A Novel Member of the Immunoglobulin Gene Superfamily Expressed ...

Vol. 269,No. 22,Issue of June 3, pp. 15601-15605,

JOURNALOF BIOLOGICAL CHEMISTRY 0 1994 by T h e American Society for Biochemistry and Molecular Biology, Inc. THE

1994

Printed in U.S.A.

A Novel Member of the Immunoglobulin Gene Superfamily Expressed in Rat Carcinoma Cell Lines* (Received for publication, January 21, 1994, and in revised form, March 7, 1994)

Corinne ChadBneau$, BBatrice LeMoullac,and Marc G. DenisQ From the Devartment o f Medical Biochemistrv, ". ZNSERM C J F 90-11, Znstitut de Biologie, 9 quai Moncousu, F-44035 NantesCbdex, France

Defined by monoclonal antibody E4, the pE4 antigen is a 66,000-Da glycoprotein whichis expressed at the cell surface of rat colon and mammary carcinomas, but only in trace amounts in normal adult rat tissues. To determine the structure of this tumor-associatedantigen and to identify its functional domains, we have cloned a cDNA coding for this protein. It encodes a 416-amino acid proteinwith an expected molecular weight for the core protein of -42,000. The predicted amino acid sequence reveals that pE4 contains the conserved amino acids and domain structures characteristicof members of the immunoglobulin gene superfamily. Comparison of this sequence with data banks revealed a significant homology with the human and mouse receptors forpoliovirus. However, pE4 is not the rat receptor for poliovirus, as different patterns were obtained by hybridization of rat genomicDNA with both probes.A major -2.2-kilobase transcript of thepE4 gene was detected in all the rat tumor cell lines tested. In contrast, barely detectablelevels of pE4 mRNAwere foundin normal adult rattissues.

sequence of a cDNA clone, t h e composition and structure of t h e encoded polypeptide, and the expression of pE4 mRNA in rat tissues and cell lines. MATERIALSANDMETHODS

