The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Masuo Kondoh - One of the best experts on this subject based on the ideXlab platform.

  • radiolabeled ccpe peptides for spect imaging of Claudin 4 overexpression in pancreatic cancer
    The Journal of Nuclear Medicine, 2020
    Co-Authors: Julia Baguna Torres, Masuo Kondoh, Michael Mosley, Veerle Kersemans, Sofia Koustoulidou, Samantha L Hopkins, S Knapp, A Chaikuad, Keisuke Tachibana, Bart Cornelissen
    Abstract:

    Overexpression of tight-junction protein Claudin-4 has been detected in primary and metastatic pancreatic cancer tissue and is associated with better prognosis in patients. Noninvasive measurement of Claudin-4 expression by imaging methods could provide a means for accelerating detection and stratifying patients into risk groups. Clostridium perfringens enterotoxin (CPE) is a natural ligand for Claudin-4 and holds potential as a targeting vector for molecular imaging of Claudin-4 overexpression. A glutathione S-transferases (GST)–tagged version of the C terminus of CPE (cCPE) was previously used to delineate Claudin-4 overexpression by SPECT but showed modest binding affinity and slow blood clearance in vivo. Methods: On the basis of the crystal structure of cCPE, a series of smaller cCPE194–319 mutants with putatively improved binding affinity for Claudin-4 was generated by site-directed mutagenesis. All peptides were conjugated site-specifically on a C-terminal cysteine using maleimide-diethylenetriamine pentaacetate to enable radiolabeling with 111In. The binding affinity of all radioconjugates was evaluated in Claudin-4–expressing PSN-1 cells and HT1080-negative controls. The specificity of all cCPE mutants to Claudin-4 was assessed in HT1080 cells stably transfected with Claudin-4. SPECT/CT imaging of BALB/c nude mice bearing PSN-1 or HT1080 tumor xenografts was performed to determine the Claudin-4–targeting ability of these peptides in vivo. Results: Uptake of all cCPE-based radioconjugates was significantly higher in PSN-1 cells than in HT1080-negative controls. All peptides showed a marked improvement in affinity for Claudin-4 in vitro when compared with previously reported values (dissociation constant: 2.2 ± 0.8, 3 ± 0.1, 4.2 ± 0.5, 10 ± 0.9, and 9.7 ± 0.7 nM). Blood clearance of [111In]In-cCPE194–319, as measured by SPECT, was considerably faster than that of [111In]In-cCPE.GST (half-life,

  • Impaired airway mucociliary function reduces antigen-specific IgA immune response to immunization with a Claudin-4-targeting nasal vaccine in mice.
    Scientific reports, 2018
    Co-Authors: Hidehiko Suzuki, Hiroshi Kiyono, Kiyohito Yagi, Takahiro Nagatake, Ayaka Nasu, Huangwenxian Lan, Koji Ikegami, Mitsutoshi Setou, Yoko Hamazaki, Masuo Kondoh
    Abstract:

    Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) targeted Claudin-4, which is expressed by the epithelium associated with nasopharynx-associated lymphoid tissue (NALT), and could be effective as a nasal vaccine delivery. Mice lacking tubulin tyrosine ligase-like family, member 1 (Ttll1-KO mice) showed mucus accumulation in nasal cavity due to the impaired motility of respiratory cilia. Ttll1-KO mice nasally immunized with C-CPE fused to pneumococcal surface protein A (PspA-C-CPE) showed reduced PspA-specific nasal IgA responses, impaired germinal center formation, and decreased germinal center B-cells and follicular helper T cells in the NALT. Although there was no change in the expression of Claudin-4 in the NALT epithelium in Ttll1-KO mice, the epithelium was covered by a dense mucus that prevented the binding of PspA-C-CPE to NALT. However, administration of expectorant N-acetylcysteine removed the mucus and rescued the PspA-specific nasal IgA response. These results show that the accumulation of mucus caused by impaired respiratory cilia function is an interfering factor in the C-CPE-based Claudin-4-targeting nasal vaccine.

