A novel four transmembrane spanning protein, CLP24 A hypoxically regulated cell junction protein Jonathan Kearsey, Silvere Petit, Catherine De Oliveira and Fabien Schweighoffer ExonHit Therapeutics, Paris, France A novel hypoxically regulated intercellular junction protein (claudin-like protein of 24 kDa, CLP24) has been identified that shows homology to the myelin protein 22/epithelial membrane protein 1/claudin family of cell junction proteins, which are involved in the modulation of paracellular per- meability. The CLP24 protein contains four predicted transmembrane domains and a C-terminal protein–protein interaction domain. These domains are characteristic of the four transmembrane spanning (tetraspan) family of pro- teins, which includes myelin protein 22, and are involved in cell adhesion at tight, gap and adherens junctions. Expression profiling analyses show that CLP24 is highly expressed in lung, heart, kidney and placental tissues. Cellular studies confirm that the CLP24 protein localizes to cell–cell junctions and co-localizes with the b-catenin adherens junction-associated protein but not with tight junctions. Over-expression of CLP24 results in decreased adhesion between cells, and functional paracellular flux studies confirm that over-expression of the CLP24 protein modulates the junctional barrier function. These data therefore suggest that CLP24 is a novel, hypoxically regu- lated tetraspan adherens junction protein that modulates cell adhesion, paracellular permeability and angiogenesis. Keywords: adherens; angiogenesis; claudin; DATAS; hyp- oxia. Endothelial and epithelial cell sheets line all the cavities of the body and are linked by specialized adhesive junctions that provide a selective barrier for the passage of plasma proteins, circulating cells, water and/or solutes. Two types of adhesive junctions, namely tight junctions and adherens junctions, play a major role in controlling this paracellular barrier function [1,2]. Tight junctions are required at the apical face of the cell junctions in order to maintain a selective paracellular barrier. Adherens junctions are located below the tight junction at the apical junction and are required for tight junction formation and the maintenance of barrier integrity. Adhesion junctions also contribute to vascular morphogenesis in endothelial cells [3]. Adherens junctions undergo changes following a reduction in oxygen levels (hypoxia), in order to allow the initiation of an angiogenic response that requires increased vascular per- meability, endothelial cell proliferation and migration [3,4]. ln addition to providing barrier and morphological func- tions, these cell junctions are targeted by a variety of signaling processes involved in normal physiology (cell growth and differentiation) and pathology [2,5]. All cell junctions, including tight and adherens junctions, are composed of transmembrane proteins that show structural, but often little sequence, homology. These transmembrane proteins comprise four transmembrane domains, together with extracellular loop regions that interact adhesively with complementary molecules in adja- cent cells to form the junction. This structural family of proteins (tetraspan proteins) includes connexins/innexins and peripheral myelin protein 22 (PMP22)/epithelial membrane protein 1 (EMP1)/claudin members, which are involved in gap and tight junctions, respectively [6,7]. Claudins have been shown to be one of the structural adhesive components of tight junctions [2]. PMP22 was originally isolated as a growth arrest specific transcript (Gas 3), induced following serum deprivation of fibroblasts [8]. PMP22 was subsequently shown to be a major component of myelinated fibers in the peripheral nervous system and associated with tight junctions [6,9,10]. Mutations of the gene encoding PMP22 cause Charcot–Marie–Tooth disease Type 1A, Dejerine–Sottas syndrome and hereditary neuro- pathy [11]. Both Charcot–Marie–Tooth disease and Dejerine–Sottas syndrome are sensorineural peripheral polyneuropathies, the most commonly inherited disorder of the peripheral nervous system [12]. Sequence similarity and co-localization studies show that PMP22 is a tight junction associated transmembrane protein in both neur- onal and non-neuronal cells [6]. Thus, the PMP22 gene product is a dual-function protein, involved in both tight junctions adhesion and cell proliferation. This study describes the identification and characteriza- tion of a novel transmembrane junctional protein with structural homology to the tetraspan family of proteins. This gene was identified in a screen for hypoxically regulated genes in endothelial cells that could provide angiogenic therapeutic targets. Sequence analyses show that this novel Correspondence to F. Schweighoffer, ExonHit Therapeutics, 65 Bd Masse ´ na, 75013 Paris, France. Fax: + 33 1 53 94 77 04, Tel.: + 33 1 53 94 77 69, E-mail: fabien.schweighoffer@exonhit.com Abbreviations: CLP24, claudin-like protein of 24 kDa; EGFP, green fluorescent protein; EMP1, epithelial membrane protein 1; EST, expressed sequence tag; HKG, housekeeping gene; HMEC, human microvascular endothelial cells; PDZ, protein–protein interaction domain; PMP22, myelin protein 22; TMHMM, transmembrane hidden Marckoff model: ZO-1, Zona Occluden-1. Note: a website is available at http://www.exonhit.com/ (Received 11 February 2004, revised 19 April 2004, accepted 26 April 2004) Eur. J. Biochem. 