Cell Culture-The rat colon tumor cell lines PROb (DHD/K12/TRb) and WI32054M were obtained from the European Collection of Animal Cell Cultures (ECACC) and grown in complete medium (RPMI1640,2 mM L-glutamine, 100 unitdm1 penicillin, and 100 &ml streptomycin) supplemented with 5% fetal calf serum under standard conditions. Isolation of a Fragment of the Gene Encoding pE4-A specific probe for pE4 was synthesized by PCR' amplification of PROb genomic DNA using oligonucleotides deduced from the NH,-terminal amino acid sequence (2). Primers are presented in Fig. 1.Thirty cycles of PCR were performed with 250 ng of PROb genomic DNAin 1x PCR buffer (10 mM Tris-HC1, pH 8.3, 50 mM KCl, 1.5 mM MgCl,, 0.1% (w/v)gelatin) and 40 pmol of each primer as follows: 94 "C for 10 s; 47-57 "C for 10 s; 72 "C for 1 s. The amplified fragment was blunt-ligated in the SmaI restriction site of the pBluescript plasmid (Stratagene, La Jolla, CA). cDNA Library Construction and Screening-A PROb cDNA library (4) was plated at a density of lo4 plaque forming units/l50-mm dish. Lifts were done with colony plaque screening hybridization transfer membrane discs (DuPont NEN). The nylon filters were prehybridized for 4-6 h at 65 "C in 2 x SSC (0.3 M NaCl, 0.015 M sodium citrate, pH 7.0), 5% SDS, 10% dextran sulfate. Hybridization was performed for 20 h at 62 "C in thesame solution containing -lo6 cpdml of probe labeled pE4 is a rat carcinoma-associatedantigenidentifiedby by nick translation. Following hybridization, the membranes were monoclonal antibody E4, which was raised against a rat colonwashed with 1x SSC and 1%SDS at room temperature, 1x SSC and 1% carcinoma cell line (1). pE4 is a cell membrane glycoprotein, SDS at 62"C,0.2 x SSC and 0.1% SDS at 62"C, then exposedto with an apparent molecular weight as determined by SDS- Amersham Hyperfilm-MP. The insert of the isolated phages was amplified byPCR using oligonucleotides specific for AgtlO (Clontech, Palo polyacrylamide gel electrophoresis of -66,000 (1, 2). NH,-terminal amino acid sequencing did not allowus to identify pE4 to Alto, CA). DNA Sequencing-The amplified cDNAofclone 58.3 was digested rat any known protein(2). This protein is expressed in all the with HindIII, and the fragments subcloned in the SmaI cloning site colon carcinoma cell lines tested, i.e. in PROb cells, in all the of the pBluescript SK plasmid. Sequence analysis was performed by clonesisolatedfrom a singleadenocarcinoma (3), and in the dideoxy chain termination method with synthetic oligonucleotide WB2054M cells, as determined by immunofluorescence analyprimers specificfor the plasmid (T3,T7) or designedfrom partial sis.' p E 4 could also be detected on rat mammary carcinomacell sequencing. The sequences were determined from both strands. The lines (MAD-B106 a n d 13762), a n d o n R F L B rat fetal lung cells final sequence was confirmed by direct sequencing ofPCR products without subcloning. (1). In contrast, a very low expression was found on normal adult rat colon a n d lung, and no expression could be detected on Southern Blot Analysis of GenomicDNA-High molecular weight DNA prepared from normal rat liver was digested with BamHI or the other normal rat tissues tested (1). HindIII, electrophoresed on a 0.8% agarose gel (10pg/lane) and transTo investigate the role of p E 4 in ontogenesis and malignancy, ferred to Genescreen PlusTMhybridization membrane (DuPont NEN). and to develop animal models to study the usefulness of this The nylon filter was prehybridized for 4 4 h at 65 "C in 2 x SSC, 5% SDS, 10% dextran sulfate (5). Hybridization was performed for16-20 h tumor-associated antigen in tumor therapy, we have cloned at 65 "C in the same solution containing lo6 cpdml of probe labeled by pE4 mRNA. In the present study, we describe the nucleotide random priming. Following hybridization, the membrane was washed with 2 x SSC at room temperature, 1 x SSC and 1%SDS at 65 "C, 0.5 * This work was supported in part by Grant 415 fromthe Association x SSC and 0.1% SDS at 65 "C, and then exposed at -70 "C. The -2kilobase insert amplified by PCR from phage 58.3 was usedas a probe pour la Recherche contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This for pE4. A -1.8-kilobase fragment of the human poliovirus receptor article must therefore be hereby marked "aduertisement"in accordance gene, generated by digestion of plasmid pMEM.2OAKpn-4 with SmaI with 18 U.S.C. Section 1734 solely to indicate this fact. and XbaI, was also used as a probe and hybridized at 62 "C. The nucleotide sequence(s) reportedin this paper has been submitted RNA Extraction and Northern Blot Analysis-Total cytoplasmicRNA to the GenBankTMIEMBL Data Bank with accession number(s) L12025. was extracted as described (5). Total RNA (-20 1.18)were electrophore$ Recipient of a research fellowship from the Ligue Departementale sed on a 1%agarose gel containing 2.2 M formaldehyde, and transferred de Lutte contre le Cancer. 8 To whom correspondence and reprintrequests should be addressed. Tel.: 33-40-08-41-50;Fax: 33-40-08-40-82. The abbreviations used are: PCR, polymerase chain reaction; bp, C. Chadeneau, B. LeMoullac, and M. G. Denis, unpublished data. base pair(s).

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Novel Member of the Immunoglobulin Gene Superfamily

to GeneScreen PlusTM nylon membrane. For pE4 gene detection, the membranes were hybridized as described for Southern blots.

