Note: Descriptions are shown in the official language in which they were submitted.
CA 02765075 2011-12-09
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EGFR AND PAR2 REGULATION OF INTESTINAL PERMEABILITY
Federal Funding Legend
This invention was made with government support under Grant number DK048373
awarded by the National Institute of Health. The government has certain rights
in this
invention.
Cross-Reference to Related Application
This international application claims benefit of priority under 35 U.S.C.
119(e) of
provisional U.S. Serial No. 61/185,662, filed June 10, 2009, now abandoned,
the entirety of
which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the fields of cell biology and intestinal
permeability.
More specifically, the present invention relates to EGFR and PAR2 regulation
of intestinal
permeability.
Description of the Related Art
Increased hygiene leading to a reduced exposure to various microorganisms has
been implicated as a cause for the 'epidemic' of allergic, inflammatory, and
autoimmune
diseases recorded 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 permeability (IP), has been proposed in the pathogenesis of these
diseases (2-4).
Intestinal permeability, together with antigen sampling by enterocytes and
luminal
dendritic cells, regulates molecular trafficking between the intestinal lumen
and the
submucosa, leading to either tolerance or immunity to non-self antigens (5).
However, the
dimensions of the paracellular space (10 to 15 A) suggest that solutes with a
molecular
radius exceeding 15 A (-3.5 kDa) (including proteins) are normally excluded
from this
uptake route. The intercellular tight junctions (TJs) tightly regulate this
paracellular antigen
trafficking.
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Tight junctions are dynamic structures operative in several key functions of
the
intestinal epithelium under both physiological and pathological circumstances
(3). However,
despite major progress in the knowledge on the composition and function of
intercellular
tight junctions, the mechanism(s) by which they are regulated is(are) still
incompletely
understood.
The discovery of Vibrio cholerae zonula occludens toxin (Zot), a toxin that
increases
tight junction permeability, led to the identification of its eukaryotic
counterpart, zonulin, as
the only physiological mediator known to reversibly regulate intestinal
permeability by
modulating intercellular tight junctions (6, 7). Human zonulin is a -47 kDa
protein that
increases intestinal permeability in non-human primate intestinal epithelia
(7), participates in
intestinal innate immunity (8), and is overexpressed in autoimmune disorders
in which tight
junction dysfunction is central, including celiac disease (CD) (9, 10) and
type 1 diabetes
(T1 D) (11).
Haptoglobin (Hp) is an acute-phase response protein, synthesised mainly in the
liver
as well as arterial walls, endometrium and peritoneum. The core function of
haptoglobin is
as a haemoglobin (Hb) binding protein, required for terminal processing and
disposal of free
haemoglobin, mostly in the reticular endothelial system of the liver. This
system allows the
iron present in the Hb moiety to be conserved.
Haptoglobin has a tetrameric structure comprising two alpha. and two beta.
chains,
linked by disulphide linkages. The beta. chain (245 amino acids) has a mass of
about 40
kDa (of which approximately 30% w/w is carbohydrate) and is shared by all
phenotypes. The
.alpha. chain exists in two forms:.alpha.1, (83 amino acids, 9 kDa) and
.alpha.2 (142 amino
acids, 17.3 kDa) and therefore haptoglobin occurs as three phenotypes,
referred to as Hpl-
1, Hp2-1 and Hp2-2. Hpl-1 contains two .alpha.1 chains, Hp2-2 contains two
.alpha.2
chains, and Hp2-1 contains one .alpha.1 and one .alpha.2 chain. Hp 1-1 has a
molecular
mass of 100 kDa, or 165 kDa when complexed with Hb. Hpl-1 exists as a single
isoform,
and is also referred to as Hp dimer. Hp2-1 has an average molecular mass of
220 kDa and
forms linear polymers. Hp2-2 has an average molecular mass of 400 kDa and
forms cyclic
polymers. Each different polymeric form is a different isoform.
Haptoglobin is a potential treatment for renal disorders caused by haemolysis.
It is
potentially useful therapeutically as a means of removing free haemoglobin;
the complexes
thus formed having potential additional benefits as anti-inflammatory,
antioxidant or
angiogenic agents. However, haptoglobin is considered difficult to isolate in
large amounts
whilst retaining its biological activity.
There is a recognized need in the art for a functional characterization of pre-
haptoglobin 2 as well as methods of regulating intestinal permeability. The
present invention
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fulfills this long standing need in the art.
SUMMARY OF THE INVENTION
While zonulin's role as an intestinal permeating modulator in health and
disease has
been described functionally, its biochemical characterization has remained
elusive. The
present invention shows that through proteomic analysis of human sera, zonulin
is identical
to pre-haptoglobin (HP)2, a molecule that, to date, has only been regarded as
the inactive
precursor for HP2, one of the two genetic variants (together with HP1) of
human pre-
haptoglobins. The present invention demonstrates the functional
characterization of zonulin
as pre-haptoglobin 2, a multifunctional protein that, in its intact single
chain precursor form,
appears to regulate intestinal permeability by transactivating the EGFR via
PAR2 activation,
while in its cleaved two-chain form acts as a Hb scavenger.
Thus, in one embodiment of the present invention, there is provided a method
of
treating an autoimmune disease, comprising the steps of increasing
transepithelial electrical
resistance leading to decreased cell permeability.
In another embodiment of the present invention, there is provided a method of
treating an autoimmune disease in an individual in need of such treatment,
comprising the
steps of inhibiting epidermal growth factor receptor; and inhibiting PAR2.
In yet another embodiment of the present invention, there is provided a method
of
treating celiac disease in an individual in need of such treatment, comprising
the steps of:
administering an antibody directed against single chain zonulin thereby
inhibiting epidermal
growth factor receptor and inhibiting PARZ.
Other and further aspects, features and advantages of the present invention
will be
apparent from the following description of the presently preferred embodiments
of the
invention given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features, advantages and
objects of the invention, as well as others which will become clear, are
attained and can be
understood in detail, more particular descriptions and certain embodiments of
the invention
briefly summarized above are illustrated in the appended drawings. These
drawings form a
part of the specification. It is to be noted, however, that the appended
drawings illustrate
preferred embodiments of the invention and therefore are not to be considered
limiting in
their scope.
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Figure 1 shows Western blotting using zonulin cross-reacting anti-Zot
polyclonal Ab
on CD patient sera that were depleted of albumin and immunoglobulins. 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 kDa and 9
kDa bands (lane 3).
