Molecular Characterization and Developmental Expression of NORPEG, a Novel Gene Induced by Retinoic Acid*

R. Krishnan KuttyDagger §, Geetha KuttyDagger , William SamuelDagger , Todd DuncanDagger , Christy C. Bridges, Amira El-Sherbeeny, Chandrasekharam N. Nagineni||, Sylvia B. Smith**, and Barbara WiggertDagger

From the Dagger  Biochemistry Section, Laboratory of Retinal Cell and Molecular Biology, and the || Immunology and Virology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740 and the  Departments of Cellular Biology and Anatomy and of Ophthalmology, Medical College of Georgia, Augusta, Georgia 30912-2000

Received for publication, August 15, 2000, and in revised form, October 12, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have characterized NORPEG, a novel gene from human retinal pigment epithelial cells (ARPE-19), in which its expression is induced by all-trans-retinoic acid. Two transcripts (~3 and ~5 kilobases in size) have been detected for this gene, which is localized to chromosome band 5p13.2-13.3. Placenta and testis showed the highest level of expression among various human tissues tested. Six ankyrin repeats and a long coiled-coil domain are present in the predicted sequence of the NORPEG protein, which contains 980 amino acid residues. This ~110-kDa protein was transiently expressed in COS-7 cells as a FLAG fusion protein and immunolocalized to the cytoplasm. Confocal microscopic analysis of the NORPEG protein in ARPE-19 cells showed threadlike projections in the cytoplasm reminiscent of the cytoskeleton. Consistent with this localization, the expressed NORPEG protein showed resistance to solubilization by Triton X-100 and KCl. An ortholog of NORPEG characterized from mouse encoded a protein that showed 91% sequence similarity to the human NORPEG protein. The expression of Norpeg mRNA was detected in mouse embryo at embryonic day 9.5 by in situ hybridization, and the expression appears to be developmentally regulated. In adult mouse, the highest level of expression was detected in the seminiferous tubules of testis.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The retinal pigment epithelium (RPE)1 is a monolayer of highly differentiated cells located between the choroid and neural retina in the eye (1, 2). It provides nutrients to photoreceptor cells and contains melanin pigments that absorb excess light radiation. RPE cells are polarized: the basolateral membrane side encounters choroidal circulation, whereas the apical microvillus membrane side faces photoreceptors. These cells are indispensable for the regeneration of 11-cis-retinal, the visual chromophore, and carry out the phagocytosis and degradation of photoreceptor outer segment discs undergoing circadian shedding (1, 3). The RPE also plays a critical role in infectious and inflammatory diseases affecting the retina (4), and macular degeneration, a major visual system disorder associated with aging, has been attributed to RPE dysfunction (5, 6).

Several cell lines derived from human RPE have been recently established (7-9). ARPE-19, a rapidly growing cell line established by Dunn et al. (8), is noted for its ability to retain many structural and functional characteristics of intact RPE. ARPE-19 cells exhibit a distinctly epithelial morphology, noticeable pigmentation, and polarized distribution of cell-surface markers (8, 10). They express several genes specific for the RPE; assimilate photoreceptor outer segment discs by phagocytosis; and show polarized expression of reduced folate transporter-1, folate receptor-alpha , and Na+-K+-ATPase (8, 11, 12). They are also capable of expressing growth factors and cytokines (10, 13).

To study gene expression in the RPE, we have constructed a cDNA library with poly(A)+ RNA preparations isolated from ARPE-19 cells. A cDNA clone identified during the initial characterization of this library was found to represent a novel human gene. We have further characterized this gene and shown that its expression is regulated by retinoic acid. We have also characterized an ortholog of this gene from mouse, and its expression, when analyzed by in situ hybridization, appears to be developmentally regulated.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and cDNA Cloning-- Human retinal pigment epithelial cells (ARPE-19), a rapidly growing human RPE cell line, were grown at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin as described previously (12). Cells were grown on 100-mm culture dishes and treated with all-trans-retinoic acid when they were 60-80% confluent.

Total RNA was isolated from ARPE-19 cells using RNAzol B (Tel-Test, Inc., Friendswood, TX). Poly(A)+ RNA fractions were separated using oligo(dT)-cellulose and used for cDNA synthesis and library construction using a ZAP Express cDNA synthesis kit and ZAP Express cDNA Gigapack III Gold cloning kit (Stratagene, La Jolla, CA). Poly(A)+ RNA preparations from ARPE-19 cells or mouse testis were used for reverse transcriptase-PCR. The RNA preparations were reverse-transcribed with an oligo(dT) primer, and the first strand cDNA preparations were used as template for PCR. Rapid amplification of cDNA ends (RACE) was performed using 5'-and 3'-RACE systems obtained from Life Technologies, Inc. (14). Sequencing of cDNA clones and PCR amplification products was performed on an Applied Biosystems 370A DNA sequencer using the Taq dideoxy termination sequencing kit (PerkinElmer Life Sciences).

DNA and protein sequences were analyzed using the BLAST similarity search program supported by NCBI. Multiple sequence alignment was performed using the ClustalW program (15). The Paircoil program was employed for predicting coiled-coil regions (16). ExPASy proteomic tools were used for analyzing protein physicochemical properties.

Chromosomal Localization-- A human genomic P1 library was screened by PCR using primers 5'-TGACCCAGCTCAAACAAC and 5'-ACCATTGCATCAGTCATAGCAGC, designed from the NORPEG cDNA sequence (Genome Systems, Inc., St. Louis, MO). The identity of one of the P1 clones thus obtained was verified by PCR using different sets of primers and by sequencing some of the amplification products. Purified DNA from this clone was labeled with digoxigenin-dUTP by nick translation and used as a probe for in situ hybridization. The labeled probe was combined with sheared human DNA and hybridized to metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2× SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluorescein-conjugated anti-digoxigenin antibodies, followed by counterstaining with 4,6-diamidino-2-phenylindole.