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GRRATYGCNGTNCRRGTNYTNWSNXXXWSNACNGGNTTYYTNGGNGGNWSNACNGTNYT 5'-GARATWGCDGTSCARGTSCT-Y

~"CCNAAFMYCCNCCGTCGTGTCACG-5'

e - -Probe 2 ---_--- Probe 1 ---> Synthesis of a pE4-specific Probe-Monoclonal antibody E4 recognizes an epitope which is at least partially glyco~ylated.~D GAGATAGCGGTCCAGGTGCTCTCCAATTCGACCGACCGGGTTCTTGGGCGGCAGCACAGTGC Therefore, it could not be used to screen a phage expression E E I A V Q V L S E S T G F L G G S T V L library. FIG. 1. Sequences used in the amplificationof a fragment of To isolate cDNAs encoding pE4, we initially constructed a A g t l O cDNA library using mRNA from PROb cells (4). This the pE4 gene. A, NH,-terminal amino acid sequence of the purified protein. B , nucleotide sequence deduced from the protein sequence. C , library was first screened with a mixture of degenerated syn- probes designed from the amplification of rat genomic DNA. D, sethetic oligonucleotides, the sequence of which were designed quence of a 58-bp fragment amplified and subcloned, and E,deduced from the NH,-terminal amino acid sequence of purified pE4. amino acid sequence. The asparagine residue, not identified by protein Several clones were isolated. Allof them were false positive sequencing, is double underlined. clones as determined by sequencing. Homology with the Immunoglobulin GeneSuperfamily-A In order to obtain a more specific probe, we applied the mixed search of the GenBank data base (release 78) disclosed a sigoligonucleotide-primed amplification ofcDNA procedure re- nificant homology to human receptor for poliovirus (9). The ported by Lee et al. (6). Two probes were designed from the overall homology was 25.9% at the amino acid level. This hoavailable sequence information (Fig. 1). Probe 1 corresponded mology was situated in two regions of about 40-50 amino acids to the first7 amino acids (sense primer, degeneracy = 96), and each (amino acids 109-156 and 248-293) with -64 and -69% probe 2 to amino acids 12-19 (antisense primer, degeneracy = homology, respectively, at the amino acid level (Fig. 3). A simi64). These primerswere used to amplify PROb genomic DNA (a lar homology was observed when comparingclone 58.3 with the variation of the MOPAC procedure in which cDNA is used as a mouse receptor for poliovirus (22.8%; Ref. 10). template). Different annealing temperatureswere tested, rangThe receptor for poliovirus is a member of the immunoglobuing from 47 to 57 "C. The PCR products were electrophoresed lin superfamily. Like this receptor, the pE4 protein presents on a 3% agarose gel stained with ethidium bromide. The ex- conserved residues that are characteristicof this superfamily pected 58-bp DNA fragment was present when the annealing (11).They are shown on Fig. 3. Among them, cysteine residues temperature was 47, 50, and 55 "C but not a t a n annealing are particularly important, as they stabilize the basic immutemperature of 57 "C. This 58-bp DNAfragment was subcloned, noglobulin loop (11).The presence of these cysteines predicts and several clones were sequenced. The presence between the that pE4 comprises three immunoglobulin-like domains, comprimers of a nucleotide sequence encoding the 4 amino acids posed of 74, 43, and 48 aminoacids, respectively (Fig. 4). The identified by protein sequencing (Ser-X-Ser-Thr)allowed us to most NH,-terminal domain would be of the V-type due to the identify the product as a fragment of the pE4 gene.None of the longer distance between cysteine residues,while the two others six subclones sequenced from the PCR performed at an anneal- would be of the C-type. The otherconserved residues (shown on ing temperatureof 50 "C had thepredicted sequence while 3 of Fig. 3) are believed to be involved in the formation of the the 11 subclones sequenced from the PCR performed at a n p-strands forming the immunoglobulin domains. annealing temperature of 55 "C presented the expected nucleDNA Blot Analysis-In order to determine whether pE4 otides (Fig. 1).Amino acid 9 was found to be an asparagine as could represent the rathomolog of the poliovirus receptor, we previously postulated (2). digested rat genomic DNA with BamHI and Hind111 and hyIsolation and Characterization of a cDNA Encoding pE4bridized the membrane witha pE4 probe (Fig. 5 A ) or a human The PROb cDNA library was then screened by hybridization poliovirus receptor probe (Fig. 5B).The hybridization patterns with the nick-translated 58-bp DNA fragment to isolate the were clearly different, demonstrating that pE4 is not the rat corresponding cDNA. Screening of 100,000 clones resulted in homolog of the cloned human poliovirus receptor gene. Howthe identification of five positive clones. One of these, 58.3, was ever, the possibility that pE4 is an alternativepoliovirus recepsubcloned and sequenced. This cDNA (GenBank accession tor cannot be completely excluded. number L12025) is 1862-bp long, with a 1248-bp open reading Only a few fragments of genomic DNA were detected by the frame starting with an ATG at position 64, encoding a 416- 58.3 cDNA probe, indicating that pE4 is encoded by a unique amino acid protein (Fig. 2). gene. The NH,-terminal sequence of pE4 determined by microseRNA Blot Analysis-The pE4 cDNA was also used for hyquencing was localized between residues 34 and 53, allowing us bridization with blots of size-fractionated total RNA isolated to identify this cDNA. The predicted protein has a 33-amino from cancer cells and normalrat tissues. ThecDNA hybridizes acid signal sequence which is in complete agreement with the to two mRNA species of -2,200 and -3,500 nucleotides in -3,-1 rule (7), followed by 383 amino acids of the mature pro- PROb and WB2054Mcolon carcinoma cells (Fig. 61, and in tein, with an expected core polypeptide M,of about 42,370. This MAD-B106 mammary cancer cells (datanot shown). The is in agreement with thesize of the deglycosylated P E ~Nine . ~ smaller species was 2-5-fold more abundant than the larger potential N-glycosylation sites were found, which is to be exspecies. Under identical experimental conditions, no hybridizapected when taking into account the -30% carbohydrate con- tion signal was detected with RNA isolated from normal rat tent of mature pE4, whose oligosaccharide chains areall linked colon, lung, liver, spleen, and kidney. In contrast, a signal was to asparagine via N-glycosidic bonds3 Following Edman deg- detected with RNA prepared from lung containing PROb meradation of the protein, asparagine 42 was not identified (2). tastasis (Fig. 6). However, expression of the pE4 gene is not This would suggest that thisamino acid is glycosylated in uiuo. completely absent in normal adult rat colon, as a faint signal Finally, the sequence has no hydrophobic domain of suffi- was seenfollowing prolonged exposure of the film (several days cient length (8) to indicate that pE4 could be a membraneuersus 4-6 h for the cell lines). spanning protein.

