abc50, a novel human atp-binding cassette protein found in tumor necrosis factor-α-stimulated...

ABC50, a Novel Human ATP-Binding Cassette Protein Found inTumor Necrosis Factor-a-Stimulated Synoviocytes

Manon Richard,* Regen Drouin,† and Andre D. Beaulieu*,1

*Laboratoire de Recherche sur l’Arthrite et l’Inflammation, Department of Medicine, Faculty of Medicine, Centre Hospitalier deL’Universite Laval, 2705 Boulevard Laurier, Sainte-Foy, Quebec, Canada G1V 4G2; and †Unite de Recherche en GenetiqueHumaine et Moleculaire, Research Center, CHUQ, Pavillon Saint-Francois d’Assise and Division of Pathology, Department

of Medical Biology, Faculty of Medicine, Universite Laval, Sainte-Foy, Quebec, Canada

Received May 26, 1998; accepted July 16, 1998

We have used the recently developed technique ofdifferential display polymerase chain reaction to seekfor new genes modulated by tumor necrosis factor-a(TNF-a) in cultured synoviocytes. One PCR fragmentwas shown to correspond to a new gene that wasmapped by high-resolution fluorescence in situ hy-bridization to band 6p21.33. The cDNA of this gene wascloned, and the deduced amino acid sequence re-vealed consensus motifs for the nucleotide bindingfolds of the ATP-binding cassette (ABC) family of pro-teins. However, a hydropathy curve showed that thepolypeptide does not contain the transmembrane do-mains that are typical of the subfamily of ABC trans-porters and are associated with transporter/channelfunctions. The new gene, called ABC50, is the firsthuman and mammalian ABC protein found to lacktransmembrane domains. Homology with some yeastABC proteins suggests that ABC50 codes for a newhuman ribosomal protein involved in translation ofmRNA. It could therefore play a role in the enhance-ment of protein synthesis that follows TNF-a treat-ment of synoviocytes and thus participate in the in-flammatory processes mediated by this cytokine.Furthermore, since TNF-a also modulates the expres-sion of MHC class I genes, and these genes are knownto map to 6p21.33, it is hypothesized that ABC50 andMHC class I are part of the same chromatin expressiondomain. © 1998 Academic Press

INTRODUCTION

Rheumatoid arthritis (RA) is characterized by in-flammation of the joints and destruction of cartilageand bone (Arend and Dayer, 1995; Feldmann et al.,1996; Firestein and Zvaifler, 1997; Koch et al., 1995).

The cytokine tumor necrosis-a (TNF-a) plays an impor-tant role in RA since it appears to be at the top of acascade of cytokines that induce inflammatory and de-structive effects on the joints (Arend and Dayer, 1995;Feldmann et al., 1996; Koch et al., 1995; Maini et al.,1995). The importance of TNF-a is outlined by thepositive results obtained in clinical trials on the use ofantibodies against TNF-a (Arend and Dayer, 1995;Feldmann et al., 1996; Koch et al., 1995; Maini et al.,1995) and of soluble TNF-a receptors (Arend andDayer, 1995; Moreland et al., 1997) for the treatment ofRA. The TNF-a-stimulated fibroblast-like synoviocytesare responsible for the production of many of the cyto-kines and enzymes involved in inflammation and tis-sue destruction (Arend and Dayer, 1995; Firestein andZvaifler, 1997; Koch et al., 1995).

The family of ATP-binding cassette (ABC) proteins iswidespread among both prokaryotes and eukaryotes(Higgins, 1992). All of the known mammalian and hu-man ABC proteins have transmembrane domains andare responsible for the transport of a variety of mole-cules across biological membranes (Decottignies andGoffeau, 1997; Higgins, 1992; Tusnady et al., 1997).Among these proteins, many have a great impact onhuman health (Decottignies and Goffeau, 1997; Tus-nady et al., 1997). Examples are MRP and MDR-1,which cause multidrug resistance in cancer patients byparticipating in drug secretion (Chen et al., 1986; Za-man et al., 1994); TAP1 and TAP2, which form a com-plex involved in antigen presentation and associatedwith many diseases of autoimmune nature (Decottig-nies and Goffeau, 1997; Hill and Ploegh, 1995; Kelly etal., 1992); and CFTR, a chloride channel involved incystic fibrosis (Harris, 1992; Riordan et al., 1989).

To understand the effects of TNF-a on synoviocytesbetter, we undertook studies to search for new genesmodulated by TNF-a in synoviocytes. We used differ-ential display polymerase chain reaction (DDPCR) (Li-ang and Pardee, 1992) to produce cDNA fragmentscorresponding to TNF-a-modulated genes. In thisstudy, we describe a new gene that codes for a polypep-

Sequence data from this article have been deposited with theDDBJ/EMBL/GenBank Data Libraries under Accession No.AF027302.

1 To whom correspondence should be addressed. Telephone: (418)654-2299. Fax: (418) 654-2126. E-mail: [email protected].

GENOMICS 53, 137–145 (1998)ARTICLE NO. GE985480

1370888-7543/98 $25.00

Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

tide that, on the basis of its unique structural features,corresponds to a new type of human and mammalianABC protein.

