whom correspondence should be addressed NEI-LRCMB, Bldg. 6, Rm. 339, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-0439;Fax: 301-402-0750.
The retinal pigment epithelium (RPE)’ is a monolayer epithelium interposed between the vascular-rich choroid and the neural retina. This ocular tissue is in close contact with the photoreceptor rod and cone outer segments and theMuller cells (retinal glial cells) extending its microvilli through the interphotoreceptor matrix around the outersegments. These anatomical features suggest two roles for the R P E control of the exchanges between the neural retina and the systemic blood supply of the choroid and interactive functions with photoreceptors and Muller cells. Amongthe many roles of the RPE cell which have been characterized, some are essential to thesurvival of the photoreceptor and hence, to vision itself. RPE, for example, phagocytizes periodically the tips of the outer segments ( l ) , a process whose defect leads to retinal degeneration (2). RPE is the site of many enzymes involved in retinoid metabolism, including retinyl ester synthetase (3) and 1ecithin:retinol acyltransferase (4),retinyl ester hydrolase (5, 6),retinol isomerase (7), and11-cis retinol dehydrogenase (8), as well as theRPE/retina-specific cellular retinaldehydebinding protein (9), in ion transport (10) and in digestion of the phagosomes and detoxification of the photoreceptor byproducts (11).The RPE also mediates the selective uptake of docosahexaenoic acid (22:6 n-3) from the bloodstream (12) and its selective transfer to the photoreceptor cells. RPE participates in the elaboration of the interphotoreceptor matrix (13) and may play a role in retinal development (14, 15). Despite their variety, there is still little molecular characterization of RPE functions. Efforts have been made to isolate RPE-specific proteins. Monoclonal antibodies have been generated (for review see Ref. 161, certain proteins of the RPE have been purified (17) or partially purified (4,18), andcertain cDNA clones have been isolated (19-21), although not strictly RPE-specific. Recently, we described an RPE-specific protein that is associated with the microsomal membranes of the RPE cell and is conserved in higher vertebrate species (22). Despite the fact that its role in RPE is still unclear, we believe that it could be involved in a function related to the photoreceptor outer segment metabolism since it begins to be expressed in RPE cells when the outer segments start to develop (22) and as such is a valuable cell-specific marker. In this paper, we report the cDNA and deduced amino acid sequences of the bovine 65-kDa protein,which we propose to name RPE65, its overexpression in Escherichia coli and show that it is posttranscriptionally regulated in vitro. ‘The abbreviations used are: RPE, retinal pigment epithelium; PAGE, polyacrylamide gel electrophoresis; MOPS, I-morpholinepropanesulfonic acid; bp, base pair(s); IPTG,isopropyl 1-thio-0-D-galactopyranoside; RACE, rapid amplification of cDNA ends.
15751
15752
Retinal Pigment Epithelium-specific Microsomal Protein EXPERIMENTALPROCEDURES
Materials-Guanidine thiocyanate and formaldehyde were from Fluka (Buchs, Switzerland), and TritonX-100 was from Pierce Chemical Co. Sonicated salmon sperm DNA, oligo(dT)-cellulose, T4 polynucleotide kinase, RNase block 11, and Moloney murine reverse transcriptase were from Stratagene (La Jolla, CA). When not specified,DNA bands were purified from agarose gels using QIAEX (Qiagen, Diisseldorf, Germany). Dispase, terminal transferase, arandom-primed DNA labeling kit and Sephadex G-25 Quick Spin columns were from Boehringer Mannheim. Vent DNA polymerase and restriction endonucleases were from New England Biolabs (Beverly, MA). Oligonucleotides were synthesized ona model 392 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA) and purified using Sephadex G-25 Quick Spin columns. Protein Analysis and Microsequencing-The RPE65 was isolated from bovine RPE as described previously (22). Briefly, RPE cells brushed off the Bruch's membrane or pellets of cultured RPE cells were homogenized in TBS (150 mM NaCl, 10 mM Tris-C1, pH 7.5) containing 5 mM EDTA and 0.25 mM phenylmethylsulfonyl fluoride (Sigma) and centrifuged at 10,000 X g for 20 min. The supernatant (salt-soluble extract) was reserved and thepellet resuspended in the same buffer containing 1%Triton X-100. After centrifugation at 10,000 X g for 20 min, the resulting supernatant (detergent-soluble extract) and the salt-soluble extract were analyzed in12%SDSPAGE (23). Gels were stained with 0.1% Coomassie Blue in 40% methanol, 10% acetic acid or electroblotted onto nitrocellulose (24). Immunoblots were reacted with the monoclonal antibody RPE9 (25) which recognizes the RPE65, a t a dilution of 1:8,000. The secondary antibody was an