DISCUSSION C. ChadBneau, G . Cornu, K. Meflah, and M. G. Denis, manuscript in preparation.

Using degenerated oligonucleotides derived from the NH,terminal sequence of the protein, we were first able to obtain a

Novel Member of the Immunoglobulin Gene Superfamily

15603

TTGCCGCTCGCTGCTAGCTTGGATCCGCGTGGACTACAGGCCCCACTC 75 M A P L 4 GCCGGTGCCTCTCGCTCCCGGGTGTGGTCAGCGGGGCTACTGAGGCT~T~T~TGTCCTGCTTTACGCTCCAG 150

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AAAGCGGGTGGGGAGATAGCTGTGCAGGTGCTCTCCAATTCGACC~~~CTTCTT~A~TCTACAGTCTTGCAC 225

K A G G E I A V Q V L S N S T G F L G G S T V L H 54 TGTAGTCTGGCTTCCARAGACAATGTGACAATCACTCAGCTAACAT~~~ATGAAGAGGGATCCAGATGGATCACCC 300 C S L A S K D N V Z I T Q L T W M K R D P D G S P 79 TTCCGTGCCTGTCTTCCACCCCAAGAAffiGCCCAGCATCTCTGATCCAGAGA~TGAAGTTCTTffiTTGCCAAG 375 F R A C L P P Q E G P S I S D P E R V K F L V A K 104 G T G T A C G A G G A T C T G A G G A G C A T C T C T G G C C A T C T C G A T G T 450 V Y E D L R N A S L A I S N L R V E D E G I Y E C 129 CAGATTGCCACGTTCCCCACAGGCAGT~GAGCGCCAATGTCTGGCTGAAffiTGTTCGCCCGACCT~CACA 525 Q I A T F P T G S K S A N V W L K V F A R P K N T 154 GCAGAGGCCCTGGAGCCCTCTCCCACCTTGATGCCGCAGGACGT~CC~TGCATCTCTGCTGATGGTCACCC~ 600 A E A L E P S P T L M P Q D V A K C I S A D G H P 179 C C T G G A C G A A T C A C G T G G T C C T C G A A T G T G G R A G C T A 675 P G R I T W S S N V N G S Y R E M K E T G S S R A 204 CCACCACAGTTATCAGCTACCTCTCCATGGTGCCTTCTAGCCAGGCAGATGGCACGAACATCACCTGCACAGTGG 750 P P Q L S A T S P W C L L A R Q M A R T S P A Q W 229 AACATGAAAGCTTCCAGGAGCCGGACCAGCAGGCCATTGATCCTTTCCCTACCTTATCCACCCGAAGTGTCCATC 825 N M K A S R S R T S R P L I L S L P Y P P E V S I 254

