CLMP, a Novel Member of the CTX Family and a New Component of Epithelial Tight Junctions

The CTX family is a growing group of type I transmembrane proteins within the immunoglobulin superfamily (IgSF). They localize to junctional complexes between endothelial and epithelial cells and seem to participate in cell-cell adhesion and transmigration of leukocytes. Here, we report the identification of a new member of the CTX family. This protein, which was designated CLMP (coxsackie- and adenovirus receptor-like membrane protein), is composed of 373 amino acids including an extracellular part containing a V- and a C2-type domain, a transmembrane region and a cytoplasmic tail. CLMP mRNA was detected in a variety of both human and mouse tissues and cell lines. The protein migrated with an Mr of around 48 on SDS-PAGE and was predominantly expressed in epithelial cells within different tissues. In cultured epithelial cells, CLMP was detected in areas of cell-cell contacts. When exogenously expressed in polarized MDCK cells, CLMP was restricted to the subapical area of the lateral cell surface, where it co-localized with the tight junction markers ZO-1 and occludin. Also endogenous CLMP showed association with tight junctions, as analyzed in polarized human CACO-2 cells. This suggested a role for CLMP in cell-cell adhesion and indeed, overexpressed CLMP induced aggregation of non-polarized CHO cells. Furthermore, CLMP-expressing MDCK cells showed significantly increased transepithelial resistance, indicating a role for CLMP in junctional barrier function. Thus, we conclude that CLMP is a novel cell-cell adhesion molecule and a new component of epithelial tight junctions. We also suggest, based on phylogenetic studies, that CLMP, CAR, ESAM, and BT-IgSF form a new group of proteins within the CTX family. The CTX family is a growing group of type I transmembrane proteins within the immunoglobulin superfamily (IgSF). They localize to junctional complexes between endothelial and epithelial cells and seem to participate in cell-cell adhesion and transmigration of leukocytes. Here, we report the identification of a new member of the CTX family. This protein, which was designated CLMP (coxsackie- and adenovirus receptor-like membrane protein), is composed of 373 amino acids including an extracellular part containing a V- and a C2-type domain, a transmembrane region and a cytoplasmic tail. CLMP mRNA was detected in a variety of both human and mouse tissues and cell lines. The protein migrated with an Mr of around 48 on SDS-PAGE and was predominantly expressed in epithelial cells within different tissues. In cultured epithelial cells, CLMP was detected in areas of cell-cell contacts. When exogenously expressed in polarized MDCK cells, CLMP was restricted to the subapical area of the lateral cell surface, where it co-localized with the tight junction markers ZO-1 and occludin. Also endogenous CLMP showed association with tight junctions, as analyzed in polarized human CACO-2 cells. This suggested a role for CLMP in cell-cell adhesion and indeed, overexpressed CLMP induced aggregation of non-polarized CHO cells. Furthermore, CLMP-expressing MDCK cells showed significantly increased transepithelial resistance, indicating a role for CLMP in junctional barrier function. Thus, we conclude that CLMP is a novel cell-cell adhesion molecule and a new component of epithelial tight junctions. We also suggest, based on phylogenetic studies, that CLMP, CAR, ESAM, and BT-IgSF form a new group of proteins within the CTX family. CTX 1The abbreviations used are: CTX, cortical thymocyte marker in Xenopus; TER, transepithelial resistance; CLMP, coxsackie- and adenovirus receptor-like membrane protein; aa, amino acids; CAR, coxsackie- and adenovirus receptor; IgSF, immunoglobulin superfamily; JAM, junctional adhesion molecule; CHO, Chinese hamster ovary. (cortical thymocyte marker in Xenopus) was originally discovered as a developmentally regulated type-I transmembrane protein specifically expressed by a large fraction of cortical thymocytes in Xenopuslaevis (1.Chretien I. Robert J. Marcuz A. Garcia-Sanz J.A. Courtet M. Du Pasquier L. Eur. J. Immunol. 