Evolutionarily Conserved ELOVL4 Gene Expression in the Vertebrate Retina

Pamela S. Lagali,1 Jiafan Liu,2 Rajesh Ambasudhan,2 Laura E. Kakuk,2 Steven L. Bernstein,3 Gail M. Seigel,4 Paul W. Wong,1,5 and Radha Ayyagari2,5

PURPOSE. The gene elongation of very long chain fatty acids-4 plays a critical role in retinal cell biology. (Invest Ophthalmol (ELOVL4) has been shown to underlie phenotypically hetero- Vis Sci. 2003;44:2841–2850) DOI:10.1167/iovs.02-0991 geneous forms of autosomal dominant macular degeneration. In this study, the extent of evolutionary conservation and the targardt macular dystrophies are inherited disorders of the existence and localization of retinal expression of this gene Sretina that are characterized by impaired central vision, was investigated across a wide variety of species. cellular atrophy of retinal and retinal pigment epithelial (RPE) METHODS. Southern blot analysis of genomic DNA and bioinfor- cells initiating in the macular region, and the presence of matic analysis using the human ELOVL4 cDNA and protein yellow fundus flecks surrounding the atrophic area resulting sequences, respectively, were performed to identify species in from the subretinal accumulation of lipofuscin-like material.1–4 which ELOVL4 orthologues and/or homologues are present. Both autosomal recessive and autosomal dominant forms of Retinal RNA and protein extracts derived from different spe- Stargardt’s disease (STGD) have been described.5,6 cies were assessed by Northern hybridization and immunoblot Classical or recessive Stargardt’s disease (STGD1; OMIM techniques to assess evolutionary conservation of gene expres- 248200; http://www.ncbi.nlm.nih.gov/omim, provided by the sion. Immunohistochemical analysis of tissue sections pre- National Institutes of Health, Bethesda, MD) is the most com- pared from various mammalian retinas was performed to de- mon form of hereditary macular dystrophy, occurring at an termine the distribution of ELOVL4 and homologous proteins incidence of approximately 1 in 10,000 live births, and ac- within specific retinal cell layers. counts for roughly 7% of all retinal dystrophies.7 A distinguish- RESULTS. The existence of ELOVL4 sequence orthologues and ing clinical feature of the disease is the dark or silent choroid homologues was confirmed by both Southern blot analysis and observed on fluorescein angiography of the fundus, resulting in silico searches of protein sequence databases. Phylogenetic from the accumulation of lipofuscin. Mutations in the ABCA4 (ABCR) gene on human chromosome 1p32 have been found to analysis places ELOVL4 among a large family of known and 8,9 putative fatty acid elongase proteins. Northern blot analysis underlie STGD1. This gene encodes a retina-specific adeno- revealed the presence of multiple transcripts corresponding to sine triphosphate–binding cassette transport protein found in ELOVL4 homologues expressed in the retina of several differ- photoreceptor cell outer segment disc membranes and func- ent mammalian species. Conserved proteins were also de- tions as an outwardly directed flippase to facilitate the shuttling tected among retinal extracts of different mammals and were of N-retinylidene-phosphatidylethanolamine from the interior to the exterior of the outer segments for subsequent retinoid found to localize predominantly to the photoreceptor cell layer 10,11 within retinal tissue preparations. recycling by RPE cells after phototransduction. ABCR mutations have also been implicated in the etiology of a variety of CONCLUSIONS. The ELOVL4 gene is highly conserved throughout other retinal diseases, including retinitis pigmentosa, cone–rod evolution and is expressed in the photoreceptor cells of the dystrophy, and age-related macular degeneration (AMD).9,12,13 retina in a variety of different species, which suggests that it Cases of autosomal dominant Stargardt’s macular dystrophy (adSTGD) are comparatively rare, and a number of genetic loci for diseases with typical adSTGD traits have been mapped, 14,15 1 including STGD3 and adMD (OMIM 600110) on 6q14 and From the Department of Biological Sciences, University of Al- 16 berta, Edmonton, Alberta, Canada; 2Department of Ophthalmology and STGD4 (OMIM 603786) on 4p. Forms of adSTGD are clini- Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, Ann cally quite similar to the phenotype characteristic of recessive Arbor, Michigan; the 3Department of Ophthalmology, University of Stargardt’s disease; however, there are a number of key differ- Maryland, Baltimore, Maryland; and the 4Department of Ophthalmol- entiating features. The most notable differences between ogy, State University of New York at Buffalo, Buffalo, New York. STGD1 and STGD3/adMD-associated phenotypes in particular 5Contributed equally to the work and therefore should be consid- are the later and more variable age of onset and the absence of ered equivalent authors. the dark choroid observed in fluorescein angiograms of pa- Supported by grants from the Foundation Fighting Blindness- tients with dominant STGD.15,17,18 Canada (PWW), the Foundation Fighting Blindness (RA), Research to Prevent Blindness (RA), research Grant EY13198 (RA) and core Grant Using a positional candidate approach, we and others re- EY07003 from the National Institutes of Health, a doctoral research cently identified the gene defect corresponding to the STGD3/ award from the Canadian Institutes of Health Research, and the E. A. adMD locus responsible for the disease phenotype in five 19 Baker Foundation of the Canadian National Institute for the Blind (PSL). related pedigrees throughout North America. The mutation, Submitted for publication September 26, 2002; revised January 10, a 5-bp deletion, lies in the elongation of very long chain fatty 2003; accepted February 5, 2003. acids-4 gene (ELOVL4), a novel member of a family of genes Disclosure: P.S. Lagali, None; J. Liu, None; R. Ambasudhan, that encode enzymatic components of the microsomal fatty None; L.E. Kakuk, None; S.L. Bernstein, None; G.M. Seigel, None; acid elongation system referred to as fatty acid elongases P.W. Wong, None; R. Ayyagari, None (ELOs). The ELOVL4 gene consists of six exons and encodes a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- 2.9-kb full-length mRNA transcript that in turn encodes a 314- amino-acid protein that contains the three typical features that ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 20,21 Corresponding author: Paul W. Wong, Department of Biological define a fatty acid elongase : five putative transmembrane Sciences, G-502 Biological Sciences Center, University of Alberta, Ed- segments, a single HXXHH histidine box redox center motif, monton, Alberta, T6G 2E9, Canada; [email protected]. and a carboxyl-terminal dilysine targeting signal thought to be