MATERIALS AND METHODS

Preparation and TNF-a treatment of synoviocytes. Synovialmembranes were obtained from both healthy donors and patientswith RA during the course of surgical interventions for carpal tunnelsyndrome. Pieces of synovial tissues were incubated overnight at37°C in 5% CO2 and collagenase (Life Technologies, Gaithersburg,MD) used at a concentration of 4 mg/ml in PBS. Culture medium(minimum essential medium; ICN Pharmaceuticals, Inc., CostaMesa, CA) supplemented with 2 g/L sodium bicarbonate, 50 U/mlpenicillin, 50 mg/ml streptomycin, 2 mM glutamine, 0.05% B-mer-captoethanol (Life Technologies), and 10% characterized fetal calfserum (HyClone Laboratories, Inc., Logan, UT) was added. Disheswere incubated for 24 h. The pieces of synovial membranes werediscarded, and individual cells that remained on the surface of theculture dish were cultured at 37°C in 5% CO2. Cells were passed with0.05% typsin–EDTA (Life Technologies). After three passages, theremaining cells were considered to be pure fibroblast-like synovio-cytes.

For TNF-a stimulation, dishes containing cells at 85 to 95% con-fluency were treated with recombinant human TNF-a (Knoll Phar-maceuticals, Whippany, NY) at a concentration of 103 units/ml for 2to 48 h.

RNA extraction and Northern blot. Untreated and TNF-a-stimu-lated synoviocytes were harvested with 0.05% trypsin–EDTA andwashed with phosphate-buffered saline. Total synoviocyte RNA wasprepared using the TRIzol Reagent (Life Technologies) following therecommended protocol.

For Northern blots, 10 mg of total RNA from untreated and stim-ulated cells was migrated on agarose gels containing formaldehyde(Sambrook et al., 1989) and vacuum transferred (Sambrook et al.,1989) using a vacuum blotting unit (Pharmacia Biotech AB, Upp-sala, Sweden) on Hybond N membranes (Amersham Internationalplc, Little Chalfont, England).

DNA extraction and Southern blot. Plasmid DNA extractionswere performed using a standard protocol (Ish-Horowicz and Burke,1981). The preparations were migrated on agarose gels and trans-ferred onto Hybond N1 membranes by capillary transfer (Sambrooket al., 1989).

Northern and Southern hybridizations. Probe N4G6a is a 706-bpPCR fragment obtained by differential display PCR (described be-low) that corresponds to bases 2436 to 3141 in Fig. 3. Probe 7/59 is a288-bp PCR product obtained with the primers 59-CATCCTCCT-CATCACTG-39 and 59-GAGAGAGCACGAGACCC-39. The PCR con-ditions were as described below for the production of the enrichedlibrary. The probe starts at position 147 and ends at position 434 inFig. 3. Probe 8/59 was produced by a SacI (Promega Corp., Madison,WI) digestion of clone 8. It contains 40 bp originating from pBlue-script, followed by 327 bp corresponding to positions 59 to 385 in Fig.3. Probe G3PDH is a 579-bp PCR product obtained with the primers59-ACCAGCGCTGCTTTTAACTCT-39 and 59-CAGTAGAGGCAGG-GATGATGTTCT-39 (DDBJ/EMBL/GenBank Accession No. M17851).Probe IL-8 is a 236-bp EcoRI/PstI digestion of the IL-8 cDNA (Ac-cession No. U54995). All the probes were cut from a low-melting-point agarose gel (Life Technologies) using a standard protocol (Sam-brook et al., 1989) and used without further purification for labeling.

Probes were labeled by random priming (Prime a Gene LabelingSystem, Promega Corp.), using [a32P]dCTP (Amersham Interna-tional plc), and hybridized to the membranes using a standard pro-tocol (Sambrook et al., 1989). Membranes used for Fig. 2 were pur-chased from Clontech Laboratories, Inc. (Palo Alto, CA) andcontained 2 mg of poly(A)1 RNA from various tissues. They werehybridized according to the recommended protocol. All membraneswere exposed on X-OMAT AR films (Eastman Kodak Company,Rochester, NY).

Differential display PCR. Differential display PCR was per-formed as recommended by Liang and Pardee (1992). Total RNAfrom synoviocytes, either untreated or stimulated with TNF-a for18 h, was used. Briefly, RNA was treated with DNase to eliminateDNA contamination using the MessageClean kit (GeneHunter Corp.,Nashville, TN). Total RNA was then reverse transcribed to cDNAand amplified. The PCR amplifications were performed with theRNAmap kits A and B (GeneHunter Corp.) using AmpliTaq DNApolymerase (Perkin–Elmer, Foster City, CA) and [35S]dATP fromAmersham International plc. Products of the DDPCR were migratedon a 6% polyacrylamide gel and exposed overnight to an X-OMAT ARfilm. Bands corresponding to modulated fragments were cut out fromthe dried gel, eluted, and reamplified using the same primer pairs.Reamplified PCR products were hybridized to Northern blots ofstimulated and untreated synoviocytes to assess their modulation bythe TNF-a treatment.

