Physiological, Pathological, and Therapeutic Implications of Zonulin-Mediated Intestinal Barrier Modulation

The anatomical and functional arrangement of the gastrointestinal tract suggests that this organ, beside its digestive and absorptive functions, regulates the trafficking of macromolecules between the environment and the host through a barrier mechanism. Under physiological circumstances, this trafficking is safeguarded by the competency of intercellular tight junctions, structures whose physiological modulation is mediated by, among others, the recently described protein zonulin. To prevent harm and minimize inflammation, the same paracellular pathway, in concert with the gut-associated lymphoid tissue and the neuroendocrine network, controls the equilibrium between tolerance and immunity to nonself antigens. The zonulin pathway has been exploited to deliver drugs, macromolecules, or vaccines that normally would not be absorbed through the gastrointestinal mucosal barrier. However, if the tightly regulated trafficking of macromolecules is jeopardized secondary to prolonged zonulin up-regulation, the excessive flow of nonself antigens in the intestinal submucosa can cause both intestinal and extraintestinal autoimmune disorders in genetically susceptible individuals. This new paradigm subverts traditional theories underlying the development of autoimmunity, which are based on molecular mimicry and/or the bystander effect, and suggests that the autoimmune process can be arrested if the interplay between genes and environmental triggers is prevented by re-establishing intestinal barrier competency. Understanding the role of zonulin-dependent intestinal barrier dysfunction in the pathogenesis of autoimmune diseases is an area of translational research that encompasses many fields. The anatomical and functional arrangement of the gastrointestinal tract suggests that this organ, beside its digestive and absorptive functions, regulates the trafficking of macromolecules between the environment and the host through a barrier mechanism. Under physiological circumstances, this trafficking is safeguarded by the competency of intercellular tight junctions, structures whose physiological modulation is mediated by, among others, the recently described protein zonulin. To prevent harm and minimize inflammation, the same paracellular pathway, in concert with the gut-associated lymphoid tissue and the neuroendocrine network, controls the equilibrium between tolerance and immunity to nonself antigens. The zonulin pathway has been exploited to deliver drugs, macromolecules, or vaccines that normally would not be absorbed through the gastrointestinal mucosal barrier. However, if the tightly regulated trafficking of macromolecules is jeopardized secondary to prolonged zonulin up-regulation, the excessive flow of nonself antigens in the intestinal submucosa can cause both intestinal and extraintestinal autoimmune disorders in genetically susceptible individuals. This new paradigm subverts traditional theories underlying the development of autoimmunity, which are based on molecular mimicry and/or the bystander effect, and suggests that the autoimmune process can be arrested if the interplay between genes and environmental triggers is prevented by re-establishing intestinal barrier competency. Understanding the role of zonulin-dependent intestinal barrier dysfunction in the pathogenesis of autoimmune diseases is an area of translational research that encompasses many fields. The intestinal epithelium is the largest mucosal surface, providing an interface between the external environment and the mammalian host. Its exquisite anatomical and functional arrangements and the finely-tuned coordination of digestive, absorptive, motility, neuroendocrine, and immunological functions are testimonial of the complexity of the gastrointestinal (GI) system. Also pivotal is the regulation of molecular trafficking between the intestinal lumen and the submucosa via the paracellular space. The dimensions of the paracellular space are estimated to be between 10 and 15 Å, suggesting that under physiological circumstances, solutes with a molecular radius exceeding 15 Å (∼3.5 kDa) will be excluded from this uptake route. Macromolecule trafficking is dictated mainly by intestinal paracellular permeability, the regulation of which depends on the modulation of intercellular tight junctions (TJs). A century ago, TJs were conceptualized as a secreted extracellular cement forming an absolute and unregulated barrier within the paracellular space. The contribution of the paracellular pathway