  • pro chemotherapeutic effects of antibody against extracellular domain of Claudin 4 in bladder cancer
    Cancer Letters, 2015
    Co-Authors: Masaomi Kuwada, Yoshitomo Chihara, Yi Luo, Yukiko Nishiguchi, Rina Fujiwara, Takamistu Sasaki, Kiyomu Fujii, Hitoshi Ohmori, Kiyohide Fujimoto, Masuo Kondoh
    Abstract:

    Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the Claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.

  • mutated c terminal fragments of clostridium perfringens enterotoxin have increased affinity to Claudin 4 and reversibly modulate tight junctions in vitro
    Biochemical and Biophysical Research Communications, 2011
    Co-Authors: Azusa Takahashi, Hiroshi Uchida, Masuo Kondoh, Takao Hamakubo, Yohei Kakamu, Kiyohito Yagi
    Abstract:

    Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the Claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a Claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve Claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to Claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to Claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.

  • mucosal vaccination using Claudin 4 targeting
    Biomaterials, 2010
    Co-Authors: Hideki Kakutani, Masuo Kondoh, Takao Hamakubo, Hidehiko Suzuki, Masahiro Fukasaka, Kiyohito Yagi
    Abstract:

    Mucosa-associated lymphoid tissue (MALT) plays pivotal roles in mucosal immune responses. Efficient delivery of antigens to MALT is a critical issue for the development of mucosal vaccines. Although Claudin-4 is preferentially expressed in MALT in the gut, a Claudin-4-targeting approach for mucosal vaccination has never been developed. In the present study, we found that Claudin-4 is expressed in nasal MALT, and we prepared a fusion protein of ovalbumin (OVA) as a model antigen with a Claudin-4-binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) (OVA-C-CPE). Nasal immunization with OVA-C-CPE, but not a mixture of OVA and C-CPE, induced the production of OVA-specific serum IgG and nasal, vaginal and fecal IgA. Deletion of the Claudin-4-binding region in OVA-C-CPE attenuated the induction of the immune responses. OVA-C-CPE immunization activated both Th1 and Th2 responses, and nasal immunization with OVA-C-CPE showed anti-tumor activity in mice inoculated with OVA-expressing thymoma cells. These results indicate that the Claudin-4-targeting may be a potent strategy for nasal vaccination.

Kiyohito Yagi - One of the best experts on this subject based on the ideXlab platform.

  • Impaired airway mucociliary function reduces antigen-specific IgA immune response to immunization with a Claudin-4-targeting nasal vaccine in mice.
    Scientific reports, 2018
    Co-Authors: Hidehiko Suzuki, Hiroshi Kiyono, Kiyohito Yagi, Takahiro Nagatake, Ayaka Nasu, Huangwenxian Lan, Koji Ikegami, Mitsutoshi Setou, Yoko Hamazaki, Masuo Kondoh
    Abstract:

    Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) targeted Claudin-4, which is expressed by the epithelium associated with nasopharynx-associated lymphoid tissue (NALT), and could be effective as a nasal vaccine delivery. Mice lacking tubulin tyrosine ligase-like family, member 1 (Ttll1-KO mice) showed mucus accumulation in nasal cavity due to the impaired motility of respiratory cilia. Ttll1-KO mice nasally immunized with C-CPE fused to pneumococcal surface protein A (PspA-C-CPE) showed reduced PspA-specific nasal IgA responses, impaired germinal center formation, and decreased germinal center B-cells and follicular helper T cells in the NALT. Although there was no change in the expression of Claudin-4 in the NALT epithelium in Ttll1-KO mice, the epithelium was covered by a dense mucus that prevented the binding of PspA-C-CPE to NALT. However, administration of expectorant N-acetylcysteine removed the mucus and rescued the PspA-specific nasal IgA response. These results show that the accumulation of mucus caused by impaired respiratory cilia function is an interfering factor in the C-CPE-based Claudin-4-targeting nasal vaccine.

  • mutated c terminal fragments of clostridium perfringens enterotoxin have increased affinity to Claudin 4 and reversibly modulate tight junctions in vitro
    Biochemical and Biophysical Research Communications, 2011
    Co-Authors: Azusa Takahashi, Hiroshi Uchida, Masuo Kondoh, Takao Hamakubo, Yohei Kakamu, Kiyohito Yagi
    Abstract:

    Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the Claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a Claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve Claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to Claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to Claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.

