Regulation of heterotypic claudin compatibility

Tissue barrier function is directly mediated by tight junction transmembrane proteins known as claudins. Cells that form tight junctions typically express multiple claudin isoforms which suggests that heterotypic (head-to-head) binding between different claudin isoforms may play a role in regulating paracellular permeability. However, little is known about motifs that control heterotypic claudin compatibility. We found that although claudin-3 and claudin-4 were heteromerically compatible when expressed in the same cell, they did not heterotypically interact despite having extracellular loop (EL) domains that are highly conserved at the amino acid level. Claudin-1 and -5, which were heterotypically compatible with claudin-3, did not heterotypically bind to claudin-4. In contrast, claudin-4 chimeras containing either the first EL domain or the second EL domain of claudin-3 were able to heterotypically bind to claudin-1, claudin-3, and claudin-5. Moreover, a single point mutation in the first extracellular loop domain of claudin-3 to convert Asn(44) to the corresponding amino acid in claudin-4 (Thr) produced a claudin capable of heterotypic binding to claudin-4 while still retaining the ability to bind to claudin-1 and -5. Thus, control of heterotypic claudin-claudin interactions is sensitive to small changes in the EL domains.     

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.     

The first extracellular domain of claudin-7 affects paracellular Cl- permeability

Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl(-) and increases paracellular permeability to Na(+). To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl(-) permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl(-) permeability.     

Molecular Determinants of the Interaction between Clostridium perfringens Enterotoxin Fragments and Claudin-3

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116–319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10–13, 15, 16, 18–20, and 22. Substitution peptide mapping identified the central motif ¹⁴⁸NPL¹⁵⁰VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn¹⁴⁸ and Leu¹⁵⁰ of full-length claudin-3 in the binding. CPE-(116–319) and CPE-(194–319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116–319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116–319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116–319) and CPE-(194–319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.The clinical use of many promising drug candidates is impeded by unacceptable pharmacokinetics (1). The ability of a drug to pass through tissue barriers is a major determinant for its delivery. In epithelia and endothelia, the paracellular route is blocked by tight junctions (TJ).⁴ Different approaches have been used to enhance transcellular drug delivery. These include the use of influx transporters, blocking of efflux transporters, or receptor-mediated endocytosis (2). Alternative approaches aim to enhance paracellular permeation of drugs by loosening the TJ (3, 4). This strategy has the advantage that it could improve the delivery of structurally unrelated drugs, and the drug itself does not have to be modified. Although different TJ modulators have been described, most of these are based on surfactants or chelators (3). These often have low tissue specificity and cause severe side effects, e.g. exfoliation of cells, which irreversibly compromise the barrier functions (5, 6). Fewer side effects may be obtained by more specific modulation of a molecular key component of the TJ (7).TJ consist of transmembrane proteins, mainly the tetraspan proteins of the claudin family, as well as occludin and tricellulin (8). Other molecules associated with TJ include membrane-bound scaffolding and signaling proteins (9). However, claudins (Cld) are the major functional constituent of TJ (10). Claudins tighten the paracellular space, selectively for tissue, size, and charge. The tissue-specific combination of the claudin subtypes present in heteropolymers is assumed to determine the permeability properties of TJ (11). It was therefore proposed that tissue-specific drug delivery via the paracellular route would be possible by modulation of the barrier-function of claudins in a subtype-specific manner (7).A subset of claudins, e.g. Cld3 and -4 but not -1 and -2, have been shown to be receptors for Clostridium perfringens enterotoxin (CPE) with high association constants of about 10⁸ m⁻¹ (12). CPE causes one of the most common food-borne diseases (13). It consists of two functional domains, an N-terminal region that mediates the cytotoxic effect and the C-terminal region (CPE-(184–319)), which binds to extracellular loop 2 (ECL2) of Cld3 but not of Cld1 nor to the ECL1 of Cld3 (12). Treatment of epithelial monolayers with non-cytotoxic CPE-(184–319) increases paracellular permeability (14). CPE-(184–319) enhanced drug absorption in rat jejunum 400-fold relative to sodium caprate, which is in clinical use (15). Thus, CPE is a promising tool to specifically modulate claudins, the key constituents of TJ, and thereby to enhance paracellular drug delivery. In addition, some studies have suggested the use of CPE for the chemotherapy of tumors overexpressing claudins (16–18).Cld1 and -5 are potential targets for transepidermal and brain drug delivery, respectively (19, 20). However, it has been reported that these claudins do not interact with CPE (12). Modification of CPE could enhance and/or shift its claudin-subtype specificity. Therefore, the design of CPE-based TJ modulators could permit efficient claudin subtype-specific modulation, which would also be tissue-specific modulation of TJ. To achieve this, an understanding of the molecular mechanism of the CPE-claudin interaction is a necessary prerequisite. In this study, we identify the residues within the ECL2 of Cld3 that are involved in interaction with CPE.     