1996; 26: 780-791Crossref PubMed Scopus (57) Google Scholar). It is structurally organized in an extracellular part containing one variable (V-type) and one constant (C2-type) immunoglobulin domain (separated from each other by a J segment), a transmembrane region and a cytoplasmic tail. Being the first identified member, CTX has been regarded as the prototype of a new subclass of proteins belonging to the large immunoglobulin superfamily (IgSF) of transmembrane proteins (1.Chretien I. Robert J. Marcuz A. Garcia-Sanz J.A. Courtet M. Du Pasquier L. Eur. J. Immunol. 1996; 26: 780-791Crossref PubMed Scopus (57) Google Scholar, 2.Chretien I. Marcuz A. Courtet M. Katevuo K. Vainio O. 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Other recently identified members are the endothelial cell-selective adhesion molecule (ESAM) (16.Hirata K. Ishida T. Penta K. Rezaee M. Yang E. Wohlgemuth J. Quertermous T. J. Biol. Chem. 2001; 276: 16223-16231Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and the brain- and testis-specific immunoglobulin superfamily (BT-IgSF) (17.Suzu S. Hayashi Y. Harumi T. Nomaguchi K. Yamada M. Hayasawa H. Motoyoshi K. Biochem. Biophys. Res. Commun. 2002; 296: 1215-1221Crossref PubMed Scopus (58) Google Scholar). These genes/proteins share characteristics with CTX such as a specific exon/intron organization, in which both the V and the C2 domains are encoded by splicing of two half-domain exons, and an extra pair of cysteine residues flanking the C2 domain. These characteristics have therefore been regarded as hallmarks of the CTX family (2.Chretien I. Marcuz A. Courtet M. Katevuo K. Vainio O. Heath J.K. White S.J. Du Pasquier L. Eur. J. Immunol. 1998; 28: 4094-4104Crossref PubMed Google Scholar). The only exception to this basic structure is JAM-A, which lacks the extra pair of cysteine residues. The CTX-like proteins are localized to cell-cell contacts between epithelial and endothelial cells within various tissues of the body. Among the specific members, A33 is exclusively expressed in epithelial cells within the gastrointestinal tract (6.Heath J.K. White S.J. Johnstone C.N. Catimel B. Simpson R.J. Moritz R.L. Tu G.F. Ji H. Whitehead R.H. Groenen L.C. Scott A.M. Ritter G. Cohen L. Welt S. Old L.J. Nice E.C. Burgess A.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 469-474Crossref PubMed Scopus (154) Google Scholar, 7.Johnstone C.N. Tebbutt N.C. Abud H.E. White S.J. Stenvers K.L. Hall N.E. Cody S.H. Whitehead R.H. Catimel B. Nice E.C. Burgess A.W. Heath J.K. Am. J. Physiol. Gastrointest Liver Physiol. 2000; 279: G500-G510Crossref PubMed Google Scholar), where it sublocalizes to the basolateral cell membranes. 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Immunol. 2003; 273: 87-111Google Scholar), was recently identified as being a transmembrane component of the tight junction (TJ) in polarized epithelial cells (24.Cohen C.J. Shieh J.T. Pickles R.J. Okegawa T. Hsieh J.T. Bergelson J.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15191-15196Crossref PubMed Scopus (554) Google Scholar). The JAMs, having attracted much attention because of their possible role in transmigration of leukocytes across the endothelial barrier to sites of inflammation, are localized to the cell surface of endothelial and epithelial cells, but have also been detected on circulating blood cells (14.Aurrand-Lions M. Johnson-Leger C. Wong C. Du Pasquier L. Imhof B.A. Blood. 2001; 98: 3699-3707Crossref PubMed Scopus (220) Google Scholar). The extracellular part of JAM-A interacts with other JAM-A molecules in a homophilic manner (25.Bazzoni G. Martinez-Estrada O.M. Mueller F. Nelboeck P. Schmid G. Bartfai T. Dejana E. Brockhaus M. J. Biol. 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Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2128) Google Scholar), does not seem to be an essential component of these complexes because the TJs in occludin -/- mice are not affected morphologically, and the barrier function of intestinal epithelium is normal (32.Saitou M. Furuse M. Sasaki H. Schulzke J.D. Fromm M. Takano H. Noda T. Tsukita S. Mol. Biol. Cell. 2000; 11: 4131-4142Crossref PubMed Scopus (933) Google Scholar). The CTX-like proteins, CAR and JAM, represent a newly discovered family of type I transmembrane proteins that also sublocalize to endothelial and epithelial TJs. These proteins are single spanning membrane Ig-like adhesion molecules, which mediate homophilic adhesion (25.Bazzoni G. Martinez-Estrada O.M. Mueller F. Nelboeck P. Schmid