CTRP5 Is a Membrane-Associated and Secretory Protein in the RPE and Ciliary Body and the S163R...

CTRP5 Is a Membrane-Associated and Secretory Proteinin the RPE and Ciliary Body and the S163R Mutation ofCTRP5 Impairs Its Secretion

Md Nawajes A. Mandal,1 Vidyullatha Vasireddy,1 G. Bhanuprakash Reddy,1,2

XiaoFei Wang,3 Sayoko E. Moroi,1 Bikash R. Pattnaik,1 Bret A. Hughes,1

John R. Heckenlively,1 Peter F. Hitchcock,1 Monica M. Jablonski,3 and Radha Ayyagari1

PURPOSE. In a prior study, a S163R mutation in the comple-ment-1q tumor necrosis factor–related protein 5 (CTRP5/C1QTNF5) was reported to be associated with early-onset longanterior zonules (LAZ) and late-onset retinal degeneration(L-ORD). The ocular tissues involved in the phenotype are theretinal pigment epithelium (RPE) in the posterior segment andciliary epithelium (CE) and lens in the anterior segment. Thepurpose of this study was to characterize the spatial and tem-poral expression of the mouse Ctrp5 gene, determine tissueand subcellular localization, and study the effect of the S163Rmutation.

METHODS. The expression of the Ctrp5 gene in the mouse wasstudied by quantitative (q)RT-PCR and in situ hybridization.CTRP5 protein expression and distribution were studied byWestern blot analysis, immunohistochemistry, and immuno-electron microscopy. Cellular location of wild-type and mutantCTRP5 in MDCK and COS-7 cells was determined by immuno-fluorescence and immunoblot analysis.

RESULTS. A significant level of Ctrp5 expression was detected inthe adult mouse in the ciliary body (CB) and RPE, and expres-sion started at a very early stage of embryogenesis. Immuno-histochemical analysis showed CTRP5 protein in the apicalprocesses of the RPE and forming a hexagonal lattice associ-ated with the RPE lateral membranes. In the ciliary body,CTRP5 was localized to the apical aspects of the CE, the regionbetween the bilayered ciliary epithelial cells. The membraneassociation of CTRP5 in the RPE and CE was further confirmedby immunoelectron microscopy. Furthermore, cultured cells

were used to show that the CTRP5 is a secretory protein andthat its secretion is impaired by the S163R mutation.

CONCLUSIONS. CTRP5, a secretory and membrane-associatedprotein, is localized to the lateral and apical membranes of theRPE and CB. Impaired secretion of the mutant protein mayunderlie the pathophysiology of L-ORD and LAZ. (Invest Oph-thalmol Vis Sci. 2006;47:5505–5513) DOI:10.1167/iovs.06-0312

Macular degenerations form a heterogeneous group of dis-eases affecting the macula and causing loss of central

vision. These diseases are clinically characterized by geo-graphic atrophy or neovascularization of the choroid and maybe accompanied by accumulation of lipofuscin or developmentof drusen in the central area of the retina.1 Age-related maculardegeneration (AMD) alone affects more than 20% of the U.S.population older than 65 years.2–4

Genetic studies of familial forms of macular degenerationhave thus far revealed 17 mapped loci, and genes for 10 ofthese have been cloned.5 In addition, several genes associatedwith susceptibility to AMD have been reported (FBLN5,ABCA4, TLR4, APOE, CFH, BF, LOC387715, and C2).6–13 Re-cent findings have also suggested potential involvement ofcomplement pathway in the development of AMD.14,15 Thereis significant phenotypic overlap among the late-onset maculardegenerations, which include Sorsby’s fundus dystrophy, adult-onset foveomacular dystrophy, late-onset retinal degeneration(L-ORD), and AMD.16–18 The functional roles of the genesassociated with macular degenerations and the pathophysiol-ogy of these diseases are not well understood. Some of thegenes associated with monogenic macular degenerations areexpressed in photoreceptors (ELOVL4, RDS, RPGR, ABCA4,and FSCN2) and are thought to have a role in maintaining thestructure and/or physiological function of photoreceptorcells.19–23 The remaining genes associated with the maculardegenerations are expressed in the RPE, with most beingextracellular matrix or plasma membrane components(CTRP5, EFEMP1, FIBL-6, TIMP3, and VMD2).24–28 A foundermutation, S163R, in a conserved domain of the CTRP5 gene hasbeen reported in families with L-ORD.18 Recently, we reportedthe same mutation in a family (UM:H389) with a complexdominant phenotype of late-onset progressive macular degen-eration (L-ORD) and early-onset long anterior lens zonules(LAZ).24 This gene is expressed in the RPE and ciliary body, thetwo tissues of neuroectodermal origin that are involved in thepathologic courses of L-ORD and LAZ, respectively.24

In the human and mouse genomes, the CTRP5 gene is veryclosely associated with the membrane frizzled-related protein(MFRP) gene, and the complete CTRP5 open reading frame(ORF) is reported to reside in the 3� untranslated region (UTR)of MFRP and, therefore, is thought to be dicistronic.29,30 Arecessive mutation in the Mfrp gene causes retinal degenera-tion in the rd6 mouse.29,31 In addition, null mutations in the

From the 1Department of Ophthalmology and Visual Sciences,University of Michigan, Ann Arbor, Michigan; the 2National Institute ofNutrition, Hyderabad, India; and the 3Retinal Degeneration ResearchCenter, Ophthalmology, University of Tennessee Health Sciences Cen-ter (UTHSC), Memphis, Tennessee.