alkaline phosphatase-conjugated goat anti-mouse IgG (GIBCO-BRL) used at a dilution of 1:3,000. For microsequencing, a bovine RPE detergent-soluble extract was run on a preparative 12 X 16-cm 12% sodium dodecyl sulfate (SDS)polyacrylamide gel and blotted onto nitrocellulose. The 65-kDa band was visualized by 0.1% Ponceau S and excised from the blot. The nitrocellulose-bound 65-kDa band was digested in situ with trypsin (26), and the digest was separated by high performance liquid chromatography using a Vydac C18 column. Late eluting peptides were pooled, digested with chymotrypsin, and separated as before. The resultant purified peptides from both digests were subjected to automated Edman degradation on an Applied Biosystems model 477A gas phase Sequencer with phenylthiohydantoin derivatives analyzed by an online model 120Aphenylthiohydantoin analyzer. Sequencing was carried out by the Harvard Microchemical Facility (Cambridge, MA). Cultures of Bovine RPE Cells-Freshly harvested bovine eyes briefly sterilized with 70% ethanol were opened at the equator and the vitreous and retina removed. While submerged in complete balanced saline solution (Hanks' salts, including calcium and magnesium), 10-mm2large pieces of RPE-choroid were cut from the posterior wall of the eye and washed several times in this solution. Sheets of RPE cells were then obtained by incubation of the RPE-choroid pieces in 1.6% Dispase (w/v) for 30 min at 37 "C with gentle agitation and seeded ina culture medium (27) containing 0.1 mM Ca", a condition that enhanced migration of proliferating cells away from the original explants. As the cells approached confluence, the [Ca"] was raised to 0.5 mM, and by 1 month after the initial plating the RPE cultures exhibited hexagonal packing and numerous fluid-filled domes, indicative of the polarization characteristic of differentiated epithelial cells (28). After 7 weeks of primary culture, the cells were washed twice in cold balanced saline solution, once in phosphatebuffered saline supplemented with 0.1% ovalbumin, scraped, and pelleted at low speed centrifugation. Pellets of cells were used either for protein analysis or RNA extraction. RNA Extraction and Northern Blot Analysis-Bovine eyes freshly obtained from the abattoir were opened at the equator and the vitreous and retina removed. One ml of guanidine thiocyanate solution (29) was introduced into the eye cup, and the RPE cells were rapidly detached with a sterile 4.8-mm brush. For cultured RPE, the cell pellets were directly resuspended in the guanidine thiocyanate solution. Other tissues including the iris and the retina were dissected and directly placed in the guanidine thiocyanate solution. The RNA was then extracted following the APCG single-step method (29). For Northern blot analysis, 20 pg of total RNA was resolved by electrophoresis through a 0.8% agarose gel in MOPS-formaldehyde. The RNA was transferred to 0.20-pm nitrocellulose in 10 X SSC and cross-linked to themembrane by UV irradiation. The 1,143-bpEcoRI insert of clone pPE3 (see Fig. 2) was labeled with 32Pby random
priming (30) and used as a probe to hybridize the blot in 50% formamide, 10% dextran sulfate, 20 mM sodium phosphate, pH 6.7,5 X Denhardt's solution, 5 X SSC, 0.1% SDS, 100 pg/ml salmon sperm DNA overnight a t 42 "C. Final washing was in 0.1 X SSC, 0.2% SDS a t 68 "C. After stripping off the pPE3 probe, the blot was also hybridized with a 0-actin cDNA probe, under identical conditions. cDNA Cloning and Sequencing-Poly(A)+ RNA was selected by oligo(dT)-cellulosechromatography using standard methods. Approximately 10 pg of fresh bovine RPE poly(A)+RNA wasused to generate a cDNA library in XZap I1 (Stratagene). The library was screened with the 84-nucleotide guessmer oligonucleotide 461 derived from the chymotryptic peptide CH-72 (see Fig. 3). 11.5 p~ oligonucleotide 461 was 5' end labeled at a specific activity of 7.4 X 10' cpm/pg with 32P using T4 polynucleotide kinase. Nitrocellulose plaque lift replicates were hybridized with this probe in 5 X Denhardt's solution, 6 X SSC, 20 mM sodium phosphate, pH 6.7, 0.2% SDS, 10% dextran sulfate, 50 pg/ml salmon sperm DNA at 37 "C for 24 h. Final washing was in 2 X SSC, 0.2% SDS at 63 "C. Three positive clones were isolated, plaque purified, and pBluescript plasmids rescued by in vivo excision (Stratagene). To test whether the isolated clones hybridized with other RPE65-derived oligonucleotides, the EcoRI inserts were separated in a l%agarose gel, blotted, UV cross-linked and incubated with 32P-labeled51 (20-mer)and 362 (38-mer) oligonucleotides,under the same conditions as with 461 except for final washing (10 min at 60 "C and 10 min at 55 "C for 51 and 362, respectively). Plasmids were purified, and the DNA was sequenced by the dideoxy chain termination method (31) using