G. Bartfai T. Dejana E. Brockhaus M. J. Biol. Chem. 2000; 275: 30970-30976Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 33.Honda T. Saitoh H. Masuko M. Katagiri-Abe T. Tominaga K. Kozakai I. Kobayashi K. Kumanishi T. Watanabe Y.G. Odani S. Kuwano R. Brain Res. Mol. Brain Res. 2000; 77: 19-28Crossref PubMed Scopus (195) Google Scholar) and are involved in barrier function. In this study, we present the identification and characterization of CLMP (CAR-like membrane protein), an additional member of the CTX family. We show that CLMP, is expressed in epithelial cells within different tissues, co-localizes with the tight junction proteins ZO-1 and occludin in polarized MDCK cells, can mediate cell aggregation, and can regulate transepithelial resistance across polarized epithelial cells, supporting the notion that CLMP is involved in epithelial cell-cell adhesion. Bioinformatics—Multiple sequence alignments of the CTX-like proteins were performed using the ClustalW algorithm and the Blossum 62 substitution matrix (BioEdit Sequence Alignment Editor). The most conserved regions within the J segment and the C2 domain were used as search strings to identify novel related genes in the nonredundant and EST databases in the NCBI and the Celera human and mouse databases using the TBLASTN program. This resulted in the identification of a human "full insert sequence" (fis) clone termed HRC08561. This clone was originally isolated from primary human renal epithelial cells as part of the NEDO human cDNA sequencing project and filed in Genbank (NCBI) as a full-length cDNA sequence with the accession number AK026068, potentially encoding an unnamed protein product of 373 amino acids (accession number BAB15347). By searching the fully assembled mouse genomic Celera data base with the TBLASTX program with the BAB15347 amino acid sequence as a query, a genomic sequence potentially encoding a mouse orthologue to human BAB15347 was identified. Phylogenetic relationships were examined by distance matrix analysis and a tree was constructed in the TreeView program. cDNA Cloning and Sequencing—The human fis HRC08561 clone was kindly provided by Dr. Hiroko Hata at the Department of Virology, Institute of Medical Science, University of Tokyo. Standard PCR technique using primers: 5′-CACCATGTCCCTCCTCCTTCTC-3′, 5′-TCAGACCGTTTGGAAGGC-3′ (the translation initiation and termination signals are underlined) and a DNA polymerase with 5′ → 3′ proofreading activity (PFU DNA Polymerase, Promega, Madison, WI) was used to amplify the coding region of the cDNA. The amplified sequence was ligated into the mammalian expression vector pcDNA3.1D/V5-His-TOPO (Invitrogen AB, Stockholm, Sweden) by standard TOPO-cloning procedures. The coding part of the mouse cDNA sequence was isolated from a mouse 17-day embryo MATCHMAKER cDNA library (Clontech, Palo Alto, CA) by standard PCR technique, using the two primers: 5′-CACCATGTCCCTCTTCTTCCTC-3′ and 5′-TCAGACAGTTTGGAAGGC-3′. The amplified region was, similarly to the coding part of the human cDNA, cloned into the mammalian expression vector pcDNA3.1D/V5-His-TOPO. DNA sequencing was performed using the Thermo Sequenase dye terminator cycle sequencing pre-mix kit (Amersham Biosciences) and an automatic DNA sequencer (ABI PRISM 310 Genetic Analyzer, PerkinElmer, Stockholm, Sweden) as described by the manufacturer. Cell Lines and Transfection—HEK293 (293), CACO-2, BEAS-2B, MDCK, and CHO cells were obtained from American Type Culture Collection (ATCC). The human glioma cell line T98G was originally established from a patient tumor classified as glioblastoma multiforme, malignancy grade IV (34.Collins V.P. Int. Rev. Exp. Pathol. 1983; 24: 135-202PubMed Google Scholar). All cells except CHO and MDCK cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal calf serum, 2 mm l-glutamine, and 100 units/ml penicillin/100 units/ml streptomycin (Invitrogen). CHO and MDCK cells were cultured in EMEM (Invitrogen) supplemented with 10% fetal calf serum, 2 mm l-glutamine, and 100 units/ml penicillin and 100 units/ml streptomycin. Transfections were performed with the LipofectAMINE 2000 Reagent (Invitrogen) according to conditions that were recommended by the manufacturer. Northern Blot Analysis—Northern blot hybridization was performed on human or mouse multiple tissue Northern blots (MTN, Clontech) with