Tripathi, A. et al. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc. Natl Acad. Sci. USA 106, 16799-16804

Mucosal Biology Research Center, Center for Vascular and Inflammatory Diseases and Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 20201, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2009; 106(39):16799-804. DOI: 10.1073/pnas.0906773106
Source: PubMed
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.

Full-text

Available from: Alessio Fasano
Identification of human zonulin, a physiological
modulator of tight junctions, as prehaptoglobin-2
Amit Tripathi
a
, Karen M. Lammers
a
, Simeon Goldblum
a
, Terez Shea-Donohue
a
, Sarah Netzel-Arnett
b
,
Marguerite S. Buzza
b
, Toni M. Antalis
a,b
, Stefanie N. Vogel
a,c
, Aiping Zhao
a
, Shiqi Yang
a
, Marie-Claire Arrietta
c
,
Jon B. Meddings
d
, and Alessio Fasano
a,1
a
Mucosal Biology Research Center,
b
Center for Vascular and Inflammatory Diseases and Department of Physiology, and
c
Department of Microbiology
and Immunology, University of Maryland School of Medicine, Baltimore, MD 20201; and
d
Department of Medicine, University of Alberta, Edmonton,
Alberta, Canada T6G 2V2
Communicated by Maria Iandolo New, Mount Sinai School of Medicine, New York, NY, June 25, 2009 (received for review December 16, 2008)
Increased intestinal permeability (IP) has emerged recently as a
common underlying mechanism in the pathogenesis of allergic, in-
flammatory, and autoimmune diseases. The characterization of zonu-
lin, 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 haptoglo-
bin-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 transac-
tivation 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
2
- and
-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.
autoimmune diseases epidermal growth factor receptor
gut permeability proteinase-activated receptor 2 celiac disease
I
ncreased hygiene leading to a reduced exposure to various
microorgan isms has been implicated as a cause for the ‘‘epi-
demic’’ of allergic, inflammatory, and autoimmune diseases
rec orded in industrialized countries during the past 3–4 decades
(1). Apart from genetic makeup and exposure to environmental
triggers, a third key element [i.e., increased intestinal perme-
abilit y (IP)] has been proposed in the pathogenesis of these
diseases (2–4). IP, together w ith antigen sampling by enteroc ytes
and luminal dendritic cells, regulates molecular trafficking be-
t ween the intestinal lumen and the submucosa, leading to either
tolerance or immunit y to non–self-antigens (5). However, the
dimensions of the paracellular space (10–15 Å) suggest that
solutes with a molecular radius exceeding 15 Å (3.5 kDa)
(including proteins) are normally excluded from this uptake
route. The intercellular tight junctions (TJs) tightly regulate this
paracellular antigen trafficking. TJs are now appreciated to be
extremely dynamic structures operative in several key functions
of the intestinal epithelium under both physiological and patho-
logical circumst ances (3). However, despite major progress in
our knowledge regarding the composition and function of in-
tercellular TJs, the mechanism(s) by which they are regulated
is(are) still incompletely understood. The disc overy of Vibrio
cholerae zonula occludens toxin (Zot), a toxin that increases TJ
per meability, led us to the identification of its eukaryotic
c ounterpart, zonulin, as the only physiological mediator known
to regulate IP reversibly by modulating intercellular TJs (6, 7).
Human zonulin is a 47-kDa protein that increases IP in
nonhuman primate intestinal epithelia (7), participates in intes-
tinal innate immunity (8), and is overexpressed in autoimmune
disorders in which TJ dysfunction is central, including celiac
disease (CD) (9, 10) and type 1 diabetes (T1D) (11). Although
zonulin’s role as an intestinal per meating modulator in health
and disease has been described functionally, its biochemical
characterization has remained elusive. Through proteomic anal-
ysis of human sera, we report herein that zonulin is identical to
the precursor of haptoglobin-2 (pre-HP2), a molecule that, to
date, has only been regarded as the inactive precursor for HP2,
one of the two genetic variants (together w ith HP1) of human
HPs (see Fig. S1). Our studies demonstrate the previously
undescribed functional characterization of zonulin as pre-HP2,
a multifunctional protein that, in its intact single-chain precursor
for m, appears to regulate IP by transactivating the epidermal
growth factor receptor (EGFR) via proteinase-activating recep-
tor 2 (PAR
2
) activation, whereas in its cleaved 2-chain form, it
acts as an Hb scavenger.