Sequenase (U. S. Biochemical Corp.) by use of both nested deletions (32) and internal sequencing primers in both directions (Lark Sequencing Technologies, Houston, TX). To obtain the 5' and 3' ends of the RPE65 cDNA, we used the RACE procedure (33) with slight modifications. Oligonucleotides 3' end of pPEl were synthesized. matching the 5' end of pPE3 and the The first strand of cDNA was synthesized in a 20-pl reaction volume containing 50 mM Tris-C1, pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCI,, a 0.5 mM concentration of each dNTP, 20 pM primer, 1 unit of RNase block 11, 20 units of Moloney murine reverse transcriptase, and 1pg of poly(A)+ RNA and incubated at 37 "C for 1 h. The unhybridized RNA was then digested with 0.4 unit of RNase H at 37 "C for 20 min. For the 5' end, the first strand synthesis primer and the dNTPs wereremovedby electrodialysis ina biodialyzer (Sialomed, Columbia, MD) closed bytwo 50-kDa molecular mass cutoff membranes in 1 X TAE for 10 min at 100 V. The 5' RACE cDNA was then poly(A)+tailed with 25 units of terminal transferase in a 30-pl volume of the appropriate buffer (0.2 M potassium cacodylate, 25 mM Tris-C1, pH 6.6, 1.5 mMCoC12, 0.25 mg/ml bovine serum albumin) containing0.2 mM dATP and incubated a t 37 'C for 10 min. The 3' and 5' first strand cDNAs were finally diluted to 250500 p1 in 10 mM Tris, pH 8.0, 1 mM EDTA (TE). The rest of the procedure followed the standard RACE method (33) except that the second strand synthesis was performed separately from the subsequent amplification steps using a low concentration of primer (0.1 pM/pl) to minimize mispriming. RACE products were purified by gel electrophoresis, extracted with QIAEX, digested with the appropriate restriction enzyme, subcloned in pBluescript I1 SK (Stratagene), and sequenced as described above. E. coli Expression of 65-kDa Protein-A single cDNA spanning the RPE65 open reading frame was obtained by reverse transcription coupled to DNA amplification and cloned in the prokaryotic expression vector pKK223-3 (Pharmacia LKB Biotechnology Inc.) using a ligation-independent cloning procedure (34). Briefly, antisense 5' >
AATATTATGAAAATCTCAGGATTTTTTGAACAGTCCATG < 3' and sense 5' > AATATTATGAAAATCTTAAGCAGCCAAGT'I" GAACATCCAGCTGGT< 3' primers with identical 15-nucleotide 5' ends were generated to amplify the open reading frame cDNA. The sense primer contained 9 nucleotides (underlined) interposed between the initiation codon and the second codon (AGC) of the original 65kDa sequence which have been added to optimize the translation (35). The first strand of cDNA was synthesized from 1 pg of bovine RPE poly(A)+ RNA under the same conditions as for the RACE procedure (see above) and subsequently brought to a 5OO-pl volume with TE. The second strand of cDNA was synthesized in a 100-pl reaction volume containing 10 mMKC1,20mM Tris-C1, pH 8.8, 10 mM (NH,),SO,, 2 mM MgSO,, 0.1%Triton X-100, 10 pg of bovine serum albumin, a 0.2mM concentration of each dNTP, 5 pM sense primer, 2 p1 of cDNA, and 4 units of Vent DNA polymerase in a onestep program (3 min at 95 "C, 5 min a t 60 "C, 40 min at 72 "C). After completion of the second strand cDNA synthesis a 100 PM concentration of each primer was added, and the reactions were amplified
Retinal Pigment Epithelium-specific Microsomal Protein for 40 cycles (30 s a t 95 "C, 30 s a t 64 "C, 2 min a t 72 "C). EcoRIdigested pKK223-3 was amplified in the same buffer containing an additional 6 mMMgSO., using primers with the 5' 15 nucleotides complementary to the 5' ends of the RPE65 primers (30cycles: 30 s at 95 "C, 1 min at 65 "C, 4 min a t 72 "C). Amplified RPE65 cDNA and pKK223-3 were ethanol precipitated, diluted in1 X Tris-borateEDTA (TBE)/Ficoll, andpurified on a model 230A high performance micropreparative electrophoresis system (Applied Biosystems) using 1 and 0.5% agarose gels, respectively. Fractions containing the purified DNAfragment were pooled, ethanolprecipitated,and resuspended in water. Cohesive ends were obtained by digestion with T4 DNA polymerase and cloned (34). E. coli JM105 colonies containing the properly oriented RPE65 expression plasmid were grown in 2 X YT medium supplemented with 125 pg of ampicillin/ml and transcription of the insert induced with 2 mM IPTG at midlog phase. Aliquots of the cultureswere harvested at various time points,diluted in sample buffer, and analyzed by SDS-PAGE and immunoblotting. Computer Sequence Analysis-Sequencing management, searches for similarities, and protein structure analysiswere performed using the Genetics Computer Group Package (UW Biotechnology Center, Madison, WI) (36). The hydropathy profile of the deduced amino acid sequence was analyzed with the Kyte and Doolittle (37) hydropathy index using a window size of 9. Helical structure was represented as a wheel diagram (38).
