Proc. Natl. Acad. Sci. USA Vol. 96, pp. 511–516, January 1999 Cell Biology Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands KAZUMASA MORITA*†,MIKIO FURUSE*, KAZUSHI FUJIMOTO‡, AND SHOICHIRO TSUKITA*§ Departments of *Cell Biology, †Dermatology, and ‡Anatomy, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan Communicated by Setsuro Ebashi, National Institute for Physiological Sciences, Okazaki, Japan, November 9, 1998 (received for review September 4, 1998) ABSTRACT Two related integral membrane proteins, well as fence functions of TJs (24–27). Recently, gene knock- claudin-1 and -2, recently were identified as novel components out of occludin was performed successfully, but, unexpectedly, of tight junction (TJ) strands. Here, we report six more occludin-deficient epithelial cells still demonstrated a well claudin-like proteins, indicating the existence of a claudin developed network of TJ strands (28). gene family. Three of these were reported previously as RVP1, Most recently, as the second and third components of TJ Clostridium perfringens enterotoxin receptor, and TMVCF, but strand, claudin-1 and -2, '22-kDa integral membrane proteins their physiological functions were not determined. Through that are structurally related (38% identical at the amino acid similarity searches followed by PCR, we isolated full length sequence level), were identified (29). These two proteins also cDNAs of mouse RVP1, Clostridium perfringens enterotoxin bear four transmembrane domains but do not show any receptor, and TMVCF as well as three mouse claudin-like sequence similarity to occludin. Furthermore, it was shown proteins and designated them as claudin-3 to -8, respectively. that claudin-1 and -2 have an ability to induce the formation All of these claudin family members showed similar patterns of networks of strandsygrooves at cell–cell contact sites when on hydrophilicity plots, which predicted four transmembrane introduced into fibroblasts lacking TJs (30). Because occludin domains in each molecule. Northern blotting showed that the induced only a small number of short strands at cell–cell tissue distribution pattern varied significantly, depending on contact sites in fibroblasts (30), these findings suggest that claudin species. Similarly to claudin-1 and -2, when these claudin-1 and -2 function as major structural components of TJ claudins were HA-tagged and introduced into cultured Madin– strands and that occludin is an accessory protein in terms of TJ Darby canine kidney cells, all showed a tendency to concen- strand formation. trate at TJs. Immunofluorescence and immunoelectron mi- So far, intensive efforts have failed to identify isotypes of croscopy with polyclonal antibodies specific for claudin-3, -4, occludin in any species. In contrast, identification of claudin-1 or -8 revealed that these molecules were exclusively concen- and -2 indicated the existence of a gene family that could be trated at TJs in the liver andyor kidney. These findings called the ‘‘claudin family.’’ Questions, then, naturally have indicated that multiple claudin family members are involved arisen as to how many members constitute this gene family and in the formation of TJ strands in various tissues. whether all of the members are directly involved in TJ forma- tion. To date, three full length cDNAs, RVP1, Clostridium The tight junction (TJ) is a specialized membrane domain at perfringens enterotoxin receptor (CPE-R), and TMVCF, with the most apical region of polarized epithelial and endothelial sequence similarity to claudin-1 and -2 cDNAs, have been cells that not only creates a primary barrier to prevent para- reported, although physiological functions have not been cellular transport of solutes (barrier function) but also restricts determined for any of their products. Rat RVP1 was isolated the lateral diffusion of membrane lipids and proteins to as a cDNA expression that was elevated in regressing ventral maintain the cellular polarity (fence function) (for reviews, see prostate by subtraction cDNA cloning (31), and its human refs. 1–4). Freeze fracture techniques demonstrate that TJs homologue also was identified recently (32, 33). Monkey CPE-R was shown to encode a receptor for the Clostridium represent a continuous network of interconnected rows of y intramembranous particles that appear as strands with com- perfringens enterotoxin (CPE) (32), and, recently, human plementary grooves, which are thought to be directly involved mouse CPE-R as well as human RVP1 products were shown both in barrier and fence functions of TJs (5, 6). To date, to specifically bind to CPE (32). The human TMVCF (trans- several unique peripheral membrane proteins have been iden- membrane protein deleted in Velo-cardio-facial syndrome) gene was localized to chromosome 22q11, which frequently is tified as concentrated at the cytoplasmic surface of TJs: ZO-1 y (7–9), ZO-2 (10, 11), ZO-3 (12, 13), cingulin (14), 7H6 antigen deleted in Velo-cardio-facial DiGeorge syndrome patients (15), and