Northern Blot Analysis-- RNA preparations were subjected to agarose gel electrophoresis in the presence of formaldehyde, transferred to a Nytran membrane (Schleicher & Schüll), and hybridized with a cDNA probe labeled with 32P (17). Hybridization was carried out using QuickHyb hybridization solution (Stratagene) containing labeled probe (~2 × 106 cpm/ml) and denatured salmon sperm DNA (0.1 mg/ml) at 68 °C for 1 h. The blots were then washed under stringent conditions and exposed to Kodak XAR film or analyzed using a PhosphorImager (STORM 860, Molecular Dynamics, Inc., Sunnyvale, CA).

Peptide Antibodies-- Antibodies were produced against two synthetic peptides designed from the predicted sequences of the human and mouse NORPEG proteins. The first peptide (NORPEG-28, CENGDAEKVASLLGKKGAS) contained a cysteine residue at the N terminus in addition to amino acids 28-45 of the human or mouse NORPEG protein. The second peptide (NORPEG-961, DVQKVLKQILTMCKNQSQKK) contained amino acids 961-980 of the human or amino acids 960-979 of the mouse NORPEG protein. The peptides were synthesized, purified to >90% by high pressure liquid chromatography, conjugated to keyhole limpet hemocyanin, and used for immunization of rabbits (Bethyl Laboratories, Inc., Montgomery, TX). The antisera (anti-NORPEG-28 and anti-NORPEG-961) were purified using immunoaffinity columns prepared with peptides linked to agarose using cyanogen bromide. The affinity-purified antibody preparations were passed over keyhole limpet hemocyanin immunosorbent to remove keyhole limpet hemocyanin immunoreactivity.

Expression of FLAG Fusion Protein in COS-7 Cells-- Poly(A)+ RNA preparations isolated from ARPE-19 cells were reverse-transcribed with 5'-AGAGCTCTCCTGTGCTGA (corresponding to bp 3465 to 3448 of NORPEG cDNA) as a primer using Superscript II reverse transcriptase (Life Technologies, Inc.). The reaction mixture was treated with RNase, and an aliquot was subjected to PCR with high fidelity Pfu Turbo DNA polymerase (Stratagene) employing 5'-GTGGAGCAGCCAGCTGGGTC and 5'-CATCAGGACCAGACCTC as sense and antisense primers, respectively. The amplification product, corresponding to bp 16-3327 of NORPEG cDNA, was cloned into the pCR-Blunt vector (Invitrogen, Carlsbad, CA). This plasmid DNA was used as a template for PCR with primers 5'-cttgcggccgcCATGAAGAGCTTGAAAGCG and 5'-agctggatccCTTCTTTTGAGACTGGTTTT. These primers contained the restriction enzyme sites (underlined) NotI and BamHI, respectively (extra nucleotides added to create restriction sites are shown in lowercase letters). Pfu Turbo DNA polymerase was used as the enzyme. The PCR product, containing bp 111-3051 of NORPEG cDNA, was digested with restriction enzymes and cloned into the NotI and BamHI sites of the pFLAG-CMV-2 expression vector (Sigma). The fusion protein containing the FLAG epitope at the N terminus was transiently expressed in COS-7 cells (CRL-1651, American Type Culture Collection). The cells were transfected with plasmid DNA using LipofectAMINE reagent (Life Technologies, Inc.) and allowed to grow for 72 h.

The extracts prepared from transfected cells were analyzed for expressed FLAG fusion protein by Western blotting using anti-FLAG M2 monoclonal antibody (Sigma) or antibody preparations against NORPEG. SDS-polyacrylamide gel electrophoresis was carried out using NUPAGE 4-12% BisTris gels and MOPS/SDS running buffer (Novex, San Diego, CA), and then the protein bands from the gels were electroblotted onto nitrocellulose membranes. The WesternBreeze chromogenic immunodetection system (Novex), which utilizes alkaline phosphatase-conjugated secondary antibody, was used for detecting immunoreactivity.

The expression of FLAG fusion protein was also analyzed by immunofluorescence microscopy. COS-7 cells were grown to 60-80% confluence on 8-well glass chamber slides (Nalge Nunc, Naperville, IL) and transfected with the NORPEG construct using LipofectAMINE reagent as described above. The cells were then allowed to grow for 48 h before fixing for 5 min in 1:1 methanol/acetone mixture precooled to -20 °C. The slides were incubated in the presence of anti-FLAG M2 or anti-NORPEG-28 antibody for 1 h at 37 °C. Immunoreactivity was detected using the appropriate fluorescein-conjugated secondary antibody.

Laser Scanning Confocal Microscopic Analysis of the NORPEG Protein in ARPE-19 Cells-- The ARPE-19 cells were grown on Nunc 8-well chamber slides coated with laminin and were allowed to differentiate for at least 4 weeks (12). The cells were fixed with ice-cold methanol and blocked with blocking solution (Novex) for 1 h at room temperature. Cells were incubated with anti-NORPEG-28 antibody preparations for 3 h in a humidified chamber at room temperature and then were washed in antibody wash solution (Novex). Incubation with 0.1% pre-bleed serum or with buffer only served as a negative control. After rinsing, all samples were incubated overnight at 4 °C with fluorescein-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a dilution of 1:100. Cells were optically sectioned (z series) using a Nikon Diaphot 200 laser scanning confocal imaging system (Molecular Dynamics, Inc.). Images were analyzed using the Image Display 3.2 software package (Silicon Graphics, Mountain View, CA).

