U.S. patent application number 12/908709 was filed with the patent office on 2011-07-28 for peptide antagonists of zonulin and methods for use of the same.
This patent application is currently assigned to University of Maryland, Baltimore. Invention is credited to Alessio FASANO.
Application Number | 20110183923 12/908709 |
Document ID | / |
Family ID | 22432094 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110183923 |
Kind Code |
A1 |
FASANO; Alessio |
July 28, 2011 |
PEPTIDE ANTAGONISTS OF ZONULIN AND METHODS FOR USE OF THE SAME
Abstract
Peptide antagonists of zonulin are disclosed, as well as methods
for the use of the same. The peptide antagonists bind to the zonula
occludens receptor, yet do not physiologically modulate the opening
of mammalian tight junctions.
Inventors: |
FASANO; Alessio; (West
Friendship, MD) |
Assignee: |
University of Maryland,
Baltimore
Baltimore
MD
|
Family ID: |
22432094 |
Appl. No.: |
12/908709 |
Filed: |
October 20, 2010 |
Related U.S. Patent Documents
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11842738 |
Aug 21, 2007 |
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12908709 |
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11500429 |
Aug 8, 2006 |
7531504 |
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11842738 |
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11074727 |
Mar 9, 2005 |
7189696 |
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11500429 |
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10648642 |
Aug 27, 2003 |
6936689 |
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11074727 |
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10095450 |
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6670448 |
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Aug 3, 1998 |
6458925 |
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10095450 |
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Current U.S.
Class: |
514/21.7 |
Current CPC
Class: |
A61P 1/00 20180101; A61P
35/00 20180101; C07K 7/08 20130101; C07K 7/06 20130101; A61P 1/06
20180101; C07K 14/4716 20130101; A61P 37/08 20180101; C07K 14/4705
20130101; A61P 43/00 20180101; A61K 38/00 20130101; A61P 29/00
20180101; A61P 1/04 20180101; A61P 31/00 20180101; A61P 37/02
20180101 |
Class at
Publication: |
514/21.7 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61P 1/00 20060101 A61P001/00; A61P 1/06 20060101
A61P001/06 |
Goverment Interests
[0001] The development of the present invention was supported by
the University of Maryland, Baltimore, Md. The invention described
herein was supported by funding from the National Institutes of
Health (NIH DK-48373). The Government has certain rights.
Claims
1-17. (canceled)
18. A method for inhibiting permeability of the small intestine,
comprising: administering to the small intestine of a subject in
need thereof an effective amount of a peptide, the amino acid
sequence of the peptide consisting essentially of SEQ ID NO:15 so
as to act as an inhibitor of the paracellular pathway.
19. The method of claim 18, wherein the subject has celiac
disease.
20. The method of claim 18, wherein the subject has inflammatory
bowel disease.
21. The method of claim 18, wherein the peptide is administered in
a gastroresistant tablet.
22. The method of claim 18, wherein the peptide is administered in
a gastroresistant capsule.
23. The method of claim 22, wherein the capsule comprises
gastroresistant beads.
24. The method of claim 18, wherein the peptide is administered in
a liquid oral dosage composition.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to peptide antagonists of
zonulin, as well as methods for the use of the same. Said peptide
antagonists bind to the zonula occludens receptor, yet do not
physiologically modulate the opening of mammalian tight
junctions.
BACKGROUND OF THE INVENTION
I. Function and Regulation of Intestinal Tight Junctions
[0003] The tight junctions ("tj") or zonula occludens (hereinafter
"ZO") are one of the hallmarks of absorptive and secretory
epithelia (Madara, J. Clin. Invest., 83:1089-1094 (1989); and
Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al,
Chapter 11, pages 125-138 (1990). As a barrier between apical and
basolateral compartments, they selectively regulate the passive
diffusion of ions and water-soluble solutes through the
paracellular pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol.
22):C749-C758 (1987)). This barrier maintains any gradient
generated by the activity of pathways associated with the
transcellular route (Diamond, Physiologist, 20:10-18 (1977)).
[0004] Variations in transepithelial conductance can usually be
attributed to changes in the permeability of the paracellular
pathway, since the resistances of enterocyte plasma membranes are
relatively high (Madara, supra). The ZO represents the major
barrier in this paracellular pathway, and the electrical resistance
of epithelial tissues seems to depend on the number of
transmembrane protein strands, and their complexity in the ZO, as
observed by freeze-fracture electron microscopy (Madara et al, J.
Cell Biol., 101:2124-2133 (1985)).