CPE对体外培养前列腺癌细胞株的抑制作用

目的:探讨产气荚膜梭菌肠毒素(CPE)对前列腺癌细胞株在体外培养过程中的抑制作用。方法:以细胞免疫荧光染色观察人前列腺癌PC-3细胞株和22RV1细胞株表达Claudin-3情况;并分别用生长曲线测定法及MTT法等测定CPE对PC-3和22RV1细胞活性的影响;分析CPE对肿瘤的生长抑制作用。结果:应用Claudin-3单抗进行免疫荧光染色,证实体外培养的人前列腺癌细胞株22RV1可以特异性高表达Claudin-3,而在PC-3上有弱表达;用相关方法测定经过不同浓度的CPE处理后的前列腺癌细胞株22RV1细胞增殖明显降低,有明显抑制作用;而对PC-3则未产生明显细胞毒性作用。结论:CPE在体外可通过特异性作用于Claudin-3高表达的前列腺癌细胞株对前列腺癌细胞产生抑制作用。     

Regulation of tight junctions by sex hormones in normal human endometrial epithelial cells and uterus cancer cell line Sawano

The number of patients with uterine endometrial carcinoma, the cause of which involves sex hormones, has recently been growing rapidly because of increases in life expectancy and obesity. Tight junction proteins claudin-3 and ?4 are receptors of Clostridium perfringens enterotoxin (CPE) and increase during endometrial carcinogenesis. In the present study of normal human endometrial epithelial (HEE) cells and the uterus cancer cell line Sawano, we investigate changes in the expression of tight junction proteins including claudin-3 and ?4, the fence and barrier functions of the tight junction and the cytotoxic effects of CPE by sex hormones. In primary cultured HEE cells, treatment with progesterone (P4) but not estradiol (E2), induced claudin-1, ?3, ?4 and ?7 and occludin, together with the downregulation of the barrier function but not the fence function. In Sawano cells, claudin-3 and ?4 were upregulated by E2 but not by P4, together with a disruption of both the barrier and fence function. In primary cultured HEE cells, claudin-3 and ?4 were localized at the apicalmost regions (tight junction areas) and no cytotoxicity of CPE was observed. In Sawano cells, claudin-3 and ?4 were found not only in the apicalmost regions but also at the basolateral membrane and the cytotoxicity of CPE was enhanced by E2. Thus, tight junctions are physiological regulated by sex hormones in normal HEE cells during the menstrual cycle suggesting that safer and more effective therapeutic methods targeting claudins in uterine cancer can be developed.     

Structural constraints for the binding of short peptides to claudin-4 revealed by surface plasmon resonance

Claudin family transmembrane proteins play an important role in tight junction structure and function in epithelial cells. Among the 24 isoforms identified in mice and humans, claudin-4 and -3 serve as the receptor for Clostridium perfringens enterotoxin (Cpe). The second extracellular loop (Ecl2) of claudin-4 is responsible for the binding to the C-terminal 30 amino acids of Cpe (Cpe30). To define the structural constraints for the claudin-4/Cpe30 interaction, a surface plasmon resonance (SPR) method was developed. GST fusions with claudin-4 revealed that Ecl2 with the downstream transmembrane domain of claudin-4 reconstituted the basic structural requirement for optimal binding activity to Cpe30, with affinity in the nanomolar range. Two 12-mer peptides selected by phage display against claudin-4-transfected CHO cells and a 12-mer Cpe mutant peptide also showed significant affinity for claudin-4 with this SPR assay, suggesting that a short peptide can establish stable contact with Ecl2 with nanomolar affinity. Alignment of these short peptides unveiled a common Ecl2 binding motif: . Whereas the short peptides bound native claudin-4 on transfected CHO cells in pull-down assays, only the larger Cpe30 peptide affected trans-epithelial electrical resistance (TER) in peptide-treated Caco-2BBe monolayers. Importantly, Cpe30 retained its binding to claudin-4 when fused to the C terminus of influenza hemagglutinin, demonstrating that its binding activity can be maintained in a different biochemical context. These studies may help in the design of assays for membrane receptor interactions with soluble ligands, and in applying new targeting ligands to delivering attached "cargo" proteins.     