of the GI tract to the general economy of molecule trafficking between environment and host, therefore, was judged to be negligible. It is now apparent that TJs are extremely dynamic structures involved in several key functions of the intestinal epithelium both under physiological and pathological circumstances. TJ modulation has also been exploited for the delivery of drugs and macromolecules normally not absorbed through the GI tract. Given the structural and functional characteristics of intercellular TJs and the protean nature of the intestinal content, the gut mucosa represent the battlefield where friends (ie, nutrients and enteric microflora) and foes (ie, pathogenic microorganisms and their toxins) must be selectively recognized to reach an ideal balance between tolerance and immune response to nonself antigens. Antigen trafficking through TJs and their sampling by the gut-associated lymphoid tissue (GALT) are among the key strategies applied to reach this balance.This review is mainly focused on the role of zonulin, a modulator of intercellular TJs, on the pathophysiology of intestinal permeability and its use for therapeutic applications. For a more complete overview on the topic, the reader is referred to an excellent review article recently published in The American Journal of Pathology.1Turner JR Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application.Am J Pathol. 2006; 169: 1901-1909Abstract Full Text Full Text PDF PubMed Scopus (442) Google ScholarStructural Composition of Intercellular TJsThe intercellular TJs are complex structures composed by a multiprotein complex anatomically and functionally connected with the underlying apical actomyosin ring (Figure 1). Among the TJ proteins identified so far are the transmembrane proteins occludin, the claudins, and junctional adhesion molecules (JAMs), and cytoplasmic plaque proteins ZO-1, ZO-2, ZO-3, cingulin, and 7H6.2Nusrat A Turner JR Madara JL Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells.Am J Physiol. 2000; 279: G851-G857Google Scholar In vitro, the electrical resistance is a measure of charge flow across the membrane and thus reflects the permeability of the paracellular shunt pathway and the tightness of the intercellular TJ. TJ assembly is the result of a complex cascade of biochemical events that ultimately lead to the formation of an organized network of TJ elements, the composition of which has been only partially characterized. An in depth discussion of the complexity of TJ composition, the protein-protein interactions that govern their assembly and competency, and the multiple signaling pathways involved in the orchestration of all these events goes beyond the scope of this review. Therefore, only a general overview of the TJ structural composition will be covered herein to provide proper background and rationale to the main focus of the review.Transmembrane TJ ProteinsTo date, multiple proteins that make up the TJ strands have been identified and include occludin3Furuse M Hirase M Itoh M Nagafuchi A Yonemura S Tsukita S Tsukita S Occludin: a novel integral membrane protein localizing at tight junctions.J Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2096) Google Scholar and members of the claudin family,4Furuse M Fujita K Hiiragi T Fujimoto K Tsukita S Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.J Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1683) Google Scholar a group of at least 20 tissue-specific proteins. The JAMs, proteins that belong to the Ig superfamily, have been described as additional components of the TJ fibrils.5Martin-Padura I Lostgalio S Schneemann M Williams L Romano M Fruscella P Panzeri C Stoppacciaro A Ruco L Vill A Simmons D Dejana E Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration.J Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1132) Google Scholar JAMs reportedly bind to ZO-1, aiding its localization to the junctional complex.6Ebnet K Suzuki A Ohno S Vestweber D Junctional adhesion molecules (JAMs): more molecules with dual functions?.J Cell Sci. 2004; 117: 19-29Crossref PubMed Scopus (387) Google ScholarOccludin is a transmembrane TJ phosphoprotein of ∼65 kDa.7Anderson JM Van Itallie CM Tight junctions and the molecular basis for regulation of paracellular permeability.Am J Physiol. 1995; 269: G467-G475PubMed Google Scholar It is expressed in TJs of both epithelial and endothelial cells. The proposed folding topology places both NH2 and COOH terminals within the cytoplasm, allowing the polypeptide to pass out and back inside twice within the NH2 terminal half.7Anderson JM Van Itallie CM Tight junctions and the molecular basis for regulation of paracellular permeability.Am J Physiol. 