Lars Winkler

Formation of tight junction: determinants of homophilic interaction between classic claudins

Claudins are the critical transmembrane proteins in tight junctions. Claudin‐5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin‐claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin‐5 mutants trans‐fected into tight junction‐free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans‐interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild‐type‐like trans‐ and cis‐interaction; the disjunction type (blocked trans‐interaction); the intracellular type (disturbed folding). Combining site‐directed mutagenesis, live‐cell imaging‐, electron microscopy‐, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation.— Piontek, J., Winkler, L., Wolburg, H., Müller, S. L., Zuleger, N., Piehl, C., Wiesner, B., Krause, G., Blasig, I. E. Formation of tight junction: determinants of homophilic interaction between classic clau‐dins. FASEB J. 22, 146–158 (2008)

On the self-association potential of transmembrane tight junction proteins

Abstract.Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

Transmembrane proteins of the tight junctions at the blood-brain barrier: structural and functional aspects.

The blood-brain barrier (BBB) is formed by microvascular endothelial cells sealed by tetraspanning tight junction (TJ) proteins, such as claudins and TAMPs (TJ-associated marvel proteins, occludin and tricellulin). Claudins are the major components of the TJs. At the BBB, claudin-5 dominates the TJs by preventing the paracellular permeation of small molecules. On the other hand, TAMPs regulate the structure and function of the TJs; tricellulin may tighten the barrier for large molecules. This review aims at integrating and summarizing the most relevant and recent work on how the BBB is influenced by claudin-1, -3, -5, -12 and the TAMPs occludin and tricellulin, all of which are four-transmembrane TJ proteins. The exact functions of claudin-1, -3, -12 and TAMPs at this barrier still need to be elucidated.

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.

Structure and Function of Extracellular Claudin Domains

Most claudins are tight junction (TJ)–forming proteins. However, their interaction on the molecular level remains unresolved. It is hypothesized that the extracellular loops specify these claudin functions. It is assumed that the first extracellular loop (ECL1) is critical for determining the paracellular tightness and the selective paracellular ion permeability, and that the second extracellular loop may cause narrowing of the paracellular cleft. Using a combination of site‐directed mutagenesis and homology modeling for the second extracellular loop (ECL2) of claudin‐5, we found several amino acids important for claudin folding and/or trans‐interaction to claudins in neighboring cells. These sensitive residues are highly conserved within one group of claudins, whereas the corresponding positions in the remaining claudins show a large sequence variety. Further functional data and analysis of sequence similarity for all claudins has led to their differentiation into two groups, designated as classic claudins (1–10, 14, 15, 17, 19) and nonclassic claudins (11–13, 16, 18, 20–24). This also corresponds to conserved structural features at ECL1 for classic claudins. Based on this, we propose a hypothesis for different pore‐forming claudins. Pore formation or tightness is supported by the spatial encounter of a surplus of repulsing or attracting amino acid types at ECL1. A pore is likely opened by repulsion of equally charged residues, while an encounter of unequally charged residues leads to tight interaction. These considerations may reveal the ECLs of claudins as decisive submolecular determinants that specify the function of a claudin.

Analysis of RovA, a Transcriptional Regulator of Yersinia pseudotuberculosis Virulence That Acts through Antirepression and Direct Transcriptional Activation

The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.

Sodium caprate transiently opens claudin-5-containing barriers at tight junctions of epithelial and endothelial cells.

Claudin-5 is a tight junction (TJ) protein which limits the diffusion of small hydrophilic molecules. Thus, it represents a potential pharmacological target to improve drug delivery to the tissues protected by claudin-5-dependent barriers. Sodium caprate is known as an absorption enhancer which opens the paracellular space acting on TJ proteins and actin cytoskeleton. Its action on claudin-5 is not understood so far. Epithelial and endothelial systems were used to evaluate the effect of caprate on claudin-5 in TJ-free cells and on claudin-5 fully integrated in TJ. To this aim, confocal microscopy on live and fixed cells and isolated mouse brain capillaries, Western blotting and permeability assays were employed. Caprate reversibly reduced claudin-5 trans-interactions in TJ-free human embryonic kidney-293 cells expressing claudin-5-YFP. It decreased the membranous claudin-5 and the F-actin content in Madin-Darby canine kidney-II cells expressing Flag-claudin-5, thereby increasing the permeability to the small molecule lucifer yellow. Interestingly, zonula occludens protein 1 (ZO-1), which links transmembranous TJ proteins to the actin cytoskeleton, was not affected by caprate treatment. Similarly, endogenous claudin-5 in the membrane of brain endothelia was displaced together with F-actin, whereas ZO-1 remained unaffected. Caprate transiently opens the paracellular space, reducing the intercellular claudin-5/claudin-5 interactions and the polymerized actin at the perijunctional region of endothelial and epithelial cells. In conclusion, the study further elucidates the cellular effects of caprate at the tight junctions.

A strategy for enrichment of claudins based on their affinity to Clostridium perfringens enterotoxin.

BackgroundClaudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes.ResultsImmunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE116–319 fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material.ConclusionCPE116–319 provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery

The blood–brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin‐5, which we selected as a target to modulate BBB permeability. For this purpose, drug‐enhancer peptides were designed based on the first extracellular loop (ECL) of claudin‐5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin‐5) size‐selectively (≤40 kDa) and reversibly (12–48 h) increased the permeability of brain endothelial and claudin‐5–transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin‐5 was downregulated and redistributed from cell–cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 μmol/kg i.v.) confirmed that, for both C5C2 and a d‐amino acid derivative, brain uptake of Gd–diethylene‐triamine penta‐acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between β‐sheets flanked by α‐helices, formed by claudin‐5 ECLs, and the peptides. In conclusion, we identified claudin‐5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin‐5.

Trictide, a tricellulin-derived peptide to overcome cellular barriers

The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis‐stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin‐1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super‐resolution stimulated‐emission depletion microscopy revealed a tricellulin‐free area at the tricellular region. Cis‐interactions (as measured by fluorescence resonance energy transfer) of tricellulin–tricellulin (tTJs), tricellulin–claudin‐1, tricellulin–marvelD3, and occludin–occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a β‐sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.