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

Abstract

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 (PAR(2)) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR(2) or the use of PAR(2)(-/-) mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its alpha(2)- and beta-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.

Fig. 1.

WB analysis using zonulin cross-reacting anti-Zot polyclonal Ab on CD patient sera samples that were depleted of albumin and immunoglobulins. Three main patterns were detected: sera showing an 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).

Fig. 2.

Coomassie and Western immunoblotting of purified human homozygote HP1–1 and HP2–2 both untreated and after deglycosylation with PGNase. (A) Coomassie staining of untreated HPs showed a shared glycosylated β-chain migrating at a MW of ≈52 kDa, whereas the α of HP1–1 (α₁) and of HP2–2 (α₂) migrated at the predicted MWs of 9 and 18 kDa, respectively. Deglycosylation with PGNase caused a shift of the β-chain to a MW of ≈36 kDa (complete deglycosylation) or higher (incomplete deglycosylation). As expected, no shifts were observed in the nonglycosylated α₁- and α₂-chains. (B) WB of purified human homozygote HP1–1 and HP2–2 both untreated and after deglycosylation with PGNase run in triplicate on a single gel, transferred, and then separately subjected to WB analysis using polyclonal anti-Zot (Left), monoclonal anti-HP (Center), or polyclonal anti-HP (Right) Ab. The polyclonal Ab tested recognized both the α₁- and α₂-chains (lanes 1 and 2), whose pattern of reactivity did not change after deglycosylation of both HP1–1 and HP2–2 protein preparations (lanes 3 and 4). Conversely, deglycosylation caused the expected gel mobility shift of the β-chain in both HP1–1 and HP2–2 detected by either the anti-HP monoclonal (Center, lanes 3 and 4) or anti-HP polyclonal (Right, lanes 3 and 4) Ab. The zonulin cross-reacting anti-Zot Ab recognized an extra ≈45-kDa band in HP2–2 but not in HP1–1 that did not shift after deglycosylation (arrows). MS/MS analysis and N-terminal sequencing identified this ≈47-kDa band as pre-HP2.

Fig. 3.

Zonulin increased IP in C57BL/6 WT mice in a dose- and time-dependent manner. Zonulin was applied to the luminal side of C57BL/6 WT intestinal segments at increasing concentrations. Trypsin-cleaved pre-HP2 was applied at a rate of 200 μg/mL. Starting at 60 min postexposure, zonulin induced a significant drop in TEER when applied at concentrations ≥40 μg/mL (P value ranging from 0.03–0.036). Data are mean values ± SEM from 4 independent experiments.

Fig. 4.

(A) Zonulin at increasing concentrations was incubated on serum-starved Caco-2 cells. The cells were lysed, immunoprecipitated using anti-EGFR Ab, and processed for WB using anti-phospho EGFR (PY-Plus) Ab. To ensure equal loading, the blots were stripped and reprobed for EGFR. Zonulin caused a dose-dependent increase in EGFR phosphorylation that reached a plateau at 15 μg/mL. (B) Zonulin at 50 μg/mL was incubated either alone (lane 2) or in the presence of 5 μM of the EGFR-selective PTK inhibitor AG1478 (lane 3) on serum-starved Caco-2 cells. Cells exposed to media (lane 1) or AG1478 alone (lane 4) were used as additional controls. Zonulin caused an increase in EGFR phosphorylation that was completely abolished by the PTK inhibitor AG1478 (n = 3 experiments). (C) Zonulin, either alone or in the presence of 5 μM AG1478, was applied to the luminal side of C57BL/6 WT intestinal segments at a concentration of 50 μg/mL, and TEER was measured at baseline (open bars) and 90 min postincubation (closed bars). Zonulin caused a significant drop in TEER that was prevented by the presence of AG1478 (n = 4 mice for each group). t₀, time point t = 0. (D) The zonulin-induced EGFR phosphorylation was significantly reduced following treatment with 2-chain mature HP2 (50 μg/mL; lane 3) compared with single-chain zonulin (lane 2). Lane 1 shows EGFR phosphorylation in cells treated with media alone.