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At least 19 recordsLinked to original sources
PubMed2000

Occludin 1B, a variant of the tight junction protein occludin.

Occludin and claudin are the major integral membrane components of the mammalian tight junction. Although more than 11 distinct claudins have been identified, only 1 occludin transcript has been reported thus far. Therefore, we searched by reverse transcription-PCR for occludin-related sequences in Madin-Darby canine kidney (MDCK) mRNA and identified a transcript encoding an alternatively spliced form of occludin, designated occludin 1B. The occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Analysis of the MDCK occludin gene revealed an exon containing the 193-base pair sequence between the exons encoding the original N terminus and the distal sequence, suggesting that occludin and occludin 1B arise from alternative splicing of one transcript. To assess the expression and distribution of occludin 1B, an antibody was raised against its unique N-terminal domain. Immunolabeling of occludin 1B in MDCK cells revealed a distribution indistinguishable from that of occludin. Furthermore, occludin 1B staining at cell-to-cell contacts was also found in cultured T84 human colon carcinoma cells and in frozen sections of mouse intestine. Immunoblots of various mouse tissues revealed broad coexpression of occludin 1B with occludin. The wide epithelial distribution and the conservation across species suggests a potentially important role for occludin 1B in the structure and function of the tight junction.

Alternative Splicing↗
PubMed1997

Phosphorylation of occludin correlates with occludin localization and function at the tight junction.

Multiple forms of occludin were found in Madin-Darby canine kidney (MDCK) cells. In the absence of cell-to-cell contacts achieved by incubating cells in low-calcium growth medium, a cluster of lower-molecular-weight (LMW) occludin bands (approximately 65,000-68,000) was present in both MDCK I and II cells. On formation of tight junctions, achieved by changing the low-calcium growth medium to normal-calcium growth medium, a cluster of higher-molecular-weight (HMW) bands (approximately 72,000-75,000 for MDCK I cells and approximately 70,000-73,000 for MDCK II cells) was also expressed. The HMW occludin bands could be eliminated by phosphatase treatment. Therefore, the HMW forms of occludin appeared to be the hyperphosphorylated product of the LMW forms. These HMW forms were Triton X-100 insoluble, which correlated with their localization at the tight junctions. Furthermore, depletion of tight junction-localized occludin by an occludin extracellular domian peptide (20) correlated with a decrease in the HMW forms of occludin. In conclusion, phosphorylation of occludin may be a mechanism by which occludin localization and function are regulated.

Animals↗
PubMed1999

Connexin-occludin chimeras containing the ZO-binding domain of occludin localize at MDCK tight junctions and NRK cell contacts.

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.

Animals↗
PubMed1998

Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group.

Blood-retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, but the molecular changes that cause this pathology are unclear. Occludin is a transmembrane component of interendothelial tight junctions that may regulate permeability at the BRB. In this study, we examined the effects of vascular endothelial growth factor (VEGF) and diabetes on vascular occludin content and barrier function. Sprague-Dawley rats were made diabetic by intravenous streptozotocin injection, and age-matched animals served as controls. After 3 months, BRB permeability was quantified by intravenous injection of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), Mr 66 kDa, and 10-kDa rhodamine-dextran (R-D), followed by digital image analysis of retinal sections. Retinal fluorescence intensity for FITC-BSA increased 62% (P < or = 0.05), but R-D fluorescence did not change significantly. Occludin localization at interendothelial junctions was confirmed by immunofluorescence, and relative protein content was determined by immunoblotting of retinal homogenates. Retinal occludin content decreased approximately 35% (P < or = 0.03) in the diabetic versus the control animals, whereas the glucose transporter GLUT1 content was unchanged in rat retinas. Additionally, treatment of bovine retinal endothelial cells in culture with 0.12 nmol/l or 12 nmol/l VEGF for 6 h reduced occludin content 46 and 54%, respectively. These data show that diabetes selectively reduces retinal occludin protein expression and increases BRB permeability. Our findings suggest that the elevated VEGF in the vitreous of patients with diabetic retinopathy increases vascular permeability by downregulating occludin content. Decreased tight junction protein expression may be an important means by which diabetes causes increased vascular permeability and contributes to macular edema.

Animals↗
PubMed2003

Occludin phosphorylation: identification of an occludin kinase in brain and cell extracts as CK2.

In epithelial and endothelial cells, tight junctions limit paracellular flux of ions, proteins and other macromolecules. However, mechanisms regulating tight junction function are not clear. Occludin, a tight junction protein, undergoes phosphorylation changes in several situations but little is known about occludin kinases. A recombinant C-terminal fragment of occludin is a substrate for a kinase in crude extracts of brain. This activity was purified about 10000-fold and identified as CK2 (casein kinase 2) by peptide mass fingerprinting, immunoblotting and mutation of CK2 sites within the occludin sequence. CK2 is therefore a candidate kinase for regulation of occludin phosphorylation in vivo.

Amino Acid Sequence↗
PubMed1994

Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

Animals↗
PubMed1996

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

Animals↗
PubMed1997

COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos.