271, 2584–2592 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04186.x protein is most closely related to PMP22, a claudin cell junction-associated family member. This novel gene prod- uct has therefore been called claudin-like protein of 24 kDa (CLP24). The protein product of the CLP24 gene contains four transmembrane spanning domains together with a C- terminal protein–protein interaction (PDZ) domain. CLP24 is most highly expressed in lung, heart, kidney and placenta, showing little similarity to the expression patterns of other PMP22/EMP1/claudin members. However, this is not unexpected as distinct tissue-distribution profiles are observed for all the PMP22/EMP1/claudin family members, which allows regulation of paracellular specificity between different endothelial cell types [2,13]. Expression studies using recombinant CLP24-enhanced green fluorescent protein (EGFP) demonstrated that CLP24 localizes to cell junctions. Co-localization studies were performed using recombinant CLP24-EGFP together with antibodies against either the cell adhesion molecule, b-catenin, or the tight junction Zona Occluden-1 (ZO-1) associated protein. These experiments demonstrated that CLP24 was localized to regions of the membrane associated with adherens junctions, but with little association to the tight junction components at the apical face. Over-expres- sion of CLP24 increased paracellular permeability across an endothelial monolayer, confirming that CLP24 acts as a structural component in cell junctions. These data therefore suggest that a novel, although distantly related, member of the claudin/PMP22 family of proteins has been identified. However, CLP24 appears to be distinct from many PMP22/ EMP1/claudin members, in that CLP24 influences paracel- lular permeability through its interaction with adherens, rather than tight junction components. Materials and methods Cell culture Immortalized human microvascular endothelial cells [HMEC-1; CDC (Centre for Disease Control and Preven- tion), Atlanta, GA, USA) were cultured in MCDB-131 medium (Sigma) supplemented with 15% (w/v) heat- inactivated fetal bovine serum (Invitrogen), 2 m ML -glut- amine (Invitrogen), 100 UÆmL )1 penicillin (Invitrogen), 100 lgÆmL )1 streptomycin (Invitrogen), 10 ngÆmL )1 human recombinant EGF (Invitrogen) and 1 lgÆmL )1 hydrocorti- sone. MDCK (a canine normal kidney cell line; ATCC) and Calu-6 (a human, lung carcinoma cell line; ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with Glutamax (Invitrogen), 10% (w/v) heat-inactivated fetal bovine serum, 100 UÆmL )1 penicillin, and 100 lgÆmL )1 streptomycin. For hypoxic treatments, cells were grown in an atmosphere of 3% O 2 in an IG750 incubator (Jouan, France), or in the presence of 100 lgÆmL )1 desferrioxamine. The HMEC-1 and Calu-6 cell lines were chosen as they both express CLP24 mRNA (data not shown for Calu-6). Differential analysis of transcripts with alternative splicing (DATAS) This technology has been previously described by Sch- weighoffer et al. [14]. Briefly, first-strand cDNAs were reverse transcribed from HMEC-1 total RNA (treated either with normoxia or hypoxia) using the Superscript II RT kit (Invitrogen) and an anchored biotinylated oligo- dT 25 . cDNAs were then treated with RNAse I followed by proteinase K and phenol/chloroform extraction. mRNA from normoxic HMEC-1 cells and cDNA from hypoxic HMEC-1 cells were mixed at a 1 : 1 molar ratio and precipitated using sodium acetate and ethanol. The recip- rocal experiment with mRNA from hypoxic HMEC-1 cells and cDNA from normoxic HMEC-1 cells was also performed. The pellet was redissolved in 80% formamide/ 0.1% SDS, and heteroduplexes were allowed to form by denaturation at 85 °C and gradual cooling to 40 °C. Heteroduplexes were then isolated using streptavidin beads (no. 112.06; Dynal) and the single-stranded RNA released by the action of RNAse H (no. 18021-071; Invitrogen). Residual cDNA was removed by extraction using strept- avidin beads and treatment with DNAse I. The isolated single-stranded RNA molecules were reverse transcribed using the Superscript II RT kit and random hexamer primers. The cDNAs were amplified by PCR (using the DOP PCR methodology [15]) with six different anchored degenerate primers, purified using spin columns and cloned (pCR II-TOPO vector, no. 45-0640; Invitrogen). Following transformation and plating onto LB (Luria–Bertani) plates containing ampicillin (100 lgÆmL )1 ), recombinant bacterial colonies were isolated and the cloned cDNA was sequenced. This library was designated HMEC-EHT1. cDNA array generation Each cDNA was amplified from a bacterial colony by performing PCR amplifications using SP6-T7 primers. Each PCR product (600 lL final volume) was concentrated and visualized on an agarose gel before spotting. The PCR products were arrayed on 8 · 12 cm nylon filters that were spotted in duplicate using a QPix robot (GenePix 4000; Axon) with the nonredundant human HMEC-EHT1 cDNA library. Arrayed nylon filters were stored at 4 °C until use. Probe labelling and hybridizations To make a single probe, 50 ng of total RNA (from normoxic or hypoxic HMEC cells) was used to generate double-stranded cDNA with the MMLV RT (Invitrogen) using an anchored oligo-dT primer [5¢-CCTATTGTTTGT GTGTGTCC-3¢ RN1-oligo(dT) 25 ]. PCR amplification, using an RN1 primer, was performed and the amplified products were measured by a fluorometric method (Pico- Green quantitation kit; Interchim). The cDNAs were labeled with redivue, stabilized [ 33 P]dCTP[aP] (Rediprime II, Amersham) and hybridized according to the manufac- turer’s instructions. After hybridization, the filters were quantified by scanning densitometry using a Biorad Mole- cular Imager and evaluated using biostatistical analyses. cDNA cloning and construct generation Total RNA from HMEC-1 and Calu-6 cell lines were reverse transcribed using the multiscribe RNA polymerase and random hexamer primers (Archive kit; Applera). PCR Ó FEBS 2004 CLP24 – a hypoxically regulated cell junction protein (Eur. J. Biochem. 