Figures 2A-2B shows Coomassie and Western immunoblotting (WB) of purified
human homozygote HP1-1 and HP2-2 both untreated and after deglycosylation with
PGNase. Figure 2A: Coomassie staining of untreated HPs showed a shared
glycosylated b
chain migrating at a MW -52 kDa, while the a of HP1-1 (al) and of HP2-2 (a2)
migrated at
the predicted MW of 8 and 18 kDa, respectively. Deglycosylation with PGNase
caused a
shift of the b chain to a MW of -36 kDa (complete deglycosylation) or higher
(incomplete
deglycosylation). As expected, no shifts were observed in the non-glycosylated
al and a2
chains. Figure 2B: 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 panel),
monoclonal anti-
HP (center panel), or polyclonal anti-HP Ab (right panel). The three Ab tested
recognized
both the al and a2 chains (all panels, 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 R
chain in both
HP1-1 and HP2-2 detected by either the anti-HP monoclonal (center panel, lanes
3 and 4) or
anti-HP polyclonal Ab (right panel, lanes 3 and 4). 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.
Figure 3 shows that zonulin increased intestinal permeability in C57BU6 WT
mice in
a dose-and time-dependent manner. Zonulin was applied to the luminal side of
C57BU6
WT intestinal segments at 5, 10, 25 and 50 pg/well. Trypsin-cleaved pre-HP2
was applied at
50 pg/well. Starting at 60 min post-exposure, zonulin induced significant drop
in TEER when
applied at concentrations ? 10 g/well (P value ranging from 0.03 to 0.036).
Data are mean
values SEM from 4 independent experiments.
Figures 4A-4D show the effect of zonulin on mouse gastrointestinal
permeability in
vivo. Zonulin (closed bars) (170 mg/mouse) increases both mouse small
intestinal (Figure
4A) and gastroduodenal (Figure 4B) permeability as compared to BSA-treated
controls
(open bars). The differences in lacman ratio (small IP) 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
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HP2 (dotted bars) (170 mg/mouse) caused no changes in either small intestinal
or
gastroduodenal permeability. The effect of zonulin was completely reversible,
since both
small intestinal (Figure 4C) and gastroduodenal (Figure 4D) permeability
returned to pre-
challenge values within 48 h. The differences in Iacman or sucrose fractional
excretion are
shown as percentage of permeability change between the value of 2 d after the
challenge
and the challenge day. *Lacman P<0.0024 compared to both BSA control and 2-
chain HP2;
*Sucrose P<0.0049 compared to both BSA control and 2-chain HP2 (n = 10 for
each group
of treatment).
Figures 5A-5D show the effect of zonulin on EGFR phosphorylation. Figure 5A:
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 re-
probed for EGFR. Zonulin caused a dose-dependent increase in EGFR
phosphorylation that
reached a plateau at 3 ml/ml. Figure 5B: Zonulin at 10 ml/well 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). Figure
5C: Zonulin
10 mg/ml, either alone or in the presence of 5 M of AG1478, was applied to
the lumina)
side of C57BL/6 WT intestinal segments at a concentration of 10 g/well and
TEER
measured at baseline (open bars) and 90 min post-incubation (closed bars).
Zonulin caused
a significant drop in TEER that was prevented by the presence of AG1478 (n = 4
mice for
each group). Figure 5D: The zonulin-induced EGFR phosphorylation was
significantly
reduced following treatment with two-chain mature HP2 (10 ml/ml) (lane 3)
compared with
single chain zonulin (lane 2). Lane 1 shows EGFR phosphorylation in cells
treated with
media alone.
Figures 6A-6B illustrate the effects of zonulin on EGFR phosphorylation and
IP.
Figure 6A: Zonulin-induced EGFR phosphorylation was decreased when PAR2 was
silenced. PAR2 expression was silenced in Caco-2 using two different PAR2
siRNAs. Cells
were then treated with zonulin (10 mg/ml) or media control, lysed,
immunoprecipitated using
anti-EGFR Ab, and processed for WB with anti-phospho-EGFR PY-plus Ab. Zonulin-
mediated EGFR phosphorylation was prevented by PAR2 silencing. Equivalent
protein
loading and transfer was confirmed by stripping and reprobing the blots for
EGFR. Figure
6B: Zonulin did not increase intestinal permeability in PAR2" mice. Segments
of small
intestine from both C57BL/6 WT and PAR2 _/" mice were mounted onto the
microsnapwell
system, exposed for 30 min to medium alone or to the medium containing 10 mg
the purified
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recombinant zonulin, and transepithelial electrical resistance (TEER)
monitored at time 0
(open bars) and after 90 min incubation (closed bars). The zonulin-induced
drop in TEER
observed in wild-type mice was ablated in PAR2 1" mice (n = 5).
DETAILED DESCRIPTION OF THE INVENTION
The following abbreviations may be used herein. Ab: Antibodies; EGFR:
Epidermal
Growth Factor Receptor; HP: Haptoglobin; IP: Intestinal Permeability; PAR:
Proteinase
Activating Receptor; TJ: Tight Junctions; WB: Western Blot; CD: celiac
disease.
As used herein, the term "a" or "an", when used in conjunction with the term
"comprising" in the claims and/or the specification, may refer to "one", but
it is also
consistent with the meaning of "one or more", "at least one", and "one or more
than one".
Some embodiments of the invention may consist of or consist essentially of one
or more
elements, method steps, and/or methods of the invention. It is contemplated
that any
device, compound, composition, or method described herein can be implemented
with
respect to any other device, compound, composition, or method described
herein.
As used herein, the term "or" in the claims refers to "and/or" unless
explicitly
indicated to refer to alternatives only or the alternatives are mutually
exclusive, although the
disclosure supports a definition that refers to only alternatives and
"and/or".
As used herein, the term "contacting" refers to any suitable method of
bringing one
or more of the compounds described herein with or without one or more other
therapeutic
agents into contact with one or more cells. For in vivo applications, any
known method of
administration is suitable as described herein.
As used herein, the terms "effective amount", "pharmacologically effective
amount"
or "therapeutically effective amount" are interchangeable and refer to an
amount that results
in an effect against cells in vitro or an improvement. Those of skill in the
art understand that
the effective amount may improve the patient's or subject's condition, but may
not be a
complete cure.
As used herein, the term "subject" refers to any target of the treatment.