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FIG.2. Mapping of RPE65 cDNA clones. The restriction map of RPE65 cDNA is shown at the top ( B ,BarnHI; E, EcoRI; N , NcoI; P , PstI; X , XbaI);the open reading frame is in bold. The distribution of the five independent cDNA clones, pPE1,3,4,5, and 6, of RPE65 is represented. kB,kilobases.
were generated and are shown in Fig. 2. The RACE clone pPE4 overlapped with pPE3 by about 100 bp; pPE5 overlapped with pPE4 by about 60 bp; and pPE6overlapped with pPEl by almost its entire length except for the additional 3' sequence beyond the polyadenylation signal. Both strands of pPE1, pPE3, pPE4, pPE5,and pPE6were sequenced, including several individual replicates of the RACE-derived clones. The complete nucleotide sequence of RPE65 cDNA and the deduced amino acid sequence are shown in Fig. 3. The 3,115 RESULTS bp of this cDNA contain an open reading frame consisting of Isolation of cDNA Clones Encoding RPE65"An 84-nucleo- 1,599 bp from the firstATG at position 53 to TGA at position tide guessmer (oligonucleotide 461) was deduced from the 1,651. We assume that this ATG functions as an initiation amino acid sequence of the chymotryptic peptide CH-72 (see site for translation, since it is the first one, considering that Fig. 3) and used to screen 75,000 plaques of an unamplified there is an A at position -3 (39) and that an in-frame TGA RPE cDNA library generated in the vector XZap11. Three termination codon is found at position -9. The 3"untransclones (pPE1, pPE2, and pPE3)were isolated, plaque purified, lated region minus the poly(A) tail is 1,457 bp long.An and the pBluescript phagemids were rescued by in vivo exci- AATAAA polyadenylation signal is found 17 nucleotides from sion (Stratagene). These clones hybridized strongly with oli- upstream from the poly(A) tail; an ATTAAA, a possible gonucleotide 461 on Southern blot (Fig. lB), and in addition, polyadenylation signal for a shorter transcript(see below), is clone pPE3 hybridized with two other RPE65peptide-derived found at nucleotide 2,759. The combined average percentage oligonucleotides (Fig. 1, C and D),362 deduced from chymo- of As and Ts of the RPE653"untranslated region is 70.28%. tryptic peptide CH-86A, and 51 from tryptic peptide NT-9 It is interesting to note that this AT-rich 3"untranslated (see Fig. 3). The EcoRI insert of clone pPEl contains an region contains two identical dodecanucleotides TTATTinternal EcoRI site as shown by the two fragments of 1,289 TAGATAA found at positions 1,837 and 2,713. When comand 433 bp obtained following digestion (Figs. lA and 2). pared with GenBank(release 73.1) using FASTA (40) no DNA sequencing of the EcoRI insert of these clones revealed significant similarities with other sequences were detected. that pPEl and pPE3 were authentic RPE65 cDNA clones This sequence has been deposited in GenBank under the (Fig. 2) as several peptides matched with their deduced amino accession number L11356. Analysis of the Amino Acid Sequence of RPE65"The open acid sequence, while pPE2 was a foreign cDNA. pPE1 and reading frame codes for a protein of 533 amino acids with a pPE3 overlapped by 433 bp. RACE was performed to obtain calculated molecular weight of 60,940. Three asparagine resthe 5' end of the cDNA and thefew bases downstream of the polyadenylation signal in the 3'-untranslatedregion. Two 5' idues are potential sitesfor N-linked glycosylation. Six tryptic RACE (pPE4 and pPE5) and one 3' RACE (pPE6) fragments and 11 chymotryptic peptides obtained by microsequencing from protease digestion of RPE65 matched the deduced amino acid sequence of the cDNA and are underlined and designated A B C D in Fig. 3. A total of 231 amino acid residues obtained by 1 2 3 1 2 3 2 2 bp n n n n nn microsequencing was assigned, accounting for 43.33% of the amino acid sequence deduced from the cDNA, thereby corroborating its validity. The predicted isoelectric point of RPE65 is 6.41. No signal peptide is found in thissequence. The hydropathy 603 profile shows no evidence for