1995; 269: G467-G475PubMed Google Scholar Occludin has recently been shown to function as a cell-cell adhesion molecule and appears to participate in maintaining the intramembrane diffusion barrier.8Mitic LL Anderson JM Molecular architecture of tight junctions.Annu Rev Physiol. 1998; 60: 121-142Crossref PubMed Scopus (619) Google Scholar Occludin was previously considered to be involved in establishing the seal at the sites of junctional strands.9Mitic LL Van Itallie Cereijido M Anderson JM Occludin and claudins: transmembrane proteins of the tight junction. Tight Junctions. CRC Press, Inc., Boca Raton2001: 213-230Google Scholar However, discrepancies between the presence of fibrils and occludin expression in several tissues, including human testis and endothelial cells, led to the discovery of the claudins as integral membrane TJ proteins.4Furuse M Fujita K Hiiragi T Fujimoto K Tsukita S Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.J Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1683) Google ScholarThe claudins are 20- to 25-kDa tetraspan membrane proteins that each contain two extracellular loops with variably charged amino acid residues and short intracellular tails.9Mitic LL Van Itallie Cereijido M Anderson JM Occludin and claudins: transmembrane proteins of the tight junction. Tight Junctions. CRC Press, Inc., Boca Raton2001: 213-230Google Scholar The claudins determine the barrier properties of the paracellular pathway because they can completely tighten the intercellular space to solutes, and they can form paracellular ion pores. It is assumed that the extracellular loops specify these claudin functions.JAMs are characterized by the molecular structure composed of two extracellular immunoglobulin loops, a single transmembrane domain, and a cytoplasmic domain with a PDZ-binding motif.5Martin-Padura I Lostgalio S Schneemann M Williams L Romano M Fruscella P Panzeri C Stoppacciaro A Ruco L Vill A Simmons D Dejana E Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration.J Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1132) Google Scholar JAMs interact with several ligands in both homophilic and heterophilic manners by the extracellular domain and also bind to various PDZ domain-containing proteins at the C terminus. Through these protein-protein interactions, JAMs are implicated in diverse biological functions at the TJ, including cell-cell adhesion, junctional assembly, regulation of paracellular permeability, and leukocyte transmigration.10Hirabayashi S Hata Yutaka Gonzales-Mariscal L JAM family proteins: tight junction proteins that belong to the immunoglobulin superfamily. Tight Junctions. CRC Press, New York2006: 43-63Google Scholar, 11Laukoetter MG Nava P Lee WY Severson EA Capaldo CT Babbin BA Williams IR Koval M Peatman E Campbell JA Dermody TS Nusrat A Parkos CA JAM-A regulates permeability and inflammation in the intestine in vivo.J Exp Med. 2007; 204: 3067-3076Crossref PubMed Scopus (368) Google ScholarCytoplasmic TJ ProteinsThe cytoplasmic plaque of the TJ includes multiple proteins that have been defined at the molecular level as well as several others that await further characterization. Through interactions with each other and with cytoskeletal proteins, these scaffold elements (ZO-1, ZO-2, ZO3, and ZO-1-associated protein kinase or ZAK) functionally couple integral membrane TJ proteins to actin microfilaments.12Citi S The cytoplasmic plaque proteins of the tight junction.in: Cereijido M Anderson JM Tight Junctions. CRC Press, Inc., Boca Raton2001: 231-264Google Scholar TJ plaque proteins also appear to be direct targets and effectors of different signaling pathways. Additional cytoplasmic plaque proteins have been localized to the TJ, including 7H6,9Mitic LL Van Itallie Cereijido M Anderson JM Occludin and claudins: transmembrane proteins of the tight junction. Tight Junctions. CRC Press, Inc., Boca Raton2001: 213-230Google Scholar Rab 13 and 3b,13Zahraoui A Joberty G Arpin M Fontaine JJ Hellio R Tavitian A Louvard D A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalized with the tight junction marker Zo-1 in polarized epithelial cells.J Cell Biol. 1994; 124: 101-115Crossref PubMed Scopus (203) Google Scholar Gαi-2,14Denker BM Saha C Khawaja S Nigam SK Involvement of a heterotrimeric G protein alpha subunit in tight junction biogenesis.J Biol Chem. 1996; 271: 25750-25753Crossref PubMed Scopus (120) Google Scholar PKC,15Dodane V Kachar B Identification of isoforms of G proteins and PKC that colocalize with tight junctions.J Membr Biol. 1996; 149: 199-209Crossref PubMed Scopus (113) Google Scholar symplekin (126.5 kDa),16Keon BH Schafer S Kuhn C Grund C Franke WW Symplekin, a novel type of tight junction plaque protein.J Cell Biol. 1996; 134: 1003-1018Crossref PubMed Scopus (267) Google Scholar ZA-1TJ, and cingulin (140-kDa phosphoprotein).17Citi S Sabannay H Jakes R Geiger B Kendrich-Jones J Cingulin, a new peripheral component of tight junctions.Nature. 