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responsible for the retrieval of transmembrane proteins to the through 6. PCR cycling parameters used were as follows: 1 cycle of endoplasmic reticulum (ER) from the Golgi apparatus.22 The denaturation at 94°C for 3 minutes; 30 cycles of denaturation at 94°C 5-bp deletion in the gene identified in dominant STGD/adMD- for 1 minute, primer annealing at 45°C for 1 minute, primer extension affected pedigrees causes a reading frame shift that results in at 72°C for 3 minutes; and 1 cycle of extension at 72°C for 7 minutes. premature termination of the translation product, leading to Probes were purified with a gel extraction kit (QIAquick; Qiagen Inc., substitutions in amino acids 264-271 and the loss of the 51 Mississauga, Ontario, Canada) and radiolabeled with [␣32P]-dCTP (Am- carboxyl-terminal residues in the mutant protein. This muta- ersham Biosciences, Baie d’Urfe´, Quebec, Canada) by random primer tion has subsequently been verified in affected members labeling using a DNA labeling system (Multiprime; Amersham Bio- present in other branches of the same family.23 In addition, a sciences). Hybridization of each probe to the zooblot was performed in novel complex mutation in the ELOVL4 gene has been de- hybridization solution (Hybrisol II; Serologicals Corp., Norcross, GA) at scribed in an unrelated family that manifests diverse macular 42°C for 16 hours using 106 cpm of labeled, purified probes per dystrophy phenotypes.24 milliliter of hybridization solution. The blot was then washed twice for Thus, it is clearly established that the ELOVL4 gene plays a 15 minutes in 2ϫ sodium chloride/tri-sodium citrate buffer (SSC), once critical role in the pathogenesis of macular degeneration. How- for 30 minutes in 2ϫ SSC and 0.1% sodium dodecyl sulfate (SDS), and ever, the precise nature of this role is at present unknown. once for 10 minutes in 0.1% SSC with 0.1% SDS all at 42°C, followed by Clues to the importance of this gene in retinal cell biology have exposure to autoradiograph film (X-Omat; Eastman Kodak, Rochester, also come from the identification of both mouse and yeast NY) at Ϫ70°C for 24 hours. homologues, implying evolutionarily conserved gene expression. This limited assessment of conservation, its implied role RNA Isolation and Northern Blot Analysis in fatty acid metabolism, and its association with retinal degen- Total RNA was isolated from primate ocular tissues and retinal tissue eration suggests that ELOVL4 is an essential retina-expressed derived from various mammalian species using extraction reagent gene. To further characterize this gene in normal retinal biol- (Trizol; Invitrogen), according to the manufacturer’s protocol. Five ogy, we used nucleic acid hybridization and bioinformatic and micrograms of each total RNA sample was electrophoresed on a 1% immunodetection analyses to examine the sequence conserva- agarose-formaldehyde gel and subsequently blotted onto a nylon mem- tion and expression of ELOVL4 gene products across a wide brane (Pall Corp., Mississauga, Ontario, Canada) by capillary transfer. range of species. We report that ELOVL4 represents a highly Northern blots were consecutively hybridized with probes corre- evolutionarily conserved gene that is expressed in the photo- sponding to the human ELOVL4 and ␤-actin coding regions. The receptor cell layer within the retina of every mammalian spe- ELOVL4 probe used was the 743-base pair cDNA fragment described cies examined. earlier. The ␤-actin probe was amplified by PCR from a plasmid clone with primers 5Ј-GTCCACACCCGCCGCCAGCTCACCAT-3Ј and 5Ј-ACT- METHODS GGTCTCAAGTCAGTGTACAGGTA-3Ј to generate a 1698-bp nearly full- length cDNA product. Radiolabeled probes were prepared and hybrid- Animal Tissues and Donor Eyes izations and washes were performed as just described at either 42°Cor