A: Western blotting using zonulin cross-reacting anti-Zot polyclonal antibodies on CD patient sera. Three main patterns were detected: sera showing a 18-kDa immunoreactive band and a fainter ∼45-kDa band (lane 1), sera showing only a 9-kDa band (lane 2), and sera showing both the 18- and 9-kDa bands (lane 3). B: cartoon showing the structure of both pre-haptoglobin (HP) 1 and pre-HP2 and their mature proteins. HPs evolved from a complement-associated protein (mannose-binding lectin-associated serine protease, MASP), with their α-chain containing a complement control protein (CCP), while the β-chain is related to chymotrypsin-like serine proteases (SP domain) containing an epidermal growth factor-like motif. The gene encoding the α2-chain of pre-HP2 originated in India almost 2 million years ago through a chromosomal aberration (unequal crossing over) of HP1. Pre-HPs are translated as single-chain precursor proteins. Pre-HPs may be proteolytically cleaved intracellularly into α- and β-chains that remain disulfide linked, referred to as cleaved, two-chain mature HPs. The two-chain mature HPs are abundant plasma glycoproteins and are composed of their α- and β-subunits covalently associated by three disulfide bonds in which the carbohydrate groups are exclusively linked to the β-chain. [Modified from Camarca et al. (29).]
Evolutionary tree of HP gene. The appearance of the gene encoding HP1 has been mapped ∼450 millions years ago, soon after the split between bony fish, reptiles, and mammals. HP2 appeared much later, 500, 000 years after, then chimpanzee and human split 2.5 millions years ago.
Differences in HP gene clustering on chromosome 16 between human and chimpanzee. The HP gene complex is located on chromosome 16 and is composed by different variants in chimpanzee versus humanoid. In chimpanzee, HP1 is followed by HP-related gene (HPR) and HP chimpanzee (HPCh). The 2-gene cluster of the human was formed after the separation of the human and chimpanzee lineages by an unequal homologous crossover that deleted most of the HPCh. HP2 is found only in humans and originated 2 millions years ago through a chromosomal aberration (unequal crossover) in a humanoid in India who was heterozygous α-1F/α-1S (103).
Caco2-derived trypsin IV cleaves zonulin in its two subunits. Western immunoblotting of recombinant zonulin before (lane 1) and after (lanes 6–8) incubation on Caco-2 cells. The intensity of the ∼18-kDa band (identified by NH2-terminal sequencing as the a2 chain) increased after 120-min incubation in Caco2 cells. Media (lanes 2 and 3) and bovine serum albumin (BSA; lanes 4 and 5) were also tested to confirm antibody specificity.
Effect of zonulin on gastrointestinal permeability in vivo. Zonulin (closed bars) increases both small intestinal (A) and gastroduodenal (B) permeability compared with BSA-treated controls (open bars). The differences in lacman ratio (small intestinal permeability) and sucrose fractional excretion (gastroduodenal permeability) are shown as percentage of change in permeability between the measurements on the challenge day and 3 days before challenge. Mature two-chain HP2 (dotted bars) caused no changes in either small intestinal or gastroduodenal permeability. The effect of zonulin was completely reversible, since both small intestinal (C) and gastroduodenal (D) permeability returned to prechallenge values within 48 h. The differences in lacman or sucrose fractional excretion are shown as percentage of permeability change between the value of 2 days after the challenge and the challenge day. *Lacman P < 0.0024 compared with both BSA control and 2-chain HP2; **Sucrose P < 0.0049 compared with both BSA control and 2-chain HP2 (n = 10 for each group of treatment). [Modified from Tripathi et al. (159).]
Proposed mechanisms through which zonulin activates EGFR. Zonulin can activate EGFR through direct binding (1) and/or through PAR2 transactivation (2). This second mechanism can be mediated by either Src signaling (2a) or by the release of MMPs and/or ADAMS that in turn will activate Pro-HB-EGF. Processing of zonulin into its two-chain mature form, for example, via proteolytic cleavage by intestinal tryptase IV, induces conformational changes in the molecule that abolish its ability to bind to EGFR (3), but instead enables a different function (e.g., Hb binding), and it becomes an inflammatory marker.
Stimuli causing polarized zonulin release from intestinal epithelial cells. A–C: anti-zonulin immunoflourescence staining of human intestinal Caco2 cells. Cells exposed to gliadin/PT-gliadin (B) react by packaging preformed zonulin in vesicles (arrows) that gradually approached the cell membrane and then released their zonulin content in the cell medium within a few minutes of the exposure to gliadin. No packaging was detected in nonstimulated cells (control, A) or cells incubated with PT-casein (C). The nucleus is in blue (DAPI), cytoskeleton in red (RITC), and zonulin in green (FITC). Magnification ×100. D: polarized zonulin secretion of intestinal cells exposed to either bacteria or PT-gliadin. Both rat (IEC6) and human (Caco2 and T84) intestinal epithelial cells exposed to either nonpathogenic bacteria or gliadin secrete large amounts of zonulin in the bath medium compared with the amount of zonulin measured in media of cells exposed to control. This secretion was detected only when the triggers were added to the luminal (apical) aspect of the cell monolayers.