[0005] There is abundant evidence that ZO, once regarded as static
structures, are in fact dynamic and readily adapt to a variety of
developmental (Magnuson et al, Dev. Biol., 67:214-224 (1978); Revel
et al, Cold Spring Harbor Symp. Quant. Biol., 40:443-455 (1976);
and Schneeberger et al, J. Cell Sci., 32:307-324 (1978)),
physiological (Gilula et al, Dev. Biol., 50:142-168 (1976); Madara
et al, J. Membr. Biol., 100:149-164 (1987); Mazariegos et al, J.
Cell Biol., 98:1865-1877 (1984); and Sardet et al, J. Cell Biol.,
80:96-117 (1979)), and pathological (Milks et al, J. Cell Biol.,
103:2729-2738 (1986); Nash et al, Lab. Invest., 59:531-537 (1988);
and Shasby et al, Am. J. Physiol., 255(Cell Physiol., 24):C781-C788
(1988)) circumstances. The regulatory mechanisms that underlie this
adaptation are still not completely understood. However, it is
clear that, in the presence of Ca.sup.2+, assembly of the ZO is the
result of cellular interactions that trigger a complex cascade of
biochemical events that ultimately lead to the formation and
modulation of an organized network of ZO elements, the composition
of which has been only partially characterized (Diamond,
Physiologist, 20:10-18 (1977)). A candidate for the transmembrane
protein strands, occludin, has recently been identified (Furuse et
al, J. Membr. Biol., 87:141-150 (1985)).
[0006] Six proteins have been identified in a cytoplasmic
submembranous plaque underlying membrane contacts, but their
function remains to be established (Diamond, supra). ZO-1 and ZO-2
exist as a heterodimer (Gumbiner et al, Proc. Natl. Acad. Sci.,
USA, 88:3460-3464 (1991)) in a detergent-stable complex with an
uncharacterized 130 kD protein (ZO-3). Most immunoelectron
microscopic studies have localized ZO-1 to precisely beneath
membrane contacts (Stevenson et al, Molec. Cell Biochem.,
83:129-145 (1988)). Two other proteins, cingulin (Citi et al,
Nature (London), 333:272-275 (1988)) and the 7H6 antigen (Zhong et
al, J. Cell Biol., 120:477-483 (1993)) are localized further from
the membrane and have not yet been cloned. Rab 13, a small GTP
binding protein has also recently been localized to the junction
region (Zahraoui et al, J. Cell Biol., 124:101-115 (1994)). Other
small GTP-binding proteins are known to regulate the cortical
cytoskeleton, i.e., rho regulates actin-membrane attachment in
focal contacts (Ridley et al, Cell, 70:389-399 (1992)), and rac
regulates growth factor-induced membrane ruffling (Ridley et al,
Cell, 70:401-410 (1992)). Based on the analogy with the known
functions of plaque proteins in the better characterized cell
junctions, focal contacts (Guan et al, Nature, 358:690-692 (1992)),
and adherents junctions (Tsukita et al, J. Cell Biol.,
123:1049-1053 (1993)), it has been hypothesize that tj-associated
plaque proteins are involved in transducing signals in both
directions across the cell membrane, and in regulating links to the
cortical actin cytoskeleton.
[0007] To meet the many diverse physiological and pathological
challenges to which epithelia are subjected, the ZO must be capable
of rapid and coordinated responses that require the presence of a
complex regulatory system. The precise characterization of the
mechanisms involved in the assembly and regulation of the ZO is an
area of current active investigation.
[0008] There is now a body of evidence that tj structural and
functional linkages exist between the actin cytoskeleton and the tj
complex of absorptive cells (Gumbiner et al, supra; Madara et al,
supra; and Drenchahn et al, J. Cell Biol., 107:1037-1048 (1988)).
The actin cytoskeleton is composed of a complicated meshwork of
microfilaments whose precise geometry is regulated by a large cadre
of actin-binding proteins. An example of how the state of
phosphorylation of an actin-binding protein might regulate
cytoskeletal linking to the cell plasma membrane is the
myristoylated alanine-rich C kinase substrate (hereinafter
"MARCKS"). MARCKS is a specific protein kinase C (hereinafter
"PKC") substrate that is associated with the cytoplasmic face of
the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages 438-443
(1992)). In its non-phosphorylated form, MARCKS crosslinks to the
membrane actin. Thus, it is likely that the actin meshwork
associated with the membrane via MARCKS is relatively rigid
(Hartwig et al, Nature, 356:618-622 (1992)). Activated PKC
phosphorylates MARCKS, which is released from the membrane (Rosen
et al, J. Exp. Med., 172:1211-1215 (1990); and Thelen et al,
Nature, 351:320-322 (1991)). The actin linked to MARCKS is likely
to be spatially separated from the membrane and be more plastic.