Thus, the present invention is directed to a method of treating an autoimmune
disease, comprising the steps of increasing transepithelial electrical
resistance leading to
decreased cell permeability. This method would be applicable to any autoimmune
disease
in which decreased cell permeability is desired. Representative cells include
but are not
limited to small intestinal cells or gastroduodenal cells. In one aspect, such
cell would have
a decreased expression of zonulin mRNA. In a related aspect, this method
further comprises
the step of inhibiting epidermal growth factor receptor. A person having
ordinary skill in this
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art would readily recognize known techiques to inhibit epidermal growth factor
receptor to
use in this method. A preferred embodiment is in which epidermal growth factor
receptor is
inhibited by administering an antibody directed against single chain zonulin.
In another
related aspect, this method further comprises the step of inhibiting PAR2. A
person having
ordinary skill in this art would readily recognize known techiques to inhibit
PAR2.
In preferred embodiments of this method of the present invetnion, PAR2 is
inhibited
using an antibody directed against single chain zonulin or using an siRNA. In
another related
aspect, this method further comprises the step of avoiding zonulin release by
gliadin through
CXCR3 receptor binding.. Representative autoimmune diseases which may be
treating
using this method of the present invention include but are not limited to T1
D, systemic lupus
erythematosus, celiac disease, ankylosing spondylitis, multiple sclerosis,
rheumatoid
arthritis, Crohn's disease, and schizophrenia.
The present invention is further directed to a method of treating an
autoimmune
disease in an individual in need of such treatment, comprising the steps of
inhibiting
epidermal growth factor receptor; and inhibiting PAR2. Using this method,
transepithelial
electrical resistance is increased leading to decreased cell permeability.
Cell permeability
may be decreased in any cell including but not limited to small intestinal
cells or
gastroduodenal cells. Typically, such cell will exhibit decreased expression
of zonulin
mRNA. Epidermal growth factor receptor and PAR2 may be inhibited as described
above.
In an additional aspect, this method further comprising the step of inhibiting
gliadin using any
technique known to those of ordinary skill in this art, including anti-gliadin
antibodies.
Representative diseases which may be treated using this method of the present
invention
include but are not limited to autoimmune disease such as T1 D, systemic lupus
erythematosus, celiac disease, ankylosing spondylitis, multiple sclerosis,
rheumatoid
arthritis, Crohn's disease, and schizophrenia.
The present invention is further directed to a method of treating celiac
disease in an
individual in need of such treatment, comprising the steps of: administering
an antibody
directed against single chain zonulin thereby inhibiting epidermal growth
factor receptor and
inhibiting PAR2. Using this method of the present invention, transepithelial
electrical
resistance is increased leading to decreased cell permeability. Representative
cells include
small intestinal cells or gastroduodenal cells but this method could be useful
in many cell
types. In a related aspect of this method, PAR2 is further inhibited using an
siRNA. In
addition, this method may further comprise the step of inhibiting gliadin.
The following example(s) are given for the purpose of illustrating various
embodiments of the invention and are not meant to limit the present invention
in any
fashion.
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Increased intestinal permeability (IP) has emerged as a common, underlying
mechanism in the pathogenesis of allergic, inflammatory, and autoimmune
diseases. The
characterization of zonulin, the only physiological mediator known to
reversibly regulate
intestinal permeability has remained elusive. Through proteomic analysis of
human sera, the
present invention identified human zonulin as the precursor for haptoglobin-2
(pre-HP2).
While mature HP is known to scavenge free hemoglobin to inhibit its oxidative
activity, no
function has ever been ascribed to its uncleaved precursor form. The present
invention
demonstrates that the single chain zonulin contains an EGF-like motif that
leads to
transactivation of EGF receptor (EGFR) via Proteinase Activated Receptor
(PAR)2
activation. Activation of these two receptors was coupled to increased
intestinal permeability.
siRNA-induced silencing of PAR2 or the use of PAR2 _1_ mice prevented loss of
barrier
integrity. Proteolytic cleavage of zonulin into its a2 and b subunits
neutralized its ability to
both activate EGFR and increase intestinal permeability. Quantitative gene
expression
revealed that zonulin is overexpressed in the intestinal mucosa of subjects
with celiac
disease. This is the first example of a molecule that in its precursor form
exerts a biological
activity that is distinct from the function of its mature form. These results,
therefore,
characterize zonulin as a novel ligand that engages a key signalosome involved
in the
pathogenesis of human immune-mediated diseases that can be targeted for
therapeutic
interventions.
EXAMPLE 1
Human serum samples
Human sera from both healthy and CD volunteers were obtained from the Center
for
Celiac Research serum bank. All samples were depleted of albumin and IgG using
commercially available kits (EnchantTM Life Science kit; Pall Corporation, Ann
Arbor, MI,
USA) and IgG ImmunoPure immobilized protein G plus (PIERCE, Rockford, IL,
USA),
respectively). The albumin- and IgG-depleted sera were analyzed by SDS-PAGE, 2-
D
electrophoresis, and WB analysis.
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EXAMPLE 2
Human Haptoglobins
HP1-1 and HP2-2 extracted from human plasma were purchased from Sigma (St.
Louis, MO, USA). HP SDS-PAGE, both mono- and two-dimensional gel
electrophoresis WB,
and mass-spectrometry analyses were performed. HP deglycosylation was
performed by
addition of N-glycosidase F (PNGase F) according to the manufacturer's
instructions (Sigma,
St Louis, MO, USA).
EXAMPLE 3
SDS/PAGE and WB Analysis
Albumin- and IgG-depleted sera (50 mg per well), human HP1-1 (1 mg per well),
and
human HP2-2 (1 mg per well) were resolved by SDS/PAGE under both denaturing
and
nondenaturing conditions on 18% or 12% SDS/PAGE Tris-Glycine gels
(Invitrogen),
respectively. The denaturing condition required addition of 30 mL of Laemmli
buffer to the
samples, followed by a 5-min boiling step before SIDS/PAGE. Proteins were
either stained
with SimplyBlue SafeStain solution (Invitrogen) or transferred onto a PVDF
membrane
(Millipore) and probed with either 5 mg/mL affinitypurified rabbit polyclonal
anti-Zot IgG Ab,
which were previously shown to cross-react with purified human zonulin (1)
using the
ImmunoPure IgG (Protein A) Purification Kit (PIERCE), or with 2 mg/mL mouse
monoclonal
anti-human HP (Sigma) or 1 mg/mL rabbit polyclonal anti-human HP (Sigma) as
the primary
Ab. HRP-labeled polyclonal anti-rabbit IgG (1:5,000; Amersham) or anti-mouse
IgG
(1:10,000; Sigma) was used as a secondary Ab. Bands were detected with ECL
Plus
reagents (Amersham).