Immunohistochemistry-- To examine the expression of the NORPEG protein, testes and eyes from 5-6-week-old albino (ICR) mice were enucleated, frozen immediately in Tissue-Tek OCT (Miles Laboratories, Elkhart, IN), and sectioned at 10-µm thickness. Immediately before the experiment, sections were fixed in ice-cold acetone for 5 min. Endogenous peroxidase activity was quenched by incubating the sections for 15 min with the peroxidase solution. Sections were washed in phosphate-buffered saline and blocked for 1 h with blocking solution. Sections were incubated with anti-NORPEG-28 or anti-NORPEG-961 antibody preparations. Immunoreactivity was detected by peroxidase staining using reagents from Dako Corp. (Carpinteria, CA).

In Situ Hybridization-- In situ hybridization was performed by Phylogeny Inc. following the procedure of Lyons et al. (18) as described before (19). The cRNA probes were prepared from linearized cDNA templates (bp 2232-2619 region of mouse Norpeg cDNA cloned into the pBluescript II KS+ vector) using an in vitro transcription kit (Ambion Inc.) and 35S-UTP (Amersham Pharmacia Biotech) and subjected to alkali hydrolysis to give a mean size of 70 bases. Hybridization was carried out at 52 °C in a solution containing 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM NaPO4, dextran sulfate, 1× Denhardt's solution, 50 µg/ml total yeast RNA, and ~60,000 cpm/µl 35S-labeled cRNA probes (antisense or sense). The sections were then subjected to stringent washing at 65 °C in 50% formamide, 2× SSC, and 10 mM dithiothreitol. Following washing and RNase A treatment, the slides were dehydrated, dipped in Kodak NTB-2 nuclear track emulsion, and exposed for 2-3 weeks in light-tight boxes with desiccant at 4 °C. Photographic development was carried out using Kodak D-19. The slides were counterstained lightly with toluidine blue and analyzed using both the light- and dark-field optics of a Ziess Axiophot microscope.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification and Characterization of NORPEG cDNA from Human and Mouse-- A cDNA library was constructed using poly(A)+ RNA fractions isolated from the human RPE cell line ARPE-19. During the characterization of this library, we found a cDNA clone whose sequence did not show any similarity to published sequences at the time of its isolation. The complete cDNA sequence for this novel gene was obtained using 5'- and 3'-RACE techniques and by reverse transcriptase-PCR. The 4925-bp cDNA sequence for the gene, which we termed NORPEG (novel retinal pigment epithelial cell gene; GenBankTM/EBI accession number AF155135), shows an open reading frame with an initiation codon at position 112 and a stop codon (TAA) at position 3052, thus leaving a long 3'-untranslated region. It should be noted that a cDNA clone (mRNA for the KIAA1334 protein; GenBankTM/EBI accession number AB037755) as well as a partial cDNA clone (DKFZp564G013; GenBankTM/EBI accession number AL050011) matching the sequence reported here recently appeared in the data base.

We have also obtained the cDNA sequence for an ortholog of NORPEG that is expressed in mouse testis employing reverse transcriptase-PCR and RACE techniques. The mouse Norpeg cDNA (GenBankTM/EBI accession number AF274866) is 4879 bp long and contains an open reading frame with an initiation codon at bp 221 and a stop codon (TAA) at position 3158. The mouse Norpeg cDNA showed 81% sequence identity to the human NORPEG cDNA.

The predicted human and mouse NORPEG proteins contain 980 and 979 amino acid residues, respectively (Fig. 1A). They show 84% identity and 91% similarity. The human NORPEG protein is predicted to have a molecular mass of 110,042 Da, a pI of 5.83, and six potential N-glycosylation sites. In comparison, the mouse homolog is predicted to have a molecular mass of 108,852 Da, a pI of 5.88, and four potential N-glycosylation sites. The domain organization of the NORPEG protein is shown in Fig. 1B. Using BLAST analysis, the N-terminal regions of both human and mouse NORPEG protein sequences were found to be similar to several ankyrin repeat-containing proteins. Six ankyrin repeats spanning amino acids 18-240 were identified by ProfileScan for the human NORPEG protein. Alignment of the six repeats with the consensus sequence for the ankyrin motif is shown in Fig. 1C. In mouse, the six ankyrin repeats spanned amino acids 20-242. BLAST analysis also showed that a large segment of both the human and mouse NORPEG proteins (amino acids 400-900) shares similarity with coiled-coil helical domains of several proteins, including restin (Reed-Steinberg cell-expressed intermediate filament-associated protein), myosin heavy chain, and golgin-245 (20-22). Further analysis indicated 11 coiled-coils between amino acids 341 and 947 for the human NORPEG protein (Fig. 1D). In comparison, the mouse NORPEG protein contains 10 coiled-coils between amino acids 341 and 946 (data not shown).



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Fig. 1.   Predicted amino acid sequences and structural characteristics of human and mouse NORPEG proteins. A, predicted amino acid sequences for human and mouse NORPEG proteins. Identical amino acid residues are indicated by asterisks, and evolutionarily conserved amino acid residues are indicated by colons and periods. B, domain organization of the NORPEG protein. aa, amino acids. C, alignment of six ankyrin repeats of the human NORPEG protein with the ankyrin (ANK) repeat consensus sequence (the last sequence shown in the alignment; X is any amino acid residue). D, Paircoil analysis of the human NORPEG protein sequence shows 11 coiled-coils between amino acids 341 and 947.