Occludin is the only known integral membrane protein localized at the points of membrane- membrane interaction of the tight junction. We have used the Xenopus embryo as an assay system to examine: (a) whether the expression of mutant occludin in embryos will disrupt the barrier function of tight junctions, and (b) whether there are signals within the occludin structure that are required for targeting to the sites of junctional interaction. mRNAs transcribed from a series of COOH-terminally truncated occludin mutants were microinjected into the antero-dorsal blastomere of eight-cell embryos. 8 h after injection, the full-length and the five COOH-terminally truncated proteins were all detected at tight junctions as defined by colocalization with both endogenous occludin and zonula occludens-1 demonstrating that exogenous occludin correctly targeted to the tight junction. Importantly, our data show that tight junctions containing four of the COOH-terminally truncated occludin proteins were leaky; the intercellular spaces between the apical cells were penetrated by sulfosuccinimidyl-6-(biotinamido) Hexanoate (NHS-LC-biotin). In contrast, embryos injected with mRNAs coding for the full-length, the least truncated, or the soluble COOH terminus remained impermeable to the NHS-LC-biotin tracer. The leakage induced by the mutant occludins could be rescued by coinjection with full-length occludin mRNA. Immunoprecipitation analysis of detergent-solubilized embryo membranes revealed that the exogenous occludin was bound to endogenous Xenopus occludin in vivo, indicating that occludin oligomerized during tight junction assembly. Our data demonstrate that the COOH terminus of occludin is required for the correct assembly of tight junction barrier function. We also provide evidence for the first time that occludin forms oligomers during the normal process of tight junction assembly. Our data suggest that mutant occludins target to the tight junction by virtue of their ability to oligomerize with full-length endogenous molecules.

Animals↗
PubMed1997

Possible involvement of phosphorylation of occludin in tight junction formation.

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest Mr were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher Mr bands were selectively recovered in the NP-40-insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40-soluble and -insoluble fractions into the lowest Mr band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher Mr bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40-insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti-chicken occludin mAb, Oc-3, did not recognize the predominant lower Mr bands of occludin (non- or less phosphorylated form) but was specific to the higher Mr bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower Mr bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40-insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.

Animals↗
PubMed2005

Epithelial transport and barrier function in occludin-deficient mice.

BACKGROUND AND AIMS: This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model. METHODS: Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. RESULTS: In occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic occludin+/- or homozygotic occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin-/- mice. CONCLUSION: Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.

Animals↗
PubMed2001

Shear stress regulates occludin content and phosphorylation.

Previous studies determined that shear stress imposed on bovine aortic endothelial cell (BAEC) monolayers increased the hydraulic conductivity (L(P)); however, the mechanism by which shear stress increases L(P) remains unknown. This study tested the hypothesis that shear stress regulates paracellular transport by altering the expression and phosphorylation state of the tight junction protein occludin. The effect of shear stress on occludin content was examined by Western blot analysis. Ten dyn/cm(2) significantly reduced occludin content in a time-dependent manner such that after a 3 h exposure to shear, occludin content decreased to 44% of control. Twenty dyn/cm(2) decreased occludin content to 50% of control and increased L(P) by 4.7-fold after 3 h. Occludin expression and L(P) depend on tyrosine kinase activity because erbstatin A (10 microM) attenuated both the shear-induced decrease in occludin content and increase in L(P). Shear stress increased occludin phosphorylation after 5 min, 15 min, and 3 h exposures. The shear-induced increase in occludin phosphorylation was attenuated with dibutyryl (DB) cAMP (1 mM), a reagent previously shown to reverse the shear-induced increase in L(P). We conclude that shear stress rapidly (< or = 5 min) increases occludin phosphorylation and significantly decreases the expression of occludin over 1-4 h. Alterations in the occludin phosphorylation state and occludin total content are potential mechanisms by which shear stress increases L(P).

Animals↗
PubMed1997

Occludin confers adhesiveness when expressed in fibroblasts.

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

Amino Acid Sequence↗
PubMed2001

Occludin regulates actin cytoskeleton in endothelial cells.

Occludin is a major membrane component of tight junctions of endothelial cells, though the role of this molecule is not fully understood. RLE cells, derived from rat lung endothelial cells, express a negligible level of occludin with clear expression of E-cadherin and ZO-1 at cell junctions. Introduction of occludin by transfection induced clear junctional expression of occludin with few or no changes of expression of E-cadherin and ZO-1. The paracellular barrier function, as determined by transelectrical resistance and flux of non-ionic small molecules, was not detectably upregulated. When cells expressing occludin were cocultured with RLE cells null for occludin, clear junctional expression of occludin was observed irrespective of the expression of occludin on the apposing cells. Cortical actin was developed at the site of these occludin positive cell junctions. Treatment of cells with an actin depolymerizing agent, mycalolide B, abolished junctional expression of occludin together with E-cadherin and circumferential actin. ZO-1 showed relative resistance to this actin depolymerizing treatment and was maintained at the cell junctions, though fragmentation of immunoreactivity was detectable. Collectively, junctional expression of occludin was not associated with paracellular barrier function in this cell line. There was, however, a close correlation of occludin with the actin cytoskeleton, indicating a role of occludin as an important molecule in the regulation of the actin cytoskeleton in endothelial cells.