Genetic ablation of Slc4a10 alters the expression pattern of transporters involved in solute movement in the mouse choroid plexus

Mutational changes of one transporter can have deleterious effects on epithelial function leaving the cells with the options of either compensating for the loss of function or dedifferentiating. Previous studies have shown that the choroid plexus epithelium (CPE) from mice lacking the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger (NCBE) encoded by Slc4a10 leads to retargeting of the Na(+)/H(+) exchanger 1 (NHE1) from the luminal to the basolateral plasma membrane. We hypothesized that disruption of NCBE, the main basolateral Na(+) importer in the CPE, would lead to a compensatory increase in the abundance of other important transport proteins in this tissue. Aquaporin-1 (AQP1) abundance was 42.7% lower and Na,K-ATPase 36.4% lower in the CPE of Slc4a10 knockout mice, respectively. The NHE1 binding ezrin cytoskeleton appeared disrupted in Slc4a10 knockout mice, whereas no changes were observed in cellular polarization with respect to claudin-2 and appearance of luminal surface microvilli. The renal proximal tubule constitutes a leaky epithelium with high transport rate similar to CPE. Here, Slc4a10 knockout did not affect Na,K-ATPase or AQP1 expression. CPE from AQP1 knockout mice has a secretory defect similar to Slc4a10 mice. However, neither NCBE nor Na,K-ATPase expression was affected in CPE from AQP1 knockout mice. By contrast, the abundance of Na,K-ATPase and NBCe1 was decreased by 23 and 31.7%, respectively, in AQP1 knockout proximal tubules, while the NHE3 abundance was unchanged. In conclusion, CPE lacking NCBE seems to spare the molecular machinery involved in CSF secretion rather than compensate for the loss of the Na(+) loader. Slc4a10 knockout seems to be more deleterious to CPE than AQP1 knockout.     

Manner of interaction of heterogeneous claudin species within and between tight junction strands

In tight junctions (TJs), TJ strands are associated laterally with those of adjacent cells to form paired strands to eliminate the extracellular space. Claudin-1 and -2, integral membrane proteins of TJs, reconstitute paired TJ strands when transfected into L fibroblasts. Claudins comprise a multigene family and more than two distinct claudins are coexpressed in single cells, raising the questions of whether heterogeneous claudins form heteromeric TJ strands and whether claudins interact between each of the paired strands in a heterophilic manner. To answer these questions, we cotransfected two of claudin-1, -2, and -3 into L cells, and detected their coconcentration at cell-cell borders as elaborate networks. Immunoreplica EM confirmed that distinct claudins were coincorporated into individual TJ strands. Next, two L transfectants singly expressing claudin-1, -2, or -3 were cocultured and we found that claudin-3 strands laterally associated with claudin-1 and -2 strands to form paired strands, whereas claudin-1 strands did not interact with claudin-2 strands. We concluded that distinct species of claudins can interact within and between TJ strands, except in some combinations. This mode of assembly of claudins could increase the diversity of the structure and functions of TJ strands.     

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to <Emphasis Type="Italic">Clostridium perfringens</Emphasis> enterotoxin via claudin-4

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and −4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.     

Claudin-2 promotes breast cancer liver metastasis by facilitating tumor cell interactions with hepatocytes

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.     

Epidermal growth factor modulates claudins and tight junctional functions in ovarian cancer cell lines

Ovarian adenocarcinomas, like human ovarian surface epithelial cells, form functional tight junctions. Tight junction molecules claudin-3 and claudin-4, which are the receptors of Clostridium perfringens enterotoxin (CPE), are abnormally upregulated in epithelial ovarian cancers of all subtypes including, mucinous cystadenocarcinoma and serous cystadenocarcinoma. Clostridium perfringens enterotoxin may be a novel tumor-targeted therapy for ovarian cancers. In epithelial ovarian cancers, overexpression of epidermal growth factor receptor has been observed and the exogenous ligand EGF induces epithelial-mesenchymal transition in ovarian surface epithelium. Epidermal growth factor (EGF) signaling modulates expression of claudins with changes of fence and barrier functions in various cell types. However, the regulation of tight junctions by EGF in ovarian cancers remains unclear. In the present study, to investigate the mechanisms of the regulation of tight junctions in ovarian cancers, ovarian cancer cell lines mucinous cystadenocarcinoma (MCAS) and serous cystadenocarcinoma (HUOA) were treated with EGF. Epidermal growth factor downregulated claudin-3 in MCAS and claudin-4 in HUOA by inducing degradation of the proteins with changes in structures and functions of tight junctions via the MEK/ERK or PI3K/Akt signaling pathway. In addition, in HUOA but not MCAS, EGF downregulated the cytotoxic effect of CPE via claudin-4. Thus, there were different mechanisms for regulation of claudins by EGF between subtypes of epithelial ovarian cancer cells in vitro. These results indicate that EGF may affect claudins and tight junctional functions in ovarian cancer cells during cancer progression.     