Supported by grants from the National Eye Institute EY13198 andFoundation Fighting Blindness (RA), a special scholar award fromResearch to Prevent Blindness (RA); National Eye Institute core grantsEY07060 and EY07003 to the University of Michigan, Department ofOphthalmology and Visual Sciences; National Eye Institute GrantP30EY13080 (MMJ); an unrestricted grant from Research to PreventBlindness to Ophthalmology at UTHSC; and a fellowship from IndianCouncil of Medical Research (GBR).

Submitted for publication March 22, 2006; revised June 26 andAugust 28, 2006; accepted October 19, 2006.

Disclosure: M.N.A. Mandal, None; V. Vasireddy, None; G.B.Reddy, None; X.-F. Wang, None; S.E. Moroi, None; B.R. Pattnaik,None; B.A. Hughes, None; J.R. Heckenlively, None; P.F. Hitch-cock, None; M.M. Jablonski, None; R. Ayyagari, None

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be marked “advertise-ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Radha Ayyagari, Department of Ophthal-mology and Visual Sciences, W.K. Kellogg Eye Center, 1000 WallStreet, Ann Arbor, MI 48105; [email protected].

Investigative Ophthalmology & Visual Science, December 2006, Vol. 47, No. 12Copyright © Association for Research in Vision and Ophthalmology 5505

MFRP gene are reported to be associated with extremely small-sized eyes, nanophthalmos, and hyperopia in humans.32 How-ever, the functional role of CTRP5 and its relationship to MFRPin the development and maintenance of normal physiology ofthese tissues is not known.

In the present study, we characterized the Ctrp5 gene interms of its spatial and temporal pattern of expression andtissue distribution to gain insight into the role of this protein inocular physiology. We found that CTRP5 is both associatedwith the cell membrane and secreted. We also observed thatthe L-ORD mutant of CTRP5 is associated with impaired secre-tion, which could be one of the major contributing factors ofdisease development.

MATERIALS AND METHODS

Animals and Tissue Collection

The maintenance and care of the C57BL/6 and BALBc mice used in thestudy were in accordance with the NIH Guidelines for the Care andUse of Laboratory Animals and the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research. All the animals weremaintained on a 12-hour light–dark cycle, and tissues were collected atthe end of the dark cycle at the appropriate age. We collected theretina, lens, ciliary body and iris, posterior segment (containing RPE,choroid, and sclera), and other tissues, including brain, heart, lungs,liver, spleen, kidney, skeletal muscle, testes, skin, and uterus from four33-week-old C57BL/6 mice, to isolate RNA and measure expression ofthe Ctrp5 gene. Mouse embryonic cDNAs at embryonic days (E)7 toE17 were obtained from BD-Clontech (Mountain View, CA). Eight to 10eyes from four to five C57BL/6 mice were collected at postnatal days(P)1, P3, P5, P10, P15, and P30 (P1E–P30E) to study the expression ofthis gene during postnatal development. Ten-micrometer-thick cryo-sections of C57BL/6 embryos at E14.5 and E18.5 and adult BALBcmouse (90-day-old) eyes were prepared as described earlier.19

Real-Time qRT-PCR

Quantitative (q)RT-PCR was performed, and the data were analyzedaccording to previously published protocols.24,33 Primers for qRT-PCRwere designed from regions of the Ctrp5 sequence that spanned atleast one intron (Figs. 1A–1C). We used the expression of four genes—Gapdh, Hgprt, �-actin, and RpL19—as control genes to normalizeCtrp5 gene expression.33 Sequences of the primers used can be ob-tained from the authors by request. Mean (�SEM) relative expressionlevels were calculated by analyzing at least three independent sampleswith replica reactions and presented on a scale that represents theexpression over the housekeeping gene Hgprt.

In Situ Hybridization

A cDNA clone, IMAGE: 876157, containing the full-length codingsequence of mouse Ctrp5 (1.3 kb) was used to generate probes for insitu hybridization (ISH; Fig. 1C). The Ctrp5 riboprobes (sense andantisense) were generated with a digoxigenin (DIG)-RNA labeling kit(Roche Diagnostics Corp., Indianapolis, IN). BlastN (National Centerfor Biotechnology, Bethesda, MD) analysis of the probe sequenceshowed no significant homology to other known genes except theCTRP5 sequence from different species. ISH was performed accordingto previously published protocols.34,35

Antibodies and Their Source

To raise antibodies against the CTRP5 protein, we selected twoantigenic peptides, 15GSPPLDDNKIPSLCPGH31 and 99SVPPRSAF-SAKRSESR114. These peptides showed no homology with any otherknown proteins and had 100% homology with the human andmouse sequences (Fig. 1D). Antibodies were raised in rabbitsagainst these two peptides and purified using protein G-agaroseaffinity column. Both antibody species showed similar results in

Western blot and immunohistochemical analyses. Monoclonal anti-ezrin antibodies (clone 3C12; Sigma-Aldrich, St. Louis, MO); mono-clonal anti-TIMP3 antibodies (Chemicon, Temecula, CA); monoclo-nal anti ZO-1 antibodies, anti-Xpress, anti-V5, and secondaryantibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 555(Invitrogen-Molecular Probes, Carlsbad, CA); anti-rabbit, and anti-mouse secondary antibodies conjugated to horseradish peroxidase(HRP; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were ob-tained from the commercial sources indicated.