Results
Characterization of Zonulin from CD Human Sera. Because zonulin is
detected in human sera by a zonulin cross-reacting anti-Zot
Ab-based ELISA (7–10) and is increased in patients with CD
c ompared with normal c ontrols (10), we initially used Western
blot (WB) analysis to detect zonulin immunoreactivity of pro-
teins in albumin- and IgG-depleted sera f rom CD subjects. These
sera displayed 2 major protein bands with apparent molecular
weights (MWs) of 18 and 9 kDa (Fig. 1). Three distinct patterns
of reactivity were identified in CD sera: an 18-kDa protein band
(Fig. 1, lane 1), a 9-kDa protein band (Fig. 1, lane 2), and both
9- and 18-kDa protein bands (Fig. 1, lane 3). Of note, a 45-kDa
band was detected only in sera that displayed the single 18-kDa
band (Fig. 1, lane 1) but was not detected in sera with either the
9-kDa band or both bands (Fig. 1, lanes 2 and 3). Two-
dimensional gel electrophoresis (2-DE) of sera from CD patients
who expressed the 18-kDa band revealed 2 zonulin immunore-
active spots [see supporting information (SI) Text and Fig. S1 A
and B] that were subjected to MS/MS analysis. The 18-kDa spot
was identified as the
2
-chain of HP2 (accession no. GI:223976)
and the 9-kDa spot as the
1
-chain of HP1 (accession no.
Author contributions: S.G., T.S.-D., T.M.A., S.N.V., M.-C.A., J.B.M., and A.F. designed re-
search; A.T., K.M.L., S.N.-A., M.S.B., A.Z., S.Y., M.-C.A., and A.F. performed research; S.N.-A.,
M.S.B., and A.F. contributed new reagents/analytic tools; A.T., K.M.L., S.G., T.S.-D., S.N.-A.,
M.S.B., T.M.A., S.N.V., A.Z., S.Y., M.-C.A., J.B.M., and A.F. analyzed data; and S.G., T.S.-D.,
T.M.A., S.N.V., J.B.M., and A.F. wrote the paper.
Conflict of interest statement: A.F. and S.N.V. have financial interest in Alba Therapeutics,
a company involved in the development of treatments of CD alternative to the GFD.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. E-mail: afasano@mbrc.umaryland.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0906773106/DCSupplemental.
www.pnas.orgcgidoi10.1073pnas.0906773106 PNAS
September 29, 2009
vol. 106
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MEDICAL SCIENCES
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GI:3337390). A diag ram show ing the structure of 2-chain HP1
and HP2 and their precursors is presented in Fig. S1C. A random
screen ing of 14 sera from CD patients revealed that 7% were
HP1 homozygous, 57% were HP1/HP2 heteroz ygous, and 36%
were HP2 homozygous (Fig. S1D).
Characterization of Zonulin from Human HP Preparations. To confirm
the identity of the immunoreactive bands recognized by the
polyclonal zonulin cross-reacting anti-Zot IgG Ab in human CD
sera, commercially purified preparations of human HP from
subjects homozygous for either HP1 (HP1–1) or HP2 (HP2–2)
were simultaneously resolved on a single gel by SDS/PAGE and
analyzed by Coomassie stain ing (Fig. 2A). As expected, the
1
-chain of HP1–1 exhibited a MW of 9 kDa (Fig. 2A, lane 1),
whereas the
2
-chain of HP2–2 had a MW of 18 kDa (Fig. 2A,
lane 2). Because of its glycosylation, the
-chain exhibited a MW
of 52 kDa in both HP1–1 and HP2–2 preparations (Fig. 2A,
lanes 1 and 2). After a 3-h deglycosylation reaction with N-
glyc osidase F (PGNase F), the
-chain of both HP1–1 and
HP2–2 ran as multiple bands below 52 kDa, presumably attrib-
ut able to varying degrees of deglyc osylation (Fig. 2A, lanes 3 and
4). As anticipated, after glycosidase treatment, no changes in gel
mobilit y for either the
1
-chain of HP1–1 (Fig. 2A, compare
lanes 1 and 3) or the
2
-chain of HP2–2 (Fig. 2A, compare lanes
2 and 4) were evident.