Human ocular tissue was obtained from eyes donated to the Michigan 55°C for blots containing multispecies RNA samples or at 65°C for Eye Bank (Ann Arbor, MI) and through the National Disease Research primate RNA blots. For increased specificity of probe binding, multi- ␮ Interchange (NDRI, Philadelphia, PA). Archival human retinal sections species blots were blocked with 0.5 g of denatured human Cot-1 were obtained from the Ocular Pathology Laboratory at the University DNA (Invitrogen) added to the hybridization solution. ELOVL4-probed of Rochester (Rochester, NY). Rhesus monkey eyes were kindly pro- blots were exposed to film for 18 to 48 hours. vided by John Cogan (Bureau of Biologics, Bethesda, MD). Ocular dissections were performed as previously described.25 Mouse, Production of ELOVL4 Antibodies Sprague-Dawley rat, and feline eyes were obtained from normal control A synthetic 15-amino-acid peptide (SIADKRVENWPLMQS) correspond- animals in the University of Michigan animal facility and dissected ing to amino acids 31-46 of the human ELOVL4 protein was conjugated immediately after harvesting. Bovine eyes were obtained from a local to keyhole limpet hemocyanin and subcutaneously injected into rab- abattoir and stored on ice after harvesting. Dissections were performed bits for the production of polyclonal antibodies (Research Genetics, within 3 hours after death. The care and handling of animals was in Huntsville, AL). Preimmune serum was also collected from the rabbits compliance with the ARVO Statement for the Use of Animals in Oph- before immunization for use in control experiments. Antibodies were thalmic and Vision Research. affinity purified using a column containing immobilized peptide antigen. Southern Blot Analysis A commercially available zooblot containing 8 ␮gofEcoRI-digested Preparation of Retinal Protein Extracts genomic DNA isolated from various species (human, monkey, rat, Volumes of retinal tissue samples were estimated, and an equal volume mouse, dog, cow, rabbit, chicken, and yeast) was purchased (BD of 2ϫ lysis buffer (100 mM Tris-HCl [pH 7.4], 300 mM NaCl, 1% Triton Biosciences-Clontech, Palo Alto, CA). A 376-bp DNA probe corre- X-100, 1 tablet of a protease inhibitor cocktail dissolved in 10 mL of sponding to exon 6 of the human ELOVL4 gene was generated from a water [Complete; Roche Diagnostics Corp., Indianapolis, IN]) was genomic DNA template by polymerase chain reaction (PCR), with added. Tissues were homogenized using a sonicator (Sonifier 250; primers 5Ј-GAAGATGCCGATGTTGTTAAAAG-3Ј and 5Ј-GTCAACAA- Branson Ultrasonics, Danbury, CT), with samples pulsed for 10 sec- CAGTTAAGGCCCA-3Ј. PCR cycling parameters used were as follows: 1 onds at 2-minute intervals for 20 minutes at 4°C. Homogenates were cycle of denaturation at 94°C for 3 minutes; 30 cycles of denaturation spun at 5000 rpm for 3 to 5 minutes in a microcentrifuge to precipitate at 94°C for 30 seconds, primer annealing at 55°C for 30 seconds, insoluble material, and the supernatants were further analyzed for primer extension at 72°C for 40 seconds; and 1 cycle of extension at protein content. Total protein concentration was estimated with the 72°C for 3 minutes. A probe corresponding to ELOVL4 coding regions bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, was also generated by total cDNA synthesis with a commercial kit Rockford, IL). (Invitrogen Canada, Inc., Burlington, Ontario, Canada) and 5 ␮gof human retina total RNA as template, followed by PCR using 100 ng total cDNA template with primers 5Ј-GTGTGGAAAATTGGCCTCTG-3Ј SDS-PAGE and Immunoblot Analysis and 5Ј-CATGGCTGTTTTTCCAGCTT-3Ј to amplify a 743-bp cDNA frag- Retinal protein extracts (approximately 200 ␮g of total protein) were ment containing protein-encoding sequences found within exons 2 added to an equal volume of 2ϫ sodium dodecyl sulfate–polyacrylam-