Cloning of PCR fragments. The PCR fragments were ligated tovectors PCR2 or PCR2.1 using the TA Cloning Kit from InvitrogenCorp. (San Diego, CA) following the recommended protocol. Compe-tent cells of Escherichia coli strain DH5a were prepared according tothe protocol of Hanahan and transformed with the ligation productsby heat shock (Hanahan, 1983). Bacterial colonies were screened forpositive clones by hybridization.

Sequencing of cDNA. Cloned cDNA fragments were produced asdescribed above and sequenced by the dideoxy-mediated chain-ter-mination method (Sanger et al., 1977) using the T7 polymerasesequencing kit (Pharmacia Biotech AB). Various vector-derived andinternal primers were used. Sequences were migrated on 6% poly-acrylamide gels (Sambrook et al., 1989) and exposed to Kodak BI-OMAX MR films.

Cloning of the cDNA using a homemade library. We used anavailable homemade neutrophil library for the cloning of the cDNA ofthe new gene. The preparation of the library will be describedelsewere (D. Heitz et al., manuscript in preparation). Briefly, neu-trophils were isolated from human blood and stimulated with granu-locyte–macrophage colony-stimulating factor. Poly(A)1 RNA fromthese cells was used for reverse transcription using both oligo(dT)and random primers. The cDNA fragments were cloned in pBlue-script and transformed into E. coli SURE bacteria (StratageneGmbH, Heidelberg, Germany). Each screening was made on 5 to 8 3105 colonies by standard methods (Sambrook et al., 1989). Hybrid-izations were performed as described above.

Preparation of a library enriched for fragments of the new gene. Aplasmid DNA extract from a pool of clones of the neutrophil librarywas amplified by PCR using the gene-specific primer 59-CATCCTC-CTCATCACTG-39 and either one of the two vector primers: universalprimer 59-GTAAAACGACGGCCAG-39 or reverse primer 59-GTC-CTTTGTCGATACTG-39. Amplification was made in 500 mM KCl,100 mM TRIS, pH 8.3, 15 mM MgCl2, and 2 mM each dNTP, usingprimers at 0.5 mM and AmpliTaq DNA polymerase (Perkin–ELmer)at 0.05 U/ul. Amplification conditions were: (1) 95°C for 5 min; (2) 35repeats of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and (3) 72°Cfor 10 min. The products were cloned as described above. Clones thathybridized with probe 8/59 were sequenced.

Rapid amplification of cDNA ends (RACE). The MarathonRACE-ready spleen cDNA (Clontech Laboratories, Inc.) was used asa template for PCR amplification of the 59 end of the new gene. Therecommended protocol was followed, and the internal primer 59-TCCTGGAGCACTTTCTCTTCTTCCCC-39 was used with primerAP1 from Clontech Laboratories. Nested PCR was performed withthe internal primer 59-GCCTGTTTATCTTCTACTGCC-39 and AP2from Clontech Laboratories. The PCR products from both rounds ofamplification were cloned as described above and screened for posi-tive clones by hybridization with probe 8/59. Positive clones weresequenced.

Anchored PCR. We used a protocol similar to the one describedby Ausubel et al. (1997). A mRNA extract from TNF-a-stimulatedsynoviocytes was purified by phenol and chloroform extractions fol-

138 RICHARD, DROUIN, AND BEAULIEU

lowed by ethanol precipitation. The RNA was denatured at 65°C for10 min and reverse-transcribed using avian myeloblastosis virusreverse transcriptase (Boehringer Mannheim GmbH, Mannheim,Germany) at 50°C for 1 h under the recommended conditions. Theproduct was ethanol precipitated, resuspended in water, denaturedat 100°C for 2 min, and placed on ice. A poly(A)1 tail was added to thecDNA using terminal transferase (Promega Corp.) at 37°C for 1 h.The enzyme was denatured by a 10-min incubation at 70°C.

The tailed product was used for a first round of PCR using theinternal primer 59-TCCTGGAGCACTTTCTCTTCTTCCCC-39 and amix of all four T12MN (GeneHunter Corp.). Buffer, enzyme, andprimer concentrations were as described for the preparation of thespecific library, but AmpliTaq DNA polymerase was replaced withExpand High Fidelity enzyme mix (Boehringer Mannheim GmbH).Amplification conditions were: (1) 94°C for 3 min; (2) 9 repeats of94°C for 30 s, 50°C for 30 s, and 72°C for 30 s; (3) 24 repeats of 94°Cfor 30 s, 50°C for 30 s, and 72°C for 30 s with an extension of 5 s percycle at 72°C; and (4) 72°C for 7 min. A second PCR amplification wasperformed under the same conditions using AmpliTaq DNA polymer-ase (Perkin–Elmer) with, on one side, the nested primer 59-GCCT-GTTTATCTTCTACTGCC-39 and the mix of T12MN on the otherside. The product was cloned as described above, and clones thathybridized with probe 8/59 were sequenced.