Fig. 2B presents immunoblots of commercially available pu-
rified homozygous HP1–1 and HP2–2 proteins both before and
af ter deglycosylation. Proteins were run simultaneously on a
single gel and immunoblotted with polyclonal zonulin cross-
reacting anti-Zot Ab (Fig. 2B Left), monoclonal antiglyc osylated
-chain HP (Fig. 2B Center), or polyclonal anti-HP Ab (Fig. 2B
Right). Anti-Zot Ab reacted strongly with both the HP1–1
1
-chain and the HP2–2
2
-chain (Fig. 2B Left, lanes 1 and 2,
respectively) and revealed an additional band at 45 kDa
present in the HP2–2 but not the HP1–1 preparations (Fig 2B
Left, arrows). As expected, the monoclonal anti-HP Ab, raised
against the 52-kDa HP
-glyc osylated subunit, rec ognized only
the
-chain of either HP1–1 or HP2–2 (Fig. 2B Center, lanes 1
and 2, respectively), whereas the polyclonal anti-HP Ab recog-
n ized epitopes of the
1
-,
2
-, and
-chains of both HP1–1 and
HP2–2 (Fig. 2B Right, lanes 1 and 2, respectively). Fig. 2B also
shows immunoblotted HP1–1 and HP2–2 preparations after
deglyc osylation using the same 3 Ab. The pattern of reactivity of
the 3 Ab tested for the nonglycosylated 9-kDa
1
-subun it and the
18-kDa
2
-subun it did not change after deglycosylation (Fig. 2B,
lanes 3 and 4, respectively). However, deglycosylation caused the
ex pected gel mobility shift of the
-chain in both HP1–1 and
HP2–2. The monoclonal anti-HP Ab (Fig. 2B Center, lanes 3 and
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
-chain migrating at a MW of 52 kDa, whereas the
of HP1–1 (
1
) and of HP2–2 (
2
) migrated
at the predicted MWs of 9 and 18 kDa, respectively. Deglycosylation with PGNase caused a shift of the
-chain to a MW of 36 kDa (complete deglycosylation)
or higher (incomplete deglycosylation). As expected, no shifts were observed in the nonglycosylated
1
- and
2
-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
1
- and
2
-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
-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.
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www.pnas.orgcgidoi10.1073pnas.0906773106 Tripathi et al.
Page 2
4) rec ognized only 2 inc omplete deglycosylated
-chain bands,
whereas the polyclonal anti-HP Ab also recogn ized the com-
pletely deglycosylated 36-kDa
-chain (Fig. 2B Right, lanes 3
and 4). The 45-kDa band that was present only in the HP2–2
preparation and recognized by anti-Zot Ab did not show any
change in gel mobility on deglycosylation, but it appeared less
intense (Fig. 2B Left, lane 4). MS/MS analysis and NH
2
-ter minal
sequencing of this 45-kDa protein band performed on 2 distinct
samples analyzed at dif ferent times identified this protein as the
human pre-HP2 (accession no. P00738). The combined MS/MS
analyses covered a total of 49.8% of nonoverlapping protein and
13 unique peptides spann ing the entire protein sequence. There-
fore, in addition to
1
- and
2
-chains, the anti-Zot Ab recogn ized
the uncleaved single-chain pre-HP2 but not the
-chain. These
results suggest that the anti-Zot Ab used to measure ser um
zonulin by ELISA should supposedly detect the highly abundant
HP1 and HP2 proteins as well as pre-HP2. However, the amount
of serum zonulin detected by ELISA is in the ng/mL range (11),
whereas the entire HP pool in ser um is in the mg/mL range (12).
To address this apparent discrepanc y, we repeated the WB
analysis of both human sera and purified HPs under nondena-
turing conditions using anti-Zot Ab (see SI Tex t). The WB
showed a series of bands in HP2–2 phenot ype sera (Fig. S2A)
and in commercially purified HP2–2 (Fig. S2B), although no
bands were detected in either HP1–1 phenotype sera (Fig. S2A)
or in commercial purified HP1–1 (Fig. S2B). Conversely, the
anti-HP polyclonal Ab, which did not recognize the uncleaved
pre-HP2 (Fig. 2B), detected bands in both commercially purified
HP1–1 and HP2–2 preparations. Combined, these data suggest
that under nondenaturing conditions, the anti-Zot Ab detect
only the single-chain pre-HP2 but not the 2-chain mature HPs,
further supporting the notion that the single-chain pre-HP2, but
not its cleaved 2-chain mature form, corresponds to the zonulin
molecule.
Functional Analysis of Recombinant Zonulin. The primary transla-
tion product of the mammalian HP2 mRNA transcript is a
polypeptide that dimerizes cotranslationally and is proteolyti-
cally cleaved while still in the endoplasmic reticulum by the
serine Cr1-like protease (Cr1LP) (13). Conversely, zonulin is
detect able in human serum as uncleaved pre-HP2 (Fig. 2 and
Fig. S2). To confirm the identification of zonulin as the single-
chain pre-HP2 and not the cleaved mature 2-chain HP2, we
ex pressed recombinant pre-HP2 by inserting the pre-HP2 cDNA
into an insect cell vector and expressed it using a baculovirus
ex pression system. We obtained highly purified recombinant
pre-HP2 that was recognized by the anti-Zot polyclonal Ab
similar to Fig. 2B and that mig rated at an apparent MW of 53
kDa because of the 6xHis tag attached at the C-terminus (Fig.