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ide gel electrophoresis (SDS-PAGE) loading buffer (100 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol) and electrophoresed on a 12% poly- acrylamide gel. Proteins were transferred to a nitrocellulose membrane (Sigma-Aldrich Corp., St. Louis, MO) by electroblotting (Mini Trans-Blot Protein Blotting Cell; Bio-Rad Laboratories, Hercules, CA). Immuno- blots were blocked with 3% skim milk powder for 1 hour at room temperature, followed by incubation with the ELOVL4 polyclonal antiserum diluted 1:1000 in 3% skim milk at room temperature for 1 hour in the presence or absence of 20 ␮g of competing ELOVL4 peptide. Blots were subsequently labeled with a 1:5000 dilution of horseradish peroxidase (HRP)–conjugated goat anti-rabbit secondary antibody (Pierce Biotechnology) and subjected to immunodetection by enhanced chemiluminescence (ECL Western Blotting Detection Re- agents; Amersham Biosciences, Piscataway, NJ).

Bioinformatic Analysis ELOVL4 homologues and orthologues were identified by performing protein homology searches of sequence databases with the translated ELOVL4 mRNA sequence (GenBank accession no. AF277094; http:// www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information [NCBI], Bethesda, MD) and the BLASTP program also provided in the public domain by NCBI (http://www.ncbi.nlm.nih.gov/blastp).26 Proteins identified by the BLASTP search program with greater than 25% identity to the human ELOVL4 amino acid sequence were classified as homologues. Sequence alignments and phylogenetic analysis were performed using the Clustal W (versions 1.8, 1.82)27 and PHYLIP28,29 programs and Command Line applications for sequence analysis and phylogeny (http://www.cbr.nrc.ca/ all provided in the public domain through Canadian Bioinformatics Resources, National Research Council Can- ada, Halifax, Nova Scotia, Canada).

Immunohistochemistry Human Retina Sections. Whole human eyes were paraffin embedded and cut into 4-␮m tissue sections. Human tissue sections were permeabilized in 0.25% Triton X-100 for 5 minutes and rinsed in phosphate-buffered saline (PBS). Endogenous peroxidase was FIGURE 1. Southern blot analysis of genomic DNA derived from quenched with a 5-minute incubation in 3% hydrogen peroxide. Tis- various species demonstrating evolutionary conservation of the sues were rinsed twice for 5 minutes in PBS and blocked for 15 ELOVL4 gene. Human, monkey, rat, and mouse DNA samples were minutes in 5% goat serum. After two additional 5-minute rinses in PBS, analyzed with a radiolabeled probe corresponding to the human sections were incubated with a 1:100 dilution of ELOVL4 polyclonal ELOVL4 gene exon 6 sequence, whereas an ELOVL4 cDNA fragment antiserum or preimmune serum as a negative control for 1 hour at was used to probe the canine, bovine, rabbit, chicken, and yeast DNA room temperature. Tissues were subsequently rinsed three times for 5 samples. minutes in PBS and then incubated with 1 ␮g/mL of biotinylated goat anti-rabbit immunoglobulin secondary antibody (Vector Laboratories, dilution of the preimmune serum in place of the ELOVL4 antibodies. Burlingame, CA) for 1 hour at room temperature. After a final PBS rinse, Results were visualized by light microscopy. tissues were incubated with HRP-conjugated avidin (Vectastain Elite kit; Vector Laboratories) for 20 minutes. Tissue sections were equili- brated in 0.05 M Tris-HCl [pH 7.4], then developed with a diamino- RESULTS benzidine (DAB) kit (Pierce Biotechnology), and the brown- or black- colored reaction product was visualized by light microscopy. For DNA Multispecies Conservation of ELOVL4 DNA counterstaining, sections were incubated with 30% methyl green for Sequences and mRNA Expression 50 seconds. To detect the presence of ELOVL4 sequence homologues Animal Retina Sections. Mouse, feline, and rat specimens and/or orthologues in a number of different species, we were perfused with 4% paraformaldehyde, and eyes were extracted probed a Southern blot containing genomic DNA isolated from and fixed overnight at 4°C. The bovine eyes obtained from an abattoir human, monkey, rat, mouse, dog, cow, rabbit, chicken, and were immersed in 4% paraformaldehyde overnight. The anterior seg- yeast with ELOVL4-derived sequences. Hybridization signals ment of the eye was removed. The eye cup was rinsed in buffer and were detected in all the species examined (Fig. 1), suggesting embedded in paraffin. Specimens were sectioned at 5 to 7 ␮m. Tissue that the ELOVL4 gene is conserved across a variety of eukary- sections were dewaxed in xylene and rinsed in PBS. Rehydrated sec- otic species. tions were treated with proteinase K (500 ng/mL), as previously de- Retinal expression of the ELOVL4 gene has already been scribed.30 Immunohistochemical analysis was performed with a kit demonstrated in human tissue samples.19,23 To investigate the (Vectastain ABC kit; Vector Laboratories, Inc.), according to the man- expression of ELOVL4 mRNA in additional human eye tissues, ufacturer’s protocol. Rat and cat sections were analyzed with the we performed Northern blot analysis. The approximately 2.9- ELOVL4 polyclonal antiserum at a 1:400 dilution, and mouse and cow and 2.4-kb transcript sizes previously reported for the human sections were analyzed with the ELOVL4 affinity-purified antibodies at ELOVL4 gene were consistently observed in different periph- a 1:10,000 dilution. Control experiments were performed with a 1:400 eral retina samples (Fig. 2); however, we detected two addi-