Chromosomal Localization and Gene Structure of Human NORPEG-- The chromosomal localization of the human NORPEG gene was determined by PCR screening of a chromosome mapping panel (NIGMS Human Genetic Mutant Cell Repository at the Coriell Institute for Medical Research, Camden, NJ) with oligonucleotides 5'-TGACCCAGCTCAAACAAC and 5'-ACCATTGCATCAGTCATAGCAGC. These primers spanned the NORPEG cDNA sequence region of bp 2249-2462. A 214-bp amplification product, whose identity was verified by DNA sequencing, was observed when a DNA preparation from a chromosome 5 monochromosomal somatic cell hybrid was used as the template. Further confirmation of the localization of the NORPEG gene to human chromosome 5 was obtained by fluorescence in situ hybridization. The DNA preparation obtained from a genomic P1 clone containing the NORPEG gene was used as the probe. The probe hybridized to the short arm of chromosome 5 (Fig. 2A). The localization was further verified by co-hybridizing this probe with a probe specific for the cri-du-chat locus, which has been previously been mapped to the long arm, 5q22 (Fig. 2B). Measurements of 10 hybridized chromosomes showed that the NORPEG gene is located 29% of the distance from the centromere to the telomere of chromosome arm 5p; this is an area corresponding to band 5p13.2-13.3 (Fig. 2C). Also in support of this localization, several genomic clones designated as chromosome 5 clones that appeared recently in the data base (high throughput genomic sequencing) were found to contain segments of DNA sequences of NORPEG cDNA. Comparison of the cDNA sequence of the NORPEG gene with genomic clone sequences (AC008950, AC016602, AC025269, AC025754, AC025769, AC026801, AC055815, and AC008690) allowed us to determine the exon/intron boundaries in this gene. As illustrated in Fig. 2D, the NORPEG gene contains 18 exons and spans >120 kb.



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Fig. 2.   Chromosomal localization and gene structure of human NORPEG. A, fluorescence in situ hybridization analysis using a NORPEG probe (indicated by arrows) showed that the gene is localized to human chromosome 5. B, co-hybridization of the NORPEG probe (indicated by arrows) and a probe specific for 5q22 (indicated by arrowheads) to chromosome 5. C, idiogram of chromosome 5 showing the subchromosomal localization of NORPEG to the 5p13.2-13.3 region (indicated by the arrowhead). D, scale model of the NORPEG gene structure. Exons are represented by rectangles; horizontal lines represent intron sequences. Introns >10 kb are broken by diagonal hatch marks. The size of intron 3, identified by a question mark, is unknown.

Expression of NORPEG mRNA in Human Tissues and Cells and Its Regulation by Retinoic Acid-- RNA preparations obtained from ARPE-19 cells were analyzed by Northern blotting using a cDNA probe for the NORPEG gene. As shown in Fig. 3A, a strong signal was observed at ~5 kb, with a weak signal at ~3 kb. Hybridization of a human tissue RNA blot with this probe showed that the gene is also highly expressed in placenta and testis (data not shown). Therefore, RNA preparations from these two sources were also subjected to Northern blot analysis (Fig. 3A). Like the ARPE-19 cells, placenta showed a highly intense signal at ~5 kb, with a weak signal at ~3 kb. Interestingly, a reverse trend was observed in testis; the intense signal was at ~3 kb, and the weak signal was at ~5 kb. These two distinct mRNA species were subjected to further analysis. Blots containing RNA preparations from ARPE-19 cells and testis were analyzed with probes representing various segments of the NORPEG cDNA. Several probes generated from bp 1-3000 of the cDNA hybridized with both the ~5- and ~3-kb messages, whereas those generated from the bp 3124-4774 region hybridized only with the ~5-kb message. As shown in Fig. 3B, a probe representing the bp 2559-3000 region hybridized with both the ~5- and ~3-kb messages, whereas a probe representing the bp 3124-3666 region hybridized only with the ~5-kb message. Thus, it appears that the ~3-kb message lacks most of the long 3'-untranslated region present in the ~5-kb message. NORPEG mRNA was also found to be highly expressed in several human cancer cell lines (Fig. 3C).



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Fig. 3.   Expression of human NORPEG mRNA and its induction by all-trans-retinoic acid. A, Northern blot analysis showed the presence of two NORPEG transcripts ~3 and ~5 kb in size. The ~3-kb message is abundant in testis (lane T), whereas the ~5-kb message is abundant in placenta (lane P) and ARPE-19 cells (lane A). Left and right panels are x-ray films of the same blot exposed for 3 and 16 h, respectively. B, the ~3-kb message lacks the long 3'-untranslated region found in the ~5-kb message. A cDNA probe (left panel) made from the coding region of NORPEG (bp 2559-3000) hybridized with both the ~5- and ~3-kb mRNA species from the ARPE-19 cells (lane A) and testis (lane T); but a cDNA probe (right panel) made from the 3'-untranslated region (bp 3124-3666) hybridized only with the ~5-kb mRNA. C, NORPEG mRNA, predominantly the ~5-kb form, was detected in several of the cancer cell lines analyzed: promyelocytic leukemia HL-60 (lane 1), HeLa cell S3 (lane 2), chronic myelogenous leukemia K-562 (lane 3), lymphoblastic leukemia MOLT-4 (lane 4), Burkitt's lymphoma Raji (lane 5), colorectal adenocarcinoma SW480 (lane 6), lung carcinoma A549 (lane 7), and melanoma G361 (lane 8). D, NORPEG mRNA expression in ARPE-19 cells was increased following all-trans-retinoic acid treatment. Total RNA preparations obtained from ARPE-19 cells treated for 48 h with various concentrations of all-trans-retinoic acid were analyzed by Northern blotting. The upper panel shows a blot probed with a NORPEG cDNA probe, whereas the lower panel shows 18 S and 28 S rRNA bands on an ethidium bromide-stained gel. The chart shows the relative amount of radioactivity associated with bands on the Northern blot as analyzed with a PhosphorImager.