When MARCKS is dephosphorylated, it returns to the membrane where
it once again crosslinks actin (Hartwig et al, supra; and Thelen et
al, supra). These data suggest that the F-actin network may be
rearranged by a PKC-dependent phosphorylation process that involves
actin-binding proteins (MARCKS being one of them).
[0009] A variety of intracellular mediators have been shown to
alter tj function and/or structure. Tight junctions of amphibian
gallbladder (Duffey et al, Nature, 204:451-452 (1981)), and both
goldfish (Bakker et al, Am. J. Physiol., 246:G213-G217 (1984)) and
flounder (Krasney et al, Fed. Proc., 42:1100 (1983)) intestine,
display enhanced resistance to passive ion flow as intracellular
cAMP is elevated. Also, exposure of amphibian gallbladder to
Ca.sup.2+ ionophore appears to enhance tj resistance, and induce
alterations in tj structure (Palant et al, Am. J. Physiol.,
245:C203-C212 (1983)). Further, activation of PKC by phorbol esters
increases paracellular permeability both in kidney (Ellis et al, C.
Am. J. Physiol., 263 (Renal Fluid Electrolyte Physiol.
32):F293-F300 (1992)), and intestinal (Stenson et al, C. Am. J.
Physiol., 265(Gastrointest. Liver Physiol., 28):G955-G962 (1993))
epithelial cell lines.
II. The Blood-Brain Barrier
[0010] The blood-brain barrier (BBB) is an extremely thin
membranous barrier that is highly resistant to solute free
diffusion, and separates blood and the brain. In molecular
dimensions, the movement of drugs or solute through this membrane
is essentially nil, unless the compound has access to one of
several specialized enzyme-like transport mechanisms that are
embedded within the BBB membranes. The BBB is composed of multiple
cells rather than a single layer of epithelial cells. Of the four
different types of cells that compose the BBB (endothelial cells,
perycites, astrocytes, and neurons) the endothelial cell component
of the capillaries represents the limiting factor for the
permeability of the BBB. The capillary endothelium in vertebrate
brain and spinal cord is endowed with tj which closes the
interendothelial pores that normally exist in microvascular
endothelial barriers in peripheral tissues. Ultimately, endothelial
tj are responsible for the limited permeability of the BBB.
III. Zonula Occludens Toxin
[0011] Most Vibrio cholerae vaccine candidates constructed by
deleting the ctxA gene encoding cholera toxin (CT) are able to
elicit high antibody responses, but more than one-half of the
vaccines still develop mild diarrhea (Levine et al, Infect. Immun.,
56(1):161-167 (1988)). Given the magnitude of the diarrhea induced
in the absence of CT, it was hypothesized that V. cholerae produce
other enterotoxigenic factors, which are still present in strains
deleted of the ctxA sequence (Levine et al, supra). As a result, a
second toxin, zonula occludens toxin (hereinafter "ZOT") elaborated
by V. cholerae and which contribute to the residual diarrhea, was
discovered (Fasano et al, Proc. Natl. Acad. Sci., USA, 8:5242-5246
(1991)). The zot gene is located immediately adjacent to the ctx
genes. The high percent concurrence of the zot gene with the ctx
genes among V. cholerae strains (Johnson et al, J. Clin. Microb.,
31/3:732-733 (1993); and Karasawa et al, FEBS Microbiology Letters,
106:143-146 (1993)) suggests a possible synergistic role of ZOT in
the causation of acute dehydrating diarrhea typical of cholera.
Recently, the zot gene has also been identified in other enteric
pathogens (Tschape, 2nd Asian-Pacific Symposium on Typhoid fever
and other Salomellosis, 47(Abstr.) (1994)).
[0012] It has been previously found that, when tested on rabbit
ileal mucosa, ZOT increases the intestinal permeability by
modulating the structure of intercellular tj (Fasano et al, supra).
It has been found that as a consequence of modification of the
paracellular pathway, the intestinal mucosa becomes more permeable.
It also was found that ZOT does not affect Na.sup.+-glucose coupled
active transport, is not cytotoxic, and fails to completely abolish
the transepithelial resistance (Fasano et al, supra).