probes corresponding to a 446-nucleotide PCR fragment of the human CLMP cDNA or to a 487-nucleotide PCR fragment of the mouse CLMP cDNA, respectively. The cDNA probes were generated and labeled by PCR-amplification (human primers: 5′-ACAGGCATAGTGGCTGGA-3′, 5′-GACTCCTAGGAAAGCGTG-3′; mouse primers: 5′-GACAGGCATAGTGGCAGGA-3′, 5′-GCAAGCACAAGTCAAGTC-3′) in the presence of [α-32P]dCTP (3000 Ci/mmol, Amersham Biosciences). Hybridization and washing steps were performed under conditions that were recommended by the manufacturer and blots were exposed to a phosphorimaging screen, which was developed using a Fujifilm Bio-Image Analyzer BAS1500 (Fuji Photo Film Co., Ltd., Tokyo, Japan). A human β-actin probe was obtained from the manufacturer and was used under standard conditions. Antibodies—Two peptides, corresponding to 18 amino acids (N-GTHTEIKRVAEEKVTLPC-C) in the N-terminal part (NP1) or to 18 amino acids (N-CAETTPSMIPSQSRAFQTV-C) of the very C-terminal part (CP1) of human CLMP were synthesized (an extra cysteine residue that was added to the N-terminal part of the CP1 peptide is underlined). The peptides were coupled to Imject Maleimide-activated mcKLH carrier (Pierce) via the cysteine residues. Rabbits were immunized with 500 μg of peptide in complete Freund's adjuvant (Invitrogen) and boosted with 500 μg of peptide in incomplete Freund's adjuvant (Invitrogen) every 4 weeks. The antiserum was affinity-purified against the peptide used for immunization using the SulfoLink Kit (Pierce) and quantified by a spectrophotometer at 280 nm. The mouse monoclonal antibodies against E-cadherin (clone 36) and occludin (clone 19) were obtained from BD Transduction Laboratories (Stockholm, Sweden). The mouse monoclonal antibody against ZO-1 was obtained from Zymed Laboratories. The polyclonal rabbit antibody against calnexin was produced in the laboratory. Immunoblotting—Cell lysates were prepared by solubilization in lysis buffer (1% Triton X-100, 20 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5 mm EDTA (pH 8.0), 100 KIE/ml Trasylol (Bayer AG, Leverkusen, Germany), and Complete protease inhibitors (Roche Applied Science). Nuclei were removed by centrifugation, and equal amounts of protein were separated on 12.5% SDS-PAGE under reducing conditions, and transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Germany). Membranes were incubated with the rabbit NP1 or CP1 antibodies, washed, and then incubated with donkey anti-rabbit Ig horseradish peroxidase (HRP, Amersham Biosciences). HRP activity was detected using enhanced chemiluminescence (ECL, Amersham Biosciences). Immunofluorescence and Confocal Microscopy—Cells to be analyzed by regular immunofluorescence microscopy were seeded on coverslips, grown until confluence and at the time for staining, fixed in 3% paraformaldehyde at room temperature for 15 min, quenched with 10 mm glycin in phosphate-buffered saline for 20 min at room temperature, washed and permeabilized with 0.1% Triton X-100 for 30 min. The cells were incubated with the CP1 or the mouse monoclonal anti-ZO-1 antibodies, washed, and incubated with an Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes, Eugene, OR). For blocking, the CP1 (10 μg/ml) antibody was incubated with an excess of CP1 peptide (60 μg/ml) for 1 h at 4 °C before staining. For visualization, cells were washed and mounted on glass slides, examined and photographed using a Zeiss Axiophot fluorescence microscope. MDCK and CACO-2 cells to be analyzed by confocal microscopy were seeded on polycarbonate filters (Cell Culture Insert, 0.4-μm pore size, BD Biosciences). Transepithelial resistence (TER) was measured over time using a Millicell-ERS Voltohmmeter (see below) until TER values between 500-700 Ω·cm2 were obtained. Cells were then fixed in ethanol at -20 °C for 20 min, washed, blocked in 5% normal goat serum, and co-incubated with the CP1 and ZO-1, occludin or E-cadherin antibodies at room temperature for 2 h. After washing and incubating with secondary Alexa Fluor antibodies (see above), filters were mounted and examined with a Zeiss LSM 510 scanning module fitted to an Axiovert 100 m microscope using a 63× oil immersion objective. Routinely, 0.15-0.20-μm thick focal planes were scanned. Immunohistochemistry—Mouse tissues to be analyzed by immunohistochemistry were paraformaldehyde-fixed, paraffin-embedded, and