CaCo-2 cells treated with Clostridium perfringens enterotoxin form multiple large complex species, one of which contains the tight junction protein occludin

The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology.     

Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectants

Clostridium perfringens enterotoxin (CPE) binds to host cell receptors, forming a small complex precursor for two large complexes reportedly having molecular masses of approximately 155 or approximately 200 kDa. Formation of the approximately 155 kDa complex causes a Ca(2+) influx that leads to apoptosis or oncosis. CPE complex composition is currently poorly understood, although occludin was identified in the approximately 200 kDa complex. The current study used heteromer gel shift analysis to show both CPE large complexes contain six CPE molecules. Ferguson plots and size exclusion chromatography re-sized the approximately 155 and approximately 200 kDa complexes as approximately 425-500 kDa and approximately 550-660 kDa respectively. Co-immunoprecipitation and electroelution studies demonstrated both CPE-binding and non-CPE-binding claudins are associated with all three CPE complexes in Caco-2 cells and with small complex and approximately 425-500 kDa complex of claudin 4 transfectants. Fibroblast transfectants expressing claudin 4 or C-terminal truncated claudin 4 were CPE-sensitive and formed the approximately 425 kDa complex, indicating claudin-induced cell signalling is not required for CPE action and that expression of a single receptor claudin suffices for approximately 425-500 kDa CPE complex formation. These results identify CPE as a unique toxin that combines with tight junction proteins to form high-molecular-mass hexameric pores and alter membrane permeability.     

Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells.

Since most in vitro studies exploring the action of Clostridium perfringens enterotoxin (CPE) utilize either Vero or CaCo-2 cells, the current study directly compared the CPE responsiveness of those two cell lines. When CPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of approximately 135, approximately 155, and approximately 200 kDa. However, confluent Transwell cultures of either cell line CPE-treated for 20 min formed only the approximately 155-kDa complex. Since those Transwell cultures also exhibited significant (86)Rb release, approximately 155-kDa complex formation is sufficient for CPE-induced cytotoxicity. Several differences in CPE responsiveness between the two cell lines were also detected. (i) CaCo-2 cells were more sensitive when CPE-treated on their basal surface, whereas Vero cells were more sensitive when CPE-treated on their apical surface; those sensitivity differences correlated with CPE binding the apical versus basolateral surfaces of these two cell lines. (ii) CPE-treated Vero cells released (86)Rb into both Transwell chambers, whereas CaCo-2 cells released (86)Rb only into the CPE-containing Transwell chamber. (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form TJs. The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming TJs. Confluent CaCo-2 Transwell cultures CPE-treated for >1 h formed the approximately 200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their TJs. Collectively, these results identify CPE as a bifunctional toxin that, in confluent polarized cells, first exerts a cytotoxic effect mediated by the approximately 155-kDa complex. Resultant damage then provides CPE access to TJs, leading to approximately 200-kDa complex formation, internalization of some TJ proteins, and TJ damage that may increase paracellular permeability and thereby contribute to the diarrhea of CPE-induced gastrointestinal disease.     

Phenotypic characterization of enterotoxigenic Clostridium perfringens isolates from non-foodborne human gastrointestinal diseases

Clostridium perfringens enterotoxin (CPE) has been implicated as an important virulence factor inC. perfringens type A food poisoning and several non-foodborne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SPOR). Recent studies have revealed genotypic differences between cpe-positive isolates originating from different disease sources, with most, or all, food poisoning isolates carrying a chomosomal cpe and most, or all, non-foodborne human GI disease isolates carrying an episomal cpe. To evaluate whether these genotypic differences cause phenotypic effects that could influence the pathogenesis of CPE-associated non-foodborne human GI illnesses, a collection of SPOR and AAD isolates has been phenotypically characterized in the current study. All cpe-positive non-foodborne disease isolates examined were found to express CPE in a sporulation-associated manner. The CPE made by these AAD and SPOR isolates was shown to have the same deduced amino acid sequence and toxicity as the classical CPE made by food poisoning isolates. All of the surveyed non-foodborne human GI disease isolates were found to classify as type AC. perfringens, since they produce alpha toxin, but not beta, iota, or epsilon toxins. Finally, no consistent clonal relationships were detected between the surveyed non-foodborne human GI disease isolates. Since, by the criteria examined, all non-foodborne human GI disease isolates examined in this study appear to be phenotypically similar to food poisoning isolates, the current results confirm that the examined AAD and SPOR isolates have enteropathogenic potential. However, given the phenotypic similarities between food poisoning, AAD, and SPOR isolates that have been demonstrated in this study, it remains unclear why the symptomology of non-foodborne human GI diseases is typically more severe and longer-lasting than that of C. perfringens type A food poisoning.     