Immunofluorescence Labeling of Tissuesand Cells

Immunostaining for CTRP5 protein was performed according to pub-lished procedures.19,36 The following dilutions of the primary antibod-ies were used: anti-CTRP5 (1:2000), anti-ezrin (1:4000), anti-TIMP3(1:2500), and anti ZO-1 (1:2000).

Madin-Darby canine kidney (MDCK) epithelial cells grown to con-fluence were fixed in absolute methanol at �10°C for 15 minutes andthen incubated for 1 hour in blocking solution containing 5% horseserum and 1% IgG-free BSA (Jackson ImmunoResearch Laboratories,West Grove, PA) in PBS (Sigma-Aldrich, St. Louis, MO), and furtherincubated for 1 hour at room temperature in primary antibody dilutedin PBS containing 0.1% BSA. After PBS washes, the cells were incubatedfor 1 hour at room temperature in a secondary antibody diluted as justdescribed and then were washed and mounted in anti-fade mountingmedium containing DAPI (ProlongGold; Invitrogen). Images were cap-tured using a confocal microscope (LSM510; Carl Zeiss Meditec, Inc.,Dublin, CA).

Immunoelectron Microscopy

The electron microscopic immunohistochemical analysis was per-formed as previously described,37 by using formalin-fixed retinal sec-tions from mouse and human (65 years of age) eyes embedded in resin(Unicryl; Electron Microscopy Sciences, Fort Washington, PA). Thinsections were collected on 200-mesh nickel grids. The sections wereblocked with 5% normal goat serum (Vector Laboratories, Burlingame,CA) and incubated in anti-CTRP5 affinity-purified antibodies (1:50 di-lution) at 4°C overnight, followed by incubation with a gold-conju-gated secondary antibody (1:10 dilution, 10-nm gold particles; Sigma-Aldrich). Sections were rinsed and poststained with uranyl acetate andlead citrate before viewing and photography with an electron micro-scope (JEM1200EX II; JEOL, Tokyo, Japan).

Generation of CTRP5 Constructs and Expressionin COS-7 Cells

Primers with BamHI and NotI restriction sites were designed to am-plify full-length coding regions of CTRP5 using the human IMAGEclone 5262504 as a template. The sequences of these primers areavailable from the authors on request. The full-length CTRP5 fragmentwas ligated into the pcDNA3B vector (Invitrogen) to generate a con-struct with a V5 tag at the C-terminal end (CTRP5-V5; Fig. 1E). AV5-tagged human mutant of CTRP5 (S163R) was generated by site-directed mutagenesis using a kit (Invitrogen) according to the manu-facturer’s protocol (Fig. 1F). All cloned DNA fragments were com-pletely sequence verified. COS-7 and MDCK cells were transfectedwith expression constructs or control vectors.38 Mock transfectionswere performed using transfection reagents only. After 48 hours oftransfection, cells and media were harvested for protein isolation.

Western Blot Analysis

Cell lysates were prepared in lysis buffer (50 mM Tris-HCl [pH 7.4],0.15 M NaCl, 1 mM EDTA, 0.1% Triton X-100, and 0.1% [wt/vol] SDS),containing protease inhibitor cocktail (Sigma-Aldrich). Total proteinextract was prepared from mouse whole eye, mouse liver, mouselungs, and human RPE-choroid in lysis buffer containing proteaseinhibitor cocktail (Complete; Roche, Indianapolis, IN).

5506 Mandal et al. IOVS, December 2006, Vol. 47, No. 12

Membrane and hydrophilic fractions of the proteins from trans-fected cells were isolated (Mem-PER Eukaryotic Membrane ProteinExtraction Kit; Pierce Biotechnology, Inc., Rockford, IL). In this pro-tocol, plasma membranes are readily purified from crude mixtures bythe technique of aqueous two-phase partition. The cocktail is incu-bated at 37°C to separate the hydrophobic proteins from the hydro-philic proteins through phase partitioning. When cultured mammaliancells are used for plasma membrane protein isolation, this procedure isreported to produce membrane fractions with more than 90% purity.39

All Western blot analyses were performed with reduced proteins (byadding 0.5% �-mercaptoethanol [BME] and 5 mM dithiothreitol [DTT] inSDS sample buffer) separated on 10% Tris-glycine gels except in somecases when samples were prepared with only SDS sample buffer. Forcompetition experiments, antibodies were preadsorbed with the specificpeptides for 1 hour at 37°C followed by Western blot analysis.

RESULTS

Spatial and Temporal Expression of the Ctrp5Gene in Mouse Tissue

Tissue Distribution of Ctrp5. Analysis of various tissuesfrom 33-week-old C57BL/6 mice revealed highest levels ofCtrp5 gene transcripts in the posterior segment of the eye,followed by the iris-ciliary body (Fig. 2A). Very low or minimallevels of Ctrp5 transcripts were detected in the mouse brain,retina, lens, liver, lungs, heart, spleen, kidney, skeletal muscle,skin, testes, and uterus (Fig. 2A). The quality of cDNA isolatedfrom retina and lens tissue expressing low amounts of Ctrp5was validated by measuring the expression of RPE- and CE-specific genes, Efemp1 and Timp3 to test for potential con-tamination from RPE and CE (data not shown).