271) 2585 amplification of the full-length open reading frame of CLP24 was achieved using a proofreading DNA poly- merase (Platinum Pfx DNA polymerase; Invitrogen), and by using the sense primer TTTGAATTCCCACCATG ACCGTGCAGAGACTC (containing the ATG start codon of CLP24, together with a Kozak sequence and an EcoRI restriction site), and the antisense primer, AAAG GATCCAGGCATGGTGACTCCACGTA (containing a BamHI restriction site). PCR conditions were: 94 °Cfor 30 seconds, 58 °C for 30 seconds, 68 °C for 1 min, for 35 cycles. The PCR product was cloned in the pCRII TOPO vector (Invitrogen) and sequenced using an automatic sequencer (Applied Biosystems model 3100). A C-terminal EGFP-CLP24 fusion construct was then generated by cloning the CLP24 open reading frame into vector pEGFP- N1 (BD Biosciences) using the EcoRI and BamHI restric- tion sites. Recombinant HMEC-1/CLP24-EGFP and MDCK/CLP24-EGFP cells were established after transfec- tion with the full-length CLP24 cDNA in pEGFP-N1 (BD Biosciences) and selection of stably transfected cells with 150 lgÆmL )1 and 400 lgÆmL )1 geneticin, respectively. Bioinformatic analysis Bioinformatic analyses were performed using Genetics Computer Group (GCG) software, including BLAST ,the multiple sequence alignment tool CLUSTALW ,andthe pairwise alignment tool GAP (BLOTSUM 55). In addition, membrane protein prediction ( TMHMM ), SCANSITE and PRO- SITE software have been used to characterize CLP24 [16,17]. PCR The expression of CLP24 in different tissues and cells was determined by PCR. The cDNA from a number of different human tissues (Clonetech), together with human epithelial [Calu-6 (a lung carcinoma cell line), RCC4 (a renal carcinoma cell line), NTERA-2 (a neuronal precursor epithelial cell line), H1299 (a nonsmall cell carcinoma cell line), HepG2 (a hepatocellular carcinoma cell line) and the breast cell lines MDA-MD231, MDA-MB-435, MCF7, BT549 and T-47D (ATCC) and endothelial (HMEC and HUVEC) cell lines, were characterized. PCR was performed, using standard PCR conditions [Amp- litaq (0.075 UÆmL; Applied Biosystems), anti-taq Ig (0.075 UÆmL; Invitrogen), 15 m M MgCl 2 ,1· buffer 1 (Applied Biosystems), dNTPs (0.2 m M , Invitrogen) and 0.5 m M of each primer]. Thirty-five cycles of PCR were performed using an annealing temperature of 60 °C. The primers selected for the specific expression of CLP24 were 5¢-CCCTAGCAGCGTCGGCT-3¢ and 5¢-CGTTGCGCT AACCAGGAAAG-3¢, which give an amplicon size of 1002 bp. PCR products were visualized following separation on a 1% agarose gel. Real-time quantitative RT-PCR CLP24 differential gene expression has been monitored by quantitative real-time RT-PCR (Q-RT-PCR) using Taq- Man technology. In brief, total RNA from HMEC-1 and Calu-6 cells were isolated using the Trizol kit (Invitrogen), then 5 lg of total RNA was reverse transcribed using the multiscribe RNA polymerase and random hexamer primers (Archive kit; Applera). Real-time PCR was performed on an ABI Prism 7700 Sequence Detector machine (Applera) andanalyzedusing SDS , version 1.6.3, software. The primers (Life Technologies, Inc.) and TaqMan probes (Eurogentec) for the quantification of the CLP24 transcripts were designed using the primer design software, PRIMER EXPRESS (Applera) except for human b-actin where commercially available assay reagents were used (Applera). Initial experi- ments were performed to define a housekeeping gene (HKG) whose level remained constant following hypoxic treatment. Two HKG were tested (b-actin and b2-micro- globulin) and two separate primer sets were tested for b-actin. The b-actin primers were purchased from Applied Biosystems (b-actin control reagent 401846). b2-microglob- ulin primers were: 5¢-GGACTGGTCTTTCTATCTCTTG TACTAC and 3¢-AGTCACATGGTTCACACGGC. Only minor variation across the HKG primer sets was observed between treated and nontreated samples, and the b-actin HKG was selected for further experimental analyses. Primer sequences for CLP24 were: forward primer, 5¢-CGTTTACTGTTATGTCGGTCATAT-3¢ and reverse primer, 5¢- GTTGCGCTAACCAGGAAAGC-3¢; probe sequence: CLP24,5¢-TGTCGTGGGCCAACCTCGTT CTG-3¢. Specificity of the PCR amplification was confirmed on an agarose gel. The PCR reactions were carried out using TaqMan universal PCR master mix (Applera). For CLP24, both primers were used at 150 n M and the probe at 100 n M . The TaqMan PCR reaction conditions were: 2 min at 50 °C, 10 min at 95 °C, then 40 cycles each of 15 s at 95 °C and 1 min at 60 °C. Each plate contained triplicates of the test cDNA templates, a standard curve for the individual amplicon, and no-template controls for each reaction mix. Standard curves were generated for each amplicon in order to determine PCR amplification efficiency. The Ct value is defined as the number of PCR cycles required for the fluorescence signal to exceed the detection threshold value. The fold difference (F) was calculated and normalized to the levels for the established HKG, b-actin, according the following formula that is a derivation of that defined by Plaffl et al. [18]: F ¼ f½ð1 þ E target Þ ÀCt target =ð1 þ E HKG Þ ÀCt HKG  conditionA g= f½ð1 þ E target Þ ÀCt target =ð1 þ E HKG Þ ÀCt HKG  conditionB g where: F, fold induction; E target , PCR amplification effi- ciency of the target gene; Ct target, threshold cycle of the PCR amplification of the target gene; E HKG , PCR ampli- fication efficiency of the housekeeping gene; Ct HKG, threshold cycle of the PCR amplification of the housekeep- ing gene; condition A, normoxic; condition B, hypoxic. Northern blot analysis A human Northern blot (no. 7780-1; Clontech ) was used and a 32 P-labeled random-primed DNA probe was gener- ated using the full-length CLP24 transcript and the Strip-EZ RNA Ambion kit (no. 1360). The blot was prehybridized in ULTRAhyb hybridization buffer (no. 8670, Ambion) for 2 h at 68 °C and the probe denatured at 95 °Cfor 10 min. The blot was hybridized overnight at 68 °C, washed as described by the manufacturer, and exposed to 2586 J. Kearsey et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Kodak Biomax MS film with a transcreen-HE intensifying screen. Immunofluorescence confocal microscopy The following antibodies were obtained commercially: mouse against b-catenin (no. 610153; BD Biosciences Pharmingen) and rabbit against ZO-1 (no. 61-7300; Zymed). Cells were plated on coverslips, rinsed twice