1988; 333: 272-275Crossref PubMed Scopus (401) Google ScholarGI Mucosal Immune SystemThe GALTParacellular passage of macromolecules, under either physiological or pathological circumstances, is safeguarded by the GALT (Figure 2). The GALT serves as a containment system preventing potentially harmful intestinal antigens from reaching the systemic circulation and induces systemic tolerance against luminal antigens by a process that involves polymeric IgA secretion and the induction of regulatory T cells. Another important factor for the intestinal immunological responsiveness is the major histocompatibility complex. Human leukocyte antigen (HLA) class I and class II genes are located in the major histocompatibility complex on chromosome 6. These genes code for glycoproteins, which bind peptides, and this HLA-peptide complex is recognized by certain T-cell receptors in the intestinal mucosa.18Bjorkman PJ Saper MA Samraoui B Bennett WS Strominger JL Wiley DC Structure of the human class-I histocompatibility antigen, Hla-A2.Nature. 1987; 329: 506-512Crossref PubMed Scopus (2734) Google Scholar, 19Cuvelier C Barbatis C Mielants H Devos M Roels H Veys E Histopathology of intestinal inflammation related to reactive arthritis.Gut. 1987; 28: 394-401Crossref PubMed Scopus (120) Google Scholar Susceptibility to at least 50 diseases is associated with specific HLA class I or class II alleles.Figure 2Immunofluorescence microscopy of mouse small intestine (ileum) cross section. The confocal image shows the anatomical arrangement of epithelial cells (stained in red) and immune cells (dendritic cells stained in green, B cells stained in blue, and T cells stained in white). This arrangement suggests functional interactions between epithelial and immune cells for handling nonself antigens and microbiota present in the intestinal lumen. (Image provided by Marcello Chieppa, Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The balance between immunity and tolerance is essential for a healthy intestine, and abnormal or inappropriate immune responses may result in inflammatory pathologies. There is evidence that antigen-presenting dendritic cells, key players in antigen sampling, are educated by memory T cells and subsequently induce naïve T cells,20Alpan O Bachelder E Isil E Arnheiter H Matzinger P ‘Educated’ dendritic cells act as messengers from memory to naive T helper cells.Nat Immunol. 2004; 5: 615-622Crossref PubMed Scopus (114) Google Scholar thereby supporting the role for dendritic cells in coupling innate and adaptive immune responses that affect intestinal permeability.Innate and Adaptive Immunity and Their InteractionsRecognition of antigens by dendritic cells triggers a family of pattern recognition receptors, Toll-like receptors (TLRs), which change dendritic cell phenotype and function. TLRs are the major receptors involved in the discrimination between self and nonself based on the recognition of conserved bacterial molecular patterns (Figure 3). In intestinal epithelial cells, TLRs play a role in normal mucosal homeostasis and are particularly important in the interaction between the mucosa and the luminal flora.21Rakoff-Nahoum S Paglino J Eslami-Varzaneh F Edberg S Medzhitov R Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis.Cell. 2004; 118: 229-241Abstract Full Text Full Text PDF PubMed Scopus (3241) Google Scholar There are a number of TLRs, all of which are present in the gut and respond to different stimuli resulting in different adaptive immune responses.22Cario E Gerken G Podolsky DK Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C.Gastroenterology. 2004; 127: 224-238Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 23Rumio C Besusso D Palazzo M Selleri S Sfondrini L Dubini F Menard S Balsari A Degranulation of Paneth cells via Toll-like receptor 9.Am J Pathol. 2004; 165: 373-381Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Otte JM Cario E Podolsky DK Mechanisms of cross hyporesponsiveness to toll-like receptor bacterial ligands in intestinal epithelial cells.Gastroenterology. 