S3A). The single-chain pre-HP2 was then subjected to proteo-
ly tic cleavage using a series of serine proteases. Matriptase,
urok inase, thrombin, and plasma kallikrein did not cleave pre-
HP2, whereas plasmin caused complete deg radation of the
protein (Fig. S3B). In contrast, treatment with the intestinal
serine protease trypsin led to the appearance of 2 major bands
that migrated with MWs c ompatible with the
2
- and
-subun its
of zonulin (Fig. S3B). NH
2
-ter minal sequencing of these 2 bands
showed the 2 proteins to be identical to the pre-H2
2
- and
-chains cleaved at the predicted
161
Arg cleavage site. The intact
single-chain pre-HP2 and the cleaved 2-chain mature HP2
obt ained after trypsin digestion were both tested for their
biological activities in the studies below.
Ex Vivo Effect of Recombinant Zonulin on TEER in Mouse Small
Intestine Mounted in the Microsnapwell System. Recombinant pre-
HP2 (henceforth defined as zonulin) was applied to WT
C57BL/6 murine small intestine segments mounted in micros-
napwells. Recombinant single-chain zonulin added to the mu-
c osal (luminal) aspect of mouse intestinal segments decreased
transepithelial electrical resistance (TEER) (i.e., increased per-
meabilit y) when applied at concentrations 40
g/mL (Fig. 3).
In contrast, no c onsistent TEER changes were detected when the
trypsin-cleaved 2-chain HP2 was tested (Fig. 3).
In Vivo Effect of Recombinant Zonulin on Mouse Gastrointestinal
Permeability. To establish whether zonulin might alter IP in vivo,
mice were gavaged with zonulin (170
g per mouse), and
gastroduodenal permeability and small intestine permeability
were tested using specific sugar probes (sucrose and lactulose/
mann itol, respectively) as described (14). Zonulin increased both
small intestinal and gastroduodenal permeability compared with
BSA-treated c ontrols (Table 1). Gastroduodenal per meability
and small intestine permeability each returned to baseline within
48 h following ex posure to zonulin (Table 1).
To deter mine whether the 2-chain mature HP2 affected IP,
the in v ivo ex periments described previously were repeated by
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
g/mL. Starting at 60 min postexposure, zonulin
induced a significant drop in TEER when applied at concentrations 40
g/mL
(P value ranging from 0.03–0.036). Data are mean values SEM from 4
independent experiments.
Table 1. Effect of zonulin on mouse gastroduodenal (sucrose) and small intestinal (lacman)
permeability in vivo
Treatment
Challenge Recovery After 48 h
Change in
sucrose, %
Change in
lacman, %
Change in
sucrose, %
Change in
lacman, %
Zonulin 68.44 17.52* 22.91 5.4
1.33 3.59 0.94 2.19
2-Chain HP2 8.75 6.67 0.09 4.40 0.60 6.14 0.48 1.78
BSA 0.40 2.91 0.03 1.54
*Sucrose P 0.0049 compared with both BSA control and 2-chain HP2 (n 10 for each group of treatment).
Lacman P 0.0024 compared with both BSA control and 2-chain HP2.
Tripathi et al. PNAS
September 29, 2009
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16801
MEDICAL SCIENCES
Page 3
admin istering 2-chain proteolytically cleaved protein. In contrast
to the single-chain zonulin, 2-chain HP2 (170
g per mouse)
failed to alter either gastroduodenal or small intestine perme-
abilit y c ompared with BSA-treated c ontrols (Table 1). Com-
bined, these data indicate that the single-chain zonulin, but not
its 2-chain mature HP2 form generated by proteolytic cleavage,
ret ains the reversible permeating activit y previously reported for
zonulin.
Transcriptional Expression of Zonulin in Human Duodenal Tissues.
Zonulin mRNA expression and quantification in human intestinal mucosae.
Using specific primers and the cDNA of human intestinal
biopsies f rom zonulin-positive subjects, we amplified a 686-bp
f ragment, of which 144 bp belong to the
-chain and 542 bp
belong to the
-chain of both HP1 and HP2 genes. Sequencing
of this fragment c onfirmed its identity as HP, but HP1 could
not be distinguished f rom HP2 because of the common
sequence in the amplified region. To overc ome this and
specifically to quantif y the ex pression of the zonulin gene in the
human intestine, cDNA obt ained from the intestinal muc osae
of healthy individuals (n 10), CD patients with acute-phase
disease (n 7), and CD patients w ith disease in remission
follow ing a gluten-free diet (GFD) (n 3) was analyzed by
real-time PCR using primers and probes specific for the
2
-chain. Compared with healthy indiv iduals, zonulin mRNA
ex pression was increased in the intestinal muc osae of CD
subjects with active disease (3-fold increase; P 0.05). Intes-
tinal mucosae of 3 CD subjects adhering to a GFD showed only
a 1.5-fold increase in zonulin ex pression compared with c on-
trols (Fig. S4).
Recombinant Zonulin Activates EGFR and Causes TEER Changes Through
PAR
2
.