  • mucosal vaccination using Claudin 4 targeting
    Biomaterials, 2010
    Co-Authors: Hideki Kakutani, Masuo Kondoh, Takao Hamakubo, Hidehiko Suzuki, Masahiro Fukasaka, Kiyohito Yagi
    Abstract:

    Mucosa-associated lymphoid tissue (MALT) plays pivotal roles in mucosal immune responses. Efficient delivery of antigens to MALT is a critical issue for the development of mucosal vaccines. Although Claudin-4 is preferentially expressed in MALT in the gut, a Claudin-4-targeting approach for mucosal vaccination has never been developed. In the present study, we found that Claudin-4 is expressed in nasal MALT, and we prepared a fusion protein of ovalbumin (OVA) as a model antigen with a Claudin-4-binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) (OVA-C-CPE). Nasal immunization with OVA-C-CPE, but not a mixture of OVA and C-CPE, induced the production of OVA-specific serum IgG and nasal, vaginal and fecal IgA. Deletion of the Claudin-4-binding region in OVA-C-CPE attenuated the induction of the immune responses. OVA-C-CPE immunization activated both Th1 and Th2 responses, and nasal immunization with OVA-C-CPE showed anti-tumor activity in mice inoculated with OVA-expressing thymoma cells. These results indicate that the Claudin-4-targeting may be a potent strategy for nasal vaccination.

  • a Claudin 4 modulator enhances the mucosal absorption of a biologically active peptide
    Biochemical Pharmacology, 2010
    Co-Authors: Hiroshi Uchida, Masuo Kondoh, Takeshi Hanada, Azusa Takahashi, Takao Hamakubo, Kiyohito Yagi
    Abstract:

    Biologics, such as peptides, proteins and nucleic acids, are emerging pharmaceuticals. Passage across the epithelium is the first step in the absorption of biologics. Tight junctions (TJ) function as seals between adjacent epithelial cells, preventing free movement of solutes across the epithelium. We previously found that modulation of a key TJ component, Claudin-4, is a potent method to enhance jejunal absorption when we used dextran as a model drug and the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a Claudin-4 modulator. Here, we investigated whether the Claudin-4 modulator enhances jejunal, nasal and pulmonary absorption of a biologics human parathyroid hormone derivative, hPTH(1-34). The Claudin-4 modulator enhanced nasal but not jejunal and pulmonary absorption of hPTH(1-34). C-CPE is hydrophobic with low solubility of less than 0.3 mg/ml, but deletion of 10 amino acids at the N-terminal of C-CPE increased its solubility by 30-fold. Moreover, the N-terminal truncated C-CPE bound to Claudin-4, modulated the TJ-barrier and enhanced jejunal absorption of dextran. The N-terminal-truncated C-CPE also enhanced jejunal and pulmonary absorption of hPTH(1-34). This report is the first to indicate that a Claudin-4 modulator may be a promising enhancer of the jejunal, pulmonary and nasal absorption of a peptide drug.

  • A Novel Tumor-Targeted Therapy Using a Claudin-4-Targeting Molecule
    Molecular pharmacology, 2009
    Co-Authors: Rie Saeki, Masuo Kondoh, Takao Hamakubo, Hideki Kakutani, Shin-ichi Tsunoda, Yasuhiro Mochizuki, Yasuo Tsutsumi, Yasuhiko Horiguchi, Kiyohito Yagi
    Abstract:

