Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 39; pp. 16799 - 16804
• Main Authors: Tripathi, Amit, Lammers, Karen M, Goldblum, Simeon, Shea-Donohue, Terez, Netzel-Arnett, Sarah, Buzza, Marguerite S, Antalis, Toni M, Vogel, Stefanie N, Zhao, Aiping, Yang, Shiqi, Arrietta, Marie-Claire, Meddings, Jon B, Fasano, Alessio
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 29.09.2009; National Acad Sciences
• Subjects: Animals; Autoimmune diseases; bioactive properties; Biological Sciences; Biomarkers; Celiac disease; Cells; Certificates of deposit; Cholera Toxin - chemistry; Cholera Toxin - genetics; Cholera Toxin - metabolism; Diabetes; Digestive system diseases; epidermal growth factor receptors; Gels; Gene expression; Gene expression regulation; Haptoglobins - chemistry; Haptoglobins - genetics; Haptoglobins - metabolism; Hemoglobin; Humans; intestinal mucosa; Intestinal Mucosa - metabolism; Mice; pathogenesis; Permeability; Physiology; Protein precursors; Protein Precursors - chemistry; Protein Precursors - metabolism; Proteins; proteolysis; Proteomics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Messenger - metabolism; Rodents; Small intestine; Tight junctions; Tight Junctions - metabolism; transcriptional activation
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.0906773106

Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR₂) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR₂ or the use of PAR₂⁻/⁻ mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its α₂- and β-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immune-mediated diseases that can be targeted for therapeutic interventions.