with NaCl/P i (phosphate-buffered saline) and subsequently fixed in 4% paraformaldehyde for 20 min. After washing with NaCl/P i , cells were permeabilized with 0.1% Triton for 5 min. Then, cells were blocked with Powerblock (universal blocking agent; Biogenex) for 15 min to minimize nonspe- cific binding. Cells were incubated with primary and secondary antibodies in blocking buffer for 30 min, fol- lowed by three washes with NaCl/P i after each incubation. The primary antibody was visualized using the appropri- ate Cy3-conjugated anti-mouse or anti-rabbit secondary antibodies [Cy3-conjugated F(ab¢) 2 fragments; Jackson Immuno Research). Finally, the cells were mounted with fluorescence mounting medium (Fluosave, Molecular Probes) and viewed with a confocal imaging system (model: Leica TCS SPZ and software: LCS Software, Leica, Heidelburg, Germany). Output wavelengths were 488, 543 and 633 nm, EGFP fluorescence was imaged at 510 nm. A cross-sectional image (x–z) through the junction was computer generated in order to assess the localization of proteins within tight or adherens junctions. Phase-contrast images were taken using a Nikon Elispe TE300 microscope together with a Nikon CoolPix 990 camera. Paracellular tracer flux measurement of a 3 kDa FITC-labeled dextran molecule For paracellular tracer flux assay, MDCK cells expressing either EGF alone or the CLP24-EGF fusion protein were seeded onto a cell culture 8 lm pore size insert (no. 3097; Becton Dickinson), at a density of 10 5 cells per filter, and cultured for 5–6 days [19]. To measure paracellular flux, Dulbecco’s modified Eagle’s medium, containing 0.5 mgÆmL )1 of 3 kDa FITC-labeled dextran (Molecular Probes), was added to the apical compartment and aliquots from the basal compartment were collected over an 8 h period (37 °C). The amount of FITC-dextran that diffused from the apical to the basolateral side of the cellular layer was measured by using a fluorimeter (Fluoroscan Ascent FL; Thermolabsystem). The permeability coefficient was calculated using the following formula: DF=Dt ¼ PÃS, where DF/Dt is the rate of the increase of the fluorescence signal in the basal chamber, P is the permeability (cmÆmin )1 ) coefficient, and S is the surface of the insert. Results Hypoxic regulation of CLP24 The aim of this study was to identify novel genes regulated by hypoxia. Macroarray expression profiling studies were performed using RNA isolated from hypoxic (3% O 2 ,16h) and normoxic HMEC-1 in order to identify differentially regulated cDNA transcripts within the HMEC-EHT1 cDNA library. One of the differentially expressed tran- scripts identified in this study was found to be homologous to an expressed sequence tag (EST) sequence that represents a novel uncharacterized gene (GenBank accession number: NM_024600). This transcript was upregulated in HMEC-1 cells treated with hypoxia. An induction of 2.93-fold (± 0.85) was observed in hypoxic (3% O 2 ,16h)HMEC- 1 cells compared to normoxic HMEC-1 cells. Further confirmation of hypoxic regulation was obtained through independent experiments using the chemical agent desfer- rioxamine (100 l M for 16 h) to induce a hypoxic response in HMEC-1 cultures. This treatment resulted in a 3.16-fold (± 0.30) induction of CLP24 in hypoxic cells compared to sham-treated HMEC-1 cells. Independent validation of the macroarray data for CLP24 was obtained using real-time quantitative PCR on total RNA from normoxic and hypoxic HMEC-1 and Calu-6 (lung carcinoma) cell lines. Gaseous hypoxia treat- ment induced a strong upregulation of CLP24 expression in the endothelial cell line, HMEC-1, and the epithelial cell line, Calu-6, with an induction factor of 5.3- and 10.4-fold, respectively, thus confirming the hypoxic induction of the CLP24 transcript in two different cell types. Bioinformatic characterization of CLP24 RT-PCR was used to clone the full-length open reading frame of human CLP24, using cDNA from the HMEC-1 and Calu-6 human cell lines. Cloning and sequencing of independent clones from both these cell lines revealed the insertion of a cytidine residue within the open reading frame of CLP24 (position 1102) compared to the sequence deposited in GenBank (NM_024600). This extra base was found in every amplified cDNA from the two cell lines and bioinformatic analysis confirmed the presence of this extra cytidine residue in both the human genomic sequence (accession numbers AC046159 and AC096995) and in all homologous Homo sapiens ESTs analyzed. The insertion of this cytidine residue results in a frameshift in the protein coding sequence and a divergent C-terminal sequence compared with NM_024600. The correct cDNA and amino acid sequence of CLP24 isshowninFig.1. The full-length human CLP24 cDNA is 1871 nucleotides in length, containing a 226-amino acid open reading frame (621–1301) with a calculated molecular mass of 24.54 kDa and a predicted pI of 8.10. An in-frame stop codon is present 21 nucleotides upstream of the methionine start codon, and a kozak consensus sequence is also present (Fig.1).Theuseofmembraneproteinpredictionsoftware ( TMHMM ) [17] predicted the CLP24 protein to be composed of four alpha-helical transmembrane domains. The TMHMM analyses also revealed that the first extracellular loop of CLP24 is longer than the second and that CLP24 contains only a very short N-terminal cytoplasmic tail, but a longer C-terminal tail (Fig. 1). Furthermore, motif-scanning ana- lysis (scansite.mit.edu [20]), revealed a PMP22/EMP1/ claudin homologous domain within the CLP24 sequence (Fig. 1B) and a potential class 1 PDZ protein–protein interaction motif in the last 10 amino acids of the CLP24 protein [20]. A glycosylation site within the second Ó FEBS 2004 CLP24 – a hypoxically regulated cell junction protein (Eur. J. Biochem. 