Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins.

The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.

Occludin-binding single-chain variable fragment and antigen-binding fragment antibodies prevent hepatitis C virus infection.

Occludin (OCLN) is a tetraspan membrane component of epithelial tight junctions and a known receptor for hepatitis C virus (HCV). Previously, we established functional monoclonal antibodies (mAbs) that bind to each extracellular loop of OCLN and showed their ability to prevent in vitro and in vivo HCV infection. In this study, we converted these mAbs to corresponding monovalent antigen-binding fragments (Fabs) and single-chain variable fragment (scFv) antibodies. These Fab fragments and scFv antibodies demonstrate similar binding specificity and affinity to parental anti-OCLN mAbs. Moreover, Fab fragments and scFv antibodies inhibit in vitro HCV infection. The small functional monovalent OCLN-binding probes reported in our study have high potential as drug candidates and tools for biological and pharmaceutical studies of OCLN.

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection.

The role of the tight-junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C-terminal cytosolic domain in HCV infection. We expressed a series of C-terminal deletion mutants in Huh-7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN-C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans-Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN-C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN-C18. However, in the context of full-length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN-C18 in human-polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.

Phosphorylation hotspot in the C-terminal domain of occludin regulates the dynamics of epithelial junctional complexes.

The apical junctional complex (AJC), which includes tight junctions (TJs) and adherens junctions (AJs), determines the epithelial polarity, cell-cell adhesion and permeability barrier. An intriguing characteristic of a TJ is the dynamic nature of its multiprotein complex. Occludin is the most mobile TJ protein, but its significance in TJ dynamics is poorly understood. On the basis of phosphorylation sites, we distinguished a sequence in the C-terminal domain of occludin as a regulatory motif (ORM). Deletion of ORM and expression of a deletion mutant of occludin in renal and intestinal epithelia reduced the mobility of occludin at the TJs. ORM deletion attenuated Ca2+ depletion, osmotic stress and hydrogen peroxide-induced disruption of TJs, AJs and the cytoskeleton. The double point mutations T403A/T404A, but not T403D/T404D, in occludin mimicked the effects of ORM deletion on occludin mobility and AJC disruption by Ca2+ depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blocked AJC disruption. Expression of a deletion mutant of occludin attenuated collective cell migration in the renal and intestinal epithelia. Overall, this study reveals the role of ORM and its phosphorylation in occludin mobility, AJC dynamics and epithelial cell migration.

Occludin S471 Phosphorylation Contributes to Epithelial Monolayer Maturation.

Multiple organ systems require epithelial barriers for normal function, and barrier loss is a hallmark of diseases ranging from inflammation to epithelial cancers. However, the molecular processes regulating epithelial barrier maturation are not fully elucidated. After contact, epithelial cells undergo size-reductive proliferation and differentiate, creating a dense, highly ordered monolayer with high resistance barriers. We provide evidence that the tight junction protein occludin contributes to the regulation of epithelial cell maturation upon phosphorylation of S471 in its coiled-coil domain. Overexpression of a phosphoinhibitory occludin S471A mutant prevents size-reductive proliferation and subsequent tight junction maturation in a dominant manner. Inhibition of cell proliferation in cell-contacted but immature monolayers recapitulated this phenotype. A kinase screen identified G-protein-coupled receptor kinases (GRKs) targeting S471, and GRK inhibitors delayed epithelial packing and junction maturation. We conclude that occludin contributes to the regulation of size-reductive proliferation and epithelial cell maturation in a phosphorylation-dependent manner.

Claudin-binder C-CPE mutants enhance permeability of insulin across human nasal epithelial cells.

OBJECTIVE: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1. METHODS: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19. RESULTS: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126. CONCLUSION: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration.

A study on the encapsulation of an occludin lipophilic derivative in liposomal carriers.

Many peptides and proteins, although potentially useful for the treatment of various diseases, are hindered in their clinical use by poor oral absorption and rapid enzymatic degradation. One of the available solutions to these problems is to increase the lipophilicity by conjugating the peptides to lipophilic moieties, making them more able to cross the biomembranes by passive transport. Occludin is a 65-kDa integral plasma-membrane protein located at the tight junctions. This protein and the peptide derived from it have potential clinical application for drug delivery. Peptide OP90-103 (1) is a fragment of occludin that shows a very poor oral bioavailability and is highly susceptible to enzymatic degradation. The conjugation of 1 with two lipoamino acid (LAA) moieties has been shown to enhance its lipophilicity and bioavailability, as well as its enzymatic stability. The purpose of this study was to evaluate the possibility of encapsulating fluorescein modified lipidated OP90-103 (2), in unilamellar- (LUV) and multilamellar liposomes (MLV), which have a different composition and surface charge and are produced by different methods. The cell internalization of the carrier systems was evaluated in vitro.

Highly conserved cysteines are involved in the oligomerization of occludin-redox dependency of the second extracellular loop.