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FIGURE 2. Northern blot analysis of human, monkey, mouse, bovine, and porcine ocular tissues demonstrating differential expression of ELOVL4 RNA transcripts between different regions of the eye and between primate and mammalian species. Top: each lane contains 5 ␮g of total RNA extracted from the indicated tissues. Arrowheads: the four distinct transcripts detected on probing with the ELOVL4 cDNA fragment. Bottom: results of analysis of the blots with a human ␤-actin control probe. Left: positions of RNA size markers (sizes given in kilobases).

tional transcripts of roughly 4.5 and 0.25 kb. These bands logues were identified in monkey and mouse, each with persisted even after Cot-1 DNA suppression and high-strin- greater than 90% amino acid identity with the human protein. gency washing of blots. Similar hybridization signals were Ninety-one amino acids are strongly evolutionarily conserved, observed for human fovea tissue; however, the 2.9- and 2.4-kb present among eight or more of the homologous sequences transcripts appear to be absent in RPE, choroid, and ciliary examined, and representing almost one third of the entire body tissue samples (Fig. 2). This suggests that ELOVL4 or protein. These residues tend to lie in or adjacent to putatively ELOVL4-like genes are differentially expressed among different functionally significant regions of the protein such as the dioxy human eye tissues. All four transcripts were detected in rhesus iron-binding HXXHH redox center motif of the elongase en- monkey peripheral retina and macular tissues, indicating that zyme and the transmembrane domains (Fig. 3). Seventy-two ELOVL4 gene expression is highly conserved between primate distinct sequences identified to date in 18 different organisms retinal tissues. share at least 23% identity with the human ELOVL4 protein. To extend these findings to additional species, retinal RNA This significant level of protein sequence similarity between samples derived from mouse, cow, and pig were similarly such diverse species is suggestive of a critical cellular role that assessed by Northern blot analysis (Fig. 2). The approximately has been maintained throughout evolution. A phylogenetic 4.5-kb transcript appeared in all the species, whereas the tree depicting the evolutionary relationship between these 2.9-kb transcript is clearly observed in the mouse and cow sequences was generated with the Clustal W (version 1.82) and retina but was undetectable in the pig retina RNA. The 2.4-kb PHYLIP (version 3.5) programs (Fig. 4). Belonging to distinct transcript readily detected in the primate retina samples is not clades, each of the human proteins identified appears to be as easily observed in the retinal RNA from the other species more related to gene products from other species than to other under the stringent hybridization and washing conditions used. members of the human ELO protein family, whereas many of The approximately 0.25-kb transcript also appeared in all sam- the putative nonhuman elongases exhibit greater intraspecies ples, except for porcine retina. These results suggest that similarity and interspecies divergence. In most cases, the in- different species express a different complement of ELOVL4 vertebrate ELO proteins group according to species, whereas transcripts that may share varying degrees of sequence conser- the vertebrate ELOs span a number of distinct clusters (Fig. 4). vation, which in turn would lead to differences in detection Despite sharing certain functional motifs including histidine capabilities based on the hybridization conditions used. clusters and ER membrane–spanning domains,31,32 fatty acid desaturases, another class of enzymes involved in the process ELOVL4 Protein Orthologues and Homologues of fatty acid elongation,33 were not found to be represented The translated human ELOVL4 mRNA sequence (GenBank Ac- among the list of putative ELO proteins. Furthermore, no sig- cession No. AF277094) was used to search protein sequence nificant overall sequence similarity was detected between the databases to identify related proteins found in other species. human ELOVL4 protein and any of the known human desatu- Putative proteins exhibiting 25% or greater identity at the rase gene products identified to date. Therefore, fatty acid amino acid level with the human ELOVL4 protein were classi- elongases and desaturases represent two independent enzy- fied as potential orthologues or homologues. Presumed matic protein families. ELOVL4 orthologues and the best-matched sequence homologue from each of a variety of species were selected and Immunodetection of ELOVL4 Protein in aligned using the Clustal W (version 1.8) program (Fig. 3). A Mammalian Retinas wide range of species including monkey, mouse, rat, insects, fish, fungus, plant, nematode, and yeast varieties were found to To confirm the existence of ELOVL4 protein homologues in contain ELOVL4 homologous protein sequences. True ortho- multiple species, we used polyclonal antibodies raised against

