Structural basis for Clostridium perfringens enterotoxin targeting of claudins at tight junctions in mammalian gut.

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

Screening approaches against claudin-4 focusing on therapeutics through molecular docking and the analysis of their relative dynamics: a theoretical approach.

Claudin-4 (CLDN4) is a class of transmembrane protein in the family of tight junction (TJ) proteins. Overexpression of CLDN4 is reported in the case of ovarian cancer and epithelial malignancies. The current study is focused on the identification of lead compounds for CLDN4 adopting the structure-based drug design method. The Schrodinger glide is used as a molecular docking tool for the initial docking of CLDN4 with Asinex Database by performing high throughput virtual screening, top hits were identified. Then, compounds BDF 33196188 and BDE 30874918 were identified by molecular docking based on binding energy in the active site of CLDN4. Subsequently, critical residues were identified such as Asp146 and Arg158 with the least binding energy from Extra Precision method. Further, molecular dynamics simulations of claudin-4 protein were used for the optimization of best ligands with claudin-4 in a dynamic system. Molecular docking and molecular dynamics simulations predicted critically important residues ASP146 and ARG158 involved in claudin-4 binding. The hits retrieved from screening were docked into protein by relevant procedures including HTVS, SP, and XP. Finally, two molecules were identified as potential claudin-4 inhibitors. The two ligands BDF 33196188 and BDE 30874918 are suggested as potential inhibitors for CLDN4. In summary, our computational strategy established novel leads against CLDN4 from Asinex Database and recommended as anti-cancer agents.

Utility of Claudin-4 versus BerEP4 and B72.3 in pleural fluids with metastatic lung adenocarcinoma.

INTRODUCTION: Lung adenocarcinoma (LADC) is the most common occult primary in patients presenting with a malignant pleural effusion. Distinguishing metastatic LADC from reactive mesothelial cells (RMC) and malignant mesothelioma (MM) based on morphology alone has been a persistent diagnostic challenge in cytopathology. Claudin-4, a major functional constituent of tight junctions, has been shown to help distinguish LADC from RMC and MM in surgical specimens. Our goal was to further validate and assess the utility of Claudin-4 in comparison to BerEP4 and B72.3 in malignant effusions with a focus on metastatic LADC. MATERIALS AND METHODS: We evaluated 58 pleural effusions (40 LADC, 10 RCM, and 8 MM). Immunohistochemistry was performed using Claudin-4, Ber-EP4, and B72.3 on cell blocks. Staining patterns, quantity of tumor cells, and intensity of staining (weak, moderate, or strong) were assessed. RESULTS: All cases of LADC were positive for Claudin-4 with an overall sensitivity of 100% (40 of 40) and specificity of 100% (18 of 18). In addition, Claudin-4 showed the highest quantity and quality of staining with 3+ staining intensity in 73% (29 of 40) of cases, compared with 35% (14 of 40) of cases using BerEP4 and 52% (21 of 40) of cases using B72.3. The sensitivity and specificity for BerEP4 were 90% and 78%, respectively. The sensitivity and specificity for B72.3 were 87.5% and 100%, respectively. CONCLUSIONS: In this study, Claudin-4 performed superiorly compared to BerEP4 and B72.3 in distinguishing lung adenocarcinoma from RMC or MM in pleural effusions. Our results show Claudin-4 is a useful marker for distinguishing RMC and MM from lung adenocarcinoma, with high sensitivity (100%) and specificity (100%), compatible with studies shown in the literature.

Targeting claudin-4 enhances chemosensitivity in breast cancer.

Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti-claudin (CLDN)-4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF-7 (luminal subtype) and MDA-468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)-induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA-468 also showed the same level of antitumor activity and survival as MCF-7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects.

Targeting claudin-overexpressing thyroid and lung cancer by modified Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) can be used to eliminate carcinoma cells that overexpress on their cell surface CPE receptors - a subset of claudins (e.g., Cldn3 and Cldn4). However, CPE cannot target tumors expressing solely CPE-insensitive claudins (such as Cldn1 and Cldn5). To overcome this limitation, structure-guided modifications were used to generate CPE variants that can strongly bind to Cldn1, Cldn2 and/or Cldn5, while maintaining the ability to bind Cldn3 and Cldn4. This enabled (a) targeting of the most frequent endocrine malignancy, namely, Cldn1-overexpressing thyroid cancer, and (b) improved targeting of the most common cancer type worldwide, non-small-cell lung cancer (NSCLC), which is characterized by high expression of several claudins, including Cldn1 and Cldn5. Different CPE variants, including the novel mutant CPE-Mut3 (S231R/S313H), were applied on thyroid cancer (K1 cells) and NSCLC (PC-9 cells) models. In vitro, CPE-Mut3, but not CPEwt, showed Cldn1-dependent binding and cytotoxicity toward K1 cells. For PC-9 cells, CPE-Mut3 improved claudin-dependent cytotoxic targeting, when compared to CPEwt. In vivo, intratumoral injection of CPE-Mut3 in xenograft models bearing K1 or PC-9 tumors induced necrosis and reduced the growth of both tumor types. Thus, directed modification of CPE enables eradication of tumor entities that cannot be targeted by CPEwt, for instance, Cldn1-overexpressing thyroid cancer by using the novel CPE-Mut3.