hydrophobic membrane spanning segments (Fig. 4). However, comparison of the hydropathy profile with the hydrophobic moment profile (not shown) reveals two regions of high hydrophobic moment and moderate hydrophobicity, indicative of potential amphipathic aFIG. 1. Cross-hybridization of clonepPE3with three helix structures. When these two regions were analyzed with RPEBS-derived oligonucleotides. EcoRI inserts of clones pPE2, the helical wheel program, amphipathic a-helixes were obpPE3, and pPEl(lanes 2-3, respectively) were electrophoresed in an tained, respectively at the amino terminus (amino acids 5ethidium bromide 1% agarose gel (panel A), blotted, and hybridized 22) and further along the molecule (amino acids 108-125) either with peptide CH-72-derived oligonucleotide 461 (panel B ) for (Fig. 5). The amino acid sequence was compared with the allthree clones andpeptide CY-86A-derived oligonucleotide 362 (panel C) plus peptide NT-9-derived oligonucleotide 51 (panel D )for sequences contained in several data bases (Swissprot release clone pPE3 only. Note that the insert of clone p P E l (panel A, lane 23.0, NBRF release 33.0, and the daily updated Blast server at the National Center for Biotechnology Information, Na3) is split in two fragments by an internal EcoRI site. "
15754
Retinal Pigment Epithelium-specificMicrosomal Protein 1
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FIG.3. R P E 6 5 cDNA and protein sequence. Nucleotide and deduced amino acid sequences of RPE65 are shown. The nucleotide sequence was derived from sequencing both strandsof each of theoverlapping clones pPE1,pPE3, pPE4,pPE5,andpPE6(see Fig. 2). Tryptic and chymotryptic peptides resulting fromdigestion of RPE65are underlined and designated. The initiation codon is in bold, and the termination codon (TGA) is indicated by an asterisk (*). Potential N-linked glycosylation sites are indicated by a diamond (+). In the 3”untranslated region, polyadenylation signals are double underlined, and the two identical dodecanucleotides TTATTTAGATAA are underlined. This sequence is deposited in GenRank under the accession number L11356.
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1700
*
GTAGTlAGCTCTCCAATCAAATTClGTlTAGCTTTAGCClGCTGTCTATAAGGGTTTTAACTTGCAGATGCACACCGlTTTGCAGTATTTTACAGAAAGC ACAGAGTTGAGlAAGGAATTCCllTTAAAAAAGTGClTAlTT~GATAATCGTACTTCGTGAGACAGGCACATCATAACTAAAAACTCTTTATAlATTlAC AATCAAATAGAAAATGAATGTGAATTTATTAAACTGTTTTTCATTCCTATTATAAAAGTGTATTTTAGGCACCCAlCTACTCCTATTACTTTTTAACATT TAAAAGCCAAAGTCCTCTACAGCTGATATGTATATAGCTTTGCTGTGTCAAGGCAGCATCTTGGAAAAAGGCTTAGTTACTAAATGTCAAATCAAACTTC TTClCAAACCAGGGAClTTGATCTAAGACAATCATTGTAATTGTATGCACATATGTATTATTATTCAAAGATTClCATGGTTTAAACTTTAATATTTCCA
1800 1900 2000 2100 2200 TTTAATATAGGCAATTATTACTATTTCTGAlTTCACAGTAAGGAAGCCTGTTTCTAAGTCACTGAGGGClGlCAACCAAATACCllCTCAGAGATTACAT 2300 2400 TATCTATTAGAAACTTATATAACATAAAAGCAATlACATAlAGlAGTGACTATTTTTTTTAAATAGCATAAAATACTTACAAACAlAATlGCTGTTTAAA ATCAGAlTATGGGGATAATAATGTGlATGGGGGGGGTTATACTlTGTTGCCTCTTCTGCTTAATTACATTTAAGCACTGlGCACCTGACTGAAGAGAAAA 2500 TGAGAAAATAGAAAATACTGTCATTAATGTTCAGATATGAATATAGTTTAAGCAClTTTGTTTTTAAAATTATTCTTTTCTGAGATACATGTTGCAAAAA 2600 A~AATGAAGATTGACAAAAATTTTlATTCTAlATCTTAATCCTGTTTGAClAAGTAAAAACAGCTTGAAGGAACAGACTGACTlGGGTACTTGATGCATC 2700 C A T T T G A T T T C T T T A T T T A G A T A A A A T G C A A A C T C A T T T l T T A A G A T T A T A A G T A C T C A T l A A A A T A T A C T T T T C A A A G A A A A T C T A A A A T T G C A A A A A A 2800 C I ~ G A A A T l A l A A A G ~ A A A A T C A l A C A G A G C T C A T C T A A G C A C A A C T A C T G G T C T T T T C ~ T T C C C A T G l G T G G C T T T T T A A l G T A A T C A T T A l T l C2QOO 3000 TTTGGGAGTAATTCCTAGGTAAACCTAGATATClCAAAAAAAAAAAAAAAGAAAAATGCCATAAAATAACAlGGAAAATGTGACTGTAGATAAACATCAA T C T G C T G G A T T T T G T C T C T A G A T G T G G C A C G T G A A C T l A A T l C C A C A A A G A A A G C l A T T C A T A C A T l T A l T T T A A A A A T G C A A A A ~ T T A C T G A A 3100 T 3115 AATTACCTAAAAAAA
.....................................................................................