NORPEG mRNA expression in ARPE-19 cells was induced by all-trans-retinoic acid (Fig. 3D). The ~5-kb transcript showed a concentration-dependent increase when the cells were treated with retinoic acid for 48 h. A >5-fold induction of this transcript was detected in the presence of 1 µM all-trans-retinoic acid. The ~3-kb transcript, due to its low abundance in ARPE-19 cells, is not quite visible in the autoradiogram presented. This is especially true in the case of untreated cells. However, signal for the shorter transcript is clearly noticeable in lanes representing RNA samples from cells treated with >= 0.5 µM retinoic acid. Thus, it appears that the ~3-kb transcript is also induced by this treatment.

Expression of the Human NORPEG Protein in COS-7 Cells as a FLAG Fusion Protein-- The entire coding region of the human NORPEG cDNA was cloned into an N-terminal FLAG expression vector, and the FLAG fusion protein was transiently expressed in COS-7 cells. Immunofluorescence microscopic analysis of these cells using an anti-FLAG monoclonal antibody showed intense immunoreactivity in the cytoplasm (Fig. 4). A similar staining pattern was also observed when anti-NORPEG-28 antibody was used as the primary antibody.



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Fig. 4.   Expression of the human NORPEG protein in COS-7 cells as a FLAG fusion protein. The open reading frame of the human NORPEG cDNA was cloned into the pFLAG-CMV-2 expression vector. COS-7 cells were transfected with the plasmid and analyzed by immunofluorescence microscopy. The cells were fixed and treated with the indicated primary antibody and then with a fluorescein-conjugated secondary antibody. A, transfected cells treated with anti-FLAG M2 monoclonal antibody (magnification × 100). B, transfected cells treated with anti-FLAG M2 monoclonal antibody (magnification × 400). C, transfected cells treated with anti-NORPEG-28 antibody (magnification × 400). D, control cells treated with anti-FLAG M2 monoclonal antibody (magnification × 100). COS-7 cells transfected with the plasmid were also analyzed by Western blotting. E, blot probed with anti-FLAG M2 monoclonal antibody showing that the expressed protein was not solubilized by Triton X-100 and high concentrations of KCl. Cells were separated from the culture medium (lane 1) and then homogenized in Tris-buffered saline. The homogenate was centrifuged, and the supernatant (lane 2) was separated from the pellet (lane 3). Supernatants were also obtained by sequential extraction of this pellet with the following solutions: Tris-buffered saline (lane 4) and Tris-buffered saline containing 0.5% Triton X-100 (lane 5), 1% Triton X-100 (lane 6), and 1.5 M KCl plus 0.5% Triton X-100 (lane 7). The resulting final pellet was also analyzed (lane 8). Molecular mass markers are shown in lane M. F, blot probed with an anti-NORPEG-28 antibody preparation. Lane M, molecular mass markers; lanes 1 and 2, pellet and supernatant from detergent extraction, respectively. G, blot probed with an anti-NORPEG-961 antibody preparation. Lane M, molecular mass markers; lanes 1 and 2, pellet and supernatant from detergent extraction, respectively.

The expressed NORPEG protein was also detected on a Western blot as an ~110-kDa band using an anti-FLAG monoclonal antibody preparation (Fig. 4E). The immunoreactivity was associated with the pellet fraction when the cell homogenate was centrifuged. Treatment of the resulting pellet with Triton X-100 and 1.5 M KCl did not result in the solubilization of the ~110-kDa protein. The expressed protein was analyzed using antibody preparations against peptide sequences from the N-terminal (amino acids 28-45) as well as C-terminal (amino acids 961-980) regions of the NORPEG protein. Both of these antibody preparations (anti-NORPEG-28 and anti-NORPEG-961) were found to recognize the ~110-kDa band (Fig. 4, F and G). The preimmune serum, as expected, did not show any reactivity toward this band (data not shown).

Analysis of the NORPEG Protein in Differentiated Human RPE Cells-- Differentiated ARPE-19 cells are known to exhibit polarized distribution of certain proteins (10, 12). Therefore, the distribution of the NORPEG protein was analyzed in these cells. ARPE-19 cells were allowed to differentiate and then immunostained using anti-NORPEG-28 antibody and fluorescein-conjugated secondary antibody. The immunostained cells were subjected to laser scanning confocal microscopic analysis. The cells were optically sectioned in a horizontal and vertical plane. The distribution of immunoreactivity was throughout the cytoplasm of the ARPE-19 cells (Fig. 5). The x-y scans (horizontal optical sections) revealed a threadlike distribution of the NORPEG protein throughout the cytoplasm. The x-z scans (vertical optical sections) showed distribution throughout the cells, indicating that expression is not limited to the apical or basal membranes of the cells. Again, the vertical scans of the cells revealed extensive expression throughout the cytoplasm. In experiments using both antibodies, the pre-bleed controls showed minimal labeling of the cells.



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Fig. 5.   Analysis of the NORPEG protein in ARPE-19 cells by laser scanning confocal microscopy. The NORPEG protein in ARPE-19 cells was detected with anti-NORPEG-28 antibody and fluorescein-conjugated secondary antibody. A, horizontal (x-y) scan; B, vertical (x-z) scan; C, horizontal (x-y) scan of the pre-bleed control.