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FIGURE 3. Sequence alignment of the human ELOVL4 protein with known orthologues and most similar homologues identified in other species. Monkey and mouse ELOVL4 orthologues are labeled, whereas the remaining sequences represent proteins identified to date that exhibit the greatest degree of homology with human ELOVL4 in each of the organisms listed. Amino acid sequences were aligned using the Clustal W program (ver. 1.8). Hyphens represent gaps introduced to maintain the alignment. The amino acid residues are numbered to the left and right of the sequences. Identical residues among the sequences in the alignment are shown on a black background, and conservative amino acid substitutions are shaded. Asterisks: residues identical among all 12 sequences; cross: amino acids common to 8 or more sequences. Predicted transmembrane domains in human ELOVL4 are indicated with a series of dots above each of the corresponding hydrophobic stretches of amino acids. The dioxy-iron binding histidine box motif shared by fatty acid elongase enzymes is shown in bold. GenBank accession numbers for each protein sequence listed are as follows: human (Homo sapiens) ELOVL4, AAG47668; monkey (Macaca fascicularis) ELOVL4, Q95K73; mouse (Mus musculus) ELOVL4, AAG47667; fowlpox virus ORF FPV048, AAF44392; mosquito (Anopheles gambiae) homologue, EAA07022; mouse (M. musculus) elongase, NP_599016; rat (Rattus norvegicus) rELO1, BAB69887; human (H. sapiens) HELO1, AAF70631; turbot (Scophthalmus maximus) elongase, AAL69984; fruit fly(Dro- sophila melanogaster) CG5278 gene product, AAF56019; yeast (Schizo- saccharomyces pombe) homologue, T50139; nematode (Caenorhabditis elegans) CEELO1, AAF70462.

a peptide derived from the human sequence to probe immu- says performed using the ELOVL4 antibodies in the presence noblots of total protein extracts prepared from the retinas of and absence of the peptide antigen veriﬁed the speciﬁcity of various animals. A single intense band of approximately 37 kDa the antibodies for this 37-kDa band, which is consistent with was detected in mouse, bovine, feline, and rat retinal extracts the predicted molecular weight of the human ELOVL4 protein (Fig. 5). A similarly prominent band was detected in protein (36.8 kDa). Thus, we conclude that the ELOVL4 protein is extracts prepared from human retinal tissue. Competition as- present in the retinas of different mammalian species and that

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FIGURE 4. Phylogenetic tree display- ing evolutionary relationships between all known and predicted ELO protein family members found in the GenBank protein sequence database to date. Two- or three-letter species abbreviations precede the protein name or GenBank accession number for each sequence represented. Per- centage identity between each sequence and human ELOVL4 (bold)is indicated in parentheses after each name or accession number. The rooted phenogram shown was generated by aligning amino acid sequences using the Clustal W program (ver. 1.82) followed by phylogenetic analysis with PHYLIP (ver. 3.5). Spe- cies abbreviations: Hs, Homo sapiens (human); Mf, Macaca fascicularis (monkey); Mm, Mus musculus (mouse); FPV, fowlpox virus; Ag, Anopheles gambiae (mosquito); Rn, Rattus norvegicus (rat); Sm, Scoph- thalmus maximus (turbot); Dm, Drosophila melanogaster (fruit ﬂy); Pp, Physcomitrella patens (moss); Ma, Mortierella alpina (fungus); Sp, Schizosaccharomyces pombe (ﬁs- sion yeast); Sc, Saccharomyces cerevisiae (budding yeast); At, Arabi- dopsis thaliana (plant); Os, Oryza sativa (rice); Dd, Dictyostelium dis- coideum (amoebae); Ce, Caeno- rhabditis elegans (nematode); Lm, Leishmania major (trypanosome); Ig, Isochrysis galbana (microalgae).

the amino acid sequence corresponding to the peptide antigen tain ELOVL4 protein, as evidenced by the absence of immuno- used for polyclonal antibody production is conserved between staining in this subcellular region. In mouse, bovine, feline, and humans, mice, cows, cats, and rats, in which ELOVL4 protein rat retinal sections, a similar protein localization pattern was species of the identical size are detected. observed, with staining above background levels predominantly confined to the photoreceptor inner segment layer Distribution of ELOVL4 in Mammalian Retinas (Figs. 6B–E). The intensely pigmented RPE cell layer observed The localization of ELOVL4 proteins in specific cell layers in the mouse, bovine, and feline sections make it difficult to within the retinas of various mammalian species was examined ascertain whether staining due to the ELOVL4 antibodies is by immunohistochemistry. In human retina tissue sections, present in this region. However, slight RPE staining was de- strong ELOVL4 immunostaining was observed in the photore- tected in sections derived from melanin-deficient albino rats, ceptor layer, particularly within the inner segments of the indicating localization of ELOVL4 proteins to RPE cells (Fig. photoreceptor cells (Fig. 6A). Uniform staining was detected 6E). Human retina sections also exhibited increased RPE stain- across the length of this retinal layer in the sections examined ing in the presence of the ELOVL4 antiserum (Fig. 6A), which and was found in both rod and cone cells. Slight antibody is in accordance with the detection of ELOVL4 RNA transcripts reactivity was also noted in cell processes extending from the in human RPE tissue (Fig. 2). No significant immunostaining outer plexiform layer. Cell bodies were not observed to con- was observed in control sections incubated with preimmune