addition, two otherminorbands a t about 2.25 and 1.75 kilobases are detectable. Total RNA from 7-week-old primary cultures of bovine RPE cellsshowsa pattern of RPE65 transcripts identical to that of fresh bovine RPE cells, even FIG. 4. Hydrophobicity profile of RPE65. Hydrophobicity though RPE65 from these cells was undetectable on immuprofile for the predicted aminoacid sequence of RPE65 was obtained noblots using themonoclonal antibody RPES (Fig. 7). using the Kyte-Doolittleprogram with a window size of 9. Expression of RPE65in E. coli-To test if the cDNA reported herecodes forthe 65-kDa RPE-specific band bearing tional Library of Medicine, Bethesda, MD) using the FASTA the determinant recognized by monoclonal antibody RPES (40), SEQFP (41), and BLAST (42) programs. No significant on immunoblots, we generated a full-length RPE65 cDNA by extended similarity to any other protein was detected. Except for such features as potential phosphorylation sites and N - reverse transcription polymerase chain reaction and cloned it linked glycosylation sites, the protein sequence did not con- in the IPTG-inducible tac promoter vector pKK223-3. The tain other sequence motifs when compared with the Prosite recombinant RPE65 shouldbe identical to the native RPE65 except for the insertion of the tripeptide KIL between the data base. Analysis of RPE65 Messenger RNA-Total RNA from three first andsecond amino acids of the original sequence, resulting intraocular tissuesincluding bovineiris, retina, and RPE were from the optimization of the flanking sequences of the initiisolated and analyzed by Northern blotting using the 1,143- ation codon in prokaryotes (see “ExperimentalProcedures”). plasmid were grown bp EcoRI insert of cDNA clone pPE3 as a probe (Fig. 6). JM105 E. coli containing the recombinant Although no message was detectable in iris and retina (Fig. in the presence of 125 pg of ampicillin/ml, induced by the 6 B ) , a major band of 3.15 kilobases was present in the RPE addition of IPTG to 2 mM, and aliquots of the culture harvRNA, corresponding with the3,115-bp cDNA reported here. ested a t 4 and 6 h after induction. As shown in Fig. 8, a band Another band of2.85 kilobases is also clearly visible; this of apparent molecular weight similar to that of the native shorter transcript might have been generated from the atyp- protein is visible after induction and is readily detected by ical ATTAAA polyadenylation signal a t nucleotide 2,759. In RPES on theimmunoblot. This band is absentin the nonin-
A
~
P
Retinal Pigment Epithelium-specific Microsomal Protein
15755
FIG. 5. Structural analysis of the amino-terminal end of RPE65. The amino-terminal end of RPE65 was analyzed with the wheel program which displays a view down the barrel of a helical region and illustrates the aminoacids surrounding the helix. Two regions(am-
ino acids 5-22 and 108-125) were found to have an amphipathic asymmetry and are depicted here. Hydrophobic residues are boxed. H
A
B
1 2 3 4
1 2 3 4
K
D
E
S
R
1 2 3
kb
Kt,
+-
6.22 3.91
‘t --
2.80 1.90
0.87
FIG. 7. Absence of expression of RPE65 in cultured bovine RPE cells. Immunoblot fromSDS-PAGE of cultured RPE cells (same lot as those used for mRNA analysis) reacted with the RPE65specific monoclonal antibody RPES. Shown are a detergent-soluble extract of freshRPE cells ( l a n e 1 ) andsalt-soluble (lane 2) and detergent-soluble ( l o n e 3 ) extracts of cultured RPEcells (see “Experimental Procedures”). RPE65is indicated by an arrow.