FIG.2. Sequences of pE4 cDNA and protein. Nucleic acid sequence of clone 58.3 is indicated on the top line. The deduced amino acid sequence is indicated on the bottom line. The amino acid sequence determined by protein sequencing is double underlined.The potential N-glycosylation sites are underlined.

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S G Y E G N W Y I G L T N V N L I C E A R S K P P CCCACCAACTATAGCTGGAGCACGGCCACGGGTCCCCTTCCCCTTCCCAACTCCACTCATTTCCAAGG-CGGCAGTCAC P T N Y S W S T A T G P L P N S Z H F Q E N G S H

279 975 304 CTGCTAATCTCCACCGTGGATGACCTCAATAACACGATCTTTGTGTGC~GCCATCAATGCCCTAGGGTCTGffi 1050 L L I S T V D D L N N Z I F V C K A I N A L G S G 329 C A G G G C C A A G T G A C C A T C C T A G T T A R A G A G G C A T C T G A G A T G G C T A 1125 Q G Q V T I L V K E A S E I L P P K T K L R H W L 354 CATCATTGCCATCGTCTTTTGTGTCCTGATCATCGGAGTAGTAGCAGGCATTGTATTCTGG~TACAGGCGTGG 1200 H H C H R L L C P D H R S S S R H C I L E I Q A W 379 TTGTGGTCGGCAGTCCAGGACCTTAGACAGGGAGAACGTCCGCTATTCAGCAGCGAATGGCGTCTCTGTCCC~ 1275 L W S A V Q D L R Q G E R P L F S S E W R L C P K 404 CGTGGAGACGAACAACTTGAGGTGATGGTGCTGGGGTAGACAGAACTAAGG~CTTGAAGACATAAC~CTGG~ 1350 R

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FIG.3. Comparison between the amino acid sequences of the pE4(top lane) and the human poliovirus receptor (bottom lane). Identical amino acids are boxed. Cysteine residues thought to be implicated in domain formation are shaded. The positions conservedacross the immunoglobulin superfamily (11) are indicated by minus (-).

specific probe and used itt o isolate a cDNA coding for the pE4 protein. The amino acid sequence predicted from this cDNA reveals that pE4 contains the conserved amino acids and do-

main structure characteristic of members of the immunoglobulin gene superfamily. Interestingly, we previously reported (2) that computer searches of protein data bankrevealed a signifi-

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Gene Superfamily

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FIG.4. Hypothetical structure of pE4. The numbers indicate the number of residues contained in each domain. Thestars represent the potential N-glycosylation sites.

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A FIG.6. Northern blotof total RNA from colon cancercell lines and normal rat tissues with a pE4 probe. -20 pg of total RNA prepared from PROb cells ( I ) , WB2054M cells (Z), spleen (3),liver ( 4 ) , kidney (5),lung containingPROb metastasis ( 6 ) ,normal lung(7), and normal colon (8)were loaded per lane. The amount of RNA in eachwell was estimated to be equivalent as by judged ethidium bromide staining. Hybridization was performeda t high stringency.