Analysis of Norpeg mRNA Expression in Mouse during Development-- Norpeg mRNA expression was analyzed in mouse embryo by in situ hybridization at various stages of development. At 9.5 days, expression (as indicated by a high level of silver grains) was detected in branchial arch mesenchyme, forebrain, hindbrain, midbrain, and neural tube (Fig. 6). At 12.5 days, a hybridization signal was detected in the hindbrain, forebrain, lung, genital eminence, spinal ligaments around vertebrae and ribs, and around the cartilage of ribs and nasal sinuses (Fig. 7). Signal was also detected in frontonasal mass, mandibular arch, optic sulcus, spinal ganglia and hind limb bud (data not shown). At 15.5 days, expression was detected in the ventricular layer of neurons subjacent to the neocortex, around the nasal sinuses, bronchioles of the lung, kidney, and around the vertebrae of the tail (Fig. 8). Signal was also seen in the olfactory bulb (data not shown).



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Fig. 6.   In situ hybridization analysis of Norpeg mRNA expression in 9.5-day mouse embryo. Dark-field micrographs are shown. Sections probed with sense cRNA (A and B) showing low background levels of silver grains served as controls. Antisense cRNA (C, sagittal section; D, oblique sagital section of two embryos) detected Norpeg mRNA expression, as indicated by high levels of silver grains, in branchial arch mesenchyme (ba), forebrain (fb), hindbrain (hb), midbrain (mb), and neural tube (nt). d, decidua; h, heart; ys, yolk sac.



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Fig. 7.   In situ hybridization analysis of Norpeg mRNA expression in 12.5-day mouse embryo. The control sense cRNA probe (A and B) showed a low background. Antisense cRNA (C and D) detected Norpeg mRNA expression in the hindbrain (hb), forebrain (fb), lung (lu), and genital eminence (ge). Expression was also detected around the cartilage of the ribs (r) and nasal sinuses (ns) as well as in areas that appear to be spinal ligaments around the vertebrae (v) and ribs (r). h, heart; li, liver; to, tongue; si, small intestine; sc, spinal cord.



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Fig. 8.   In situ hybridization analysis of Norpeg mRNA expression in 15.5-day mouse embryo. The sense cRNA probe (A-C) showed a low background. The antisense cRNA probe (D-F) detected Norpeg mRNA expression in the ventricular layer of neurons subjacent to the neocortex (cx), around the nasal sinuses (ns), in bronchioles of the lung (lu), in kidney (k), in genital eminence (ge), and around the vertebrae of the tail (t). hb, hindbrain; mb, midbrain; h, heart; j, jaw; r, rib; bl, bladder; oc, occipital cartilage; th, thymus; sc, spinal cord; to, tongue; li, liver; v, vertebrae; si, small intestine.

Analysis of a mouse tissue RNA blot with a Norpeg cDNA probe showed that the Norpeg mRNA expression was very high in testis compared with all other tissues (data not shown). Therefore, we analyzed the expression of Norpeg mRNA in adult mouse testis by in situ hybridization (Fig. 9). The mRNA expression was very high in seminiferous tubules that contain differentiating sperm.



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Fig. 9.   In situ hybridization analysis of Norpeg mRNA expression in adult mouse testis. Antisense cRNA (A) showed high level expression of Norpeg mRNA in seminiferous tubules (st) that contain differentiating sperm. ta, tunica albuginea; epi, epididymis. The sense cRNA control probe (B) showed a low background level of silver grains as expected.

Immunohistochemical Analysis of the NORPEG Protein in Mouse Testis and Retina-- Mouse testis was subjected to immunohistochemical analysis using anti-NORPEG-28 antibody. Fig. 10A (Testis) shows a hematoxylin- and eosin-stained cross-section of mouse testis. The arrow points to one of several seminiferous tubules. Each tubule is surrounded by a lamina propria; and immediately internal to this, the tubule is lined with the supporting cells (of Sertoli). Fig. 10B (Testis) shows a similar section of testis subjected to immunohistochemistry. The supporting cells are intensely positive, as indicated by the reddish-brown precipitate. Fig. 10C (Testis) shows a section incubated with the pre-bleed control, where no positive reaction was observed.



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Fig. 10.   Immunohistochemical analysis of NORPEG expression in mouse testis and retina. Sections of testis and retina were immunostained using anti-NORPEG-28 antibody and peroxidase-conjugated secondary antibody. TESTIS: A, hematoxylin- and eosin-stained cross-section shows one of several seminiferous tubules (arrow). Each tubule is surrounded by a lamina propria; and immediately internal to this, the tubule is lined with the supporting cells (of Sertoli). B, section stained with the antibody shows that supporting cells are intensely positive as indicated by the reddish-brown color. C, no staining was observed for the section incubated with the pre-bleed control. RETINA: A, section stained with hematoxylin and eosin for comparison. rgc, retinal ganglion cells; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; is, inner segment; os, outer segment; rpe, retinal pigment epithelium. B, section stained with the antibody shows an intense positive reaction in the ganglion cell layer, in Müller cell fibers spanning the inner portion of the retina. The outer plexiform layer shows an intense positive reaction, as does the RPE. C, a higher magnification of the inner portion of the retina shows the intense positive reaction in ganglion cells and Müller cell fibers. D, a high magnification shows the positive reaction in the RPE. The photoreceptor cells of the outer nuclear layer are negative.

Fig. 10 also shows the results from the immunohistochemical analysis of mouse retina. Fig. 10A (Retina) shows a section stained with hematoxylin and eosin for comparison with the other panels of retina subjected to immunohistochemistry procedures (B-D, Retina). As shown in Fig. 10B (Retina), there is an intense positive reaction for the NORPEG protein in the ganglion cell layer and in Müller cell fibers spanning the inner portion of the retina. The outer plexiform layer shows an intense positive reaction, as does the RPE. Fig. 10C (Retina) is a high magnification of the inner portion of the retina, showing the intense positive reaction in ganglion cells and vertically oriented Müller cell fibers. Fig. 10D (Retina) is a high magnification demonstrating the positive reaction in the RPE. The photoreceptor cells of the outer nuclear layer are negative for NORPEG. There was also expression in the optic nerve (data not shown). The pre-bleed controls showed no labeling (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study has resulted in the identification and characterization of NORPEG, a novel gene localized to human chromosome 5, as well as an ortholog of this gene from mouse. The proteins encoded by human and mouse genes contain 980 and 979 amino acid residues, respectively, and share 91% sequence similarity (84% identity) between them. These proteins appear to represent a new gene family since they did not show any close similarity to published protein sequences in the data base. The closest match observed was 45% similarity (28% identity) between the N-terminal region of the NORPEG protein and the protein sequence encoded by an mRNA that is overexpressed in dog thyroid tissue following thyrotropin stimulation (GenBankTM/EBI accession number X99145).