[0013] More recently, it has been found that ZOT is capable of
reversibly opening tj in the intestinal mucosa, and thus ZOT, when
co-administered with a therapeutic agent, is able to effect
intestinal delivery of the therapeutic agent, when employed in an
oral dosage composition for intestinal drug delivery (WO 96/37196;
U.S. patent application Ser. No. 08/443,864, filed May 24, 1995;
and U.S. Pat. No. 5,665,389; and Fasano et al, J. Clin. Invest.,
99:1158-1164 (1997); each of which is incorporated by reference
herein in their entirety). It has also been found that ZOT is
capable of reversibly opening tj in the nasal mucosa, and thus ZOT,
when co-administered with a therapeutic agent, is able to enhance
nasal absorption of a therapeutic agent (U.S. patent application
Ser. No. 08/781,057, filed Jan. 9, 1997; which is incorporated by
reference herein in its entirety).
[0014] In U.S. patent application Ser. No. 08/803,364, filed Feb.
20, 1997; which is incorporated by reference herein in its
entirety, a ZOT receptor has been identified and purified from an
intestinal cell line, i.e., CaCo2 cells. Further, in U.S. patent
application Ser. No. 09/024,198, filed Feb. 17, 1998; which is
incorporated by reference herein in its entirety, ZOT receptors
from human intestinal, heart and brain tissue have been identified
and purified. The ZOT receptors represent the first step of the
paracellular pathway involved in the regulation of intestinal and
nasal permeability.
IV. Zonulin
[0015] In pending U.S. patent application Ser. No. 098/859,931,
filed May 21, 1997, which is incorporated by reference herein in
its entirety, mammalian proteins that are immunologically and
functionally related to ZOT, and that function as the physiological
modulator of mammalian tight junctions, have been identified and
purified. These mammalian proteins, referred to as "zonulin", are
useful for enhancing absorption of therapeutic agents across tj of
intestinal and nasal mucosa, as well as across tj of the blood
brain barrier.
[0016] In the present invention, peptide antagonists of zonulin
have been identified for the first time. Said peptide antagonists
bind to ZOT receptor, yet do not function to physiologically
modulate the opening of mammalian tight junctions. The peptide
antagonists competitively inhibit the binding of ZOT and zonulin to
the ZOT receptor, thereby inhibiting the ability of ZOT and zonulin
to physiologically modulate the opening of mammalian tight
junctions.
SUMMARY OF THE INVENTION
[0017] An object of the present invention is to identify peptide
antagonists of zonulin.
[0018] Another object of the present invention is to synthesize and
purify said peptide antagonists.
[0019] Still another object of the present invention is to use said
peptide antagonists as anti-inflammatory agents in the treatment of
gastrointestinal inflammation.
[0020] Yet another object of the present invention is to use said
peptide antagonists to inhibit the breakdown of the blood brain
barrier.
[0021] These and other objects of the present invention, which will
be apparent from the detailed description of the invention provided
hereinafter, have been met, in one embodiment, by a peptide
antagonist of zonulin comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:35, wherein said peptide
antagonist binds to a ZOT receptor, yet does not physiologically
modulate the opening of mammalian tight junctions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows the effect of zonulin purified from rabbit
intestine (.box-solid.), as compared to various negative controls
(Fraction 2 (.diamond.); Fraction 3 ( ); Fraction 4
(.tangle-solidup.); and Fraction 5 (.quadrature.) from a
Q-Sepharose column), on the tissue resistance (Rt) of CaCo2 cell
monolayers.
[0023] FIG. 2 shows the effect of zonulin purified from rabbit
intestine (.box-solid.), as compared to the negative control
(.quadrature.), on the tissue resistance (Rt) of rabbit ileum
mounted in Ussing chambers.
[0024] FIG. 3 shows the effect of zonulin purified from rabbit
intestine (.box-solid.), as compared to the negative controls
(zonulin+anti-ZOT antibody (.quadrature.); zonulin+anti-tau
antibody (.DELTA.); and tau (.tangle-solidup.)), on the tissue
resistance (Rt) of rabbit ileum mounted in Ussing chambers.
[0025] FIGS. 4A and 4B show the effect of zonulin purified from
either human brain (.tangle-solidup.), human intestine ( ), or
human heart (.largecircle.), as compared to the negative control
(.quadrature.), on the tissue resistance (Rt) of Rhesus monkey
jejunum (FIG. 4A) and Rhesus monkey ileum (FIG. 4B) mounted in
Ussing chambers.