anchors (15).pE4 is most likely linkedto thecell membrane via such anassociation, since it does not seem to contain a typical 23.1 kb transmembrane domain. Preliminary attempts to release pE4 from PROb cells by treatment with phosphatidylinositol phospholipase C have been unsuccessful. We have also failed to detect pE4 in culturemedium and serumof rats bearingPROb 9.4 kb tumors, indicating that this protein is not secreted by tumor cells. Hybridization of RNA revealed the presence of 2 hybridizing r, 6.6 kb species in the cell lines expressing pE4. Since a unique gene was detected by Southern blotting, the 2 signals might arise from incomplete maturation of a unique transcript,or alternative splicing. Such a splice variant might encode a transmembrane domain. The immunoglobulin superfamily includesa large numberof cellular adhesion molecules. These are thought to have wide ranging functions, and mediate a variety of homotypic and heterotypic cellular interactions. For instance, neural cell adhesion molecule and neuron-glia cell adhesion molecule play key roles during embryogenesis (16-18); T-cell receptors, CD4, CD8, expressed on thymocytes, are crucial in thedevelopment of the immune response (19); intercellular cell adhesion molFIG.5. Southern blot analysis of rat genomic DNA with pE4 ecule-l and vascular cell adhesion molecule-l are involved durand humanPVR probes. DNA was digested with BamHI or HindIII, ing inflammation and wound healing (20, 21). In addition, a electrophoresed on agarose gels, and transferred tonylon membranes. Hybridization and washes of pE4 probes (A) were performed a t high large array of adhesion molecules belonging to the immunostringency. Hybridization and washes of human PVR probe ( B ) were globulin superfamily have been implicated in tumorigenesis performed a t a lower stringency. kb, kilobase pair. and cancer metastasis. For instance, neural cell adhesion and neuron-glia cell adhesion molecules have been related to tumor cant homology (-50%) of pE4 with the murine CD4 (L3T4), cell invasion (22). Vascular cell adhesion molecule-1 has been which is also a member of the immunoglobulin superfamily demonstrated to mediate tumor cell adhesion to the vascular monolayer via binding to VLA-4 (23, 24). Intercellular cell ad(12). An additional cysteine residue at position 83 is present in the hesion molecule-1, which is normally expressed on activated putative V-type domain. Usually, a n immunoglobulin domain endothelium, is also expressed on solid tumor cells and its does not contain other cysteine residues than those stabilizing expression is correlated with metastatic potential (25). Carcithe structure of the domain. However, exceptions to this rule noembryonic antigen, which is expressed on solid tumors, has have been observed (13).In particular, a Bence Jones protein, been found to mediatehomotypic cell aggregation, and heteroNIG-58, has 2 additional cysteine residues.These cysteine resi- typic cell-cell interaction (26-28). Finally, during completion of dues would be involved in theformation of interchain disulfide this manuscript, Tang et al. (29) reported that platelet endobond in the polymers (14). In this context, it is interesting to thelial cell adhesion molecule-1, which is normally expressed note that we observed an M, -130,000 protein on Western by endothelial cells (30), is also expressed in solid tumor cells, immunoblots, and detected pE4 in a high molecular weight and is involved in tumor cell adhesion to endothelium. In this context, the demonstration thatpE4 is a member of complex by gel filtration chromatography. Thus we proposed that pE4 could be present as homodimers, or associated with this family, and, inaddition, its expression on the cell surface, support the hypothesis that it might also be involved in interother proteins in the cell membrane (2). Although mostmembers of the immunoglobulin superfamily cellular recognition and cell adhesion mechanisms, which, in are transmembrane proteins (111, some of them, such as the turn, are importantfor the development of tumors and/or mecarcinoembryonic antigen and the neural cell adhesion mol- tastasis. The availability of pE4 cDNA should enable identifiecule are linked tothemembrane via glycophospholipid cation of its function(s). A potentialrole in cell aggregation and

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Novel Member of the Immunoglobulin Gene Superfamily adhesion is being analyzed. We are also attempting to identify the pE4 gene in human tumors. Acknowledgments-We are grateful to Drs. Bibb, Wimmer, and Racaniello for the generous gift of human poliovirus receptor cDNA, and to Drs. LePendu,Lustenberger,andMeflah for criticalreading of the manuscript.

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