Top: in situ immunofluorescence microscopy of CXCR3 in human small intestinal biopsies obtained from either celiac patients or nonceliac controls. CXCR3 staining in red (RITC) is homogeneously visible on the apical side of intestinal epithelial cells of biopsies from celiac disease patients, while the CXCR3 staining is patchy in nonceliac controls. Bottom: quantitative real-time PCR of the CXCR3 gene confirmed an increased expression of the receptor compared with nonceliac controls that decreased after treatment with a gluten-free diet. Note the increased infiltrate of CXCR3-expressing immune cells in celiac disease biopsies compared with nonceliac controls. The nucleus is in blue (DAPI) and the cytoskeleton in green (FITC). Magnification ×60.
Western blotting using zonulin cross-reacting anti-Zot polyclonal antibodies on ELISA zonulin-negative and zonulin-positive sera. Sera depleted of albumin (lanes 1 and 4) or albumin + Ig (lanes 3 and 6) from both a zonulin-negative subject (HP1–1 homozygous, lanes 1–3) and a zonulin-positive subject (HP2–2 homozygous, lanes 4–6) showed the expected single α1- and α2-subunits, respectively. Conversely, the sera Ig fractions of both subjects (lanes 2 and 5) showed no immunoreactivity. The zonulin band was also visible at the expected 47-kDa size in the HP2–2 sera (arrow).
Photomicrographs of immunohistochemistry on small intestinal tissues from a healthy control and an active CD patient stained with zonulin cross-reacting anti-Zot antibodies. Zonulin is visualized both in enterocytes and in cells of the lamina propria (arrows) and is overexpressed in active CD patients compared with controls.
Diseases associated with zonulin and chromosome 16. Diseases that have been proven, suspected, or related to zonulin whose gene is located on chromosome 16, as a biomarker include autoimmune diseases, cancers, and diseases of the nervous system. The same categories of diseases have been related to other genes located on chromosome 16.
Gluten structure. Gluten is composed by a mixture of two main proteins, gliadins and glutenins, both being toxic for celiac disease patients. Glutenin forms a meshwork of fibers in which globular gliadins are entrapped. On the left, cartoons show both class of proteins and how they interact. On the right, a scan electron micrograph shows the structural interaction between gliadins and glutenins.
Mechanisms of gliadin-induced zonulin release, increased intestinal permeability, and onset of autoimmunity. The production of specific gliadin-derived peptides by digestive enzymes causes CXCR3-mediated, MyD88-dependent zonulin release (2) and subsequent transactivation of EGFR by PAR2 leading to small intestine TJ disassembly (3). The increased intestinal permeability allows non-self antigens (including gliadin) to enter the lamina propria (4), where they are presented by HLA-DQ, -DR molecules (5). The presentation of one or more gliadin peptides leads to abrogation of oral tolerance (switch to Th1/Th17 response) and a marked increase in peripheral immune responses to gliadin. Furthermore, gliadin-loaded dendritic cells migrate from the small intestine to mesenteric and/or pancreatic lymph nodes (6) where they present gliadin-derived antigens. This presentation leads to migration of CD4−CD8− γδ and CD4−CD8+ αβ T cells to the target organ (gut and/or pancreas) where they cause inflammation (7). Implementation of a gluten-free diet or treatment with the zonulin inhibitor AT1001 (8) prevents the activation of the zonulin pathway and, therefore, of the autoimmune process targeting the gut or pancreatic β-cells.
AT1001 protects against insulitis in BB-Wor diabetes-prone rats. Histological analysis (A–D) and immunohistochemistry (E–H) of the pancreata isolated from both untreated BB-Wor diabetes-prone rats that developed T1D (A, B, E, and F) and AT1001-treated rats that did not develop T1D (C, D, G, and H). The islets indicated by the arrows in A and C (magnification ×10) are shown at higher magnification (×40) in B and D. Islets from rats that developed T1D showed the typical collapsed aspect with no insulin staining (E) and clusters of preserved glucagon-producing α-cells (F). Conversely, AT1001-treated animals showed undamaged islets producing both insulin (G) and glucagon (H).
Serum zonulin levels in subjects affected by different types of MS. Serum zonulin levels were assessed in MS subjects affected by different subtypes: relapsing-remitting (RRMS) during exacerbation of the disease (n = 30), RRMS in remission (n = 14), secondary-progressive (SPMS) (n = 18) and healthy controls (n = 171). Data are presented as means ± SE. *P = 0.04 compared with controls; **P = 0.03 compared with RRMS. These data were partially presented at the American Academy of Neurology 2004 annual meeting.
Cover: Paracrine hormones, released from the vascular endothelium in response to shear stress, impact on the subintimal space and vascular smooth muscle cell function. Artwork by Paul Ricketts. See Green, Daniel J., Maria T. E. Hopman, Jaume Padilla, M. Harold Laughlin, and Dick H. J. Thijssen. Physiol Rev 97: 495–527, 2017.