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Consistent with previous reports,19,23 we detect both the presumed full-length 2.9-kb mRNA transcript and a shorter 2.4-kb transcript in human retinal tissue. However, we also consistently observe the existence of additional transcripts that bind specifically to ELOVL4 coding sequences. Nucleic acid sequence database searches using the hybridization probe sequence as bait do not reveal any other known genes that are predicted to produce the transcript sizes detected. It is possible that these novel RNA species represent the products of genes that have yet to be defined as ELOVL4 homologues. Multiple paralogues of other genes involved in fatty acid elongation, such as desaturases, have been identified in various species, giving rise to physically and catalytically distinct enzymes.34–36 Furthermore, some members of the human ELO gene family share a greater degree of sequence similarity be- FIGURE 5. Western blot analysis of retinal protein extracts derived tween them than others, suggesting the existence of protein from various mammalian species demonstrating conserved ELOVL4 “subfamilies” whose members may exhibit more similar func- protein production. The 37-kDa protein corresponding to the pre- tional characteristics as well. This observation, along with the dicted size of ELOVL4 was detected in mouse, bovine, feline, rat, and ongoing discovery of novel human elongase genes (most re- human retina tissues. Significant reduction in the intensity of the cently, the LCE gene37,38), may reveal the existence of multiple 37-kDa band was observed in human extracts when blots were incu- ELOVL4-like sequences arising from distinct genetic loci. These bated with primary antibodies in the presence of excess competing sequences may give rise to the approximately 4.5- and 0.25-kb peptide antigen (ϩ peptide), demonstrating specificity of the ELOVL4 polyclonal antibodies. transcripts appearing on our Northern blot analysis. The 2.4-kb mRNA species is thought to arise by alternative polyadenyla- tion of the nascent ELOVL4 RNA transcript.23 Alternative pro- serum instead of the ELOVL4 polyclonal antibodies (Figs. 6F–J). moter usage is another means by which different transcripts The subretinal localization of the ELOVL4 protein thus appears can be produced from a single genetic locus. Thus, both pre- to be consistent for a variety of mammalian species. and posttranscriptional processing of as yet unidentified ELOVL4-like genes and gene products may account for the DISCUSSION results of Northern blot analysis observed. As these novel transcripts are detected in the retina of different species, the Our results clearly demonstrate that the ELOVL4 gene and its mechanism by which they arise appears also to be conserved. expression are highly conserved across a diverse array of spe- All four of the transcripts are seen in tissue derived from both cies. At the genomic DNA, mRNA, and protein levels, and the central and peripheral retina; however, there is more lim- through both in silico and classic experimental techniques, we ited gene expression noted in other human ocular tissues. This have consistently observed similarities in sequence and expres- finding resembles the expression profile of other retinal sion profile among retinal tissues from many different types of genes39,40 and indicates that retina-specific gene function may animals. Expression of the ELOVL4 gene has previously been be imparted through differential rather than retina-specific assessed in human retina and other cell types at the level of gene expression. mRNA production; however, no analysis of ELOVL4 protein Bioinformatic analysis reveals that ELOVL4 protein ortho- has been performed until now. Our results confirm those of logues have been identified thus far in monkey (Umeda S, et al. previous studies that have examined ELOVL4 gene expres- ARVO Abstract 2807, 2002) and in mouse,19 exhibiting 97% sion19,23 and extend their findings. and 93% amino acid identity, respectively, with the human

FIGURE 6. Immunohistochemical analysis of mammalian retina sections demonstrating conserved localization of ELOVL4 protein in the inner segments of photoreceptor cells. Retinal sections were incubated with rabbit anti-ELOVL4 polyclonal antibodies (A–E) or preimmune serum (F–J) followed by subsequent detection and visualization with diaminobenzidine (brown) upon light microscopy. (A, F) Human retina sections; (B, G) mouse retina sections; (C, H) cow retina sections; (D, I) cat retina sections; (E, J) rat retina sections. Hu- man retina sections were counter- stained with methyl green to visualize cell nuclei (blue-green). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Bars, 50 ␮m.