The human and mouse NORPEG proteins contain identical structural domains. The C terminus of the NORPEG protein contains a large coiled-coil domain covering ~60% of the protein. This region shows similarity to coiled-coil regions of several other proteins such as restin, myosin heavy chain, and golgin-245 and could be involved in self-aggregation or interaction with other proteins (20-22). The second structural domain seen in the NORPEG protein (both human and mouse) is an ankyrin repeat region. It contains six ankyrin repeats, each composed of 33 amino acid residues, toward the N terminus. Ankyrin repeats are known to be present in a large number of proteins and are involved in protein-protein interactions (23). As many as 24 of these repeats are present in ankyrin, the protein that anchors the cytoskeleton to erythrocyte membranes (23, 24). Ikappa B, the inhibitor of nuclear factor-kappa B, and the GA-binding protein beta  (GABPbeta ), a transcription factor, contain five and four ankyrin repeats, respectively (25, 26). The presence of ankyrin repeats and the coiled-coil domain in the NORPEG protein indicates a potential for interaction with other cellular proteins.

The open reading frame of the human NORPEG cDNA was expressed as a FLAG fusion protein. It was recognized by an anti-FLAG monoclonal antibody and found to have the expected molecular mass of ~110 kDa. The fusion protein was identified as that of NORPEG based on its immunoreactivity toward antibodies raised against peptides designed from the N- and C-terminal regions of the predicted sequence. Immunofluorescence microscopic analysis indicated that the fusion protein is localized to the cytoplasm. The NORPEG protein appears to be a cytoskeletal protein based on the threadlike staining in the cytoplasm observed by laser scanning confocal microscopic analysis of ARPE-19 cells. The resistance of the expressed NORPEG protein to solubilization by Triton X-100 and 1.5 M KCl is also in support of this conclusion (27).

The functional role of the NORPEG protein remains to be elucidated. Nevertheless, the expression pattern of this gene could offer an insight into its possible function. The RPE, testis, and placenta are three sites where the highest level of NORPEG expression is observed. In the eye, RPE cells are joined by tight junctions in the region of the apical membrane. The RPE tight junctions form a portion of the blood-retinal barrier (1). In testis, Norpeg is expressed in seminiferous tubules, and the expression appears to be localized to Sertoli cells. These cells, joined at their bases by tight junctions to create a permeability barrier between the extratubular and intratubular compartments, are part of the blood-testis barrier (28). The cell type in placenta that expresses NORPEG is not yet identified. But, syncytiotrophoblast cells in this organ constitute the blood-placental barrier (29, 30). Thus, the high level of NORPEG expression in the RPE, testis, and placenta would indicate that the putative cytoskeletal protein encoded by this gene could be involved in some type of barrier function. A second feature that the RPE, testis, and placenta have in common is retinoid metabolism and transport (3, 31, 32). Therefore, it is possible that the NORPEG protein may play a key role in this process. An explanation of the developmental expression pattern of NORPEG in neuronal tissues as well as in prenatal kidney and lung will require further functional characterization of the encoded protein.

Although NORPEG is mainly expressed as an ~5-kb transcript in many tissues and cells analyzed, an ~3-kb transcript, the major form found in testis, was also detected. Both the ~5- and ~3-kb transcripts were also detected for mouse Norpeg (data not shown). The ~5-kb transcript appears to represent the cDNA sequence reported here for NORPEG. The ~3-kb transcript failed to hybridize to the cDNA probes representing the ~2-kb-long 3'-untranslated region of the NORPEG cDNA sequence. Thus, it appears that the difference in length of the 3'-untranslated regions in these two transcripts is most likely due to the use of alternative polyadenylation signals. Sequences similar but not identical to the canonical AATAAA sequence are found at several positions within the 3'-untranslated region. A large number of genes are known to generate two or more transcripts due to the presence of multiple polyadenylation sites within a 3'-terminal exon (33, 34). This process results in the formation of multiple transcripts differing in the length of their 3'-untranslated regions. These transcripts generally exhibit altered stability, translatability, and tissue-specific expression. This is due to the fact that the 3'-untranslated region is known to harbor various cis-regulatory elements controlling these parameters. The 3'-untranslated region of the ~5-kb NORPEG transcript contains five AUUUA repeats, a regulatory element implicated in mRNA instability (35). Thus, cis-regulatory elements similar to this present in the 3'-untranslated region could be responsible for the observed tissue-specific expression of the ~5-kb transcript.

The ~3- and ~5-kb transcripts of NORPEG could also be generated by a different mechanism. Several genes are known to yield multiple transcripts by alternative 3'-terminal exon usage (33). In the case of the ~5-kb transcript of NORPEG, the 3'-terminal exon consists of the last 81 bases of the coding region plus the whole 3'-untranslated region. This long exon could be replaced by an alternative short exon(s) to form the ~3-kb transcript. The results obtained by Northern blot analysis did not eliminate this type of alternative splicing. The resulting shorter transcript will encode a variant of the NORPEG protein with an altered C terminus in comparison with the normal NORPEG protein encoded by the larger transcript. Although there is no evidence for alternative splicing according to expressed sequence tags found in the GenBankTM/EBI Data Bank, further investigation is needed to rule out this possibility.