271) 2587 extracellular loop of CLP24 is also predicted. An amino acid comparison between PMP22 and claudin family members showed only a weak homology with CLP24; however, this low level of sequence homology is characteristic of a number of PMP22/EMP1/claudin family members, including VAB- 9 and MP20 [21,22]. Even though the amino acid sequence of CLP24 is only distantly related to that of PMP22/EMP1/ claudin, the predicted structure of the CLP24 protein shows a number of similarities to this tight junction protein family that support the notion that CLP24 is a cell junction- associated protein. The PMP22/EMP1/claudin family all have four transmembrane segments, most family members contain a C-terminal PDZ domain-interacting motif and the first extracellular loop is larger than the second and is believed to bridge the extracellular space (Fig. 2) [17,23,24]. These structural motifs are all present within CLP24 and, together with the observation that CLP24 is expressed in both endothelial and epithelial cell lines, provides further support for the suggestion that CLP24 is a novel member of the PMP22/EMP1/claudin four transmembrane junctional protein family; thus it has been designated CLP24 for claudin-like protein of 24 kDa. Comparison of rattus, murine, gallus, porcine and bovine ESTs reveals a high level of conservation of CLP24 nucleic acid sequence between species. The translated protein sequences were aligned using CLUSTALW software (Fig. 3). The rat, mouse and chicken sequences were found to be 89%, 87% and 81% identical to that of human, respectively. CLP24 tissue-specific expression The tissue distribution of CLP24 was characterized by Northern blot analyses (Fig. 4) and showed the presence of a 1.9kb transcript in lung, heart, kidney and placenta. aaaaaacaaccatttcctctctgctgagagccagggaaggcgagctctgc gcacacgggcgtccctgcagcagccactctgctttccaggaccggccaac tgccctggaggcatccacacaggggcccaggcagcacagaggagctgtga acccgctccacaccggccaccctgcccggagcctggcactcacagcaggc cggtgctaaggagtgtggcgcgggctcgactcccactgctgccggcctcc cgagtgactctgttttccactgctgcaggcgagaagaggcacgcgcggca caggccggcctccgcttcccgggaagacggcgcactcctggccctgggtt cttgctgctgcccaccctctgctccctgggatgggccccgaggcgagcag cttcagcacaggcctggccctgctccaggtgcaggaaggaggataaggcc gggccgagaggcggcacacctggaccatcccatgggcctccgcccgcgcc gccccgaggatgagtggtgatgtcctctagccacccctagcagcgtcggc tctccctggacgtgcggccgcggactgggacttggctttctccggataag cggcggcaccggcgtcagcgATGACCGTGCAGAGACTCGTGGCCGCGGCC MTVQRLV A A A GTGCTGGTGGCCCTGGTCTCACTCATCCTCAACAACGTGGCGGCCTTCAC V L V A L V S L I L N N V A A F T CTCCAACTGGGTGTGCCAGACGCTGGAGGATGGGCGCAGGCGCAGCGTGG S N W V C Q T L E D G R R R S V G GGCTGTGGAGGTCCTGCTGGCTGGTGGACAGGACCCGGGGAGGGCCGAGC L W R S C W L V D R T R G G P S CCTGGGGCCAGAGCCGGCCAGGTGGACGCACATGACTGTGAGGCGCTGGG P G A R A G Q V D A H D C E A L G CTGGGGCTCCGAGGCAGCCGGCTTCCAGGAGTCCCGAGGCACCGTCAAAC W G S E A A G F Q E S R G T V K L TGCAGTTCGACATGATGCGCGCCTGCAACCTGGTGGCCACGGCCGCGCTC Q F D M M R A C N L V A T A A L ACCGCAGGCCAGCTCACCTTCCTCCTGGGGCTGGTGGGCCTGCCCCTGCT T A G Q L T F L L G L V G L P L L GTCACCCGACGCCCCGTGCTGGGAGGAGGCCATGGCCGCTGCATTCCAAC S P D A P C W E E A M A A A F Q L TGGCGAGTTTTGTCCTGGTCATCGGGCTCGTGACTTTCTACAGAATTGGC A S F V L V I G L V T F Y R I G CCATACACCAACCTGTCCTGGTCCTGCTACCTGAACATTGGCGCCTGCCT P Y T N L S W S C Y L N I G A C L TCTGGCCACGCTGGCGGCAGCCATGCTCATCTGGAACATTCTCCACAAGA L A T L A A A M L I W N I L H K R GGGAGGACTGCATGGCCCCCCGGGTGATTGTCATCAGCCGCTCCCTGACA E D C M A P R V I V I S R S L T GCGCGCTTTCGCCGTGGGCTGGACAATGACTACGTGGAGTCACCATGCTG A R F R R G L D N D Y V E S P C * Agtcgcccttctcagcgttccatcgatgcacacctgctatcgtggaacag cctagaaaccaagggactccaccaccaagtcacttcccctgctcgtgcag aggcacgggatgagtctgggtgacctctgcgccatgcgtgcgagacacgt gtgcgtttactgttatgtcggtcatatgtctgtacgtgtcgtgggccaac ctcgttctgcctccagctttcctggttagcgcaacgcggctccacgacca cacgcacttcagggtggaagctggaagctgagacacaggttaggtggcgc gaggctgccctgcgctccgctttgctttgggattaatttattctgcatct gctgagaggggcaccccagccatatcttacactttggtaaagcagaaaac caggaaaattttcttaaaatatccacaatattccttgagtgagtcagaat ctatagccggttagtgatggtttcaggcagaatcgtgttcgtgtctgttt tgctcgattcctttcctaagtta aataaa tgcaagcctctgaacttctgt ctataaaaaaaaaaaaaaaaa 1 51 101 151 201 251 301 351 401 451 501 551 601 1 651 11 701 28 751 45 801 61 851 78 901 95 951 111 1001 128 1051 145 1101 161 1151 178 1201 195 1251 211 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 50 100 150 200 250 300 350 400 450 500 550 600 650 10 700 27 750 44 800 60 850 77 900 94 950 110 1000 127 1050 144 1100 160 1150 177 1200 194 1250 210 1300 226 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1871 PMP22 1 MLLLLLSIIVLHVAVLVL.LF VSTI-VSQWIVGNG HATDLWQNC 42 ml+lLl iivlh+a L LF Vsti qW v +LW +C Consensus mlvlLlgiivlhiawviL.Lf.VsTiPtdqWkvsdyvgdniiTaaaasaGLWrnC m v l + +a v L L V t W s GLWr+C CLP24 1 MTVQRLVAAAVLVALVSLILnnVAAF-TSNWVCQTLED GRRRSVGLWRSQ 49 B A Fig. 1. Nucleotide sequence and deduced amino acid sequences of human claudin-like protein of 24 kDa (CLP24). (A) Uppercase letters represent the coding sequence, lowercase letters represent 5¢-and 3¢-untranslated regions (UTR). The CLP24 sequence has an inser- tion of a C at position 1102 compared with NM_024600 (bold). This insertion within the protein coding sequence results in a divergent amino acid sequence as compared with NM_024600. The potential N-glycosylation site is marked with a circle. The protein trans- membrane domains are underlined. The protein–protein interaction domain-interacting motif is boxed. The italic region in the 3¢-UTR is a consensus polyadenylation sequence. *, Stop codon. (B) Align- ment of the conserved motifs of the CLP24 and myelin protein 22 (PMP22) amino acid sequence against the PMP22/epithelial mem- brane protein 1 (EMP1)/claudin family consensus sequence using motif-scanning analysis (http://scansite.mit.edu). Uppercase letters show conserved amino acids between PMP22/EMP1/claudin family members. N C Extracellular loop Claudins 10-21 aa CLP21 6 aa Extracellular loop Claudins 41-55 aa CLP24 68 aa Intracellular loop Claudins 21-42 aa CLP24 35 aa PDZ domain Fig. 2. Membrane folding model of claudin-like protein of 24 kDa (CLP24) and myelin protein 22 (PMP22)/epithelial membrane protein 1 (EMP1)/claudin family members. Schematic representation of the tetraspan structure of the CLP24 protein compared with PMP22/ EMP1/claudin family members. Both CLP24 and PMP22/claudin members show four transmembrane domains, a C-terminal protein– protein interaction domain (PDZ), and a characteristic extracellular loop structure. Approximate sizes (number of amino acids) of the extracellular and intracellular domains are given for the claudin/ PMP22 family and CLP24. 