Chomosomal localization of the new gene. Chromosomes wereobtained from lymphocyte cultures of human peripheral blood ob-tained from normal subjects. The cells were blocked with a high doseof thymidine and released in presence of low dose of thymidine forDAPI (49,6-diamidino-2-phenylindole) banding and low dose of Br-dUrd (5-bromo-29-deoxyuridine) for R banding as previously de-scribed (Drouin et al., 1988). The cDNA clone 7 (see Results) waslabeled with biotin-14–dATP by nick-translation according to theGibco BRL protocol (BioNick Labeling System, Gibco BRL). To con-firm the identification of the chromosome 6, a satellite probe (Oncor;Cat. No. P5009-DG.5, D6Z1) was hybridized concomitantly. FISHtechniques were performed according to the method of Lemieux et al.(1992). Denaturation of slides was carried out at 70°C in 70% form-amide, 23 SSC (0.15 M NaCl, 0.015 M sodium citrate) for 90 s.Twenty microliters of hybridization buffer [50% deionized form-amide, 10% dextran sulfate, 23 SSC, 0.1% sodium dodecyl sulfate,13 Denhardt’s (0.02% Ficol, 0.02% polyvinylpyrrolidone, and 0.02%bovine serum albumin at pH 7)] containing the labeled clone 7 probe(at a concentration of 12.5 ng/ml), 1 ml of D6Z1 probe, and 1 ml ofdenatured herring sperm DNA (10 mg/ml; Gibco BRL, Cat. No.15634-017) were added onto the slides. A coverslip was then placed,and the slides were incubated overnight in a humidified chamber at41°C. They were then washed at 37°C as follows: twice using 50%formamide in 23 SSC followed by twice in 23 SSC for 2 min.

Sites of hybridization were visualized by indirect immunofluores-cence. First, 100 ml of diluted (2% or 1 in 50) rabbit anti-biotinantibody (Enzo Diagnostic Inc., New York, NY) and diluted (2%)mouse monoclonal anti-digoxigenin antibody (Boerhinger Mann-heim) was added onto the slides. The slides were incubated in ahumidified chamber at 37°C for 30 min. Then, they were washedtwice for 5 min in PBD (Oncor; Cat. No. S1370-7) at room tempera-ture. A second incubation was carried out for 30 min at 37°C in thepresence of 100 ml of diluted (2%) biotinylated goat anti-rabbit IgG(Vector Laboratories) and of diluted (2%) digoxigeninylated sheepanti-mouse IgG (Boerhinger Mannheim); this was followed by twowashes. Third, the slides were incubated for 30 min at 37°C with 100ml of diluted (1%) streptavidin–FITC (fluorescein isothiocyanate con-jugate; Gibco BRL) and diluted (1%) sheep anti-digoxigenin–rhoda-mine Fab fragments (Boerhinger Mannheim); this was followed bytwo washes. The slides were soaked in PBS for 1 min and thenstained with DAPI II (Vysis; 125 ng/ml) for 5 min at room tempera-ture. The slides containing BrdUrd-substituted chromosomes weretreated with a solution of PBS, pH 11.5, at 4°C for 5 min and thenmounted with propidium iodide (100 mg/ml) in a PPD (p-phenylene-diamine)/glycerin (1 mg/ml PPD) solution, pH 11.5. All the pictureswere taken with an Olympus BX60 with an image analysis systemusing a black and white digital camera (IMAC-CCD 930) coupled

with the Metasystems In Situ Imaging System (ISIS 2) software,version 2.5. Separate filter sets were used for viewing the signals andchromosome banding, and the images were merged using the Imageanalysis system.

Computer analysis of DNA sequences. The DNA Strider program(Ausubel et al., 1997) was used to search for open reading frames, todeduce corresponding polypeptide sequences, to produce hydropathycurves, and to calculate the number of charged amino acids con-tained in the analyzed polypeptides. The version 8.1 of the GCGpackage (Genetic Computer Group, 1994) was used to retrieve se-quences of known accession numbers from the GenBank and SWISS-PROT (Bairoch and Apweiler, 1997) databanks. Other programsfrom the GCG package were used for DNA and protein analysis: theWORDSEARCH and FASTA programs to compare cDNA sequenceswith databanks of known genes, the MOTIFS program to find pro-tein consensus regions, and the PILEUP program to align proteinsequences and to produce dendrograms. The PSORT program (Nakaiand Kanehisa, 1992) was used to predict the cellular localization ofABC50.