The reason for the preferential expression of the ~3-kb transcript of NORPEG in testis is not known. However, testis-specific expression of transcripts with shortened 3'-untranslated regions has been reported for several genes containing multiple polyadenylation signals (36-38). The shorter transcripts are comparatively more stable since the truncation of the 3'-untranslated region effectively eliminates various elements contributing to the mRNA instability. The selection of stable transcripts is thought to be necessary to compensate for the minimal transcription occurring during later stages of spermatogenesis (38).

Interestingly, the expression of NORPEG mRNA in ARPE-19 cells is induced by all-trans-retinoic acid. The treatment resulted in the increased expression of both the ~3- and ~5-kb transcripts, indicating that the retinoid effect is at the transcriptional level. A mechanism involving the transcript stabilization mediated through the 3'-untranslated region should have resulted in the preferential increase in the longer transcript. The observed effect is similar to the transcriptional regulation reported for fibroblast growth factor (int-2) transcripts in F9 cells, in which the induction by retinoic acid is not dependent on the length of the 3'-untranslated region (39). Retinoic acid is a key regulator of many biological functions, including cell differentiation, proliferation, and development and it regulates the transcription of a number of genes by its ability to bind specific nuclear receptors, i.e. retinoic acid receptors and retinoid X receptors (40-43). These receptors mediate their effect by binding to specific DNA sequences (retinoic acid response elements and retinoid X response elements) on the target genes. It remains to be elucidated whether the observed increase in NORPEG mRNA in response to retinoic acid treatment is a direct effect mediated through the retinoid receptors interacting with retinoic acid response elements or retinoid X response elements, possibly present in the regulatory region of the NORPEG gene. Studies are currently underway to clone and characterize the promoter region of the human NORPEG gene. ARPE-19 cells do respond to all-trans-retinoic acid by increasing the expression of retinoic acid receptor-alpha and retinoid X receptor-alpha (data not shown). Also, it has been reported that retinoic acid delays the expression of RPE-specific genes in ARPE-19 cells (44).

In situ hybridization analysis of Norpeg mRNA in developing mouse embryo (9.5-15.5 days gestation) showed that it is highly expressed in nervous tissue that appears to be less differentiated, in dividing neurons, in mesenchyme surrounding developing cartilage, in spinal ligaments, and in prenatal kidney and lung. The presence of the Norpeg message in the early stages of embryonic development and its expression pattern during development suggest that this gene may play an important developmental role. Also, the fact that Norpeg expression is apparently regulated by retinoic acid is in support of this role since retinoic acid is a key player in early vertebrate development (45-47). In adult mouse, Norpeg mRNA expression remains high in testis. Interestingly, the expression is localized to seminiferous tubules that contain differentiating sperm. Retinoic acid is known to play an important role in spermatogenesis (48, 49), and since Norpeg mRNA is up-regulated by retinoic acid, this gene could be a potential target for retinoic acid in testis.

Expression of the NORPEG protein was detected in mouse retina in the RPE, Müller cells, and ganglion cells by immunocytochemistry. It will be of interest to determine whether this expression pattern is related to the regulation of the Norpeg gene by retinoic acid. Retinoic acid is produced in neural retina and the RPE in the developing mouse eye, and the RPE is the principal site of retinoic acid synthesis in postnatal and adult mouse eyes (50). Cellular retinoic acid-binding protein I has been detected in early differentiating ganglion cells (51). A retina-specific nuclear receptor, which is thought to regulate the transcription of the cellular retinaldehyde-binding protein gene through possible interaction with the retinoic acid receptor and the retinoid X receptor, is reported to be highly expressed in the RPE and Müller glial cells (52).

In summary, we have identified a novel gene, NORPEG, localized to chromosome 5 and expressed in cultured human RPE cells, where its expression is induced by all-trans-retinoic acid. This gene is also highly expressed in human placenta, testis, and several cancer cell lines. Two transcripts were detected for this gene. An ortholog of NORPEG expressed in mouse was also characterized, and its expression appears to be developmentally regulated. The NORPEG protein contains an ankyrin repeat domain as well as a long coiled-coil domain and appears to be associated with the cytoskeleton. The precise role that this retinoic acid-responsive and developmentally regulated novel gene plays in cell structure and function remains to be elucidated.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Cynthia Jaworski for analyzing the gene structure and valuable suggestions and to Dr. Paul Russell for valuable suggestions and critical reading of the manuscript.


    Note Added in Proof

While this article was in press, Peng et al. (Peng, Y., Mandai, K., Sakisaka, T., Okabe, N., Yamamoto, Y., Yokoyama, S., Mizoguchi, A., Shiozaki, H., Monden, M., and Takai, Y. (2000) Genes Cells, in press) independently characterized ankycorbin, a novel actin cytoskeleton-associated protein whose sequence is identical to that reported in this paper for mouse Norpeg protein.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The 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) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF155135 and AF274866.

** Supported by Grant NIH-EY 13089 from the National Institutes of Health and by Research to Prevent Blindness.

§ To whom correspondence and reprint requests should be addressed: LRCMB, Rm. 338, National Eye Institute, NIH, 6 Center Dr., Bethesda, MD 20892-2740. Tel.: 301-496-5809; Fax: 301-402-1883; E-mail: krishnan@helix.nih.gov.

Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007421200


    ABBREVIATIONS

The abbreviations used are: RPE, retinal pigment epithelium; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; NORPEG, novel retinal pigment epithelial cell gene; bp, base pair(s); BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase(s).


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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