2588 J. Kearsey et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Transcripts were also detected in thymus, spleen and liver, but at lower levels. A similar tissue distribution was observed by RT-PCR using a panel of cDNAs from human tissues (multiple tissue cDNA panel; Clonetech). This analysis showed CLP24 to be expressed in testis, spleen and ovary, but not in prostate (data not shown). CLP24 is expressed in a number of different tissues that contain epithelial cell tight junctions, including lung and kidney, together with tissue containing high levels of blood vessel endothelial cells, including the placenta and lung. Further confirmation of endothelium- and epithelium- specific expression was sought through expression profiling of human cell lines. As described above, CLP24 is expressed within the HMEC-1 and primary endothelial cells (HUVEC). RT-PCR expression analysis showed the CLP24 gene to be expressed in a number of the human epithelial cell lines, including the Calu-6 lung carcinoma cell line, the RCC4 renal carcinoma cell line and the NTERA-2 (NT2) neuronal precursor epithelial cell line. CLP24 is not, however, expressed ubiquitously in all epithelium-contain- ing tissues and cell lines. No CLP24 expression was detected in epithelial cell lines, including the nonsmall cell carcinoma cell line, H1299, the hepatocellular carcinoma, HepG2, or the breast carcinoma cell lines, MDA-MB-231, MDA-MB- 435, MCF7, BT-549, or T-47D (data not shown).These data therefore demonstrate that CLP24 is expressed within specific epithelial and endothelial cell populations, which is consistent with that expected for a cell junction-associated protein. Localization of exogenously expressed CLP24 To further assess whether CLP24 is a member of the PMP22/EMP1/claudin family, the subcellular localization of CLP24 was determined using recombinant CLP24 fused to a green fluorescent protein (CLP24-EGFP). Transfection and translation of this plasmid construct was used to monitor the subcellular location of CLP24 in a number of cell lines, including MDCK and HMEC-1. Analysis of stably transfected clones demonstrated that the CLP24- EGFP fluorescence signal was localized to intercellular junctions (Fig. 5A), in a similar manner to b-catenin and ZO-1. To confirm the association of CLP24 with tight junction components, co-localization experiments, using the tight junction ZO-1 or adherens junction b-catenin markers, were performed. MDCK cells were stably transfected with a CLP24-EGFP fusion construct and immunocytochemistry was performed using either anti-ZO-1 or anti-b-catenin Ig. Figure 5A shows good co-localization of CLP24-EGFP with ZO-1 and b-catenin to the intercellular junctions. b-catenin and ZO-1 are, however, associated with different compartments of the intercellular junction, namely the adherens and tight junctions, respectively. The tight junction is found at the apical face, whereas the adherens junction is more basal. The view shown in Fig. 5A looks vertically through the intercellular junction and is therefore unable to distinguish tight from adherens junction components because the tight junction is directly above the adherens junction. More precise localization studies were therefore H. sapiens 1 MTVQRLVAAAVLVALVSLILNNVAAFTSNWVCQTLEDGRRRSVGLWRSCWLVDRTRGGPSPGARAGQVDAHDCEALGWGSEAAGFQESRGTVKLQFDMMR R. norvegicus 1 MTVQKLVATAVLVALVSLILNNAAAFTPNWVYQTLEDGRKRSVGLWKSCWLVDRGKGGTSPGTRTGQVDTHDCEVLGWGSESAGFQESRGTVKLQFDMMR M. musculus 1 MTLQKLVATAVLVALVSLILNNAAAFTPNWVYQTLEDGRKRSVGLWKSCWLVDRGKGVTSPGTRTGQVDTHDCEVLGWGSESAGFQESRGTVKLQFDMMR G. gallus 1 MTVQKLVATAVLVALVSLILNNAAAFTPNWVYQTLEDGRKRSVGLWKMCWLAERSRAGASTSSRHGQGEERECEALGWGSESAGFQESRSTVKLQFDMMR S. scrofa 1 MTVXKVVATAVLVALVSLVLNNVAALTPNWVYQTLEDGRRRSVGLWRSCWLLDRXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGFQESRGTVKLQFDMMR B. taurus 1 DVRDCEALGWGSEAAGFQESRGTVKLQFDMMR H. sapiens 101 ACNLVATAALTAGQLTFLLGLVGLPLLSPDAPCWEEAMAAAFQLASFVLVIGLVTFYRIGPYTNLSWSCYLNIGACLLATLAAAMLIWNILHKREDCMAP R. norvegicus 101 ACNLVATAALAVGQITFILGLTGLPLMSPESQCWEEAMAAAFQLASFVLVIGLVTFYRIGPYTNLSWSCYLNIGACLLATLAAAMLIWNILHRREDCMAP M. musculus 101 ACNLVATAALVVGQITFILGLTGLPLMSPESQCWEEAMAAAFQLASFVLVIGLVTFYRIGPYTNLSWSCYLNIGACLLATLAVAMLIWNILHRREDCMAP G. gallus 101 ACNLIATVALTAGQLIFVLGLVEIPIISQDTQWWEEAIAAVFQLASFVLVIGLVTFYRIGPYTNLSWSCYLNIGACLLATLAAAILIWNILHRREDCMAP S. Scrofa 101 ACNLVATAALAAGQLTFVLGLTGLPLMSPDSQCWEEAMAAAFQLASFVLVIGLVTFYRIGPYTNLSWSCYLDIGACLLATLAAAMLIWNVLHRREDCMAP B. taurus 35 ACNLVATAALAAGQLTFVLGLTGLPLLSPDAQCWEEAMAAAFQLASFVLVIGLVTFYRIGPYTSLSWSCYLNIGACLLATLAAAMLIWNVLHRREDCTAP H. sapiens 201 RVIVISRSLTARFRRGLDNDYVESPC R. norvegicus 201 RVIVISRSLTARFRRGLDNDYVESPC 88.9% identity M. musculus 201 RVIVISRSLTARFRRGLDNDYVESPC 88.0% identity G. gallus 201 RVIVISRTLTARFRRGLENDYVESPC 81.4% identity S. Scrofa 201 RVIVISRSLAARFRRGLDXXXXXXXX B. taurus 135 RVIVISRSLTARFRRGLDNDYVESPC Fig. 3. Alignment of the amino acid sequences of mammalian claudin-like protein of 24 kDa (CLP24) orthologues. Alignment of the amino acid sequences of human CLP24 with rat, mouse and chicken orthologues, together with partial amino acid sequences of porcine and bovine ortho- logues. The human sequence is 89%, 87% and 81% identical to the rat, mouse and chicken sequences, respectively. ske. muscle 2.4 1.35 Brain colon heart PBL thymus spleen kidney liver smallintes. placenta lung kb Fig. 4. Northern blot analysis of human claudin-like protein of 24 kDa (CLP24). Human multiple tissue Northern blots (Clonetech) were probed with a radiolabelled DNA fragment of CLP24. ske. muscle, skeletal muscle; small intes., small intestine; PBL, peripheral blood leukocytes. Ó FEBS 2004 CLP24 – a hypoxically regulated cell junction protein (Eur. J. Biochem. 