Accession numbers for sequences compared with ABC50 in Fig. 5are DDBJ/EMBL/GenBank X97187 for ABC-C, M28668 for CFTR,M14758 for MDR1, L05628 for MRP1, X96395 for MRP2, U91318 forMRP6, U63421 for SUR1 and SWISS-PROT P21439 for MDR3,P43535 for GCN20, P40024 for YER036, P16521 for YEF3, S65245for YPL226, and S62926 for YNL014. For the determination of pro-portions of charged amino acids, the following human ABC trans-porters were added: ADLR, DDBJ/EMBL/GenBank AJ000327;PMP69, AF009746; PMP70, X58528; TAP1, X57522; TAP2, Z22935;and also ADLP and PMP35 (Mosser et al., 1993) and HWHITE(Croop et al., 1997).

RESULTS

Identification of TNF-a-Modulated mRNA by DDPCR

Cultured synoviocytes were treated with TNF-a(1000 units/ml) for 18 h, and total RNA was extracted.Differential display PCR was performed using theRNAmap system (GeneHunter Corp.) developed by Li-ang and Pardee (1992). Twenty different primer pairswere tested by combining both T12MC and T12MGwith AP1 to AP10. Seventeen fragments that appearedto be modulated by TNF-a treatment were identified.Sixteen of them were successfully reamplified and werelabeled for Northern blot hybridization. Three of themcorresponded to mRNAs that were stimulated byTNF-a. The cloning of two of these fragments is inprogress in our laboratory. The analysis of the third,N4G6a, is presented below.

The N4G6a fragment is a 706-bp amplicon that wasobtained using primers AP6 and T12MG. It presenteda stretch of A14 at one end, preceded by a consensuspolyadenylation signal, AATAAA, starting at 25 bpfrom the first A of the stretch, strongly suggesting thatthis was the 39 end of a cDNA. When compared withthe contents of DDBJ/EMBL/GenBank (Benson et al.,1997), the sequence of N4G6a was found to be homol-ogous to expressed sequence tags but no homologousgene was found, indicating that N4G6a is part of a newgene.

N4G6a Corresponds to a Ubiquitous 3.7-kb mRNA

The cloned N4G6a fragment was hybridized toNorthern blots of synoviocyte mRNA from nine differ-

139ABC50, A NOVEL HUMAN ABC PROTEIN

ent donors, including two donors with RA. In all cases,the TNF-a treatment increased the accumulation of a3.7 6 0.2-kb RNA. An example of these hybridizationsis presented in Fig. 1A. It shows a Northern blot hy-bridization of RNA obtained from synoviocytes of onehealthy donor and one RA patient, both untreated andstimulated with TNF-a for 18 h. The mRNA is ex-pressed at similar levels in normal and RA cells and itsaccumulation is similarly increased by TNF-a treat-ment. The IL-8 mRNA had been shown to be producedin TNF-a-stimulated synoviocytes and was thereforeused as a marker of the TNF-a treatment (Bedard andGolds, 1993; Rathanaswami et al., 1993). A probe forglyceraldehyde 3-phosphate dehydrogenase (G3PDH)mRNA was used to compare the RNA loading from laneto lane.

Figure 1B shows the time course of TNF-a stimula-tion in normal cells. The level of the 3.7-kb mRNA hadalready started to rise 2 h after the beginning of thetreatment and remained elevated at least for the next46 h. Again, an IL-8 probe was used as a marker of theTNF-a stimulation, and a G3PDH probe was used as aloading marker. The expression of this mRNA wastested in 16 human tissues. Figure 2 shows that thisRNA was found in all the tested tissues. The RNA isexpressed in greater abundance in the heart, skeletalmuscle, and testis (lanes 1,6,12). This suggests thatthis mRNA is ubiquitous in human tissues.

Cloning of the cDNA Corresponding to FragmentN4G6a

The mRNA detected by the probe N4G6a is alsofound in human neutrophils (data not shown). This

allowed us to use our homemade neutrophil cDNAlibrary to clone the corresponding cDNA. Probe N4G6awas used to screen this library, and six positive cloneswere obtained. The longest one, clone 7, is 2.9 kb inlength and contains all of the N4G6a sequence exceptfor 78 bp at the 39 end. Clone 7 contains an openreading frame of 2.4 kb. Probe 7/59 was prepared fromthe 59 end of clone 7 to screen the same library. Onlyone of the two clones obtained, clone 8, contained newsequence. The length of the new stretch was 40 bp. Athird probe containing these new sequences, probe 8/59,was used to screen the same library, but none of thetwo obtained clones revealed new sequence.

We used three different strategies to verify if more 59sequence of the cDNA could be cloned. First, a libraryenriched in fragments containing the 59 region of thecloned cDNA was produced. A plasmid DNA extract ofa pool of clones from the neutrophil library was used asa template for PCR amplification using primers specificto probe 8/59 on one side and specific to the pBluescriptvector on the other side. The PCR products were clonedand screened for positive fragments with probe 8/59.Second, we used spleen cDNA prepared by ClontechLaboratories and an internal primer to perform 59RACE. The mRNA detected by probe N4G6a is ex-pressed in the spleen (Fig. 2, lane 9). The use of thiscDNA enabled us to look into another library for thepresence of the 59 region, which might have been un-derrepresented in the neutrophil library. Last, an-chored PCR was performed using mRNA from synovio-cytes stimulated with TNF-a. For this experiment, thereverse transcription step was performed at 50°C tounfold any secondary structures that could impair re-verse transcription and, therefore, normal representa-tion of the 59 end of the new gene. Avian myeloblastosisvirus reverse transcriptase, which works at elevatedtemperatures, was used for this purpose.