EXAMPLE 4
2-DE Analysis and 2-DE WB
2-DE was performed using the ZOOM IPGRunner System (Invitrogen). Briefly,
albumin and IgG depleted sera were added to the commercial sample rehydration
buffer
containing urea, detergent, reducing agent, ampholyte solution, and a dye
(ReadyPrep
Rehydration/Sample buffer; Bio-Rad) in a ratio of 1:2 to rehydrate the ZOOM
STRIP pH
5.3-6.3 (Invitrogen) for 1 h at room temperature (RT). The strips were then
loaded in the
ZOOM IPGRunner Cassette (Invitrogen) to perform the isoelectric focusing
(IEF). To
fractionate samples, an IEF step voltage protocol of 200 V for 20 min, 450 V
for 15 min, 750
V for 15 min, and 2,000 V for 105 min was used. After IEF, before the 2-DE
SDS/PAGE,
strips were equilibrated for 15 min in NuPAGE LDS Sample buffer (Invitrogen)
containing
NuPAGE Sample Reducing Agent and alkylated for 15 min in NuPAGE LDS Sample
buffer
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containing freshly added iodoacetamide (125 mM; BioRad). 2-DE SDS/PAGE was run
using
NuNovex 4-20% Tris-GlycineZOOMGels (1.0 mm) in an immobilized pH gradient well
(Invitrogen). Protein bands were visualized by SimplyBlue SafeStain solution
(Invitrogen).
Protein bands were transferred onto PVDF membrane (Millipore) and probed using
affinity-
purified [Immuno-Pure IgG (Protein A) Purification Kit; PIERCE] rabbit
polyclonal zonulin
cross-reacting anti-Zot IgG (5 mg/mL) as the primary Ab and anti-rabbit IgG
(ECL Rabbit
IgG, HRP-Linked; Amersham Biosciences) as the secondary Ab. Films were
developed after
exposure of the PVDF membrane with ECL detection reagent (Amersham
Biosciences).
EXAMPLE 5
MS Analysis
In-gel tryptic digest for protein band identification was performed on gel
bands
prestained with SimplyBlue excised from the SDS/PAGE or 2-DE and analyzed by
MS/MS to
identify the protein using the protein sequencing/mass mapping facility at the
Stanford
Protein and Nucleic Acid Biotechnology Facility (Beckman Center, Stanford,
CA).
EXAMPLE 6
Expression of the Zonulin/Pre-HP2 in Insect Cells
Human full-length cDNA clone encoding for the HP2 was purchased from OriGene
(TC116954; accession no. NM_005143; OriGene Technologies, Inc.). Recombinant
baculoviruses containing WT human zonulin cDNA, with a 6xHis tag at the C-
terminus, were
constructed using pDEST8 and the Bac-to-Bac baculovirus expression system
(Invitrogen)
according to the manufacturer's protocol. Zonulin was then transferred from
the pENTR/D-
TOPO vector into the pDEST8 through recombination using Gateway technology
(Invitrogen). MAX Efficiency DH1OBac cells carrying bacmid DNA were
transformed with
pDEST8-zonulin. Recombinant bacmid was isolated from DH1OBac cells and
transfected
into Spodoptera frugiperda (Sf9) cells using Cellfectin reagent (Invitrogen)
to generate
recombinant baculoviruses. Sf9 cells were used for expression of zonulin
protein. For protein
expression of zonulin, Sf9 cells (3 x 107) were grown in suspension flasks in
SFM-900 III
medium (Invitrogen) at 27 C. Cells were infected by recombinant baculoviruses
at a
multiplicity of infection of 3. At 72 h after infection, Sf9 cells were
collected by centrifugation
for 10 min at 2,000 x g. For purification of the zonulin, phosphate buffer (pH
7.5) and NaCl
were added to the conditioned medium to final concentrations of 20mMand 0.5 M,
respectively (2). The solution was applied to a chelatingsepharose (His-bind
resin; Novagen)
column charged with Ni2' and then eluted with 200 mM imidazole and dialyzed
into PBS.
The purified human zonulin was aliquoted and stored at -80 C until use.
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EXAMPLE 7
Ex Vivo IP Studies Ex Vivo IP Studies by the Microsnapwell System
The effect of zonulin/pre-HP2 on ex vivo intestinal permeability was monitored
in the
microsnapwell system as described (3). Briefly, segments of small intestine
from C57BL/6
WT mice were mounted onto the microsnapwell system, and their luminal side was
exposed
for 30 min to medium alone or to the medium containing increasing
concentrations of the
purified recombinant zonulin. TEER was measured at time 0 and at 30-min time
intervals for
a period of 2 h using a planar electrode (Endohm SNAP electrode attached to an
Evom-G
WPI analyzer; World Precision Instruments) and expressed in Q/cm2 after
normalization. All
the TEER microsnapwell experiments were performed on mouse small intestine
with a
baseline TEER value of 77.9 3.5 Q/cm2 (n = 23). In selected experiments, the
effect of
zonulin on TEER was monitored both under basal conditions and after
pretreatment with the
EGFR tyrosine kinase inhibitor AG1478. In another set of experiments, zonulin
was tested
both in C57BU6 WT and PAR2-/- mice.
EXAMPLE 8
In Vivo IP
129/SvEv WT mice were randomized into 3 groups of 30 mice. They were
acclimatized to the experimental techniques for 3 wk, by fasting the animals
for 3 h,
gavaging the animals with a sugar probe, and placing them in metabolic cages
twice each
wk. On the day of protein challenge, the animals received either 170 mg of the
purified
single-chain zonulin in a 60-mL solution or a similar amount of purified 2-
chain cleaved HP2,
together with the sugar gavage as described (4). Mice were placed in metabolic
cages and
offered drinking water ad libitum for the following 22 h; during this time,
their urine was
collected, and the mice were then returned to conventional cages. Two days
after the drug
challenge day, mice were again placed in metabolic cages to measure their
recovery from
the treatment.
EXAMPLE 9
Knockdown of PAR2 Through RNA Interference
PAR2 expression in Caco-2 cells was silenced using 2 different PAR2 siRNAs
[HSS103471 and HSS103473 (50 nM each); Invitrogen]. The cells were transfected
following the manufacturer's instructions with the PAR2 siRNAs using
DharmaFECT1
transfection reagent (Dharmacon) in a 10-cm plate in the presence of 5% FCS
for 24 h.