Embryonic Expression of Ctrp5. Expression of this geneduring embryonic development was determined by qRT-PCR us-ing the RNA isolated from whole mouse embryos between E7 andE17. The presence of Ctrp5 transcripts was first observed at E11and continued up to E17 (Fig. 2B). Alterations observed in thelevel of Ctrp5 expression at different stages of embryonic devel-opment could be due to changes in the ratio of the tissue express-ing the gene to the total size of the embryo (Fig. 2B).

Expression in Postnatal Eye. In whole eye, the level ofCtrp5 was highest at P1 (P1E), gradually declined up to P5, andthen remained unchanged during further development up toP30 (Fig. 2C). We studied the expression of Ctrp5 in theiris-ciliary body and in the posterior segment, and abundantexpression of this gene was observed in both tissues up to 20months, the latest time point that we checked (data notshown). Further detailed analysis is necessary to investigate theage-related variation in expression of this gene in those tissuesconsidering the early-onset of abnormal lens zonules and thelate-onset of L-ORD in individuals carrying the CTRP5 muta-tion.

Cellular Pattern of Ctrp5 Expression. The specific tis-sues or cell types that express the Ctrp5 transcript were de-termined by ISH with a probe specific to the Ctrp5 sequence(Fig. 1C). In sections of eye and brain from pigmented mice at

FIGURE 1. Structure of CTRP5 genes and constructs used in this studyare presented by the following diagrams. (A) The genomic region ofthe human CTRP5 gene, which is homologous to the mouse Ctrp5gene. Black and colored boxes represent the exons. The F and Rarrows represent the region from which primers were designed forqRT-PCR analysis of Ctrp5. (B) CTRP5 transcript in which the wholeCTRP5 ORF lies in the 3� UTR of the MFRP gene (accession no.AJ862823, human; NM_145613, mouse; GenBank). (C) CTRP5 tran-script independent of MFRP (accession no.: NM_015645, human;DQ002398, mouse). Bottom red line: the region from which theprobes for ISH and Northern blot analysis were generated. (D) CTRP5protein with its defined domains. Ca and Cb are the regions fromwhich the peptide sequences were selected to raise antibodies. (E)CTRP5 protein with V5 tag at the C-terminal end. (F) The mutantCTRP5 protein (S163R) with V5 tag at the C-terminal end.

FIGURE 3. Localization of the Ctrp5 transcripts in the mouse embry-onic eye and brain by ISH. Expression of Ctrp5 transcripts in the mouseeye obtained by ISH at E14.5 (A, B, arrows), and at E18.5 (C, arrows),in the developing choroid plexus at E14.5 (Fig. 3D, arrows). (A�–D�)The corresponding sections hybridized with sense riboprobe thatserved as negative control samples. L, lens; I-CB, iris-ciliary body; cp,choroid plexus.

FIGURE 2. (A) Quantitative expression (�SEM) of Ctrp5 in differenttissues of adult mouse. (B) Expression of Ctrp5 (� SEM) during em-bryonic development at E7, E11, E15, and E17. (C) Expression profileof Ctrp5 (�SEM) during postnatal eye (P1E–P30E) development. LI,liver; LU, lungs; B, brain; I-CB, iris-ciliary body; PS, posterior segment ofeye including the RPE, choroid, and sclera; R, retina; L, lens; H, heart;SP, spleen; K, kidney; SM, skeletal muscle; S, skin; T, testis; U, uterus.

IOVS, December 2006, Vol. 47, No. 12 Impaired Secretion of Mutant CTRP5 5507

E14.5, Ctrp5 was detected (purple) in the layer of neural crestcells and cranial paraxial mesoderm that mark the anlage of theciliary body and iris stroma (Fig. 3A, arrows). At E18.5, Ctrp5expression was prominent in cells of the presumptive ciliarybody and iris (Figs. 3B, 3C, arrows). At these same embryonicages, expression was evident in the developing choroid plexuswithin the lateral ventricles of developing brain (Fig. 3D, ar-rows).

Between P3 and P15, the period of development and dif-ferentiation of the anterior chamber of the eye, the expressionof Ctrp5 is localized to ciliary processes (data not shown). Inthe eyes of 3-month-old albino BALBc mice with no pigmenta-tion in the RPE and ciliary body, significant levels of expressionof Ctrp5 were observed in the cells of the ciliary epitheliumand RPE.24