Actins↗
PubMed2001

Hepatocyte growth factor promotes epithelial morphogenesis and occludin linkage to the cytoskeleton in cultured retinal pigment epithelial cells.

BACKGROUND: Although hepatocyte growth factor (HGF) is also known as scatter factor, it induces epithelial morphogenesis in cultured bovine retinal pigment epithelial (RPE) cells. To elucidate the mechanism of epithelial morphogenesis, we investigated the influence of HGF on occludin, a major component of tight junctions. METHODS: RPE cells were plated on collagen type 1-coated chamber slides or dishes, 20 ng/ml HGF was added and the cells were incubated for 1 week. Cells were harvested at several time-points, and occludin expression was examined by immunohistochemistry. Detergent extraction protocols to identify the intensity of occludin linkage to the cytoskeleton were also used. Occludin expression was determined semiquantitatively by Western blotting. RESULTS: Fluorescence microscopy revealed that HGF treatment increased the levels of insoluble occludin at the cell borders after detergent extraction. These level of insoluble occludin and the associated epithelial morphology were maintained for more than 3 weeks after withdrawal of HGF, whereas cells not treated with HGF had a fibroblastic appearance. Western blotting also showed that insoluble occludin was more abundant in HGF-treated cells. Furthermore, immunoreactive bands of insoluble occludin were somewhat larger than those of soluble occludin, suggesting that insoluble occludin may be modified in comparison to soluble occludin. CONCLUSION: Our results suggest that HGF promotes linkage of occludin to the cytoskeleton. HGF may become a therapeutic candidate in physiological recovery of RPE cells and in preparation of RPE monolayers for transplantation.

Animals↗
PubMed2003

Tyrosine phosphorylation of occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3.

Occludin, the transmembrane integral protein of the tight junction, plays a crucial role in the molecular organization and function of tight junction. While the homotypic interaction of extracellular loops of occludin appears to determine the barrier function of tight junction, the intracellular C-terminal tail, C-occludin, interacts with other tight junction proteins such as ZO-1, ZO-2, and ZO-3 and with the actin filaments of cytoskeleton. In the present study we phosphorylated GST-fused C-occludin on tyrosine residues, in TKX1 Epicurian coli or by active c-Src in vitro. c-Src binds to occludin and phosphorylates it on tyrosine residues. The effect of tyrosine phosphorylation of C-occludin on its ability to bind ZO-1, ZO-2, ZO-3, and F-actin was evaluated. Results show that the amounts of ZO-1, ZO-2, and ZO-3 bound to tyrosine phosphorylated C-occludin were several fold less than the amounts bound to non-phosphorylated C-occludin. However, the amount of tyrosine phosphorylated C-occludin bound to F-actin was not significantly different from the amount of non-phosphorylated C-occludin bound to F-actin. These results demonstrate that tyrosine phosphorylation of occludin reduces its ability to bind ZO-1, ZO-2, and ZO-3, but not F-actin. Results also suggest that c-Src-mediated disruption of tight junction may involve tyrosine phosphorylation of occludin.

Actins↗
PubMed1999

Xenopus laevis occludin. Identification of in vitro phosphorylation sites by protein kinase CK2 and association with cingulin.

Occludin is a protein component of the membrane domain of tight junctions, and has been shown to be phosphorylated in vivo in cultured cells and Xenopus laevis embryos. However, nothing is known about the identity of specific occludin kinase(s) and occludin phosphorylation site(s). Furthermore, nothing is known about the interaction of occludin with cingulin, a cytoplasmic plaque component of tight junctions. Here we report the isolation and sequencing of a complete X. laevis occludin cDNA, and experiments aimed at mapping X. laevis occludin in vitro phosphorylation site(s) and characterizing occludin interaction with cingulin. The sequence of Xenopus occludin is homologous to that of occludins from other species, with identities ranging from 41% to 58%. Bacterially expressed domain E of Xenopus occludin (amino acids 247-493) was a good substrate for protein kinase CK2 (stoichiometry 10.8%, Km 8.4 microM) but not for CK1 kinase, protein kinase A, cdc2 kinase, MAP kinase or syk kinase. Residues Thr375 and Ser379 were identified as potential CK2 phosphorylation sites in this region based on sequence analysis. Mutation of Ser379 to aspartic acid or alanine reduced phosphorylation by CK2 by approximately 50%, and double mutation of Ser379 into aspartic acid and Thr375 into aspartic acid essentially abolished phosphorylation. Glutathione S-transferase (GST) pull-down experiments using extracts of Xenopus A6 epithelial cells showed that constructs of GST fused to wild-type and mutant forms of the C-terminal region of X. laevis occludin associate with several polypeptides, and immunoblot analysis showed that one of these polypeptides is cingulin. GST pull-down experiments using in vitro translated, full-length Xenopus cingulin indicated that cingulin interacts directly with the C-terminal region of occludin.

Amino Acid Sequence↗
PubMed1997

A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier.

Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 microM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.

Amino Acid Sequence↗