cut into 6-μm sections. Sections were pretreated in 0.01 m citric acid monohydrate (pH 6.0) at 95 °C for 20 min and blocked in TNB blocking buffer (TSA Biotin System, PerkinElmer Life Sciences, Boston, MA) supplemented with 4% bovine serum albumin, and 10% normal horse serum at room temperature for 25 min. They were then incubated with the NP1 (10 μg/ml) or CP1 (10 μg/ml) antibodies with, or without an excess of NP1 or CP1 peptide (60 μg/ml), respectively, overnight at 4 °C. After washing in TNT wash buffer, sections were incubated with a biotinylated anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA) at room temperature for 1 h. After additional washing steps, sections were incubated with Vectastain Elite ABC Reagent (Vector Laboratories) for 1 h at room temperature, rinsed in 0.05 m Tris-HCl (pH 7.5) and developed with 3,3′-diaminobenzidine (0.2 mg/ml, Sigma-Aldrich) in 0.05 m Tris-HCl at room temperature. Sections were counterstained with hematoxylin-eosin using standard procedures. Cell Aggregation Assay—CHO cells were transiently transfected with the pcDNA3.1-hCLMP or -hCAR vectors, an empty vector, or with medium only, at two time points with a 48 h interval. After a total period of 4 days, cells were detached from the plastic surface by incubation with 1 mm EDTA at 37 °C for 10 min, washed twice with Ca2+-free Hanks' balanced salt solution (HBSS), and resuspended in Ca2+-free HBSS supplemented with 2% fetal calf serum (pre-dialyzed against Ca2+-free HBSS) to a final concentration of 2 × 106 cells/ml. Single-cell suspensions were incubated in bovine serum albumin-coated Eppendorf tubes on a platform rotator at 80 rpm for 60 min at 37 °C. Cells were then gently spread out on culture plates and analyzed for aggregates in an inverted Nikon microscope. Five optic fields were examined for each sample and aggregates of more than three cells were counted as positives. Reproducibility was evaluated and found to be consistent in at least three separate experiments. TER Measurement—A number of 1 × 106 non-transfected or hCLMP-expressing MDCK cells were seeded on polycarbonate filters and grown at confluency for 3 days. TER was measured using a Millicell-ERS Voltohmmeter (Millipore, Life Science Division, Sundbyberg, Sweden). Accession Numbers—hCLMP, BK001245; mCLMP, AY259213; hCAR, AAC51234; mCAR, CAA71368; hBT-IgSF, BAC07546; mBT-IgSF, BAC07547; hESAM, AAK51065; mESAM, AAK51504; hA33, AAC50957; mA33, AAF65818; CTH, AAD17522; CTM, AAD17524; CTX, AAC59899; ChT1, AAD17523; hJAM-A, AAD42050; mJAM-A, AAC32982; hJAM-B, CAC69845; mJAM-B, CAC20704; hJAM-C, AAF81223; mJAM-C, AAF81224; hCD2, AAA51738; mCD2, AAD25889. Identification of a New Member of the CTX Family—To search for additional members of the CTX family we screened public EST and genomic databases with search strings corresponding to the most conserved regions within the J segment and the C2 domain of the previously existing members. By this approach, we identified both the human and mouse orthologues of a new homologue to CTX. Blast searches identified the CAR as the closest homologue to this protein (31% identity), which we named CAR-like membrane protein (CLMP). The amino acid (aa) sequences of human and mouse CLMP (h/mCLMP) were composed of 373 aa and showed 93% identity and 96% similarity to each other. Sequence alignments of both human and mouse CLMP, and other human CTX-like proteins, revealed that several characteristics of the family were conserved in CLMP. First, CLMP was found to share the same overall structure as the other family members with an extracellular part of 233 aa, containing a signal sequence and two Ig loops (the V and C2 domains), a transmembrane region (22 aa) and a cytoplasmic tail (118 aa) (Fig. 1A). Second, the most conserved amino acids, which were found in the second half of the V domain, in the J segment and in the C2 domain, were present in CLMP as well. Third, the four cysteine residues in the ectodomain, contributing to the intrachain disulfide bonds forming the V (Fig. 1A, C-1 and C-2) and the C2 (C-4 and C-5) domains, respectively, were conserved. Four, an extra pair of cysteine residues flanking the C2 domain (C-3 and C-6), forming an additional intra-chain disulfide-linked loop, and also considered as one of the hallmarks of the CTX family, was also found in the CLMP sequence. S