It has recently been reported that gliadin, a glyc oprotein
present in wheat and several other cereals and identified as the
env ironmental trigger responsible for the autoimmune damage
of the small intestine t ypical of CD (15), fully reproduces the
ef fects of EGF on the actin cy toskeleton (16), effects that are
very similar to those prev iously reported for zonulin (7, 10, 16).
Further more, str uctural analysis revealed that the pre-HP2
-chain includes an EGF motif that c ontains 6 spatially
c onserved c ysteine residues that for m 3 intramolecular disul-
fide bonds (Fig. S1C) necessar y for EGF-like activ ity. To
deter mine whether zonulin can activate EGFR, increasing
c oncentrations of baculov irus-derived recombinant zonulin
were added to Cac o-2 intestinal epithelial cells. The cells were
lysed, immunoprecipit ated with anti-EGFR Ab, and processed
for phosphot yrosine immunoblotting (PY-Plus). At c oncen-
trations 15
g/mL, zonulin increased t y rosine phosphoryla-
tion of EGFR (Fig. 4A and Fig. S5A ). To establish the role of
EGFR in zonulin-induced alterations in TEER further, we also
performed both the in vitro and ex vivo ex periments described
prev iously in the presence of the EGFR-selective protein
t yrosine kinase (PTK) inhibitor AG1478. Preincubation of
Cac o-2 cells for2hwithAG1478 (5
M) prevented zonulin-
induced EGFR phosphorylation on Y1068 (Fig. 4B and Fig.
S5B). Similarly, pretreatment w ith AG1478 abolished the
reduction in TEER in response to zonulin (Fig. 4C). Finally,
trypsin digestion of zonulin dramatically reduced its ability to
activate EGFR (Fig. 4D). Combined, these data suggest that
the single-chain zonulin activates EGFR and induces an
EGFR-driven decrease in TEER, whereas the cleaved 2-chain
HP2 fails both to activate EGFR and to increase IP.
Several G protein-coupled receptors, including PAR
2
(17),
transactivate EGFR (18). Because Zot and zonulin share a
similar mechanism of action (6) and the zonulin protein se-
quence cont ains a Zot-like and PAR
2
-activating peptide (AP)–
like motif in its
-chain (FCAGMS), we asked whether zonulin-
induced EGFR activation might be dependent on PAR
2
.
Ex periments in Caco-2 in which PAR
2
was silenced and exper-
iments in PAR
2
/
mice demonstrated that zonulin induced
PA R
2
-dependent transactivation of EGFR, which, in turn,
caused TEER changes (see SI Text, Figs. S6 and S7).
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
g/mL. (B) Zonulin at 50
g/mL was incubated either alone (lane 2) or in the
presence of 5
M 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
M AG1478, was applied to the
luminal side of C57BL/6 WT intestinal segments at a concentration of 50
g/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). t
0
, time point t 0. (D) The
zonulin-induced EGFR phosphorylation was significantly reduced following
treatment with 2-chain mature HP2 (50
g/mL; lane 3) compared with single-
chain zonulin (lane 2). Lane 1 shows EGFR phosphorylation in cells treated with
media alone.
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Page 4
Discussion
In the current study, we have identified zonulin as the precursor of
HP2. Mature human HPs are heterodimeric plasma glycoproteins
composed of
- and
-polypeptide chains that are covalently
associated by disulfide bonds and in which only the
-chain is
glycosylated (19). Unlike the
-chain (36 kDa), the
-chain exists
in 2 forms [i.e.,
1
(9 kDa) and
2
(18 kDa)]. The presence of
one or both of the
-chains results in the 3 phenotypes HP1–1,
HP2–1, and HP2–2. The se HP variants evolved from a mannose-
binding lectin-associated serine protease (MASP) (12, 20), with the
-chain containing a complement control protein and the
-chain
a catalytically dead chymotrypsin-like serine protease domain
(21–24). Other members of the MASP family include a series of
plasminogen-related growth factors [e.g., EGF, hepatocyte growth
factor (HGF)] involved in cell growth, proliferation, differentiation,
migration, and disruption of intercellular junctions. Despite this
multidomain structure, the only function assigned to HPs, to date,
is to bind Hb to form stable HP-Hb complexes, thereby preventing
Hb-induced oxidative tissue damage (25). No function has ever
been described for their precursor forms. HPs are unusual secretory
proteins in that their precursor proteins, instead of being cleaved in
the trans-Golgi complex, are proteolytically processed by comple-
ment Cr1LP in the endoplasmic reticulum (13). Of interest, the
endoplasmic reticulum fraction was the cellular fraction in which
the highe st zonulin concentrations were detected (9).