Synthesis, 18F-labelling and radiopharmacological characterisation of the C-terminal 30mer of Clostridium perfringens enterotoxin as a potential claudin-targeting peptide.

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290-319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290-319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290-319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.

Stochastic palmitoylation of accessible cysteines in membrane proteins revealed by native mass spectrometry.

Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. However, how specificity of palmitoylation and multiple palmitoylations in membrane proteins are determined is not well understood. Here, we profile palmitoylation states of three human claudins, human CD20 and cysteine-engineered prokaryotic KcsA and bacteriorhodopsin by native mass spectrometry. Cysteine scanning of claudin-3, KcsA, and bacteriorhodopsin shows that palmitoylation is independent of a sequence motif. Palmitoylations are observed for cysteines exposed on the protein surface and situated up to 8 Å into the inner leaflet of the membrane. Palmitoylation on multiple sites in claudin-3 and CD20 occurs stochastically, giving rise to a distribution of palmitoylated membrane-protein isoforms. Non-native sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites. These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif.

A cCPE-based xenon biosensor for magnetic resonance imaging of claudin-expressing cells.

The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection.

Claudin 4-targeted nanographene phototherapy using a Clostridium perfringens enterotoxin peptide-photosensitizer conjugate.

In this study we designed a claudin 4-directed dual photodynamic and photothermal system, in which a 30-amino acid claudin 4-binding peptide, Clostridium perfringens enterotoxin (CPE), was linked to a photodynamic agent chlorin e6 (Ce6) through a polyethylene glycol spacer (CPC) and anchored onto reduced graphene oxide (rGO) nanosheets to form CPC/rGO nanosheets. For comparison, a conjugate of polyethylene glycol and Ce6 (PC) was anchored onto the rGO nanosheets to generate PC/rGO. Both PC and CPC generated reactive oxygen species upon irradiation at 660 nm. Application of CPC/rGO to claudin 4-overexpressing U87 glioblastoma cells in vitro resulted in a significantly higher cellular uptake compared to application of PC/rGO. Upon irradiation at 660 and 808 nm, the CPC/rGO-treated U87 cells generated significantly higher reactive oxygen species and caused significantly higher temperature increase, and showed most potent anticancer effect compared to the other groups. Taken together, these results suggest that CPC/rGO is potentially useful as a tumor-specific combined phototherapy.

Cell-free methods to produce structurally intact mammalian membrane proteins.

The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin.

A synthetic peptide corresponding to the extracellular loop 2 region of claudin-4 protects against Clostridium perfringens enterotoxin in vitro and in vivo.

Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

Activation mechanism of claudin-4 by ephrin type-A receptor 2: a molecular dynamics approach.

Claudin-4 is a crucial component in the tight junction which is involved in the organization of a cellular barrier. Claudin-4 is found to be up-regulated in various malignancies and is activated by forming a complex with an ephrin A2 receptor. In this computational study, we propose a rational model for the claudin-4-ephrinA2 complex which is in agreement with the experimental result. The complex model has been obtained through protein-protein docking, interface residue scanning, in silico alanine mutations and extensive molecular dynamics simulations. The docking model envisages the important residues present in the first extracellular loop of claudin-4 that plays an active role in protein-protein interaction and stability. A 30 nanosecond molecular dynamics simulation of the complex revealed a higher stability by which the number of hydrogen bond interactions increased in the complex interface. Both the molecular dynamics simulations and in silico alanine mutations revealed the involvement of Lys65 (claudin-4) as one of the prime residues in the complex interface that is actively engaged in the binding mechanism with its counterpart. We postulate that the novel hotspot, Lys65 of claudin-4 can be targeted through structure based inhibitor design, which could alter the effect of the claudin-4-ephrinA2 binding mechanism in aggressive metastatic tumors.