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protein. In addition, a wide variety of evolutionarily divergent ER-localization signal, rendering the enzyme dysfunctional as a species also harbor ELOVL4-homologous sequences, suggest- result of aberrant protein targeting. Photoreceptor inner seg- ing the importance of this gene or similar entities in cellular ment labeling by ELOVL4 antibodies described herein is the function. As expected, regions of the protein with the highest first demonstration of subcellular localization of this protein degree of conservation coincide with predicted functional mo- and is consistent with its putative location in the ER within tifs. Highly conserved amino acid residues also exist outside of cells. these domains and indicate the significance of other protein Several of the disease genes that underlie atrophic macular regions that have yet to be defined within ELOVL4, such as degeneration phenotypes are expressed in both rod and cone potential substrate-binding, protein–protein interaction, and photoreceptors,51–53 although the cone-rich macular region is regulatory domains. the primary target in these conditions.8,54,55 It is interesting to The ELOVL4 protein is a member of a large family of known note that the ELOVL4 gene is also expressed in both types of and putative fatty acid elongases, sharing from 23% to 97% photoreceptor cells. The ABCA4, RDS, and RPGR genes, amino acid sequence identity with 72 different proteins iden- whose protein products have been detected in both rods and tified in this family to date. Results of phylogenetic analysis cones, are also mutated in disorders affecting the peripheral indicate that, in general, the intraspecies divergence of verte- retina.56–58 However, no disease-associated sequence alter- brate ELO proteins is greater than that of invertebrate ELOs. ations in ELOVL4 have as yet been reported in patients with Proteins from moss, fungus, plant, nematode, and single-celled peripheral retina disorders, despite mutational analysis of the organisms cluster according to species, whereas mammalian gene within such individuals.59 Elucidation of the pathologic ELO proteins tend to cluster according to protein family mem- mechanisms by which mutation of each of these genes leads to bership instead of species designations. This is evident for all the development of diseases affecting different regions of the the human ELO protein sequences examined, including retina awaits further study. ELOVL4, and suggests that the evolutionary divergence of ELOs It has been hypothesized that ELOVL4 may be an enzyme into subfamilies occurred before the divergence of individual responsible for catalyzing an elongation step in the synthesis of vertebrate species. This, in turn, supports the hypothesis that docosahexaenoic acid (DHA),19 the most abundant polyunsat- the genes encoding these proteins have evolved from common urated fatty acid species in vertebrate retinal lipids.60 It is also ancestral genes that play distinct roles in fatty acid biosynthesis possible that this protein is involved in the synthesis of other and are part of a highly conserved metabolic mechanism. essential fatty acids, the deficiency of which may lead to im- The exclusion of fatty acid desaturases from the extensive paired retinal function. Alteration of the lipid environment of list of ELOVL4 protein homologues identified indicates that retinal cells has been shown to influence retinal protein func- physically distinct enzymes are responsible for catalyzing dif- tion, photoreceptor cell development, and cell survival.61–66 ferent reactions in the fatty acid elongation pathway in many Thus, dysfunction of a gene that results in aberrant lipid syn- species. Clearly, the HXXHH motifs, transmembrane domains, thesis in the retina is predicted to have deleterious effects and and putative ER-targeting signals are not sufficient to impart may account for the pathogenesis observed in cases of STGD3/ significant overall amino acid sequence homology between adMD and other retinal dystrophies in which ELOVL4 muta- members of the elongase and desaturase protein families, as tions are identified. Continued characterization of the physical might be expected for enzymes that are involved in distinct and functional properties of ELOVL4 will provide valuable catalytic activities and that have requirements for recognizing assistance toward uncovering its precise biological role in the chemically different substrates. This contrasts with other lipid retina, which in turn may lead to the development of potential biosynthetic processes such as de novo fatty acid synthesis, in therapeutic strategies for individuals who have the ocular dis- which the fatty acid synthase enzyme consists of multifunc- orders arising from mutations in the ELOVL4 gene. tional polypeptides in animal cells but distinct proteins in 41 bacteria. However, it is believed that the reactions constitut- Acknowledgments ing the fatty acid desaturation and chain elongation pathway are catalyzed by a closely coordinated or concerted enzyme The authors thank Janet Wagner for providing archival human tissue 42 system. samples, Mitchell S. Gillett for preparation of animal tissue sections, To further the study of the ELOVL4 protein, we generated Austra Liepa for animal care, Miguel A. Cabrita for assistance with the and describe herein for the first time polyclonal antibodies that phylogenetic analysis, and Dushanka Deretic for technical advice. specifically recognize the expected 37-kDa ELOVL4 protein in retinal extracts from different species. Immunohistochemical studies performed with these antibodies revealed conserved References subretinal localization of the protein in photoreceptor cells and 1. Hadden OB, Gass JD. Fundus flavimaculatus and Stargardt’s dis- more specifically within the inner segments of both rods and ease. 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