Because the key biological effect of zonulin is to regulate
intercellular TJ function (7, 9–11), we studied recombinant pre-
HP2 in IP assays. In a dose- and time-dependent manner, pre-HP2
reduced TEER across murine small intestinal mucosa both ex vivo
and in vivo. The observation that zonulin lost its permeating activity
after cleavage into its 2
2
- and
-subunits further supports the
notion that zonulin and mature 2-chain HP2 exert distinct biological
functions. Whether this functional divergence relates to conforma-
tional differences between the uncleaved precursor form versus the
cleaved mature protein is under study. The importance of protein
conformation in dictating HP protein function is further supported
by the finding that zonulin cross-reactive anti-Zot Ab recognized
the HP1
1
-chain under denaturing conditions (Figs. 1A and 2B)
but failed to recognize nondenatured HP1 (Fig. S2 A and B).
Combined, these data confirm the identity of zonulin as pre-HP2.
We previously reported that the NH
2
-ter minal amino-acid
sequence of zonulin has striking similarities to the light chain of
human
-globulins (7), a similarity also noted for HP (26).
Clearance of the HP-Hb complex can be mediated by the
monoc yte/macrophage scavenger receptor CD163 (25). Clustal
W dendrogram analysis showed a region in the zonulin
-chain
just upstream of the CD163 binding site with the following
-globulin–like consensus motif: QLVE—V—P. Whether dis-
crepancies between our previously reported zonulin sequence
and the pre-HP2 sequence as related to this consensus motif are
attribut able to intraspecies variability associated with a high
zonulin mutation rate or to our sequence error at that time
remains to be established.
Zonulin contains growth factor-like repeats. Like zonulin,
growth factors affect intercellular TJ integrity (27, 28). We now
show that the single-chain zonulin, but not its cleaved mature
for m, transactivates EGFR via PAR
2
and that its effect on
TEER is prevented by pharmacological inhibition of EGFR or
siRNA-induced PAR
2
silencing. This suggests that the growth
factor motif in the single-chain zonulin, but not in the mature
2-chain HP2, has the molecular conformation required to induce
TJ disassembly by indirect transactivation via PAR
2
. Whether
the EGF-like repeat in zonulin might also directly engage the
EGFR ectodomain remains to be established.
Gliadin, the environmental trigger of CD, reportedly repro-
duces the ef fects of EGF on the actin cytoskeleton (16). These
ef fects are very similar to the effects we reported for zonulin (7).
Gliadin binds to the CXCR3 chemokine receptor (29), and this
interaction is coupled to zonulin release from both intestinal
cells (9) and whole intestinal tissues (10). Hence, it is likely that
the gliadin-related EGF effects are mediated through zonulin
release. We also have identified intestinal bacterial colon ization
as a stimulus for zonulin release (8). Gliadin and microorganisms
both cause polarized luminal secretion of zonulin (8). Therefore,
we focused our studies on early zonulin action (i.e., its activit y at
intestinal luminal side). This approach may appear counterin-
tuitive, given the observation that both EGFR and PAR
2
are
ex pressed basolaterally (3, 30). However, evidence exists that
they also are apically expressed (31). The fact that we have
demonstrated that zonulin exerts a per meating effect, both in ex
vivo and in vivo, when applied to the luminal aspect of the
intestinal mucosa does not dispute the possibility that the protein
acts basolaterally as well. When environmental triggers (i.e.,
bacteria, gluten) are present in the intestinal lumen, zonulin is
released from enterocytes, a process that is mediated, at least for
gliadin, by CXCR3 (29). Following zonulin release and the
subsequent increase in IP, these triggers can reach the submu-
c osa, where zonulin-expressing immune cells can secrete zonulin
to the basolateral side. A similar bilateral action has been
reported for mucosal mast cell protease II, another serine
protease that controls IP acting f rom both luminal and serosal
sides (32).
The role of both EGFR and PAR
2
in regulating epithelial
per meability has been prev iously reported (33, 34). However,
our study provides previously undescribed evidence that the
2 receptors work cooperatively to regulate small intestine
per meability.
We have previously reported that zonulin is up-regulated
during the acute phase of CD (9, 10). Using HP-specific primers,
we now report the previously undescribed expression of zonulin
mRNA in human intestine. Furthermore, real-time PCR exper-
iments showed that zonulin expression was increased in CD
patients compared with normal controls. The enhanced expres-
sion of zonulin correlated with disease activity, because CD
patients who were on a GFD showed mean values for zonulin
ex pression that were intermediate to those of patients with active
CD and normal controls. Interestingly, Papp et al. (35) recently
reported that a polymorphism in the HP gene represents a
previously undescribed genetic risk factor for CD development
and its clinical manifest ations.