2004; 126: 1054-1070Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar There is now evidence for a differential response to stimuli arising from TLRs located at the basolateral versus the apical surface.25Lee J Mo JH Katakura K Alkalay I Rucker AN Liu YT Lee HK Shen C Cojocaru G Shenouda S Kagnoff M Eckmann L Ben-Neriah Y Raz E Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.Nat Cell Biol. 2006; 8: 1327-1336Crossref PubMed Scopus (478) Google ScholarFigure 3TLR. Currently, at least 10 different TLRs have been described, recognizing different ligands that activate the innate immune system. TLRs 3, 7, 8, and 9 are endosomal TLRs, whereas the remaining TLRs are surface, extracellular receptors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TLRs direct immune responses by activating signaling events leading to elevated expression of factors, such as cytokines and chemokines that recruit and regulate the immune and inflammatory cells, which then either initiate or enhance host immune responses.26Akira S Takeda K Toll-like receptor signalling.Nat Rev Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6586) Google Scholar The peripheral memory T-cell response is a critical outcome of adaptive immunity, and TLRs likely are required for the generation and maintenance of memory T cells.27Pasare C Medzhitov R Toll-dependent control mechanisms of CD4 T cell activation.Immunity. 2004; 21: 733-741Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar TLRs are implicated in chronic diseases such as enteric inflammation and may have both proinflammatory and protective roles. Of interest, commensal flora acting through TLR4 positively influence the susceptibility to food antigens28Bashir ME Louie S Shi HN Nagler-Anderson C Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy.J Immunol. 2004; 172: 6978-6987PubMed Google Scholar and implicate TLRs in the regulation of intestinal permeability. This concept is supported by recent in vitro studies using intestinal epithelial cell cultures, which show that TLR-2 enhances epithelial integrity by a rearrangement of the TJ protein, ZO-1.22Cario E Gerken G Podolsky DK Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C.Gastroenterology. 2004; 127: 224-238Abstract Full Text Full Text PDF PubMed Scopus (381) Google ScholarThe Zonulin SystemTo meet the diverse physiological challenges to which the intestinal epithelial barrier is subjected, TJs must be capable of rapid and coordinated responses. This requires the presence of a complex regulatory system that orchestrates the state of assembly of the TJ multiprotein network. Although knowledge about TJ ultrastructure and intracellular signaling events has progressed significantly during the past decade, relatively little is known about their pathophysiological regulation. The discovery of zonula occludens toxin (Zot), an enterotoxin expressed by Vibrio cholerae that reversibly opens TJs,29Fasano A Baudry B Pumplin DW Wasserman SS Tall BD Ketley J Kaper JB Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.Proc Natl Acad Sci USA. 1991; 88: 5242-5246Crossref PubMed Scopus (441) Google Scholar has increased our understanding of the intricate mechanisms that regulate the intestinal epithelial paracellular pathway. Zot is a single polypeptide chain of 44.8 kDa, encoded by the cholera toxin bacteriophage (CTXϕ) present in toxigenic strains of V. cholerae.29Fasano A Baudry B Pumplin DW Wasserman SS Tall BD Ketley J Kaper JB Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.Proc Natl Acad Sci USA. 1991; 88: 5242-5246Crossref PubMed Scopus (441) Google Scholar Zot action is mediated through a cascade of intracellular events that lead to PKC-α-dependent polymerization of actin microfilaments and subsequent TJ disassembly.30Fasano A Fiorentini C Donelli G Uzzau S Kaper JB Margaretten K Ding X Guandalini S Comstock L Goldblum SE Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.J Clin Invest. 1995; 96: 710-720Crossref PubMed Scopus (296) Google Scholar Immunofluorescence binding studies have shown that Zot binding varies within the intestine, being detectable in the jejunum and distal ileum, but not in the colon, and decreasing along the villous-crypt axis.31Fasano A Uzzau S Fiore C Margaretten K The enterotoxic effect of zonula occludens toxin (Zot) on rabbit small intestine involves the paracellular pathway.Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar This binding distribution coincides with the differential intestinal epithelial barrier responsiveness and actin reorganization that occurs along the villous axis32Marcial MA Carlson SL Madara JL Partitioning of paracellular conductance along the ileal crypt-villus axis: a hypothesis based on structural analysis with detailed consideration of tight junction structure-function relationships.J Membr Biol. 1984; 80: 59-70Crossref PubMed Scopus (144) Google Scholar, 33Uzzau S Lu R Wang W Fiore C Fasano A Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines.FEMS Microbiol Lett. 2001; 194: 1-5Crossref PubMed Google Scholar as well as with the regional effect of Zot on intestinal permeability.30Fasano A Fiorentini C Donelli G Uzzau S Kaper JB Margaretten K Ding X Guandalini S Comstock L Goldblum SE Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.J Clin Invest. 1995; 96: 710-720Crossref PubMed Scopus (296) Google Scholar, 31Fasano A Uzzau S Fiore C Margaretten K The enterotoxic effect of zonula occludens toxin (Zot) on rabbit small intestine involves the paracellular pathway.Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar These combined data demonstrate that Zot regulates TJs in a rapid, reversible, and reproducible manner. Based on these observations, it has been postulated that Zot mimics an immunologically related, endogenous modulator of epithelial TJs. The combination of affinity-purified anti-Zot antibodies and the Ussing chamber assay allowed the identification of a human intestinal Zot homolog that was designated as zonulin (Figure 4).34Wang W Uzzau S Goldblum SE Fasano A Human zonulin, a potential modulator of intestinal tight junctions.J Cell Sci. 2000; 113: 4435-4440PubMed Google Scholar Affinity-purified zonulin reduced transepithelial electrical resistance compared to the media control in both monkey jejunum and ileum, but not in the colon.34Wang W Uzzau S Goldblum SE Fasano A Human zonulin, a potential modulator of intestinal tight junctions.J Cell Sci. 2000; 113: 4435-4440PubMed Google Scholar V. cholerae-derived Zot and human zonulin both act on intestinal TJs29Fasano A Baudry B Pumplin DW Wasserman SS Tall BD Ketley J Kaper JB Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.Proc Natl Acad Sci USA. 1991; 88: 5242-5246Crossref PubMed Scopus (441) Google Scholar, 35Baudry B Fasano A Ketley JM Kaper JB Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae.Infect Immun. 1992; 60: 428-434Crossref PubMed Google Scholar, 36Di Pierro M Lu R Uzzau S Wang W Margaretten K Pazzani C Maimone F Fasano A Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain.J Biol Chem. 2001; 276: 19160-19165Crossref PubMed Scopus (152) Google Scholar and display the same regional barrier responsiveness31Fasano A Uzzau S Fiore C Margaretten K The enterotoxic effect of zonula occludens toxin (Zot) on rabbit small intestine involves the paracellular pathway.Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar coincident with Zot receptor distribution within the intestine,30Fasano A Fiorentini C Donelli G Uzzau S Kaper JB Margaretten K Ding X Guandalini S Comstock L Goldblum SE Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.J Clin Invest. 1995; 96: 710-720Crossref PubMed Scopus (296) Google Scholar, 33Uzzau S Lu R Wang W Fiore C Fasano A Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines.FEMS Microbiol Lett. 2001; 194: 1-5Crossref PubMed Google Scholar These findings suggest that these two molecules could potentially interact with the same receptor(s). Comparison of the primary amino acid structures of Zot and zonulin provided insights into the structural requirements for ligand engagement of the receptor coupled to intestinal TJ regulation. The NH2-termini of zonulin and the Zot active fragment share a common octapeptide motif (GGVLVQPG) that is critical for intestinal receptor binding.34Wang W Uzzau S Goldblum SE Fasano A Human zonulin, a potential modulator of intestinal tight junctions.J Cell Sci. 2000; 113: 4435-4440PubMed Google Scholar, 36Di Pierro M Lu R Uzzau S Wang W Margaretten K Pazzani C Maimone F Fasano A Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain.J Biol Chem. 2001; 276: 19160-19165Crossref PubMed Scopus (152) Google Scholar The synthetic octapeptide motif, named AT1001, is an efficient inhibitor of both Zot and zonulin because it binds to the target receptor, but it does not activate the intracellular signaling leading to TJ disassembly and antigen trafficking.34Wang W Uzzau S Goldblum SE Fasano A Human zonulin, a potential modulator of intestinal tight junctions.J Cell Sci. 2000; 113: 4435-4440PubMed Google Scholar, 36Di Pierro M Lu R Uzzau S Wang W Margaretten K Pazzani C Maimone F Fasano A Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain.J Biol Chem. 2001; 276: 19160-19165Crossref PubMed Scopus (152) Google Scholar Zonulin cloning and characterization have revealed that it be