Although the three methods used produced manypositive clones, most of the 26 clones analyzed wereentirely contained into the known sequence. Two of

FIG. 2. Northern blot showing mRNA expression of the new genein various human tissues. Lanes 1 to 16 contain RNA from thefollowing tissues and cells: heart (1), brain (2), placenta (3), lung (4),liver (5), skeletal muscle (6), kidney (7), pancreas (8), spleen (9),thymus (10), prostate (11), testis (12), ovary (13), small intestine(14), colon (15), and peripheral blood leukocytes (16). Molecularweight markers are shown on the left.

FIG. 1. Northern blot showing mRNA expression of the newgene. Time of TNF-a stimulation is shown in hours at the top of eachlane. (A) Synoviocyte RNA from one normal individual (two leftlanes) and one RA patient (two right lanes). (B) Time course analysisof mRNA expression of the new gene in synoviocytes obtained fromone normal individual. The probe N4G6a was used to detect the newgene, and the probes IL-8 and G3PDH were used as controls. Theribosomal 28S and 18S RNAs were used as molecular weight mark-ers.


these clones, obtained by RACE amplification, con-tained new sequence. Clone A44 contained 11 newbases in 59, and clone A76 presented 58 new bases,including the 11 new bases of A44.

The available sequence information was joined toproduce a cDNA sequence containing a 2.5-kb openreading frame (ORF) listed in Fig. 3. Also shown in Fig.3 are the amino acid sequence of the ORF, the N4G6a

FIG. 3. The cDNA and amino acid sequences of ABC50. The amino acids are shown by the single-letter code under the correspondingcodons of the cDNA sequence. The four groups of bold amino acids correspond to the four regions that were identified by the programMOTIFS. They are, in 59 to 39 order: (1) the 59 nucleotide binding motif, (2) the 59 ABC transporter signature motif, (3) the 39 nucleotidebinding motif, and (4) the 39 ABC transporter signature motif. The stop codon and the polyadenylation signal are underlined. The 706 basesof N4G6a are shown in italic at the 39 end.


clone at the 39 end, the stop codon of the ORF, and thepolyadenylation signal. The sequence was submitted tothe DDBJ/EMBL/GenBank databases under AccessionNo. AF027302.

High-resolution FISH was used to find the chromo-somal localization of this gene. It was mapped to band6p21.33 (Fig. 4).

The 59 End of the Cloned cDNA Is Part of a CpGIsland

To confirm that the mRNA detected by probe N4G6acorresponds to a new gene, the entire available cDNAsequence was compared with the contents of the DDBJ/EMBL/GenBank databases. It was found to containregions homologous to many expressed sequence tagsincluding EST123147 (Accession No. F07368), whichshows at least 98% homology with the cDNA and wasmapped to 6p21 using a cell hybrid panel (Allikmets etal., 1996). It was also found homologous to a fragmentpreviously identified as a CpG island (Cross et al.,1994). Clone 57g6 (Accession No. Z61733) was found tohave strong homology with the 59 end of the clonedsequence of the 3.7-kb mRNA. Homology between thetwo DNA fragments is 96% for the first 169 bases of thenew cDNA, including one inversion and 2 uncertainbases in the sequence of clone 57g6. The homologousregion ends abruptly at position 216 of clone 57g6r. Thesequence GT is found at the junction between homolo-gous and nonhomologous sequences, suggesting thatthis could be an intron boundary. The high homologybetween the two fragments strongly suggests that the

CpG island is a segment of the genomic counterpart ofthe cloned cDNA.

The Cloned cDNA Codes for an ABC Protein

The cloned cDNA codes for a predicted polypeptide of807 amino acids that was analyzed using the programMOTIFS of the GCG package. This program recognizesthe consensus sequences of known protein domains.Four motifs were found: two “ABC transporters familysignature” motifs and two “ATP/GTP-binding site A”motifs (also called P-loop, bold in Fig. 3). These motifsare arranged in two pairs, each of which is part of anucleotide binding fold (NBF). Taken together, thesemotifs associate the predicted protein with the ABCprotein family (Higgins, 1992; Tusnady et al., 1997).The new protein was named ABC50 (denominationapproved by the HUGO/GDB Nomenclature Com-mitee).