UNLABELLED: The tight junction (TJ) marker occludin is a 4-transmembrane domain (TMD) protein with unclear physiological and pathological functions, interacting with other TJ proteins. It oligomerizes and is redox sensitive. However, oligomerization sites and mechanisms are unknown. AIMS: To identify hypoxia-sensitive binding sites, we investigated the consequences of amino-acid substitutions of highly conserved cysteines in human occludin, under normal and hypoxic incubations. RESULTS: (i) The extracellular loop 2 (ECL2) showed homophilic trans- and cis-association between opposing cells and along the cell membrane, respectively, caused by a loop properly folded via an intraloop disulfide bridge between the shielded C216 and C237. Hypoxia and reductants prevented the associations. (ii) C82 in TMD1 directly cis-associated without disulfide formation. (iii) C76 in TMD1 and C148 in TMD2 limited the trans-interaction; C76 also limited occludin-related paracellular tightness and changed the strand morphology of claudin-1. (iv) The diminished binding strength found after substituting C82, C216, or C237 was accompanied by increased occludin mobility in the cell membrane. INNOVATION: The data enable the first experimentally proven structural model of occludin and its homophilic interaction sites, in which the ECL2, via intraloop disulfide formation, has a central role in occludin's hypoxia-sensitive oligomerization and to regulate the structure of TJs. CONCLUSION: Our findings support the new concept that occludin acts as a hypoxiasensor and contributes toward regulating the TJ assembly redox dependently. This is of pathogenic relevance for tissue barrier injury with reducing conditions. The ECL2 disulfide might be a model for four TMD proteins in TJs with two conserved cysteines in an ECL.

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux.

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ~62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)-deficient enhanced green fluorescent protein (EGFP)-occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin(ΔOCEL). Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1-binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK-binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.

Acute carbonyl stress induces occludin glycation and brain microvascular endothelial barrier dysfunction: role for glutathione-dependent metabolism of methylglyoxal.

We recently demonstrated that methylglyoxal (MG) induced apoptosis of brain microvascular endothelial cells (IHECs) that was preceded by glutathione (GSH) depletion. Here, we test the hypothesis that MG induces occludin glycation and disrupts IHEC barrier function, which is prevented by GSH-dependent MG metabolism. Exposure of IHECs to MG decreased transendothelial electrical resistance (TEER) in association with MG-adduct formation. A 65-kDa MG-glycated protein corresponded to occludin, which was confirmed by immunoprecipitation. Moreover, immunofluorescence staining showed that MG disrupted the architectural organization of ZO-1. Occludin glycation and ZO-1 disruption were prevented by N-acetylcysteine (NAC). Accordingly, TEER loss was abrogated by NAC (via GSH synthesis) and exacerbated by buthionine sulfoximine (BSO; GSH synthesis inhibitor). BSO treatment attenuated D-lactate production, consistent with a role for GSH in glyoxalase I-catalyzed MG elimination. Although MG increased reactive oxygen species (ROS) generation, the ROS scavengers tempol and tiron did not block barrier disruption. This suggests that endogenously generated ROS were unlikely to be a major cause of or did not reach a threshold to elicit barrier failure as elicited by exogenous hydrogen peroxide (300-400 µM). Immunohistochemistry revealed a lower percentage of microvessels stained with anti-occludin, but a higher percentage stained with anti-MG in diabetic rat brain compared to controls. Western analyses confirmed the decrease in diabetic brain occludin expression, but an increase in glycated occludin levels. These results provide novel evidence that reactive carbonyl species can mediate occludin glycation in cerebral microvessels and in microvascular endothelial cells that contribute to barrier dysfunction, a process that was prevented by GSH through enhanced MG catabolism.

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

Computational identification of interplay between phosphorylation and O-ß-glycosylation of human occludin as potential mechanism to impair hepatitis C virus entry.

Hepatitis C virus (HCV) is one of the leading causes of liver diseases. Several host factors that facilitate the attachment and entry of HCV have been discovered, of which human occludin seems to be the most promising. Studies have shown that activity of occludin is dependent upon its phosphorylation status, and that during HCV infection deregulation of phosphorylated occludin collectively leads to a reduction in tight junction (TJ) integrity of hepatocytes and favors HCV entry. However, detailed information of the posttranslational modifications (PTMs) of occludin still remains largely unknown. In addition to phosphorylation, serine/threonine residues of several proteins are also regulated by a unique type of modification known as O-ß-glycosylation and this crosstalk serves as a functional switch. To identify the O-ß-glycosylation potential and how interplay between phosphorylation and O-ß-glycosylation can be exploited for the inhibition of HCV entry, here we report a computational analysis of PTMs of human occludin. Several conserved phosphorylation residues and kinases that can alter the ability of occludin to regulate the integrity of TJs were identified. In addition to previously reported Tyr residues, two additional Tyr residues (Tyr29 and Tyr287) were identified as target sites of Src kinase. To our knowledge, this is the first study to report the O-ß-GlcNAc potential of occludin and target sites of ERK (Ser8, Ser310, and Thr345), GSK-3 (Ser8, Ser341) and Cdk5 (Thr376). Furthermore, based on findings from this study, a potential novel interplay between phosphorylation and O-ß-glycosylation at the two Yin Yang sites (Ser408 and Ser490) is also proposed.