Distribution of CTRP5 Protein in Ocular Tissues

Characterization of Antibodies. We used affinity-purifiedpolyclonal antibodies raised against CTRP5 to detect proteinbands corresponding to the expected size of human and mouseCTRP5 (�25 kDa) in extracts of human RPE choroid andmouse eye (Fig. 4A; lanes 1 and 2, respectively) by Westernblot analysis. A second 37-kDa band was detected in both the

tissues (Fig. 4A; lane 1 and 2, respectively). Neither species ofCTRP5 was detected in mouse liver or lung extract (Fig. 4A,lanes 3 and 4, respectively). The 25-kDa band corresponds tothe expected size of CTRP5 and the additional 37-kDa bandmay represent CTRP5 proteins. A commercially available anti-body against CTRP5 protein was also reported to detect a37-kDa band (product no. 3571; ProSci Inc., Poway, CA). TheC1q group of proteins is known to exist in highly posttransla-tionally modified forms, and the 37-kDa band detected by theCTRP5 antibodies may represent a posttranslationally modifiedform of the CTRP5 protein.18 Anti-CTRP5 antibodies recog-nized bands of �29 kDa in extracts of COS-7 cells transfectedwith CTRP5-V5 and media in which these cells were grown(Fig. 4A, lanes 5 and 6, respectively). Anti-V5 antibodies rec-ognized bands of the same size when the blot containing lanes5 and 6 in Figure 4A was stripped and reprobed with V5antibodies (Fig. 4B, lanes 5�, 6�), indicating that both theantibodies recognize the same protein. Western blot analysis ofa set of duplicate blots with CTRP5 antibodies preadsorbedwith antigenic peptides indicated that the intensity of CTRP5protein bands was significantly reduced or the bands wereeliminated (Fig. 4C). When the blot containing lanes 5 and 6 ofFigure 4C was reprobed with anti-V5 antibodies, a strongpresence of V5-tagged CTRP5 protein was detected, furthersupporting the competitive blocking of the CTRP5 antibodieswith corresponding antigenic peptide (Fig. 4D, lanes 5�, 6�).Preimmune serum obtained from the same rabbit did not rec-ognize any proteins close to the size of the CTRP5 (Fig. 4E, 25or 37 kDa). The blot containing lane 5 in Figure 4E showed astrong presence of CTRP5 protein when reprobed with anti-V5antibodies (Fig. 4F, lane 5�). These observations indicate thatthe polyclonal peptide antibodies generated against CTRP5specifically recognize the CTRP5 protein.

Cellular Location of CTRP5 Protein. The expression pat-tern of CTRP5 protein in mouse embryonic and adult eye tissuewas examined by immunofluorescence microscopy. Consis-tent with the localization of the Ctrp5 transcript, the CTRP5protein was also detected in cranial paraxial mesodermal cellsthat developed into the ciliary body in the section of E14.5 eye(data not shown).

In the posterior segments of the adult mouse eye, CTRP5was observed in a hexagonal lattice on the lateral plasmamembranes of the RPE (Figs. 5A–C, arrowheads) and also at theRPE apical processes (Figs. 5B, 5E, arrows) and in the ganglioncells (Fig. 5A, arrowheads). The CTRP5 protein colocalizedwith ezrin (orange color; Figs. 5C–5E, arrows and arrowheads),a marker of the apical processes of the RPE, but not withTIMP3, a marker for Bruch’s membrane (data not shown).

Based on the qRT-PCR and ISH analyses, the other region ofthe eye expected to have significant expression of CTRP5protein is the ciliary body, a bilayer consisting of pigmentedand nonpigmented ciliary epithelial cells. Immunofluorescencemicroscopy showed that CTRP5 is localized to the apposedapical membrane surfaces of these pigmented and nonpig-mented cells (Fig. 5G, arrows). As the apical membranes ofpigmented and nonpigmented CE layers are close to eachother, it is difficult to determine whether CTRP5 is localized toone of them or both. Similar to the pattern observed in RPEcells, CTRP5 protein showed punctate distribution in CE.Antibodies raised against different epitopes showed a similarpattern of expression of CTRP5 in both RPE and ciliary body.In control experiments, no labeling was observed when theprimary antibodies were replaced by preimmune serum(Fig. 5F).

In the lens, the CTRP5 protein was distributed in apunctate pattern that appeared to be localized to the plasmamembrane of the lens epithelial cells (Figs. 5H–5K, arrows).These results indicate that the CTRP5 protein is associated

FIGURE 4. Western blot analysis using anti-CTRP5 antibodies. In allpanels: lane 1: human RPE-choroid; lane 2: mouse eye; lane 3:, mouseliver; lane 4: mouse lungs; lane 5: COS-7 cell lysate containing V5tagged CTRP5; and lane 6: media of COS-7 cells containing V5-taggedCTRP5 proteins. (A) Western blot analysis was performed with anti-CTRP5 antibodies. (B) Blot containing lanes 5 and 6 of (A) wasstripped and reprobed with anti-V5 antibodies. Lanes 5� and 6� are thesame as lanes 5 and 6 of (A). (C) Duplicate blot of (A) was probed withpreadsorbed anti-CTRP5 antibodies. (D) Blot containing lanes 5 and 6of (C) was stripped and reprobed with anti-V5 antibodies. Lanes 5� and6� are the same as lanes 5 and 6 of (C). (E) Blots probed withpreimmune serum. (F) Blot containing lane 5 of (E) was stripped andreprobed with anti-V5 antibodies. Right: molecular size (kDa).