symplekin (16). Among these, ZO-1, ZO-2, and ZO-3 (35). Furthermore, several sequences similar to claudin-1 and are related to each other and belong to the membrane- -2 were found in expressed sequence tag (EST) databases. In associated guanylate kinase homologues gene family, which this study, we isolated mouse RVP1, CPE-R, and TMVCF and contains PDZ, SH3, and GUK (guanylate kinase-like) do- three full length cDNAs that encoded members of the claudin mains (17–19). family. Similarly to claudin-1 and -2, when HA-tagged mouse By contrast, the TJ-specific integral membrane protein, i.e., RVP1, CPE-R, TMVCF, and three other claudin members the components of TJ strands, only recently were identified. As were introduced into Madin–Darby canine kidney (MDCK) the first component of TJ strand, occludin, an '65-kDa integral membrane protein bearing four transmembrane do- Abbreviations: TJ, tight junction; MDCK cells, Madin–Darby canine mains, was identified (20, 21). There is now accumulating kidney cells; CPE, Clostridium perfringens enterotoxin; CPE-R, CPE receptor; pAb, polyclonal antibody; EST, expressed sequence tag; evidence that occludin is a component of the TJ strand itself GST, glutathione S-transferase. (22, 23) and that occludin is directly involved in the barrier as Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF072127, The publication costs of this article were defrayed in part by page charge AF072128, AF087821, AF087822, AF087823, AF087824, AF087825, and AF087826). payment. This article must therefore be hereby marked ‘‘advertisement’’ in A Commentary on this article begins on page 319. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. e-mail: htsukita@ PNAS is available online at www.pnas.org. mfour.med.kyoto-u.ac.jp. 511 Downloaded by guest on September 27, 2021 512 Cell Biology: Morita et al. Proc. Natl. Acad. Sci. USA 96 (1999) cells, all of them showed a tendency to concentrate at preex- cysteine to keyhole limpet hemocyanin. These conjugated isting TJs. Furthermore, we obtained polyclonal antibodies peptides were used as antigens to generate pAbs in rabbits. The (pAbs) specific for three members of the claudin family, which antiserum specific for claudin-3, -4, or -8 was affinity-purified specifically labeled TJs in the liver andyor kidney. These with glutathione S-transferase (GST) fusion protein with the results indicate that claudin family members play a central role COOH-terminal cytoplasmic domain of claudin-3, -4, or -8, in TJ formation in various tissues. respectively. To express the GSTyclaudin fusion proteins in Escherichia coli, DNA fragments were amplified by PCR from MATERIALS AND METHODS cDNAs encoding COOH-terminal cytoplasmic domains of mouse claudins, and the amplified fragments were subcloned Antibodies and Cells. Rat anti-mouse occludin mAb into pGEX4T-1 (Pharmacia) in frame. (MOC37) was raised and characterized as described (36). Rat Immunofluorescence Microscopy and Immunoelectron Mi- anti-mouse E-cadherin mAb (ECCD2) kindly was provided by croscopy. Stable MDCK transfectants plated on glass cover- M. Takeichi (Kyoto University). Mouse anti-HA-tag mAb slips and frozen sections of mouse liver and kidney were (12CA5) was purchased from Boehringer Mannheim. MDCK stained immunofluorescently as described (20, 30). Immuno- cells were grown in DMEM supplemented with 10% fetal calf electron microscopy to examine freeze-fracture replicas was serum. performed as described (22, 29, 30). cDNA Cloning and Sequencing. Mouse liver or kidney total RNA was isolated according to the method described by 1 RESULTS Chomczynski and Sacchi (37). Poly(A) RNA was prepared from the total RNA by using oligo(dT)-cellulose (New Cloning and Characterization of cDNAs Encoding Claudin England Biolabs). First strand cDNA was prepared from this Family Members. Claudin-1 and -2 were identified (29). As poly(A)1 RNA with Superscript II reverse transcriptase mentioned above, similarity searches identified three previ- (GIBCOyBRL) and was used for PCR. ously reported full length cDNAs encoding integral membrane As described in detail in Results, 15 mouse EST clones with proteins that showed homology to claudin-1 and –2: RVP1 (31, sequence homology to claudin-1 were found, which enabled us 33), CPE-R (32, 34), and TMVCF (35), which are designated to amplify cDNAs encoding ORFs of claudin-3, -5, -6, -7, and here as claudin-3, -4, and -5, respectively. Among these, only -8 from mouse liver or kidney first strand DNA by PCR. CPE-R cDNA was isolated from mouse (ORF 5 210 amino Claudin-4 cDNA also was amplified from mouse kidney first acids; calculated molecular mass 5 22.3 kDa) whereas RVP1 strand DNA by PCR using the previously reported sequence and TMVCF cDNAs were obtained from ratyhuman and of mouse CPE-R (32). Furthermore, human claudin-5 (human human, respectively. We then searched for mouse RVP1 TMVCF) was obtained from human genomic DNA isolated (mRVP1) sequences in databases and identified four overlap- from peripheral blood cells by PCR.