271) 2589 performed using a computer-generated cross-section (x–z scan) through the cell junction (Fig. 5B). The merged images of the x–z scans (Fig. 5B) show that the ZO-1 protein signal is located at the apical face of the MDCK monolayer, as expected within polarized MDCK cells, whilst the CLP24-EGFP signal is localized throughout the intercellular junctions of the MDCK monolayer and shows co-localization with the adherens junction protein b-catenin. To further confirm this initial observation, co-localization studies were performed using an anti-claudin 1 tight junction-associated antibody. As observed with ZO-1, claudin 1 was localized at the apical surface and did not localize with EGFP-CLP24 (data not shown). CLP24 mediates cell junction interactions Overexpression of CLP24-EGFP in MDCK cells results in the expression of CLP24 at cellular junctions; however, MDCK cells expressing CLP24 display a different mor- phology to the nontransfected cells (Fig. 5C). The expres- sion of the CLP24-EGFP protein led to the disappearance of the typical cobblestone structure that results from the adhesion of confluent MDCK cells. In mixed cultures containing both CLP24-EGFP expressing cells (detectable by fluorescence microscopy) and nonexpressing cells, the nontransfected cells organized themselves into cobblestone structures, whereas the cells expressing the CLP24–EGFP protein displayed a more fibroblastic morphology, charac- teristic of a decrease in cellular adhesion. Overexpression of CLP24 alters cellular permeability Studies were performed to characterize the potential effect of CLP24 on the paracellular barrier function. Paracellular flux measurements were undertaken across a confluent monolayer of cells expressing CLP24-EGFP, compared to those obtained using control cell lines, and showed a markedly higher paracellular flux of a 2 kDa FITC-dextran tracer molecule than MDCK control cells (Fig. 6A). The observed total permeability coefficient of MDCK/CLP24- EGFP monolayers was threefold higher than those of MDCK or MDCK/EGFP monolayers (Fig. 6B). This demonstrates that the CLP24 protein is able to modulate junctional barrier function and shows both structural and functional properties that are consistent with CLP24 being a novel PMP22/EMP1/claudin family member. Discussion Angiogenesis, the growth of new blood vessels out of pre- existing capillaries, is fundamental to many physiological and pathological processes, such as cancer, ischemic diseases and chronic inflammation. Hypoxia is one of the physio- logical signals that promote angiogenesis. During this process, adherens junctions are involved in the control of vascular permeability and in vascular remodeling [25–27]. Regulation of proteins involved in adherens junctions is essential in the detachment of endothelial cells from the vessel wall and invasion into the underlying tissues, which are, in turn, essential for new vessel formation [3]. The identification of CLP24 as a claudin-related protein, which is a hypoxically regulated adherens junction component that is able to influence vascular permeability, is an important observation that documents routes through which adherens junctions may be regulated during normal pathology and disease. A CLP24-EGFP ZO-1 overlay CLP24-EGFP β ββ β-catenin overlay B CLP24-EGFP β ββ β-catenin overlay CLP24-EGFP ZO-1 overlay C Phase contrast CLP24-EGFP Fig. 5. Localization of claudin-like protein of 24 kDa (CLP24). MDCK cells were stably transfected with full-length CLP24 fused to green fluorescent protein (EGFP). The transfected clones (CLP24- EGFP in green), still mixed with nontransfected cells, were stained by indirect immunofluorescence for either Zona Occluden-1 (ZO-1) or b-catenin(bothinred).(A)CLP24–EGFPexpressionislocalizedat intercellular borders, as observed for the ZO-1 and b-catenin junction proteins. (B) Co-localization studies, using an x–z scan, demonstrate that CLP24 is targeted to the intracellular junctions and co-localizes with b-catenin (top), but not with ZO-1 (bottom). Bar: 10 lm. (C) Morphological differences observed between co-cultures of CLP24-EGFP, expressed and not expressed in MDCK cells. Expres- sion of CLP24-EGFP results in an altered morphology compared to the cells not expressing CLP24 and a loss of the cobblestone structure that results from the formation of epithelial cell junctions. 2590 J. Kearsey et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Bioinformatic characterization suggested that CLP24 is a member of the PMP22/EMP1/claudin family, which are tight junction associated proteins. However, recent studies have shown that VAB-9, a PMP22/EMP1/claudin family member from Caenorhabditis elegans (nematode), is invol- ved in adherens junctions [21]. Therefore, co-localization experiments were performed to more precisely assess the location of CLP24 expression at the apical cell junction. These studies showed that CLP24 co-localizes with adherens junction protein b-catenin, but not with the ZO-1 tight junction protein. In agreement with the fact that endothelial cell interac- tions have to be reorganized during vessel formation, over- expression of CLP24 induced morphological changes in MDCK cells that are characteristic of a decreased adhesion between cells. A similar phenotype has also been observed for the over-expression of Z0-1 adherens junction trunca- tion mutants, which results in disruption of the cobblestone structure to that of a fibroblastic morphology [28]. Func- tional studies showed that CLP24 is able to influence paracellular permeability, as observed for other PMP22/ EMP1/claudin members. Taken together, these results indicate that CLP24 is involved in cell–cell