FIGURE 5. Localization of CTRP5proteins in the adult mouse eye. Byimmunofluorescence microscopy,CTRP5 antibody (green) labeling wasdetected in the sections of mouseeye (A, arrowheads). Localization onthe hexagonal RPE membrane isshown by arrowheads (B, C) and onthe RPE apical processes by arrows(B, C). CTRP5 colocalization withezrin is shown in (C–E; arrows andarrowheads). In (A–C), apical label-ing of CTRP5 appeared to be differ-ential which is due to the variation inthe optical planes selected for cap-turing images with the confocal mi-croscope. CTRP5 immunolabeling(green) in the anterior segment ofthe same mouse eye section is shownin (G, arrows). When the anti-CTRP5antibodies were replaced with pre-immune rabbit serum, no labelingwas observed in the RPE (data notshown) and the ciliary bodies (F).(H–K) CTRP5 (green) localization tothe lens epithelia. Nuclei are stainedin red with propidium iodide, andthe boxed region in (I) is enlarged in(J) and (J) is enlarged in (K). IS, pho-toreceptor inner segment. Scale bar,50 �m. (L) CTRP5 labeling with theantibody (CTRP5b) raised againstpeptide b (Cb of Fig. 1) which issimilar to the labeling of the secondantibody (G) raised against peptide a(Ca of Fig. 1). CTRP5b antibodypreadsorbed with its peptide antigenand used for labeling mouse sectioncontaining ciliary body tissue (M).

FIGURE 6. CTRP5 localization as amembrane-associated protein. (A, ar-rows) Immunostaining of the RPEsection from albino mouse with anti-CTRP5 antibodies shows a punctatehexagonal localization of CTRP5 (B,green, arrows), and colocalizationwith ZO-1 protein on the lateralmembrane of the RPE (C, D, red,arrows, arrowheads). MDCK cellscolabeled with anti-CTRP5 (red) andanti-ZO-1 antibodies (E–G, green).Arrows: lateral membrane localiza-tions; arrowheads: apical membranelocalizations. MDCK cells transientlyproducing V5-tagged CTRP5 proteinwere labeled with anti-V5 antibodies(red). Arrows: membrane localiza-tion; arrowheads: intracellularand/or apical localization. Scale bar:50 �m.


with the plasma membrane. A significant reduction wasobserved in the immunoreactivity of CTRP5 antibodies inthe ciliary body tissue when the antibodies were pread-sorbed with antigenic peptides (Figs. 5L, 5M). Polarizeddistribution of other RPE expressed proteins has been re-ported previously.40

Further support of the plasma membrane association of theCTRP5 protein came from immunocytochemical analysis ofmouse RPE sections in which CTRP5 showed a hexagonal punc-tuate membrane localization (Fig. 6A, arrows). This pattern ofCTRP5 staining was colocalized with a known membrane-associ-ated tight junctional protein ZO-1 (Figs. 6B–6D; arrows for CTRP5and arrowheads for ZO-1). Subsequently, we tested the distribu-tion of CTRP5 in MDCK cells, prototypical epithelial cells thatform tight junctions and expresses both CTRP5 and ZO-1 proteins(tested by RT-PCR and Western blot analysis; data not shown).Immunocytochemistry of confluent MDCK cells with anti-CTRP5and anti-ZO-1 antibodies indicated that CTRP5 colocalizes withZO-1 on the lateral membranes at the site of tight junctionsbetween adjacent cells (Figs. 6E–6G, arrows). In addition, CTRP5was localized to the apical membranes of MDCK cells in a punc-tate pattern (Fig. 6E, arrowheads). When MDCK cells were trans-fected with V5-tagged CTRP5 protein and immunolabeled withanti-V5 (red) and anti-ZO-1 (green) antibodies, localization of theCTRP5 protein was found to be associated with plasma mem-brane (Fig. 6H, arrows), in addition to intracellular and/or apicallocalization (Fig. 6H, arrowheads). The transfection efficiency ofMDCK cells was found to be low.

When evaluated by immunoelectron microscopy, the anti-CTRP5 antibodies were found to label the plasma membranes ofboth the human (Fig, 7A) and mouse RPE (Fig. 7B). The highestconcentration of immunolabeling was observed at the apical sur-

face of the cells in the area where two adjacent cells juxtapose,possibly near the membrane junctions (Figs. 7A, 7B, arrows).Apical processes were also heavily labeled (Fig. 7A, arrows). Usinglight microscopy, we observed predominant CTRP5 localizationon apical membranes of CE (Fig. 5G) but could not resolvelocalization on lateral membranes. However, by immunoelectronmicroscopy we observed immunopositive labeling on the lateralmembrane between two adjacent pigmented cells of CE (Figs. 7C,human CE; 7D, mouse CE). Immunoelectron microscopy pro-vides confirmatory evidence for the localization of CTRP5 to theapical and lateral membranes of RPE and CE.

Existence of CTRP5 as a Secretory Protein andEffect of L-ORD Mutation on its Secretion

Immunohistochemical analysis of CTRP5 localization in the pre-vious section suggested association with the plasma membrane.The known primary structure of the CTRP5 protein contains asignal peptide at the N-terminal end, which suggests that it couldbe a secretary protein. CTRP5 is a member of the short-chaincollagens and the C1q-TNF superfamily of proteins. The TNFproteins are known to exist as type-II membrane proteins as wellas secreted proteins.41 Immunoelectron microscopy detected apattern of CTRP5 labeling in RPE and ciliary body tissues thatappeared to be secretory vesicles that were filled with CTRP5protein (Figs. 7A–D, arrowheads), suggesting its likely nature as asecretory protein.