PAR2 knockdown efficiency was confirmed by bothWB and real-time PCR analysis.
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EXAMPLE 10
Total RNA Extraction from Intestinal Biopsies
Total RNA was extracted using the TRizol RNA purification protocol. Briefly,
each
intestinal tissue specimen was homogenized in 1 mL of TRizol Reagent
(Invitrogen) using
the Polytron power homogenizer PT 3100 (KINEMATICA AG). RNA was extracted by
adding
0.2 mL of chloroform. After shaking the tube vigorously by hand for 15 sec,
samples were
incubated at RT for 5 min and centrifuged at 15,000 x g for 15 min at 4 C
(Marathon 21000R
centrifuge; Fisher Scientific). After transferring the RNA-rich aqueous phase
to another tube,
RNA was precipitated by adding 0.5 mL of isopropyl alcohol per 1 mL of TRizol
Reagent
used for the initial homogenization. Samples were incubated at RT for 10 min
and
centrifuged at 15,000 x g for 10 min at 4 C. After removing the supernatant,
the RNA pellet
was washed once with 75% ice-cold ethanol, adding at least 1 mL of 75% ethanol
per 1 mL
of TRizol Reagent used for the initial homogenization. The pellet was air-
dried for no more
than 2 min, dissolved in 20 mL of RNase-free water, and stored at -80 C. The
RNA
concentration was read at 260 nm by spectrophotometer (DU530, UV/vis; Beckman
Coulter). The 260:280 ratio was determined for each sample.
EXAMPLE 11
cDNA Synthesis
Two micrograms of total RNA was reversetranscribed with the High-Capacity cDNA
Archive Kit according to manufacturer's instructions (Applied Biosystems).
EXAMPLE 12
PCR Amplification of HP in Human Intestinal Biopsies
Aliquots of the cDNA were utilized for PCR of fragments specific to HP2 using
the
following primer pairs, which were specifically designed to cover different
exons: forward
primer (exon 5) 5'-ATGGCTATGTGGAGCACTCG-3' (SEQ ID NO: 1) and reverse primer
(exon 7) 5'-TACAGGGCTCTTCGGTGTCT-3' (SEQ ID NO: 2). PCR was performed with 0.1
mg of cDNA, 2.5 units of TaqDNA polymerise (Promega), 0.2 mM dNTP mix, 0.5 mM
each
primer, 5 mM MgCl2, and 1:10 volume of 10ml PCR standard buffer (Promega). The
PCR
was run in the thermal cycler (Thermo Electro Corporation). After an initial 1
min of
denaturation at 94 C, 30 cycles comprising 30 sec at 94 C (denaturation), 30
sec at 58 C
(annealing), and 30 sec at 72 C (extension) were completed, followed by a 10-
min final
extension at 72 C. The PCR products were then separated on a 2%agarose gel,
stained
with ethidium bromide, excised out of the gel, purified using a gel band
purification kit
(Amersham Biosciences), and sequenced by a 3730x1 DNA Analyzer (Applied
Biosystems).
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EXAMPLE 13
Real-Time PCR with the TagMan Procedure
Real-time PCR was performed on the cDNA from only HP2-2 or HP2-1 phenotype
subjects and was performed with HP2-specific gene primers and probes (product
ID:
Hs00978377_ml) and housekeeping 18S (product ID: Hs99999901_S1 (Applied
Biosystems). The reaction was performed with TaqMan Universal PCR Master Mix
(Applied
Biosystems, manufactured by Roche) and run on the 7500 Fast Real-Time PCR
System
(Applied Biosystems). All reactions were performed in duplicate. Relative gene
expression
was calculated using the comparative Ct method with 18S as a housekeeping
gene. The fold
change in zonulin mRNA expression in active CD patients and CD patients on a
GFD diet
relative to zonulin mRNA expression in non-CD controls after normalization to
18S mRNA
was recorded.
EXAMPLE 14
Human zonulin-preHP2 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 above. Purified single chain zonulin was subjected
to proteolytic
cleavage using the serine proteases indicated, resolved by SDS-PAGE, and then
stained
with SimplyBlueT"' SafeStain solution (Invitrogen, Carlsbad, CA, USA). For
generation of two
chain HP2, single chain zonulin was exposed to trypsin-agarose beads (Sigma T-
1763) for
20 min at 25 C. The beads were removed by centrifugation, and the
effectiveness of the
removal of trypsin confirmed by assay of trypsin peptidase activity against
the substrate Glu-
Gly-Arg-pNA (Bachem Bioscience).
EXAMPLE 15
Ex vivo and in vivo IP studies
The effect of zonulin on ex vivo and in vivo intestinal permeability was
performed as
described (8, 14) and reported above. To determine whether zonulin can
activate EGFR,
increasing concentrations of either zonulin or two-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 with anti-phospho EGFR (Y1068) Ab (Cell
Signaling
Technol. Inc.) as reported (40). Experiments were repeated in the presence of
5 M of the
EGFR-selective PTK inhibitor AG1478 (Calbiochem, Gibbstown, NJ, USA).
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EXAMPLE 16
Zonulin gene sequencing and quantification from intestinal tissue from celiac
disease (CD)
and non-CD patients
Samples of small-intestine mucosae were obtained from the second/third portion
of
the duodenum from subjects undergoing a diagnostic upper gastrointestinal (GI)
endoscopy.
Subjects included were 10 healthy controls, 7 patients with active CD at
diagnosis, 3
patients with CD on treatment with a gluten-free diet 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 RNA/ater RNA Stabilization Reagent (Qiagen, Valencia,
CA, USA)
and stored at -20 C until processed. Total RNA extraction, cDNA synthesis, and
real time
PCR are described above.
All values are expressed as mean SE (standard error). The analysis of
differences
was performed by two-tailed Student's t tests to test differences between two
groups for
either paired or unpaired varieties. Multi-variate analysis was performed
where appropriate.
Values of P <_/ 0.05 were regarded as significant.