    Carcinogenesis is often accompanied by dysfunctional tight junction (TJs), resulting in the loss of cellular polarity. Claudin, a tetra-transmembrane protein, plays a pivotal role in the barrier and fence functions of TJs. Claudin-4 is deregulated in various cancers, including breast, prostate, ovarian, and gastric cancer. Claudin-4 may be a promising target molecule for tumor therapy, but the Claudin-targeting strategy has never been fully developed. In the present study, we prepared a Claudin-4-targeting molecule by fusion of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) with the protein synthesis inhibitory factor (PSIF) derived from Pseudomonas aeruginosa exotoxin. PSIF was not cytotoxic to Claudin-4-expressing cells, whereas C-CPE-PSIF was cytotoxic. Cells that express Claudin-1, -2, and -5 were less sensitive to C-CPE-PSIF. Pretreatment of the cells with C-CPE attenuated C-CPE-PSIF-induced cytotoxicity, and mutation of C-CPE in the Claudin-4-binding residues attenuated the cytotoxicity of C-CPE-PSIF. TJ-undeveloped cells were more sensitive to C-CPE-PSIF than TJ-developed cells. It is noteworthy that polarized epithelial cells are sensitive to C-CPE-PSIF applied to the basal side, whereas the cells were less sensitive to C-CPE-PSIF applied to the apical side. Intratumoral injection of C-CPE-PSIF reduced tumor growth. This is the first report to indicate that a Claudin-4-targeting strategy may be a promising method to overcome the malignant tumors.

Patrice J Morin - One of the best experts on this subject based on the ideXlab platform.

  • possible angiogenic roles for Claudin 4 in ovarian cancer
    Cancer Biology & Therapy, 2009
    Co-Authors: Srinivasulu Chigurupati, Rachana Agarwal, Mohamed R Mughal, Mark P Mattson, Kevin G Becker, William H Wood, Yongqing Zhang, Patrice J Morin
    Abstract:

    Claudin proteins are frequently overexpressed in various tumors such as breast, prostate and ovarian cancer. While their functions in cancer have not been completely elucidated, roles in survival, adhesion, and invasion have been suggested. In order to clarify the roles of Claudins in ovarian cancer, we have performed gene expression profiling of ovarian surface epithelial cells overexpressing Claudin-4 and compared the expression patterns to the parental, non-expressing cells. Claudin-4 expression leads to the differential expression of several genes, including many that have previously been implicated in angiogenesis. In particular, angiogenic cytokines, such as IL-8, were found elevated while genes of the angiostatic interferon pathway were found down-regulated. In vitro assays show that Claudin-4-expressing cells produce factors that can stimulate angiogenesis as measured by tube formation and migration in HUVEC cells. In addition, an in vivo mouse dorsal skinfold assay confirms that cells expressing Claudin-4 secrete factors that can mediate angiogenesis in the dorsal skin of mice. Our data suggest a novel function for Claudin-4 in cancer and provide an additional rationale for its common overexpression in human tumors.

  • phosphorylation of Claudin 4 by pkce regulates tight junction barrier function in ovarian cancer cells
    Experimental Cell Research, 2007
    Co-Authors: Theresa Dsouza, Patrice J Morin, Fred E Indig
    Abstract:

    Claudin proteins belong to a large family of transmembrane proteins essential to the formation and maintenance of tight junctions (TJs). In ovarian cancer, TJ protein Claudin-4 is frequently overexpressed and may have roles in survival and invasion, but the molecular mechanisms underlying its regulation are poorly understood. In this report, we show that Claudin-4 can be phosphorylated by protein kinase C (PKC) at Thr189 and Ser194 in ovarian cancer cells and overexpression of a Claudin-4 mutant protein mimicking the phosphorylated state results in the disruption of the barrier function. Furthermore, upon phorbol ester-mediated PKC activation of OVCA433 cells, TJ strength is decreased and Claudin-4 localization is altered. Analyses using PKC inhibitors and siRNA suggest that PKCe, an isoform typically expressed in ovarian cancer cells, may be important in the TPA-mediated Claudin-4 phosphorylation and weakening of the TJs. Furthermore, immunofluorescence studies showed that Claudin-4 and PKCe are co-localized at the TJs in these cells. The modulation of Claudin-4 activity by PKCe may not only provide a mechanism for disrupting TJ function in ovarian cancer, but may also be important in the regulation of TJ function in normal epithelial cells.