To evaluate further the properties of CTRP5 protein, mem-brane and hydrophilic fractions of the lysate of COS-7 cellsexpressing the CTRP5-V5 were isolated separately, and themedia in which these cells are grown were collected. Immu-noblot analysis with anti-V5 antibodies detected the presenceof CTRP5-V5 fusion protein in the membrane and hydrophilicfractions of the transfected cell lysates as well as in the medium(Fig. 8A, lanes 1, 2, and 3). These results support that CTRP5exists not only as a membrane-associated protein but also as aprotein secreted into the medium.

When COS-7 cells were transfected with a S163R mutantconstruct of CTRP5, the expected 29-kDa band was observedin the cell extract (Fig. 8A, lane 4) but not in the media inwhich these cells were cultured (Fig. 8A, lane 5). This wasconsistent in multiple independent experiments and suggestedthat the processing or turnover of L-ORD mutant CTRP5 isaltered, which prevents its secretion and provides an impor-tant clue toward our understanding of the mechanism of dis-ease development.

By expressing the gC1q domain polypeptides of CTRP5,Hayward et al.,18 showed that gC1q peptides can form mul-timers. We expressed the complete CTRP5 protein with aC-terminal V5 tag, which is naturally processed and secreted(Fig. 8A, lanes 1–3). The Western blot analysis showed thepresence of monomeric protein in the cell extracts (Fig. 8A,lanes 1, 2). In addition, we observed a significant presence ofCTRP5 protein that did not enter into the gel but remained atthe top of the lane (Fig. 8A, lanes 1, 2). To investigate furtherthe higher-molecular-weight species of the CTRP5 protein inthe cell, in the media, and when it is mutated, the proteinextracts were subjected to two types of treatments: boilingwith only SDS, and boiling with SDS and reducing agents (BMEand DTT). Only monomeric (29 kDa) CTRP5 bands were de-tected under reduced conditions in the membrane fraction(Fig. 8B, lane 1), in the hydrophilic fraction (Fig. 8B, lane 3), inthe media (Fig. 8B, lane 5), and also in the fraction containingmutant CTRP5 (Fig. 8B, lane 7). With SDS alone, an additionalband of 50 kDa was observed in the wild-type protein fractionsthat may represent a dimeric form of the CTRP5 protein that ispartially resistant to detergent but not to the reducing agents(Fig. 8B, lanes 2, 4, 6; vertical arrows). The membrane and

FIGURE 7. Immunoelectron microscopic assignment of CTRP5 locationin human and mouse RPE and CE. Arrows: CTRP5 localization (black dots)in human (A) and mouse (B) RPE. Arrows: CTRP5 localization on themembrane between the pigmented cells of human CE (C) and mouse CE(D). The cluster of dots (arrowheads) in (A–D) may indicate the secretoryvesicle filled with CTRP5 proteins. Scale bar, 500 nm.


hydrophilic fractions of wild type CTRP5 protein seemed toform stronger higher order multimers as evidenced from thelanes having significant amount of proteins that could not enterinto the gel (Fig. 8B, lanes 1–4; horizontal arrow). Of note, thehigher-molecular-weight complex of CTRP5 was not observedin the culture medium, suggesting that the secretory form ofCTRP5 may exist as a monomer or dimer (Fig. 8B, lanes 5, 6;arrowheads). Monomeric mutant (S163R) CTRP5 was observedunder reducing conditions. In contrast to the wild type pro-tein, neither monomer nor dimer was detected under nonre-ducing conditions, and the mutant CTRP5 protein remained atthe top of the gel. These observations suggest that the wild-type CTRP5 may exist in different higher order hetero- orhomo-oligomeric complexes or posttranslationally modifiedforms and the nature of the mutant CTRP5 differs from thewild-type protein. Therefore, when mutant protein interactswith wild-type protein in the heterozygous condition, it mayalter the properties of the wild-type protein and that mayexplain the dominant negative phenotype of L-ORD. A detailedfurther investigation of the nature and function of these differ-ent forms of CTRP5 and their interaction may provide usefulinformation.

DISCUSSION

The LAZ and retinal degeneration observed in patients with theS163R mutation in CTRP5 show significant variation in age ofonset. Abnormal anterior lens zonules were observed aroundage 20, whereas retinal changes were observed in the fourth orfifth decade. Delayed dark adaptation was reported as an earlysymptom of retinal change, whereas loss of central vision,accumulation of drusen, and choroidal neovascular membrane(CNVM) were observed in the late stage of disease.42 Some ofthese patients also had glaucoma that developed at a latestage.24,42 We have established that Ctrp5 is expressed at veryearly stages of development, and continued expression is main-tained in adult mouse eyes. This protein exists in membrane-

bound and secreted forms and is expressed predominantly inRPE, CE, and lens, the tissues associated with the pathologicnature of L-ORD and abnormal lens zonules. The spatial andtemporal expression patterns of CTRP5 suggest that this pro-tein may play a critical role in the normal development andphysiology of these ocular tissues.