Enhancement of cell-cell contact by claudin-4 in renal epithelial Madin-Darby canine kidney cells

Claudin-4 regulates ion permeability via a paracellular pathway in renal epithelial cells, but its other physiological functions have not been examined. We found that hyperosmotic stress increases claudin-4 expression in Madin-Darby canine kidney cells. Here, we examined whether claudin-4 affects cell motility, cell association, and the intracellular distribution of endogenous junctional proteins. Doxycycline-inducible expression of claudin-4 did not change endogenous levels of claudin-1, claudin-2, claudin-3, occludin, E-cadherin, and ZO-1. Claudin-4 overexpression increased cell association and decreased cell migration without affecting cell proliferation. Doxycycline did not change cell junctional protein levels, cell association or cell migration in mock-transfected cells. The insolubility of claudin-1 and -3 in Triton X-100 was increased by claudin-4 overexpression, but that of claudin-2, occludin, ZO-1, and E-cadherin was unchanged. Immunocytochemistry showed that claudin-4 overexpression increases the accumulation of claudin-1 and -3 in tight junctions (TJs). Furthermore, claudin-4 overexpression increased the association of claudin-4 with claudin-1 and -3. These results suggest that claudin-4 accumulates claudin-1 and -3 in TJs to enhance cell-cell contact in renal tubular epithelial cells.     

Lipopolysaccharide Disrupts the Milk-Blood Barrier by Modulating Claudins in Mammary Alveolar Tight Junctions

Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling.     

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.     

Clostridium perfringens Enterotoxin Interacts with Claudins via Electrostatic Attraction

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr³⁰⁶, Tyr³¹⁰, Tyr³¹², and Leu³¹⁵, which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.     

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.     

Claudin-6 enhances cell invasiveness through claudin-1 in AGS human adenocarcinoma gastric cancer cells

Claudins participate in tissue barrier function. The loss of this barrier is associated to metalloproteases-related extracellular matrix and basal membranes degradation. Claudin-1 is a pro-MMP-2 activator and claudin-6 transfected AGS (AGS-Cld6) cells are highly invasive. Our aim was to determine if claudin-6 was direct or indirectly associated with MMP-2 activation and cell invasiveness. Cytofluorometry, cell fractioning, immunoprecipitation, gelatin-zymography, cell migration and invasiveness assays were performed, claudin-2, -6, -7 and -9 transfected AGS cells, anti-MMP-2, -9 and -14, anti-claudins specific antibodies and claudin-1 small interfering RNA were used. The results showed a significant (p<0.001) overexpression of claudin-1 in AGS-Cld6 cell membranes. A strong MMP-2 activity was identified in culture supernatants of AGS-Cld6. Claudin-1 co-localized with MMP-2 and MMP-14; interestingly a significant increase in cell membrane and cytosol MMP-14 expression was detected in AGS-Cld6 cells (p<0.05). Silencing of claudin-1 in AGS-Cld6 cells showed a 60% MMP-2 activity decrease in culture supernatants and a significant decrease (p<0.05) in cell migration and invasiveness. Our results suggest that claudin-6 induces MMP-2 activation through claudin-1 membrane expression, which in turn promotes cell migration and invasiveness.     

Role of each immunoglobulin-like loop of nectin for its cell-cell adhesion activity

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules that form cell-cell junctions, cooperatively with or independently of cadherins, in a variety of cells. Nectins comprise a family of four members, nectin-1, -2, -3, and -4. All nectins have one extracellular region with three Ig-like loops, one transmembrane segment, and one cytoplasmic tail. It has been shown mainly by use of cadherin-deficient L fibroblasts stably expressing each nectin that nectins first form homo-cis-dimers and then homo- or hetero-trans-dimers, causing cell-cell adhesion, and that the formation of the cis-dimers is necessary for the formation of the trans-dimers. However, kinetics of the formation of these dimers have not been examined biochemically by use of pure nectin proteins. We prepared here pure recombinant proteins of extracellular fragments of nectin-3 containing various combinations of Ig-like loops, all of which were fused to the Fc portion of IgG and formed homo-cis-dimers through the Fc portion, and of an extracellular fragment of nectin-1 containing three Ig-like loops which was fused to secreted alkaline phosphatase and formed homo-cis-dimers. We showed here by use of these proteins that the first Ig-like loop of nectin-3 was essential and sufficient for the formation of trans-dimers with nectin-1, but that the second Ig-like loop of nectin-3 was furthermore necessary for its cell-cell adhesion activity.