The human plasma levels of HPs are between 100 and 300 mg
per 100 mL, with HP2–2 ranging between 100 and 260 mg per 100
mL. Almost 8% of HPs are secreted in their proform (36),
suggesting that under physiological circumstances, 80–208
g/mL pre-HP2 is present in human plasma. Therefore, the
c oncentrations of zonulin used in this study are within physio-
logical range and are most likely indicative of the signaling
pathways activated when zonulin is up-regulated during patho-
logical processes. Besides CD, increased IP has been reported in
other autoimmune diseases, including T1D (11), systemic lupus
erythematosus (37), and ankylosing spondylitis (38), further
delineating the importance of the paracellular pathway in the
pathogenesis of autoimmune diseases. These findings, together
with the observation that zonulin is overexpressed during the
acute phase of several immune-mediated diseases and its block-
age prevents the onset of the autoimmune response, suggest that
zonulin contributes to the pathogenesis of these conditions,
open ing previously undescribed paradigms in the pathobiology
and treatment options of immune-mediated diseases.
Methods
Human Serum Samples. Human sera from both healthy volunteers and patients
with CD were obtained from the Center for Celiac Research serum bank. All
samples were depleted of albumin and IgG using commercially available kits
(Enchant Life Science kit; Pall Corporation and IgG ImmunoPure immobilized
Tripathi et al. PNAS
September 29, 2009
vol. 106
no. 39
16803
MEDICAL SCIENCES
Page 5
protein G plus; PIERCE, respectively). The albumin- and IgG-depleted sera were
analyzed by SDS/PAGE, 2-DE, and WB analysis.
Human HPs. HP1–1 and HP2–2 extracted from human plasma were purchased
from Sigma. HP SDS/PAGE, both monodimensional gel electrophoresis and
2-DE, WB, and MS analyses are described in detail in (SI Text). HP deglycosy-
lation was performed by addition of PNGase F according to the manufactur-
er’s instructions (Sigma).
Human Zonulin/Pre-HP2 Cloning and Expression in a Baculovirus Expression
System and Its Cleavage by Proteases. Recombinant zonulin/preHP2 protein
production using a baculovirus system and its purification are described in SI
Text. Purified single-chain zonulin was subjected to proteolytic cleavage using
the serine proteases indicated, resolved by SDS/PAGE, and then stained with
SimplyBlue SafeStain solution (Invitrogen). For generation of 2-chain HP2,
single-chain zonulin was exposed to trypsin-agarose beads (T-1763; Sigma) for
20 min at 25 °C. The beads were removed by centrifugation, and the effec-
tiveness of the removal of trypsin was confirmed by assay of trypsin peptidase
activity against the substrate Glu-Gly-Arg-pNA (Bachem BioScience).
Ex Vivo and In Vivo IP Studies. The effects of zonulin on ex vivo and in vivo IP
were determined as previously described (8, 14) and are reported in detail in
SI Text.
Zonulin Activation of EGFR. To determine whether zonulin can activate EGFR,
increasing concentrations of either zonulin or 2-chain mature HP2 were added
for increasing exposure times to serum-starved high EGFR-expressing Caco-2
cells. The cells were lysed and processed for WB analysis with anti-phospho
EGFR (Y1068) Ab (Cell Signaling Technology, Inc.) as previously reported (39).
Experiments were repeated in the presence of 5
M of the EGFR-selective PTK
inhibitor AG1478 (Calbiochem).
Knockdown of PAR2 Through RNA Interference. The methods used to silence
PAR2 are reported in detail in SI Text.
Zonulin Gene Sequencing and Quantification from Intestinal Tissue from Pa-
tients With and Without CD. Samples of small-intestine mucosae were obtained
from the second or third portion of the duodenum from subjects undergoing
a diagnostic upper gastrointestinal endoscopy. Subjects included were 10
healthy controls, 7 patients with active CD at diagnosis, and 3 patients with CD
on treatment with a GFD for at least 6 months. All patients had clinical
indications for the procedure and gave their informed consent to undergo an
additional biopsy for the purpose of this study. The study protocol was
approved by the Ethics Committee of the University of Maryland. The small-
intestine biopsies were immediately collected in RNAlater RNA Stabilization
Reagent (Qiagen) and stored at 20 °C until processed. Total RNA extraction,
cDNA synthesis, and real-time PCR are described in SI Text.
Statistical Analysis. All values are expressed as mean SE. The analysis of
differences was performed by 2-tailed Student’s t tests to test differences
between 2 groups for either paired or unpaired varieties. Multivariate analysis
was performed where appropriate. Values of P 0.05 were regarded as
significant.
ACKNOWLEDGMENTS. This manuscript was partially supported by National
Institutes of Health grant DK048373 (to A.F.).