interactions through adherens, rather than tight, junctions. The expression pattern of CLP24 is distinct from other claudin family members, showing expression in lung, kidney heart and placenta. It is noticeable that CLP24 is expressed within the heart, which is unusual for the claudin family members. This observation suggests that CLP24, like PMP22/EMP1/claudin family members, has a tissue-speci- fic distribution, which is linked to cell junction specificity [23,29]. Both endothelial and epithelial cells possess tight junction and adhesion structures to seal intercellular spaces. Here we show, using RT-PCR, that CLP24 is expressed in endothelial cells and in only a restricted population of epithelial cell lines. These observations suggest that CLP24 has a specific barrier function within different cell types and confirm the observations of others which show that complex interplay between different claudin family members is required to control paracellular permeability. Work by Leach [26,30] has shown dynamic regulation of both adherens and tight junction components during angiogenesis. This, together with the observation that claudin family members are deregulated during hypoxia and cancer [31–35], suggests that CLP24 could be also implicated in both normal and tumor angiogenesis. Knockout experiments in mice show that loss of the adenomatous polyposis coli (APC) or b-catenin results in reduction of cell–cell adherens junctions adhesion [36] and the b-catenin–APC complex has been shown to have a role in the proliferation and migration of vascular endothelial cells during neovascularization. As claudin family members interact with b-catenin, it can be therefore envisioned that CLP24 is involved in b-catenin signaling, and that up-regulation of CLP24 upon hypoxia might participate to the pro-antigenic deregulation of this cascade. Hypoxic stimulation results in the induction of vascular endothelial growth factor (VEGF) and endothelial hyperpermeability. Both tight junction and adherens junction molecules have been shown to influence paracellular permeability. Differ- ential expression of claudin family members results in the different permeability properties observed in different epithelial and endothelial membranes [23], whilst loss of b-catenin results in decreased cell–cell adhesion and increased paracellular permeability [25]. The identification of CLP24 as a novel adherens junction component that is induced by hypoxia and is able to reduce adhesion, thereby increasing intracellular permeability, provides additional insight into the understanding of how hypoxic stimuli induce morphological changes in endothelial cells required for an angiogenic response. A valuable additional insight into the functional role of CLP24, and a clearer under- standing of its therapeutic potential, will be provided by further characterization of CLP24 in pathologies including cardiovascular disease, neurological disorders and tumori- genesis. Acknowledgements We are grateful for the support of Prof. P. 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Biochem. 271) Ó FEBS 2004 . * Agtcgcccttctcagcgttccatcgatgcacacctgctatcgtggaacag cctagaaaccaagggactccaccaccaagtcacttcccctgctcgtgcag aggcacgggatgagtctgggtgacctctgcgccatgcgtgcgagacacgt gtgcgtttactgttatgtcggtcatatgtctgtacgtgtcgtgggccaac ctcgttctgcctccagctttcctggttagcgcaacgcggctccacgacca cacgcacttcagggtggaagctggaagctgagacacaggttaggtggcgc gaggctgccctgcgctccgctttgctttgggattaatttattctgcatct gctgagaggggcaccccagccatatcttacactttggtaaagcagaaaac caggaaaattttcttaaaatatccacaatattccttgagtgagtcagaat ctatagccggttagtgatggtttcaggcagaatcgtgttcgtgtctgttt tgctcgattcctttcctaagtta aataaa tgcaagcctctgaacttctgt ctataaaaaaaaaaaaaaaaa 1 51 101 151 201 251 301 351 401 451 501 551 601 1 651 11 701 28 751 45 801 61 851 78 901 95 951 111 1001 128 1051 145 1101 161 1151 178 1201 195 1251 211 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 50 100 150 200 250 300 350 400 450 500 550 600 650 10 700 27 750 44 800 60 850 77 900 94 950 110 1000 127 1050 144 1100 160 1150 177 1200 194 1250 210 1300 226 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1871 PMP22. of a 1.9kb transcript in lung, heart, kidney and placenta. aaaaaacaaccatttcctctctgctgagagccagggaaggcgagctctgc gcacacgggcgtccctgcagcagccactctgctttccaggaccggccaac tgccctggaggcatccacacaggggcccaggcagcacagaggagctgtga acccgctccacaccggccaccctgcccggagcctggcactcacagcaggc cggtgctaaggagtgtggcgcgggctcgactcccactgctgccggcctcc cgagtgactctgttttccactgctgcaggcgagaagaggcacgcgcggca caggccggcctccgcttcccgggaagacggcgcactcctggccctgggtt cttgctgctgcccaccctctgctccctgggatgggccccgaggcgagcag cttcagcacaggcctggccctgctccaggtgcaggaaggaggataaggcc gggccgagaggcggcacacctggaccatcccatgggcctccgcccgcgcc gccccgaggatgagtggtgatgtcctctagccacccctagcagcgtcggc tctccctggacgtgcggccgcggactgggacttggctttctccggataag cggcggcaccggcgtcagcgATGACCGTGCAGAGACTCGTGGCCGCGGCC MTVQRLV. T GCGCGCTTTCGCCGTGGGCTGGACAATGACTACGTGGAGTCACCATGCTG A R F R R G L D N D Y V E S P C * Agtcgcccttctcagcgttccatcgatgcacacctgctatcgtggaacag cctagaaaccaagggactccaccaccaagtcacttcccctgctcgtgcag aggcacgggatgagtctgggtgacctctgcgccatgcgtgcgagacacgt gtgcgtttactgttatgtcggtcatatgtctgtacgtgtcgtgggccaac ctcgttctgcctccagctttcctggttagcgcaacgcggctccacgacca cacgcacttcagggtggaagctggaagctgagacacaggttaggtggcgc gaggctgccctgcgctccgctttgctttgggattaatttattctgcatct gctgagaggggcaccccagccatatcttacactttggtaaagcagaaaac caggaaaattttcttaaaatatccacaatattccttgagtgagtcagaat ctatagccggttagtgatggtttcaggcagaatcgtgttcgtgtctgttt tgctcgattcctttcctaagtta aataaa tgcaagcctctgaacttctgt ctataaaaaaaaaaaaaaaaa 1 51 101 151 201 251 301 351 401 451 501 551 601 1 651 11 701 28 751 45 801 61 851 78 901 95 951 111 1001 128 1051 145 1101 161 1151 178 1201 195 1251 211 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 50 100 150 200 250 300 350 400 450 500 550 600 650 10 700 27 750 44 800 60 850 77 900 94 950 110 1000 127 1050 144 1100 160 1150 177 1200 194 1250 210 1300 226 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1871 PMP22