  • Claudin 3 and Claudin 4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase 2 activity
    Cancer Research, 2005
    Co-Authors: Rachana Agarwal, Theresa Dsouza, Patrice J Morin
    Abstract:

    Claudin proteins form a large family of integral membrane proteins crucial for tight junction formation and function. Our previous studies have revealed that Claudin-3 and Claudin-4 proteins are highly overexpressed in ovarian cancer. To clarify the roles of Claudins in ovarian tumorigenesis, we have generated human ovarian surface epithelial (HOSE) cells constitutively expressing wild-type Claudin-3 and Claudin-4. Expression of these Claudins in HOSE cells increased cell invasion and motility as measured by Boyden chamber assays and wound-healing experiments. Conversely, small interfering RNA (siRNA)-mediated knockdown of Claudin-3 and Claudin-4 expression in ovarian cancer cell lines reduced invasion. Claudin expression also increased cell survival in HOSE cells but did not significantly affect cell proliferation. Moreover, the Claudin-expressing ovarian epithelial cells were found to have increased matrix metalloproteinase-2 (MMP-2) activity indicating that Claudin-mediated increased invasion might be mediated through the activation of MMP proteins. However, siRNA inactivation of Claudins in ovarian cancer cell lines did not have a significant effect on the high endogenous MMP-2 activity present in these cells, showing that malignant cells have alternative or additional pathways to fully activate MMP-2. Taken together, our results suggest that Claudin overexpression may promote ovarian tumorigenesis and metastasis through increased invasion and survival of tumor cells.

  • tight junction proteins Claudin 3 and Claudin 4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas
    Clinical Cancer Research, 2003
    Co-Authors: Leticia Rangel, Rachana Agarwal, Theresa Dsouza, Ellen S Pizer, Wayne D Lancaster, Lucie Gregoire, Donald R Schwartz, Patrice J Morin
    Abstract:

    Purpose: Claudin proteins represent a large family of integral membrane proteins crucial for tight junction (TJ) formation and function. Claudins have been shown to be up-regulated in various cancers and have been suggested as possible biomarkers and targets for cancer therapy. Because Claudin-3 and Claudin-4 have been proposed to be expressed in epithelial ovarian cancer, we have performed a detailed analysis of CLDN3 and CLDN4 expression in a panel of ovarian tumors of various subtypes and cell lines. We also investigated whether high expression of Claudin-3 and Claudin-4 was associated with TJ function in ovarian cancer cells. Experimental Design: RNA was obtained from a panel of 39 microdissected epithelial ovarian tumors of various histological subtypes for real-time reverse transcription-PCR analysis. In addition, a total of 70 cases of ovarian carcinomas, ovarian cysts, and normal ovarian epithelium from a tissue array were analyzed by immunohistochemistry. Finally, a panel of cell lines was used for Western analysis of Claudin expression and TJ permeability studies. Results: Although expressed at low levels in some normal human tissues, including the ovary, CLDN3 and CLDN4 are highly up-regulated in epithelial ovarian cancers of all subtypes. Immunohistochemical analyses using our ovarian tissue array confirmed the high level of expression of Claudin-3 and Claudin-4 in the majority of ovarian carcinomas, including many tumors exhibiting cytoplasmic staining. Ovarian cystadenoma did not frequently overexpress these proteins, suggesting that the expression of these proteins is associated with malignancy. In ovarian cancer cell lines, Claudin-3 and Claudin-4 expression was not associated with functional TJs as measured by transepithelial electrical resistance. Conclusions: These results show that CLDN3 and CLDN4 are frequently up-regulated in ovarian tumors and cell lines and may represent novel markers for this disease. Overexpression of these genes in ovarian cancer also suggests interesting scenarios for the involvement of TJ in tumorigenesis. A better knowledge of the mechanisms underlying ovarian tumorigenesis will likely result in the development of novel approaches for the diagnosis and therapy of this deadly disease.

Daniel A Fletcher - One of the best experts on this subject based on the ideXlab platform.

  • Claudin 4 reconstituted in unilamellar vesicles is sufficient to form tight interfaces that partition membrane proteins
    bioRxiv, 2018
    Co-Authors: Brian Belardi, Jianghui Hou, Sungmin Son, Michael D Vahey, Jinzhi Wang, Daniel A Fletcher
    Abstract:

    Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane Claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here we address this challenge by reconstituting Claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted Claudin-4 is sufficient to form adhesive interfaces between unilamellar vesicles without accessory proteins present in vivo. By controlling the molecular composition of the inner and outer leaflets of jetted membranes, we show that Claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of Claudins and their small size can contribute to the polarization of epithelial cells.