CTRP5 is a member of the C1q-tumor necrosis factor (TNF)superfamily, which is involved in processes as diverse as hostdefense, inflammation, cell differentiation, and apoptosis.41

C1q is the key subcomponent of the classic complement acti-vation pathway involved in innate immunity and IgG- or IgM-acquired immunity. C1q is also involved in other immunologicprocesses including clearance of apoptotic cells and phagocy-tosis of bacterial cells.43 It has been hypothesized that thedeposits commonly found in AMD between Bruch’s membraneand the RPE cell layer may act as a stimulus for local activationof the complement system. The accumulation of subretinaldeposits may lead to local ischemia and activation of RPEcells,44 causing them to release angiogenic stimuli that initiatechoroidal neovascularization, a common feature of AMD. Pro-teins involved in the complement cascade and retinal autoan-tibodies were detected as constituents of drusen.14,45–47 Re-cent findings suggest that immunologic factors are involved inthe pathogenesis of age-related macular degeneration (AMD).Members of the complement system, complement factor H(CFH), C-reactive protein (CRP), factor B and complementcomponent 2 are associated with age-related macular degener-ation.10,12,48 Late-onset retinal degeneration associated with amissense mutation in the CTRP5 gene shares several pheno-typic features with AMD, including subretinal deposits, drusen,and CNV membranes. Therefore, it is plausible that CTRP5with the C1q domain also may participate in the pathogenesisof macular degeneration by activating the complement system.

The CTRP5 protein also has sequence and structural similari-ties to collagens VIII and X, which have diverse functions includ-ing cell adhesion and polarity.41,49,50 We observed the polarizedexpression of CTRP5 protein on the plasma membranes of ciliary

FIGURE 8. Characterization of CTRP5 proteins by Western blot analysis. (A) A blot probed with anti-V5 antibodies. Lane 1: wild-type CTRP5-V5,membrane fraction; lane 2: wild-type CTRP5-V5, hydrophilic fraction; lane 3: wild-type CTRP5-V5 media; lane 4: mutant CTRP5-V5 lysates; lane5, mutant CTRP5-V5 media. (B) Wild-type CTRP5-V5 from membrane, hydrophilic, media, and mutant CTRP5-V5 from whole-cell extract weresubjected to two conditions: nonreducing (boiling with SDS) and reducing (boiling with SDS, BME, and DTT). Lanes 1, 2: membrane fraction; lane3, 4: hydrophilic fraction; lanes 5, 6: media; and lanes 7, 8: mutant CTRP5-V5. Left: molecular sizes (kDa).


epithelial and retinal pigment epithelial cells (Fig. 5). The local-ization of membrane-associated CTRP5 to cell junctions and itscolocalization with the tight junction protein ZO-1 (Fig. 6) sug-gests a potential role for this protein in cell–cell communication,cell adhesion, or cell polarity. This predicted role is consistentwith the observation that the CTRP5 mutation results in abnormaladhesion between RPE and Bruch’s membrane, leading to sub-retinal deposits.18 Similarly, mutant CTRP5 expressed in the cili-ary and lens epithelium affects normal zonule anatomy and prob-ably affects the general physiology of these tissues, resulting inaltered ocular pressure with age. Additional studies are needed toestablish the relationship between CTRP5 mutations and glau-coma in L-ORD and the effects of this mutation on the function ofthe ciliary body and lens.

The TNF ligands are synthesized as type-II membrane proteins,and some of these are proteolytically cleaved and secreted. Thesoluble forms can act as either agonists or antagonists of mem-brane-bound forms.41 The TNF family of proteins plays a majorrole in inflammation, and the TNF ligands are found to shiftlocation from intracellular to membrane surface or are secretedwhen cells are exposed to proinflammatory agonists.51,52 Likeother C1q-TNF superfamily proteins, CTRP5 protein also havemembrane-associated and secreted forms (Figs. 5–8). The signif-icant presence of Ctrp5 transcripts in the choroid plexus, anintraventricular secretory epithelial formation that secrets thecerebrospinal fluid (Fig. 3D), also supports its secretory nature.53

We and others have shown that the CTRP5 interacts withMFRP.54,55 MFRP is a transmembrane protein that contains extra-cellular CUB domains, and it is thought that the CUB domain ofMFRP interacts with C1q domain of CTRP5, suggesting recruit-ment of exogenous (secretory) CTRP5 to the membrane throughthis interaction. MFRP also has an intracellular frizzled or CRDdomain and frizzled-containing proteins are shown to be involvedin various cellular signaling pathways, including Wnt signaling.56

Therefore, it is likely that either secreted, membrane-associated,or intracellular CTRP5 responds to various cellular stimuli orinflammatory signals that also involve MFRP. The absence ofsecreted CTRP5 due to the S163R mutation may affect theseresponses of RPE cells to different stimuli and generate localinflammation that leads to the formation of drusen, subretinaldeposits, and choroidal neovascularization. Impairment of secre-tion due to mutations in proteins that are constitutively secretedhas been well documented.57,58 Hayward et al.18 suggested thatthe mutant CTRP5 forms higher order complexes, which may beone of the causes of impaired secretion. Future studies designedto elucidate the functional role of CTRP5 and the fate of themutant protein are likely to advance our understanding of themechanism underlying retinal degeneration in patients withCTRP5 mutation.

Acknowledgments

The authors thank Austra Liepa for maintenance of the mice andtechnical assistance, Mitchell Gillett for tissue preparation for histo-logic analysis, Bruce Donohoe for assistance in microscopy, and LauraKakuk and Hemant Khanna for technical assistance and constructivediscussion.

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