Inactivation of the gene (cpe ) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.     

Role of occludin, a tight junction protein, in blastocoel formation, and in the paracellular permeability and differentiation of trophectoderm in preimplantation mouse embryos

Tight junctions (TJ) are critical for blastocoel formation in mammalian embryos. The present study aimed to examine the role of tight junctions in the differentiation of the trophectoderm (TE), and in the pluripotency of blastomeres, as well as in the formation and integrity of the blastocoel. We examined the effect of occludin antibody on blastocoel formation, blastocyst permeability, and expression of H19 and Oct-4, markers of TE differentiation and blastomere pluripotency, respectively. Eight-cell mouse embryos and morulae were cultured in the presence or absence of occludin antibody for 31 h. Occludin antibody inhibited blastocoel formation and increased permeability of the TE of nascent and expanding blastocysts to FITC-dextran (4 kDa), a permeability tracer. At the same time Oct-4 expression increased while expression of H19 became barely detectable. These observations indicate that occludin is involved in establishing the blastocoel, as well as in maintaining its impermeability, and that the development of tight junction is critical for TE formation in mouse embryos.     

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.     

Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin. Early events in enterotoxin action are divalent cation-independent

Clostridium perfringens type A enterotoxin (CPE) is a membrane-active cytotoxin. There are a number of recognized early steps in CPE cytotoxicity including binding of CPE to a protein receptor, insertion of CPE into membranes, and CPE-mediated induction of changes in membrane permeability for small molecules such as ions and amino acids. Further support for the existence of these early steps and further characterization of these events are presented in this report. We now report that these early steps in CPE action are largely independent of extracellular divalent cations. It is also shown that 3H-nucleotide release, known to be a later CPE effect, is Ca2+-dependent. A model for CPE cytotoxicity is suggested involving CPE action as a two-step process with Ca2+-independent early steps and Ca2+-dependent late steps.     

Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo

Tight junctions (TJs) are major components of the blood–brain barrier (BBB) that physically obstruct the interendothelial space and restrict paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS. TJs are dynamic structures whose intricate arrangement of oligomeric transmembrane and accessory proteins rapidly alters in response to external stressors to produce changes in BBB permeability. In this study, we investigate the constitutive trafficking of the TJ transmembrane proteins occludin and claudin-5 that are essential for forming the TJ seal between microvascular endothelial cells that inhibits paracellular diffusion. Using a novel, detergent-free OptiPrep density-gradient method to fractionate rat cerebral microvessels, we identify a plasma membrane lipid raft domain that contains oligomeric occludin and claudin-5. Our data suggest that oligomerization of occludin involves disulfide bond formation within transmembrane regions, and that assembly of the TJ oligomeric protein complex is facilitated by an oligomeric caveolin scaffold. This is the first time that distribution of oligomeric TJ transmembrane proteins within plasma membrane lipid rafts at the BBB has been examined in vivo. The findings reported in this study are critical to understand the mechanism of assembly of the TJ multiprotein complex that is essential for maintaining BBB integrity.     

Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1

Vibrio cholerae produces a little-studied cytotoxin, haemagglutinin/protease (HA/P), in addition to several better-characterized enterotoxins, i.e. cholera toxin (CT), zonula occludens toxin (ZOT) and accessory cholera enterotoxin (Ace). We have found recently that HA/P perturbs the barrier function of Mardin–Darby canine kidney epithelial cell line I (MDCK-I) by affecting the intercellular tight junctions (TJs) and the F-actin cytoskeleton. In the present study we have assessed more specifically how TJs are affected by HA/P by investigating the cellular localization and biochemical integrity of two well-characterized TJ-associated proteins, occludin and ZO-1. Western blot analysis showed that occludin bands of 66–85 kDa were digested by HA/P to two predominant bands of around 50 kDa and 35 kDa, and that this degradation was greatly attenuated when the specific bacterial metalloproteinase inhibitor Zincov was co-administered. Trypsin, on the other hand, did not degrade occludin when it was applied in the same way, suggesting that the degradation of occludin by HA/P is an early and specific event. The other TJ-associated protein ZO-1 was not degraded by HA/P in parallel experiments, suggesting the selectivity of HA/P-associated protein degradation. Moreover, immunofluorescence labelling and confocal microscopy showed that ZO-1, but not occludin, around cell–cell boundaries was rearranged by HA/P treatment. Since ZO-1 is located on the inside of the plasma membrane and is directly associated with occludin, the results indicate that breakdown of occludin may send signals to ZO-1 that affect its organization and the structure of the F-actin cytoskeleton. Our finding that the zinc-containing metalloprotease of V. cholerae specifically degraded occludin suggests that specific degradation of important host proteins by bacterial zinc-containing metalloproteases may be an important mechanism in microbial pathogenesis.     

Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates carrying plasmid genes versus chromosomal enterotoxin genes

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringens isolates associated with food poisoning carry a chromosomal cpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (D values) at 55 degrees C than vegetative cells of plasmid cpe isolates exhibited. Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100 degrees C than the spores of plasmid cpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomal cpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.     

The tight junctional protein occludin is found in the uterine epithelium of squamate reptiles

Occludin, an integral protein associated with the mammalian tight junction, has for the first time been identified in the uterus of squamate reptiles. The tight junction is made up of anastamosing strands and forms a selective barrier that regulates paracellular diffusion of solutes across uterine epithelium. Occludin exclusively labels tight junctional strands and is an excellent marker for tight junction permeability. Using western blotting and immunohistochemistry, occludin expression was examined in the uterine epithelium of five species of Australian skinks at different stages of gestation. More occludin was detected during late stage pregnancy/gravidity compared to the lower levels of occludin detected in vitellogenic and post-parturient females in three of the five species. We conclude that the paracellular permeability of the squamate uterine epithelium decreases as gestation progresses. As placental transport of ions and solutes to the embryo is highest during the last third of pregnancy in viviparous squamates, it is likely that a decrease in paracellular permeability is compensated by an upregulation of other transporting mechanisms such as histotrophy.     

Apical junction complex protein expression in the canine colon: differential expression of claudin-2 in the colonic mucosa in dogs with idiopathic colitis.

Canine idiopathic lymphocytic-plasmacytic colitis (LPC) is a well-recognized clinical and pathological entity in the dog, associated with altered immune cell populations and cytokine expression profiles. Clinical and experimental data indicate that alterations in the permeability of the intestinal epithelium contribute to the pathogenesis of a range of related conditions. The apical junction complex plays a significant role in regulating epithelial paracellular permeability, and we have characterized the distribution of a number of its component tight junction (ZO-1, occludin, claudin-2) and adherens junction (E-cadherin and beta-catenin) proteins in normal colon and colon from dogs with idiopathic LPC. ZO-1, occludin, E-cadherin, and beta-catenin exhibited a distribution in normal canine colon similar to that described previously in humans and rodents. In contrast to the situation in humans, claudin-2-specific labeling was observed in the normal canine colonic crypt epithelium, decreasing in intensity from the distal to the proximal crypt and becoming barely detectable at the luminal surface of the colon. There was little evidence for significant changes in ZO-1, occludin, E-cadherin, or beta-catenin expression in dogs affected by idiopathic LPC. However, claudin-2 expression markedly increased in the proximal crypt and luminal colonic epithelium in affected dogs, suggesting a role in the pathogenesis of canine LPC.     

Structure of a C. perfringens Enterotoxin Mutant in Complex with a Modified Claudin-2 Extracellular Loop 2

CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.     

Enterotoxic strains of Clostridium perfringens and sporulation

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.     