EXAMPLE 17
Characterization Of Zonulin From CD Human Sera
Since zonulin is detected in human sera by a zonulin cross-reacting anti-Zot
Ab (Ab)-
based ELISA (7-10) and is increased in patients with CD compared to normal
controls (10),
Western analysis was initially used to detect zonulin immunoreactivity of
proteins in albumin-
and IgG-depleted sera from CD subjects. These sera displayed two major protein
bands with
apparent molecular weights of 18 and 9 kDa (Fig. 1). Three distinct patterns
of reactivity
were identified in CD sera: a 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, -45
kDa band was
detected only in sera that displayed the single 18 kDa band (Fig. 1, lane 1),
but not detected
in sera with either the 9 kDa band or both bands (Fig. 1, lane 2 and 3). Two-
dimensional gel
electrophoresis (2-DE) of sera from CD patients who expressed the 18 kDa band
revealed
two zonulin immunoreactive spots that were subjected to MS/MS mass
spectrometry
analysis. The 18 kDa spot was identified as the a2 chain of HP2 (accession no.
GI:223976)
and the 9 kDa spot as the al chain of HP1 (accession no. GI:3337390). A random
screening
of 14 sera from CD patients revealed that 7% were HP1 homozygous, 57% HP1/HP2
heterozygous, and 36% HP2 homozygous.
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EXAMPLE 18
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 homozygote for either HP1 (HP1-1) or HP2 (HP2-2)
were
simultaneously resolved on a single gel by SDS-PAGE and analyzed by Coomassie
staining
(Fig. 2A). As expected, the al-chain of HP1-1 exhibited a MW of -9 kDa (Fig.
2A, lane 1),
while the a2-chain of HP2-2 was -18 kDa (Fig. 2A, lane 2). Due to its
glycosylation, the b
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-glycosidase F (PGNase F),
the b chain of
both HP1-1 and HP2-2 ran as multiple bands below 52 kDa presumably due to
varying
degrees of deglycosylation (Fig. 2A, lanes 3 and 4). As anticipated, after
glycosidase
treatment, no changes in gel mobility for either the al-chain of HP1-1 (Fig.
24, compare
lanes 1 and 3) or the a2 chain of HP2-2 (Fig. 2A, compare lanes 2 and 4) were
evident.
Figure 28 presents immunoblots of commercially available purified homozygous
HP1-1 and HP2-2 proteins both before and after deglycosylation. Proteins were
run
simultaneously on a single gel and immunoblotted with polyclonal zonulin-cross
reacting
anti-Zot Ab (Fig. 2B, left panel), monoclonal anti-glycosylated b chain HP
(Fig. 2B, center
panel), or polyclonal anti-HP Ab (Fig. 2B, right panel). Anti-Zot Ab reacted
strongly with both
the HP1-1 al chain and the HP2-2 a2 chain and revealed an additional band at -
45 kDa
present in the HP2-2, but not in the HP1-1 preparations (Fig. 2B, left panel,
lanes 2 and 1,
respectively). As expected, the monoclonal anti-HP antibody, raised against
the -52 kDa HP
b glycosylated subunit, recognized only the b chain of either HP1-1 or HP2-2
(Fig. 28, center
panel, lanes 1 and 2, respectively), while the polyclonal anti-HP Ab
recognized epitopes of
the al, a2 andb chains of both HP1-1 and HP2-2 (Fig. 23, right panel, lanes 1
and 2,
respectively). Figure 2B also shows immunoblotted HP1-1 and HP2-2 preparations
after
deglycosylation using the same three Ab. The pattern of reactivity of the
three Ab tested for
the non-glycosylated 9 kDa al and the 18 kDa a2 subunits did not change after
deglycosylation (Fig. 2B, all 3 panels, lanes 3 and 4, respectively). However,
deglycosylation
caused the expected gel mobility shift of the 3 chain in both HP1-1 and HP2-2.
The
monoclonal anti-HP Ab (Fig. 28, center panel, lanes 3 and 4) recognized only 2
incomplete
deglycosylated f chain bands, while the polyclonal anti-HP Ab recognized also
the
completely deglycosylated -36 kDa (3 chain (Fig. 28, right panel, 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 upon deglycosylation, but it appeared less
intense (Fig. 28,
left panel, lane 4). MS/MS analysis and NH2-terminal sequencing of this 45 kDa
protein band
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performed on two distinct samples analyzed at different times identified this
protein as the
human HP2 precursor (pre-HP2, accession no. P00738). The combined MS/MS
analysis
covered a total of 49.8% of non-overlapping protein and 13 unique peptides
spanning the
entire protein sequence. Therefore, in addition to al and a2 chains, the anSot
Ab
recognize the uncleaved single chain pre-HP2, but not the b chain.
These results suggest that the anti-Zot Ab used to measure serum 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), while
the entire HP pool in serum is in the mg/ml range (12). To address this
apparent
discrepancy, the WB analysis of both human sera and purified HPs was repeated
under
non-denaturing conditions using anti-Zot Ab. The WB showed a series of bands
in HP2-2
phenotype sera and in commercially purified HP2-2, while no bands were
detected in either
HP1-1 phenotype sera or in commercial purified HP1-1. Conversely, the anti-HP
polyclonal
Ab, that did not recognize the uncleaved pre-HP2, detected bands both in
commercially
purified HP1-1 and HP2-2 preparations. Combined, these data suggest that under
non-
denaturing conditions, the anti-Zot Ab detect only the single chain pre-HP2,
but not the two-
chain mature HPs, further supporting the notion that the single chain pre-HP2,
but not its
cleaved two-chain mature form, corresponds to the zonulin molecule.
EXAMPLE 19
Functional analysis of recombinant zonulin
The primary translation product of the mammalian HP2 mRNA transcript is a
polypeptide that dimerizes co-translationally and is proteolytically cleaved
while still in the
endoplasmic reticulum by the serine protease, Cr1 LP (13). Conversely, zonulin
is detectable
in human serum as uncleaved pre-HP2 (see above). To confirm the identification
of zonulin
as the single chain pre-HP2 and not the cleaved mature two chain HP2,
recombinant pre-
HP2 was expressed by inserting the pre-HP2 cDNA into an insect cell vector and
expressed
it using a baculovirus expression system. Highly purified recombinant pre-HP2
was
obtained that was recognized by the anti-Zot polyclonal Ab similarly to Figure
2B and that
migrated at an apparent MW of -53 kDa due to the 6xHis tag attached at the C-
terminus.
The single chain pre-HP2 was then subjected to proteolytic cleavage using a
series of serine
proteases. Matriptase, urokinase, thrombin, and plasma kallikrein did not
cleave pre-HP2,
while plasmin caused complete degradation of the protein. In contrast,
treatment with the
intestinal serine protease trypsin led to the appearance of two major bands
that migrated
with molecular weights compatible with the a2 and b subunits of zonulin. NH2-
terminal
sequencing of these 2 bands showed the 2 proteins to be identical the pre-H2
a2 and b
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chains cleaved at the predicted Arg161 cleavage site. The intact single chain
pre-HP2 and the
cleaved two-chain mature HP2, obtained after trypsin digestion, were both
tested for their
biological activities in the studies below.