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  • Source
    • "These cells, mainly CD4+T lymphocytes, react to long fragments (up to 30–40 amino acid residues) of gluten resistant to gastrointestinal enzymatic degradation. These gluten peptides pass through the epithelial barrier via transcellular [22] or paracellular transport [23,24], this latter favored by an increased epithelial permeability mediated by the release of zonulin, an intestinal peptide that is involved in the tight junction regulation [25]. When in the lamina propria compartment, the gluten peptides become substrate for the enzyme tissue transglutaminase type 2 (tTG2) [26]. "
    [Show abstract] [Hide abstract] ABSTRACT: Celiac disease (CD) is a common lifelong food intolerance triggered by dietary gluten affecting 1% of the general population. Gliadin-specific T-cell lines and T-cell clones obtained from intestinal biopsies have provided great support in the investigation of immuno-pathogenesis of CD. In the early 2000 a new in vivo, less invasive, approach was established aimed to evaluate the adaptive gliadin-specific T-cell response in peripheral blood of celiac patients on a gluten free diet. In fact, it has been demonstrated that three days of ingestion of wheat-containing food induces the mobilization of memory T lymphocytes reactive against gliadin from gut-associated lymphoid tissue into peripheral blood of CD patients. Such antigen-specific T-cells releasing interferon-γ can be transiently detected by using the enzyme-linked immunospot (ELISPOT) assays or by flow cytometry tetramer technology. This paper discusses the suitability of this in vivo tool to investigate the repertoire of gluten pathogenic peptides, to support CD diagnosis, and to assess the efficacy of novel therapeutic strategies. A systematic review of all potential applications of short oral gluten challenge is provided.
    Full-text · Article · Dec 2015 · Nutrients
  • Source
    • "Zonulin is a 47 kDa protein which modulates intestinal permeability by disassembling intercellular tight junctions between epithelial cells in the digestive tract (Wang et al., 2000; Fasano, 2001; Vanuytsel et al., 2013). The effect of zonulin on increased intestinal permeability is mediated through activation of EGF receptor (EGFR) via proteinaseactivated receptor 2 (PAR2) activation (Tripathi et al., 2009). Patients with type 1 diabetes, an autoimmune disease in which the finely tuned regulation of intestinal tight junctions is lost, have increased serum zonulin levels. "
    [Show abstract] [Hide abstract] ABSTRACT: Liver disease is often times associated with increased intestinal permeability. A disruption of the gut barrier allows microbial products and viable bacteria to translocate from the intestinal lumen to extraintestinal organs. The majority of the venous blood from the intestinal tract is drained into the portal circulation, which is part of the dual hepatic blood supply. The liver is therefore the first organ in the body to encounter not only absorbed nutrients, but also gut-derived bacteria and pathogen associated molecular patterns (PAMPs). Chronic exposure to increased levels of PAMPs has been linked to disease progression during early stages and to infectious complications during late stages of liver disease (cirrhosis). It is therefore important to assess and monitor gut barrier dysfunction during hepatic disease. We review methods to assess intestinal barrier disruption and discuss advantages and disadvantages. We will in particular focus on methods that we have used to measure increased intestinal permeability and bacterial translocation during experimental liver disease models. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jan 2015 · Journal of Immunological Methods
  • Source
    • "Because it has been reported that the reduction of small intestinal permeability could attenuate the colitis and protect the intestinal barrier function in the IL-10 −/− mice [16,32], MIMP may alleviate the intestinal inflammation by reducing small intestinal and colonic permeability. Zonulin is a recent discovered protein that participates in TJ between IECs in the digestive tract [19]. Zonulin was originally discovered as the target of zonula occludens toxin, which is secreted by cholera pathogen Vibrio cholerae [38]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Previous studies indicated that the micro integral membrane protein located within the media place of the integral membrane protein of Lactobacillus plantarum CGMCC 1258 had protective effects against the intestinal epithelial injury. In our study, we mean to establish micro integral membrane protein -knockout Lactobacillus plantarum (LPKM) to investigate the change of its protective effects and verify the role of micro integral membrane protein on protection of normal intestinal barrier function. Methods Binding assay and intestinal permeability were performed to verify the protective effects of micro integral membrane protein on intestinal permeability in vitro and in vivo. Molecular mechanism was also determined as the zonulin pathway. Clinical data were also collected for further verification of relationship between zonulin level and postoperative septicemia. Results LPKM got decreased inhibition of EPEC adhesion to NCM460 cells. LPKM had lower ability to alleviate the decrease of intestinal permeability induced by enteropathogenic-e.coli, and prevent enteropathogenic-e.coli -induced increase of zonulin expression. Overexpression of zonulin lowered the intestinal permeability regulated by Lactobacillus plantarum. There was a positive correlation between zonulin level and postoperative septicemia. Therefore, micro integral membrane protein could be necessary for the protective effects of Lactobacillus plantarum on intestinal barrier. Conclusion MIMP might be a positive factor for Lactobacillus plantarum to protect the intestinal epithelial cells from injury, which could be related to the zonulin pathway.
    Full-text · Article · Oct 2014 · BMC Gastroenterology
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