Platelet-derived growth factor mediates tight junction redistribution and increases permeability in MDCK cells

Increased tissue permeability is a common characteristic of a number of diseases such as pulmonary edema, inflammatory bowel disease, several kidney diseases, diabetic retinopathy, and tumors. We hypothesized that growth factors increase permeability by redistribution of tight junction proteins away from the cell border. To investigate mechanisms of growth factor-mediated permeability, we examined the effect of platelet derived growth factor (PDGF) on Madin-Darby canine kidney (MDCK) cell tight junction protein distribution and on permeability. PDGF altered the cellular distribution of occludin and ZO-1 from the cell border to the cytoplasm and increased permeability to 70 kDa dextran in a concentration-dependent manner. Treatment of MDCK cells with PDGF prior to fixation allowed binding of the lectin concanavalin A to the basement membrane of fixed cells, while binding was prevented in untreated control monolayers, implying that PDGF induced the formation of a paracellular transport pathway. Cell fractionation experiments with PDGF-treated cells revealed a novel occludin-containing low-density, detergent resistant subcellular structure, which increased in the buoyant fractions relative to occludin in the pellet in a time- and concentration-dependent manner. Immunocytochemistry revealed that a pool of internalized occludin co-labels with the early endosome marker, EEA1, suggesting that PDGF may stimulate occludin to enter an endosomal pathway. PDGF may act as a permeabilizing agent by moving tight junction proteins away from the cell border in discrete microdomains, and the effects of PDGF on permeability and tight junction protein distribution may model the regulation of epithelial and endothelial barrier properties by other peptide growth factors.     

Occludin and tricellulin facilitate formation of anastomosing tight-junction strand network to improve barrier function

Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ-strand branching points and contribute to the maintenance of epithelial barrier integrity.     

Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors.

Vascular endothelial growth factor (VEGF) may have a physiologic role in regulating vessel permeability and contributes to the pathophysiology of diabetic retinopathy as well as tumor development. We set out to ascertain the mechanism by which VEGF regulates paracellular permeability in rats. Intra-ocular injection of VEGF caused a post-translational modification of occludin as determined by a gel shift from 60 to 62 kDa. This event began by 15 min post-injection and was maximal by 45 min. Alkaline phosphatase treatment revealed this modification was caused by a change in occludin phosphorylation. In addition, the quantity of extracted occludin increased 2-fold in the same time frame. The phosphorylation and increased extraction of occludin was recapitulated in retinal endothelial cells in culture after VEGF stimulation. The data presented herein are the first demonstration of a change in the phosphorylation of this transmembrane protein under conditions of increased endothelial permeability. In addition, intra-ocular injection of VEGF also caused tyrosine phosphorylation of ZO-1 as early as 15 min and increased phosphorylation 4-fold after 90 min. In conclusion, VEGF rapidly increases occludin phosphorylation as well as the tyrosine phosphorylation of ZO-1. Phosphorylation of occludin and ZO-1 likely contribute to regulated endothelial paracellular permeability.     

Molecular size of a Na(+)-dependent amino acid transporter in Ehrlich ascites cell plasma membranes estimated by radiation inactivation

Radiation inactivation was used to estimate the molecular size of a Na(+)-dependent amino acid transport system in Ehrlich ascites cell plasma membrane vesicles. Na(+)-dependent alpha-aminoisobutyric acid uptake was measured after membranes were irradiated at -78.5 degrees C in a cryoprotective medium. Twenty-five percent of the transport activity was lost at low radiation doses (less than 0.5 Mrad), suggesting the presence of a high molecular weight transport complex. The remaining activity (approximately 75% of total) decreased exponentially with increasing radiation dose, and a molecular size of 347 kDa was calculated for the latter carrier system. Vesicle permeability and intravesicular volume were measured to verify that losses in transport activity were due to a direct effect of radiation on the transporter and not through indirect effects on the structural integrity of membrane vesicles. Radiation doses 2-3-fold higher than those required to inactivate amino acid transport were needed to cause significant volume changes (greater than 15%). Vesicle permeability was unchanged by the irradiation. The structural integrity of plasma membrane vesicles was therefore maintained at radiation doses where there was a dramatic decrease in amino acid transport. The relationship between the fragmentation of a 120-130-kDa peptide, a putative component of the Na(+)-dependent amino acid carrier [McCormick, J. I., & Johnstone, R. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881], and loss of transport activity in irradiated membranes was also examined. Peptide loss was quantitated by Western blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions

BackgroundThe intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease — actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs).MethodsEffects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry.ResultsActinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin.ConclusionAct d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice.General significanceIn line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.