  • Claudin 4 reconstituted in unilamellar vesicles is sufficient to form tight interfaces that partition membrane proteins
    Journal of Cell Science, 2018
    Co-Authors: Brian Belardi, Jianghui Hou, Sungmin Son, Michael D Vahey, Jinzhi Wang, Daniel A Fletcher
    Abstract:

    Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane Claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here we address this challenge by reconstituting Claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted Claudin-4 is sufficient to form adhesive membrane interfaces without accessory proteins present in vivo. By controlling the molecular composition of the inner and outer leaflets of jetted vesicle membranes, we show that Claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins with ectodomains as small as 5 nm but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of Claudins and their small size can contribute to the polarization of epithelial cells.

Azusa Takahashi - One of the best experts on this subject based on the ideXlab platform.

  • mutated c terminal fragments of clostridium perfringens enterotoxin have increased affinity to Claudin 4 and reversibly modulate tight junctions in vitro
    Biochemical and Biophysical Research Communications, 2011
    Co-Authors: Azusa Takahashi, Hiroshi Uchida, Masuo Kondoh, Takao Hamakubo, Yohei Kakamu, Kiyohito Yagi
    Abstract:

    Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the Claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a Claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve Claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to Claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to Claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.

  • a Claudin 4 modulator enhances the mucosal absorption of a biologically active peptide
    Biochemical Pharmacology, 2010
    Co-Authors: Hiroshi Uchida, Masuo Kondoh, Takeshi Hanada, Azusa Takahashi, Takao Hamakubo, Kiyohito Yagi
    Abstract:

    Biologics, such as peptides, proteins and nucleic acids, are emerging pharmaceuticals. Passage across the epithelium is the first step in the absorption of biologics. Tight junctions (TJ) function as seals between adjacent epithelial cells, preventing free movement of solutes across the epithelium. We previously found that modulation of a key TJ component, Claudin-4, is a potent method to enhance jejunal absorption when we used dextran as a model drug and the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a Claudin-4 modulator. Here, we investigated whether the Claudin-4 modulator enhances jejunal, nasal and pulmonary absorption of a biologics human parathyroid hormone derivative, hPTH(1-34). The Claudin-4 modulator enhanced nasal but not jejunal and pulmonary absorption of hPTH(1-34). C-CPE is hydrophobic with low solubility of less than 0.3 mg/ml, but deletion of 10 amino acids at the N-terminal of C-CPE increased its solubility by 30-fold. Moreover, the N-terminal truncated C-CPE bound to Claudin-4, modulated the TJ-barrier and enhanced jejunal absorption of dextran. The N-terminal-truncated C-CPE also enhanced jejunal and pulmonary absorption of hPTH(1-34). This report is the first to indicate that a Claudin-4 modulator may be a promising enhancer of the jejunal, pulmonary and nasal absorption of a peptide drug.

  • domain mapping of a Claudin 4 modulator the c terminal region of c terminal fragment of clostridium perfringens enterotoxin by site directed mutagenesis
    Biochemical Pharmacology, 2008
    Co-Authors: Azusa Takahashi, Hideki Kakutani, Shin-ichi Tsunoda, Yasuo Tsutsumi, Yasuhiko Horiguchi, Eriko Komiya, Makiko Fujii, Hiroyuki Mizuguchi, Takeshi Yoshida, Naoya Koizumi
    Abstract:

    A C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a modulator of Claudin-4. We previously found that upon deletion of the C-terminal 16 amino acids, C-CPE lost its ability to modulate Claudin-4. Tyrosine residues in the 16 amino acids were involved in the modulation of Claudin-4. In the present study, we performed functional domain mapping of the 16-amino acid region of C-CPE by replacing individual amino acids with alanine. To evaluate the ability of the alanine-substituted mutants to interact with Claudin-4, we carried out a competition analysis using Claudin-4-targeting protein synthesis inhibitory factor. We found that Tyr306Ala, Tyr310Ala, Tyr312Ala, and Leu315Ala mutants had reduced binding to Claudin-4 compared to C-CPE. Next, we investigated effects of each alanine-substituted mutant on the TJ-barrier function in Caco-2 monolayer cells. The TJ-disrupting activity of C-CPE was reduced by the Tyr306Ala and Leu315Ala substitutions. Enhancement of rat jejunal absorption was also decreased by each of these mutations. The double mutant Tyr306Ala/Leu315Ala lost the ability to interact with Claudin-4, modulate TJ-barrier function, and enhance jejunal absorption. These data indicate that Tyr306 and Leu315 are key residues in the modulation of Claudin-4 by C-CPE. This information should be useful for the development of a novel Claudin modulator based on C-CPE.

  • role of tyrosine residues in modulation of Claudin 4 by the c terminal fragment of clostridium perfringens enterotoxin
    Biochemical Pharmacology, 2007
    Co-Authors: Motoki Harada, Masuo Kondoh, Azusa Takahashi, Shin-ichi Tsunoda, Yasuhiko Horiguchi, Chiaki Ebihara, Eriko Komiya, Makiko Fujii, Hiroyuki Mizuguchi, Kiyohito Yagi
    Abstract:

    The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the barrier function of Claudin-4 via its C-terminal 16 amino acids. In the current study, we investigated the roles of tyrosine residues (Y306, Y310 and Y312) in this region in the modulation of TJs by C-CPE. Single mutations of Y306, Y310 and Y312 to alanine resulted in partial reduction of Claudin-4 binding. We also prepared double mutants of C-CPE to further evaluate the roles of these tyrosine residues. Replacement of Y310 and Y312 with alanine (Y310A/Y312A) partly reduced the ability of C-CPE to bind to Claudin-4. Double mutants Y306A/Y310A and Y306A/Y312A, however, lost the ability to bind to Claudin-4 and to modulate the TJ barrier. We also found that a triple mutant (Y306A/Y310A/Y312A) lost the ability to bind Claudin-4, modulate the TJ barrier, and enhance jejunal absorption in rats. These results indicate that tyrosines 306, 310, and 312 are critical for the interaction of C-CPE with Claudin-4 and for the modulation of TJ barrier function by C-CPE. This study provides information that should help in the development of Claudin modulators based on C-CPE.

  • role of c terminal regions of the c terminal fragment of clostridium perfringens enterotoxin in its interaction with Claudin 4
    Journal of Controlled Release, 2005
    Co-Authors: Azusa Takahashi, Masuo Kondoh, Yasuhiko Horiguchi, Makiko Fujii, Hiroyuki Mizuguchi, Akane Masuyama, Yoshiteru Watanabe
    Abstract:

    Claudin family proteins, which contain 4 transmembrane domains, play a pivotal role in the barrier function of tight junctions (TJs) in epithelial sheets. We previously found that a modulator of Claudin-4, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is a potent enhancer of jejunal drug absorption in rats. But the effects of C-CPE on the barrier function of TJs have never been fully understood. In the present study, we investigated the effects of C-CPE on the barrier function of TJs in Caco-2 monolayer and characterized the functional domain of C-CPE that is responsible for interaction with Claudin-4. To evaluate the effects of C-CPE on the barrier function of TJs, we measured transepithelial electric resistance (TER) in Caco-2 monolayer cells seeded onto polycarbonate filters. Treatment of Caco-2 cells with C-CPE resulted in a decrease in TER. But, deletion of the 30 C-terminal amino acids of C-CPE, which is the putative binding domain for Claudin, attenuated the decrease in TER values. Moreover, ablation of the 16 C-terminal amino acids of C-CPE also resulted in attenuation of the decrease in TER values. The C-terminal-deleted C-CPEs did not interact with Claudin-4 or the extracellular domain 2 of Claudin-4, which is the C-CPE binding site. These results suggest that the 16 C-terminal amino acids of C-CPE are responsible for the interaction of C-CPE and Claudin-4 following the disruption of TJ barrier function.