0.56 0.36
C 1 2 3 4
3.15 2.85 2.25 1.75
the molecular weight of the RPE65 after endoglycosidase F digestion (22). The fact that the recombinant protein expressed in E. coli has an apparent molecular weight in SDSFIG.6. Northern blot analysis of RPE65 messenger RNA. PAGE undifferentiable from that of the native protein conTotal RNA from bovine iris (lane I), bovine retina ( l a n e 21, fresh firms that the open reading frame presented here is fullbovine RPE cells ( l a n e 3), and cultured bovine RPE cells ( l a n e 4 ) length. was electrophoresed in an ethidium bromide 0.8% agarose MOPSOne of the biochemical features of this protein is its assoformaldehyde gel (panel A ) and the blot hybridized with a 1,143-bp EcoRI insert of bovine clone pPE3 (panel I?). The blot was stripped ciation with the microsomal membranes of the RPE cell. and then hybridized with a 8-actin probe (panel C) to verify further RPE65, mostly solubilized in thepresence of detergent, partthe integrity of the RNAs. Arrows indicate the sizes of the various itions in the detergent-rich phase of the Triton X-114 (22). messages detected. Examination of the amino acid sequence of RPE65 finds no evidence of a hydrophobic transmembrane region. A glycosyl duced culture and ina control recombinant plasmid contain- phosphatidylinositol anchor is also unlikely since RPE65 is readily solubilized in Triton X-114 and does not have any ing an inappropriate insert. signal peptide. We show here that two regions of the aminoterminal partof the protein witha high hydrophobic moment DISCUSSION are predicted to have an amphipathic a-helical structure. Such In this paper we describe the cDNA and amino acid se- amphipathic a-helicescould associate with anintegral memquences of RPE65, a protein detected only in R P E cells as brane protein,for example,or directly interact with the memshown previously by immunohistochemistry (25) and immu- brane. This latter possibility is of interest sincewe have found noblotting and which is conserved in mammals, birds, and recently2 that RPE65 binds to phospholipids and that this frogs (22). The cDNA contains an open reading frame coding property canbe exploitedto purify it by the use of immobilized for a protein of 533 amino acids, which gives a calculated artificial membrane chromatography (43). It is possible that molecular mass of about 61 kDa, although the apparent mo- phospholipid bindingis necessary to the function of this lecular weight by SDS-PAGE is 65,000. This difference is protein. However, the protein sequence has no significant probably not accounted by glycosylation, even though three similarity to sequences of known phospholipid-binding propotential N-linked glycosylation sites are present, since our teins. Given the relatively weak hydrophobicity of the hydroprevious investigations have failed to demonstrate an affinity for concanavalin A or wheatgerm agglutinin, ora decrease in * E. Tsilou, C. P. Hamel, andT. M. Redmond, in preparation.
-
1.95 kb
15756
Retinal Pigment Epithelium-specific Microsomal Protein
untranslated region of RPE65 mRNA arefound two identical dodecanucleotides UUAUUUAGAUAA. This sequence is reminiscent of theoctanucleotide UUAUUUAUfound in several UA-rich3"untranslated region from cytokine mRNAs (48) and which has been implicated in inhibition of translation (49) when assayed in Xenopus oocytes. The second dodecanucleotide a t position 2713 is alsopreceded by two triplets of Ts. Whether the 3"untranslated region of RPE65 might in fact play a role in regulating the translat,ion of its message translation and transfecwill have tobe addressed by in vitro tion of the cultured RPE cells with modified messages. It is 31 possible that explantation results in the loss of some in vivo factor that is necessary for translation of the mRNA. So far, various extracellular factors present in the retinal microenvironment have been supplied to the cultured RPEcells but 21 have not triggered expression of RPE65 at the proteinlevel. It is also important to note that several RPE65 transcriptsof various lengths exist in bovine RPE cells, another indication that, at least in that species, a particular regulation of this FIG. 8. E. coli expression of RPE65. Shown is the SDS-PAGE protein might exist. of cultures of bacterial clones containing the plasmid pKK223-3. SDS Extensive data base analyses have failed to show significant gel stained with Coomassie Blue (panel A ) and immunoblot reacted extendedsimilarity of RPE65 to any other protein. This with the RPE65-specific monoclonal antibody RPE9 (panel R ) . Cul- implies that this protein is the first described member of a tures of bacteria containing the recombinant RPE65 expression is present only in plasmid were either not induced ( l o n e 1 ) or induced (lane 2 ) with 2 new family. Given the fact that this protein mM IPTG and compared with the detergent-soluble fraction from the RPE, this observation suggests that the RPE utilizes a fresh bovine RPE cells (lone 3 ) and a recombinant clone