A subfamily of ABC proteins called ABC transport-ers contain hydrophobic regions with membrane-span-ning domains (Higgins, 1992; Tusnady et al., 1997).Investigation for the presence of the transmembranedomains in ABC50 was done by the analysis of its Kyteand Doolittle hydropathy curve. No transmembraneregion was found. In contrast, regions upstream of theNBFs were observed to be strongly hydrophilic. Thehigh hydrophilicity of ABC50 is also reflected by itselevated proportion of charged residues. This was eval-uated by calculating the proportion of aspartic acid,arginine, glutamic acid, histidine, and lysine residues(Alberts et al., 1994) contained in human ABC trans-

FIG. 4. Localization of the ABC50 gene on human chromosomes. These mitoses were stained with DAPI (partial mitosis shown in blueon the left) for G-banding or with propidium iodide (partial mitosis shown in red on the right) of BrdUrd-substituted chromosomes forR-banding (see Materials and Methods). Simultaneous visualization of the hybridization signals and the band pattern allow the precisemapping of the ABC50 gene to the band 6p21.33 (yellow-green signals, arrows). To ensure the correct identification of chromosome 6, the asatellite probe D6Z1 was hybridized simultaneously with the ABC50 probe (red signals, arrows “cen 6”). The exact band localization of theABC50 gene is shown on the schematic representation of the short arm of the chromosome 6 at the bottom, on the right (Drouin and Richer,1989).


porters and in ABC50. The charged amino acid contentof ABC50 is 36%, whereas it is only 18 to 27% for theother 17 known human ABC proteins (see Materialsand Methods for a complete list of the proteins).

The PSORT program (Nakai and Kanehisa, 1992),created to predict the cellular localization of proteins,was used to analyze the sequence of ABC50. Threenuclear localization patterns were found: KKKR atposition 73, RRKK at position 82, and KRPK at posi-tion 564.

ABC50 Shows Similarity to a Subfamily of YeastABC Proteins

A recent study of yeast ABC proteins allowed theirclassification into six clusters according to the numberand position of the transmembrane domains and ofNBFs. Cluster-specific amino acid motifs were identi-fied. A group of five yeast proteins designated by thename cluster IV were characterized by the presence oftwo NBFs along with the absence of transmembranedomain. Nuclear localization patterns were found bythe PSORT program in these proteins (Decottigniesand Goffeau, 1997). To assess the relationship betweenABC50 and this category of yeast proteins, we searchedfor cluster-specific motifs in ABC50 and found the spe-cific motif for cluster IV: SGGWR/KMR/K. This is illus-trated in Fig. 5, which presents the partial PILEUPalignement for the region of the 59 ABC transportersfamily signature for various human ABC proteins andfor the yeast proteins from cluster IV. It shows thatmost of the human ABC transporters contain the con-sensus sequence for cluster II (SGGQK/QQA/R; Decot-tignies and Goffeau, 1997) and that, unlike the otherhuman proteins, only ABC50 contains the signaturefor cluster IV.

DISCUSSION

Using the DDPCR technique (Liang and Pardee,1992), we identified a cDNA fragment corresponding toa new gene called ABC50. This gene expresses a mRNA

of 3.7 6 0.2 kb, ubiquitous in human tissues. TheDDPCR fragment was used to clone a cDNA sequenceof 3141 bp. The ABC50 cDNA contains an ORF for apolypeptide of 807 amino acids starting with a methi-onine at position 95. This ATG codon presents featuresof a start site for translation. The sequence of thesurrounding bases, ACCGCCATGC, is close to the con-sensus initiation sequence GCCA/GCCATGG (Kozak,1991). It contains both a purine in position 23 and apyrimidine in position 14. This combination is consid-ered an adequate translation signal (Kozak, 1989,1991, 1996). Furthermore, the 59 end of the sequencewas found to be homologous to a genomic fragmentpreviously identified as a CpG island (Cross et al.,1994). The CpG islands are CG-rich genomic DNA se-quences that are usually found at the 59 end of house-keeping and ubiquitous genes (Bird, 1987; Cross et al.,1994; Larsen et al., 1992). This suggests that the end ofthe cloned cDNA maps near the 59 end of the corre-sponding mRNA. Therefore, we propose that thepolypeptide corresponds to the complete protein se-quence.

The ABC50 protein contains two copies of the con-sensus motifs for the NBF of ABC proteins but does notcontain the membrane-spanning domains that charac-terize the subfamily of ABC transporters, in whichmost of the known ABC proteins, including those fromhuman origin, are comprised (Decottignies and Gof-feau, 1997; Higgins, 1992; Tusnady et al., 1997). Inaddition, ABC50 contains a very high proportion ofcharged amino acids compared with human transport-ers. We therefore conclude that ABC50 is not a trans-porter. To our knowledge, this is the first time a proteinof this type has been described in humans and inmammals (Decottignies and Goffeau, 1997).

The protein ABC50 presents many similarities witha group of yeast proteins that was identified as clusterIV (Decottignies and Goffeau, 1997). These similaritiesreside in: (1) absence of transmembrane domains, (2)presence of two NBFs, (3) sequence similarity of theNBFs, (4) identical cluster-specific motif, and (5) pres-

FIG. 5. Amino acid sequence comparison of the 59 ABC transporter family signature region from various human and yeast ABC proteins.This motif is highlighted by the use of bold letters. The numbers on the left represent amino acid positions. The cluster-specific motifs appearbetween parentheses for cluster II and braces for cluster IV.


ence of nuclear localization patterns. Nuclear localiza-tion patterns not only into nuclear but also into ribo-somal protein sequences, which move to the nucleus forassembly (Alberts et al., 1994; Nakai and Kanehisa,1992), are found. The functions of two of the five yeastproteins of cluster IV, GCN20 and YEF3, were studied.They are both involved in translation. This explainsthe nuclear/ribosomal localization patterns and sug-gests a similar role for ABC50.