EXAMPLE 20
Ex vivo effect of recombinant zonulin on TEER in mouse small intestine mounted
in the
micro-snapwell system
Recombinant pre-HP2 (from now on defined as zonulin) was applied to WT C57BL/6
murine small intestine segments mounted in microsnapwells. Recombinant single
chain
zonulin added to the mucosal (luminal) aspect of mouse intestinal segments
decreased
transepithelial electrical resistance (TEER), i.e., increased permeability,
when applied at
concentrations >_ 40 g/ml (Fig. 3). In contrast, no consistent TEER changes
were detected
when the trypsin-cleaved two chain HP2 was tested (Fig. 3).
EXAMPLE 21
In vivo effect of recombinant zonulin on mouse gastrointestinal permeability
To establish whether zonulin might alter intestinal permeability in vivo, mice
were
gavaged with the single chain recombinant pre-HP2 protein (170 mg/mouse), and
gastroduodenal and small intestinal permeability tested using specific sugar
probes (sucrose
and lactulose/mannitol, respectively) as described (14). Zonulin/preHP2
increased both
small intestinal (Fig. 4A) and gastroduodenal (Fig. 4B) permeability compared
to bovine
serum albumin (BSA)-treated controls. Gastroduodenal and small intestinal
permeability
each returned to baseline within 48 h following exposure to zonulin/preHP2
(Fig. 4C and
4D).
To determine whether the two-chain mature HP2 affected IP, the in vivo
experiments
described above were repeated by administering two-chain proteolytically
cleaved protein. In
contrast to the single chain zonulin, two-chain HP2 (170 mg/mouse) failed to
alter either
gastroduodenal or small intestinal permeability compared to BSA-treated
controls (Fig. 4A
and 4B). Combined, these data indicate that the single chain zonulin, but not
its two-chain
mature HP2 form generated by proteolytic cleavage, retains the reversible
permeating
activity reported for zonulin.
EXAMPLE 22
Zonulin mRNA expression and quantification in human intestinal mucosae
Using specific primers and the cDNA of human intestinal biopsies from zonulin
positive subjects, a 686 bp fragment was amplified, 144 bp of which belongs to
the a-chain
17
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and 542 bp belonging to the b-chain of both HP1 and HP2 genes. Sequencing of
this
fragment confirmed its identity as HP, but HP1 could not be distinguished from
HP2 because
of the common sequence in the amplified region. To overcome this and to
specifically
quantify the expression of the zonulin gene in the human intestine, cDNA
obtained from the
intestinal mucosae of healthy individuals (n = 10), CD patients (disease in
acute phase (n =
7), and CD patients disease in remission following a gluten-free diet (GFD) (n
= 3) were
analyzed by real-time PCR using primers and probes specific for the a2 chain.
Compared to
healthy individuals, zonulin mRNA expression was increased in the intestinal
mucosae of
CD subjects with active disease (3-fold increase, P < 0.05). Intestinal
mucosae of three
celiac subjects adhering to a gluten-free diet showed only 1.5 fold increase
zonulin
expression compared to controls.
EXAMPLE 23
Recombinant zonulin increases tyrosine phosphorylation of EGFR
Gliadin, a glycoprotein present in wheat and several other cereals and the
environmental trigger responsible for the autoimmune damage of the small
intestine typical
of CD (15), fully reproduces the effects of EGF on the actin cytoskeleton
(16), effects that
are very similar of zonulin (7, 10, 16). Furthermore, structural analysis
revealed that the pre-
HP-2 b chain includes an EGF motif that contains 6 spatially conserved
cysteine residues
that form 3 intramolecular disulfide bonds necessary for EGF-like activity.
To determine whether zonulin can activate EGFR, increasing concentrations of
baculovirus-derived, recombinant zonulin were added to Caco-2 intestinal
epithelial cells.
The cells were lysed, immunoprecipitated with anti-EGFR Ab, and processed for
phosphotyrosine immunoblotting (PY-Plus). At concentrations z 3 mg/ml, zonulin
increased
tyrosine phosphorylation of EGFR (Fig. 5A). To further establish the role of
EGFR in zonulin-
induced alterations in TEER, both in vitro and ex vivo experiments described
above were
performed in the presence of the EGFR-selective PTK inhibitor, AG1478. Pre-
incubation of
Caco-2 cells for 2 h with the EGFR selective protein tyrosine kinase
inhibitor, AG1478 (5
mM), prevented zonulin/preHP2-induced EGFR phosphorylation on Y1068 (Fig. 5B).
Similarly, pretreatment with AG1478 abolished TEER reduction in response of
zonulin (Fig.
5C). Finally, trypsin digestion of zonulin dramatically reduced its ability to
activate EGFR
(Fig. 5D). Combined, these data suggest that the single chain zonulin
activates EGFR and
induces an EGFR-driven decrease in TEER, whereas the cleaved two-chain HP2
fails to
both activate EGFR and to increase IP.
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EXAMPLE 24
Recombinant zonulin-induced EGFR activation and TEER changes are PAR2-
dependent
Zot active peptide FCIGRL (AT1002) has structural similarities with the PAR2-
Activating Peptide (AP), SLIGRL, and causes PAR2-dependent changes in TEER
(17), a
finding that was demonstrated in WT, but not PAR2"'- mice. Further, several G
protein
coupled receptors (GPCR), including PAR2, transactivate EGFR (18). Since Zot
and zonulin
share a similar mechanism of action (6) and the zonulin protein sequence
contains a Zot-like
and PAR2 AP-like motif in its b chain (FCAGMS), whether zonulin-induced EGFR
activation
might be PAR2-dependent was determined.
siRNA-induced silencing of PAR2 in Caco-2 cells diminished EGFR Y1068
phosphorylation in response to recombinant zonulin (10 mg/ml)(Fig. 6A),
compatible with
PAR2-dependent transactivation of EGFR.
To further establish a role for PAR2 in EGFR activation in response to
zonulin, small
intestinal barrier function was studied in the microsnapwell system using
segments isolated
from either C57BL/6 WT or PAR2 -1" mice. As anticipated, recombinant zonulin
decreased
TEER in intestinal segments from C57BL/6 WT mice, while it failed to reduce
TEER in small
intestinal segments from PAR2 -1- mice (Fig. 6B), so linking zonulin-induced
PAR2-dependent
transactivation of EGFR with barrier function modulation.