containing unique protein to accomplish one of its specific functions. an insert different from that of RPE65 and induced by IPTG (lane The role of this protein is still unknown, although we know 4). The arrow indicates the 65-kDa band. that it isexpressed in RPE cells just prior to the appearance of the photoreceptor outer segments (22). This further sugphobic face of the predicted cu-helixes of RPE65, a complete gests that RPE65 might be involved in a RPE function related embedding in the membrane through dimerization been as has to the photoreceptor outer segmentbiology. It is anticipated suggestedfor the protein Sgp50 (44) is unlikely.However, thatunderstandingthemechanisms of the regulation of polar interactions with the heads of the phospholipids or RPE65 expression in vitrowill direct us toward this function. partial embedding in the hydrophobic milieu of the memExperiments to address this point are under way. branes is possible. Further experiments using recombinant amino-terminal-truncated RPE65 will test whether such deleAcknowledgments-We thank Alex Yuen for technical assistance and Dr. Robert Kim for the generous gift of certain oligonucleotide tions impair the association with the membranes and binding primers and bovine tissue RNAs. We also thank Dr. Peggy Zelenka to phospholipids. for critical reading of the manuscript. We are grateful to the staff of Messages for the RPE65 were detected by Northern blot the Advanced Scientific Computing Laboratory ofthe Frederick analysis inRPE cells from fresh bovine eyes, but not in retina Cancer Research and Development Facility for help incomputer and iris, and have not been found in brain, liver, lung, heart, analysis and to the staff of the National Center for Biotechnology kidney, and small intestine (results not shown). This confirms Information, National Library of Medicine, for help with the BLAST that this protein is preferentially expressed in the RPE cells. network service. Messagesfor the RPE65 were alsopresent in 7-week-old REFERENCES cultured bovine RPE cells. This is of interest since we show 1. Young, R. W., and Bok, D. (1969) J. Cell Riol. 42,392-403 that, in culture, RPE cells from both bovine (this study) and 2. Mullen, R. J., and LaVail, M. M. (1976) Science 192,799-801 3. Barry, R. J., Canada, F. J., and Rando, R. R. (1989) J . Riol. 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C., Hredherg, L., and Garwin. G. G. (1982) J . Riol. Chem. 2 5 7 , quired to stabilize the protein and to target it to its proper 1:3329-133:33 10. Joseph, D. P., and Miller, S.S. (1991) J. Ph.vsiol. (Cambridge) 4 3 5 , 439cell compartment. However, the sequence of RPE65 does not 463 reveal any known signal peptide. In addition, we have shown 11. Herman. E. R. (1991) The Biochemistry of the E-ye, pp. 380-406, Plenum Press, New York. that RPE65 is not glycosylated (22), and attempts to detect Wang, N.. and Anderson R. E. (1992) Curr. Eye Res. 11, 783-791 possible phosphorylation were inconclusive.3 An alternative 12. 13. Stramm. L. E.,Wolfe, J. H., Schuchman, E. H., Haskins. M. E., Patterson, D. F., and Aguirre, G. D. (1990) Exp. Eye Res. 50,521-532 hypothesis would be that theexpression of RPE65 isregulated P. E., Ulshafer, R. J., Ludwig, H. C., Allen, C. B., and Kelley, K. at the level of translation. The abundance of the mRNA in 14. Spoerri, C. (1988) Eur. J . 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E. Tsilou, C. P. Hamel, and T. M. Redmond, unpublished results.
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"
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Microsomal Protein
15757
35. Das, A. (1990) Methods Enzymol. 182,93-112 36. Devereux, J., Haeberli, P., and Smithies, 0.(1984) Nucleic Acids Res. 12, 387-395 37. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 38. Schiffer, M.,and Edmundson, A. B. (1967) Biophys. J. 7, 121-135 39. Kozak, M. (1989) J. CeUBiol. 108, 229-241 40. Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 86,2444-2448 41. Kanehisa, M. (1982) Nucleic Acids Res. 10, 183-196 42. Altschul, S. F., Gish, W., Miller, W., Myers, E. W.,Lipman, and D. (1990) J. Mol. Biol. 215,403-410 43. Pidgeon, C., Stevens, J., Otto,S., Jefcoate, C., and Marcus, C. (1991) Anal. Biochern. 194,163-173 44. Lasky, L. A,, Singer, M. S., Dowbenko, D., Imai, Y., Henzel, W. J., Grimley, C., Fennie, C., Gillett, N., Watson, S. R., and Rosen, S. D. (1992) Cell 69,927-938 45. Mayerson, P. L.,and Hall, M. 0.(1986) J. Cell Biol. 103,299-308 46. Simmons, D. M.,Voss, J., Ingraham,H., Holloway, J., Broide, R., Rosenfeld, M., and Swanson, L. W. (1990) Genes Q Deu. 4, 695-711 ClBment. A.. CamDisi. J.. Farmer. S. R.. and Brodv. _ ,J. S. (1990) .~ ~,Proc. Natl. Acad. Sci.' U. S:A. 67; 318-322 ' 48. Ca ut ' D;. Beutler, B., Hartog, K., Thayer, R., Brown-Schimer, S., and eraml A. (1986) Proc. Natl. Acad. Sci. Li. S. A. 83, 1670-1674 49. K ~ y g V., , Marinx, O., Shaw, G . , Deschamps, andJ., Huez, G . (1989) Scrence 245,852-855
8
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