The protein YEF3 is the yeast elongation factor 3,and it stimulates elongation factor 1a-dependent bind-ing of aminoacyl-tRNA to the ribosome. Attempts tofind a functional analogue to YEF3 in higher eu-karyotes have failed (Sandbaken et al., 1990). The pro-tein GCN20 is one of the two yeast proteins that showthe highest homology with ABC50 (not shown). Thisprotein is part of a complex that regulates the expres-sion of genes according to the availability of aminoacids in yeast. The complex acts in a cascade of eventsthat lead to the increased translation of a subset ofgenes involved in amino acid synthesis under aminoacid starvation. This cascade involves phosphorylationof the yeast elongation initiation factor 2a (Rhoads,1993; Vazquez de Aldana et al., 1995). In mammals,many stress conditions cause a modification of thephosphorylation state of the elongation factor 2a(Rhoads, 1993). Elevated TNF-a concentrations re-lated to RA might be one of them. If that is the case,ABC50 could play in mammals a role similar to GCN20in yeast in the control of amino acid production. Such afunction is shared by all living cells, explaining theubiquity of ABC50 in human tissues.

The proposed involvement of ABC50 in translation issupported by the recent finding of a rat protein thatcopurifies with elongation factor 2. A partial aminoacid sequence was obtained and used to isolate a cDNAfragment from a rat library that showed more than90% homology with the cDNA for ABC50 (C. G. Proud,Department of Anatomy and Physiology, Medical Sci-ences Institute, University of Dundee, pers. comm.,January 9, 1998).

The cytokine TNF-a produces many effects on thesynoviocyte in vitro, including cell proliferation andoverproduction of cytokines and enzymes (Arend andDayer, 1995; Firestein and Zvaifler, 1997; Koch et al.,1995). The similarity between ABC50 and two yeastproteins involved in translational control suggests thatit could play a role in the stimulation of protein pro-duction and in the cell proliferation caused by TNF-ain these cells. The early augmentation of ABC50mRNA transcript (Fig. 1B) is in agreement with thisproposal.

Other reports show that TNF-a plays a role in trans-lation. TNF-a was found to induce the phosphorylationof the elongation initiation factor 4E in many celltypes, including fibroblasts (Marino et al., 1989, 1991).The cytokine TNF-a has a cytotoxic effect on thegrowth of some of these cells, and in these cases, thephosphorylation of the elongation initiation factor 4E

was associated with the inhibition of cell proliferation(Marino et al., 1989, 1991). This suggests that TNF-acould produce various effects on the cells by acting atdifferent levels of the translational control mecha-nisms.

Finally, it is noteworthy that the chromosomal local-ization of the ABC50 gene is on the short arm of chro-mosome 6 and was mapped to the same band as thehuman major histocompatibilty (MHC) class I locus(6p21.33). Furthermore, EST123147, which seems tobe identical to ABC50, was mapped just telomeric toHLA-E using a radiation hybrid panel (Allikmets et al.,1996). Since TNF-a was shown to stimulate the expres-sion of MHC class I proteins (Johnson and Pober,1991), this physical proximity could signify thatABC50 and MHC class I genes are part of the samechromatin expression domain (Kellum and Schedl,1991). This putative domain could be submitted toTNF-a modulation. This, however, remains to beshown by future studies to test this specific hypothesis.

In conclusion, we cloned the cDNA of ABC50, a newgene that codes for a human ABC protein. The mRNAof this gene accumulates in vitro in synoviocytes fol-lowing TNF-a stimulation. This suggests that it couldbe involved in the inflammation related to RA. Thechromosomal proximity of ABC50 with genes of theclass I MHC supports the proposition for its involve-ment in immunological processes. Stuctural features ofthe corresponding protein suggest that it is differentfrom known human ABC proteins and that it could bea ribosomal protein involved in translation.

ACKNOWLEDGMENTS

We are grateful to Dr. Andre Leveillee for providing synovialmembranes; Claude Potvin for the preparation of the neutrophilcDNA library and for helpful discussion; Dr. Souad El Ouakfaoui, Dr.Dominique Heitz, Dr. Fatiha Chandad, and Dr. Chistopher G. Proudfor helpful discussion; and Barbara Leclerc and Marc Bronsard forexcellent technical assistance. This work was supported by Grant7604 from the Medical Research Council of Canada and by a fellow-ship to Manon Richard from the Arthritis Society. Regen Drouinholds a scholarship from the Cancer Research Society, Inc./MedicalResearch Council of Canada.

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abc50, a novel human atp-binding cassette protein found in tumor necrosis factor-α-stimulated...

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