The present invention identified zonulin as the precursor of HP2. Mature human
HPs
are heterodimeric plasma glycoproteins composed of a and b polypeptide chains
that are
covalently associated by disulfide bonds and in which only the b chain is
glycosylated (19).
Unlike the b chain (36 kDa), the a chain exists in two forms, i.e., Ot,1 (-9
kDa) and 01.2 (-18
kDa). The presence of one or both of the 2 chains results in the three
phenotypes, HP1-1,
HP2-1, and HP2-2. These HP variants evolved from a mannose-binding lectin-
associated
serine protease (MASP) (12, 20), with the a chain containing a complement
control protein
and the b 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 (EGF,
HGF, etc.) 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
complement Cl r-like
protease (Cr1 LP) in the endoplasmic reticulum (13). Of interest, the
endoplasmic reticulum
fraction was the cellular fraction in which the highest zonulin concentrations
were detected
(9).
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Since the key biological effect of zonulin is to regulate intercellular TJ
function (7, 9-
11), recombinant pre-HP2 was exanubed in intestinal permeability assays. Pre-
HP2 dose-
and time-dependently 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
two a2 and b subunits further supports the notion that zonulin/pre-HP-2 and
mature two-
chain HP2 exert distinct biological functions. 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 al chain under denaturing conditions (Fig. 1A
and 2B), but
failed to recognize non-denatured HP1. Combined, these data confirm the
identity of zonulin
as pre-HP2.
The NI-1-terminal amino acid sequence of zonulin has striking similarities
with the
light chain of human g globulins (7), a similarity also noted for HP (26).
Clearance of the HP-
Hb complex can be mediated by the monocyte/macrophage scavenger receptor,
CD163
(25). Clustal W dendogram analysis showed a region in the zonulin b chain just
upstream of
the CD163 binding site with the following gamma globulin-like consensus motif:
QLVE---V---
P. Discrepancies between the previously reported zonulin sequence and this pre-
HP2
consensus motif may be due to intra-species differences.
Zonulin contains growth factor-like repeats. Like zonulin, growth factors
affect
intercellular tight junction integrity (27, 28). The present invention shows
that the single
chain zonulin, but not its cleaved mature form, transactivates EGFR via PAR2
and that its
effect on TEER is prevented by pharmacological inhibition of EGFR or siRNA-
induced PAR2
silencing. This suggests that the growth factor motif in the single chain
zonulin, but not in the
mature two-chain HP2, has the molecular conformation required to induce tight
junctions
disassembly by indirect transactivation via PAR2.
Gliadin, the environmental trigger of CD, reportedly reproduces the effects of
EGF on
the actin cytoskeleton (16). These effects are very similar to the effects
reported for zonulin
(7). Gliadin binds to the CXCR3 chemokine receptor (29) and this interaction
is coupled to
zonulin-pre-HP2 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.
Intestinal bacterial colonization is also a stimulus for zonulin release (8).
Gliadin and
microorganisms both cause polarized, Iuminal secretion of zonulin (8).
Therefore, studies
were focused on early zonulin action, i.e., its activity at intestinal luminal
side. This approach
may appear counterintuitive, given the observation that both EGFR and PAR2 are
expressed
basolaterally (3, 30). However, evidence exists that they also are apically
expressed (31).
The fact that zonulin exerted a permeating 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
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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 subsequent
increase in
intestinal permeability, these triggers can reach the submucosa where zonulin-
expressing
immune cells can present zonulin to the basolateral side. A similar bilateral
action has been
reported for mucosal mast cell protease II, another serine protease that
controls intestinal
permeability acting both from luminal and serosal sides (32).
The role of both EGFR and PAR2 in regulating epithelial permeability has been
previously reported (33, 34). However, the present invention provides the
first evidence that
the two receptors work cooperatively to regulate small intestinal
permeability.
It has been reported that zonulin is upregulated during the acute phase of CD
(9, 10).
Using HP-specific primers, the present invention reports for the first time
the expression of
zonulin mRNA in human intestine. Furthermore, real time PCR experiments showed
that
zonulin expression was increased in CD patients compared to normal controls.
The
enhanced expression of zonulin correlated with disease activity as CD patients
who were on
a gluten-free diet showed mean values for zonulin expression that were
intermediate to
active CD patients and normal controls. Interestingly, Papp and co-workers
recently reported
that a polymorphism in the HP gene represents a novel genetic risk factor for
CD
development and its clinical manifestations (35).
The human plasma levels of pre-HPs are between 100 and 300 mg/100ml, with HP2-
2 ranging between 100-260 mg/100 ml (36). Almost 8% of HPs are secreted in
their pro-form
(37), suggesting that under physiological circumstances 80-208 mg/ml of pre-
HP2 are
present in human plasma. Therefore, the concentrations of zonulin used herein
are within
physiological range and are most likely indicative of the signaling pathways
activated when
zonulin is upregulated during pathological processes. Besides CD, elevated
levels of zonulin
have been reported in other autoimmune diseases, including T1D (11), systemic
lupus
erythematosus (38), and ankylosing spondylitis (39), further delineating the
importance of
the zonulin 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 blockage prevents the onset of the autoimmune
response, suggest that zonulin contributes to the pathogenesis of these
conditions, opening
new paradigms in the pathobiology and treatment options of immune-mediated
diseases.
The following references were cited herein:
1. Rook GA, Stanford JL (1998) Immunol Today 19:113-116.
2. Arrieta et al., (2006) Alterations in intestinal permeability. Gut 55:1512-
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3. Fasano et al., (2005) Nat Clin Pract Gastroenterol Hepatol 2:416-422.
21
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4. Wapenaar et at., (2008) Gut 57:463-467.
5. Rescigno et al., (2008) Curr Opin Immunol 20:669-675.
6. Fasano A (2000) Ann N Y Acad Sci 915:214-222.
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8. El et at., (2002) Gastroenterology 123:1607-1615.
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10. Fasano et al., (2000) Lancet 355:1518-1519.
11. Sapone et at., (2006) Diabetes 55:1443-1449.
12. Bowman BH, Kurosky A (1982) Adv Hum Genet 12:189-4.
13. Wicher KB, Fries E (2004) Proc Natl Acad Sci U S A 101:14390-14395.
14. Meddings JB, Swain MG (2000) Gastroenterology 119:1019-1028.
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