[0026] FIGS. 5A and 5B show the effect of zonulin purified from
either human heart (.tangle-solidup.) or human brain
(.quadrature.), as compared to the negative control (.box-solid.),
on the tissue resistance (Rt) of rabbit jejunum (FIG. 5A) and
rabbit ileum (FIG. 5B) mounted in Ussing chambers.
[0027] FIG. 6 shows a comparison of the N-terminal sequence of
zonulin purified from rabbit and various human tissues.
[0028] FIG. 7 shows a comparison of the N-terminal sequences of
zonulin purified from various human tissues and IgM heavy chain
with the N-terminal sequence of the biologically active fragment
(amino acids 288-399) of ZOT.
[0029] FIG. 8 shows the effect of ZOT, zonulin.sub.h, either alone
(closed bars), or in combination with the peptide antagonist FZI/0
(open bars) or in combination with FZI/1 (shaded bars), as compared
to the negative control, on the tissue resistance (Rt) of rabbit
ileum mounted in Ussing chambers. N equals 3-5; and * equals
p<0.01.
DETAILED DESCRIPTION OF THE INVENTION
[0030] As discussed above, in one embodiment, the above-described
object of the present invention have been met by a peptide
antagonist of zonulin comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:35, wherein said peptide
antagonist binds to ZOT receptor, yet does not physiologically
modulate the opening of mammalian tight junctions.
[0031] The size of the peptide antagonist is not critical to the
present invention. Generally, the size of the peptide antagonist
will range from 8 to 110, amino acids, preferably from 8 to 40
amino acids, more preferably will be 8 amino acids.
[0032] The peptide antagonists can be chemically synthesized and
purified using well-known techniques, such as described in High
Performance Liquid Chromatography of Peptides and Proteins:
Separation Analysis and Conformation, Eds. Mant et al, C.R.C. Press
(1991), and a peptide synthesizer, such as Symphony (Protein
Technologies, Inc); or by using recombinant DNA techniques, i.e.,
where the nucleotide sequence encoding the peptide is inserted in
an appropriate expression vector, e.g., an E. coli or yeast
expression vector, expressed in the respective host cell, and
purified therefrom using well-known techniques.
[0033] The peptide antagonists can be used as anti-inflammatory
agents for the treatment of gastrointestinal inflammation that
gives rise to increased intestinal permeability. Thus, the peptide
antagonists of the present invention are useful, e.g., in the
treatment of intestinal conditions that cause protein losing
enteropathy. Protein losing enteropathy may arise due to: [0034]
Infection, e.g., C. difficile infection, enterocolitis,
shigellosis, viral gastroenteritis, parasite infestation, bacterial
overgrowth, Whipple's disease; [0035] Diseases with mucosal erosion
or ulcerations, e.g., gastritis, gastric cancer, collagenous
colitis, inflammatory bowel disease; [0036] Diseases marked by
lymphatic obstruction, e.g., congenital intestinal
lymphangiectasia, sarcoidosis lymphoma, mesenteric tuberculosis,
and after surgical correction of congenital heart disease with
Fontan's operation; [0037] Mucosal diseases without ulceration,
e.g., Menetrier's disease, celiac disease, eosinophilic
gastroenteritis; and [0038] Immune diseases, e.g., systemic lupus
erythematosus or food allergies, primarily to milk (see also Table
40-2 of Pediatric Gastrointestinal Disease Pathophysiology
Diagnosis Management, Eds. Wyllie et al, Saunders Co. (1993), pages
536-543; which is incorporated by reference herein in its
entirety).
[0039] Hence, in another embodiment, the present invention relates
to a method for treatment of gastrointestinal inflammation
comprising administering to a subject in need of such treatment, a
pharmaceutically effective amount of a peptide antagonist of
zonulin, wherein said peptide antagonist comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, wherein said peptide
antagonist binds to the ZOT receptor in the intestine of said
subject, yet does not physiologically modulate the opening of tight
junctions in said intestine.
[0040] To this end, the peptide antagonists can be administered as
oral dosage compositions for small intestinal delivery. Such oral
dosage compositions for small intestinal delivery are well-known in
the art, and generally comprise gastroresistent tablets or capsules
(Remington's Pharmaceutical Sciences, 16th Ed., Eds. Osol, Mack
Publishing Co., Chapter 89 (1980); Digenis et al, J. Pharm. Sci.,
83:915-921 (1994); Vantini et al, Clinica Terapeutica, 145:445-451
(1993); Yoshitomi et al, Chem. Pharm. Bull., 40:1902-1905 (1992);
Thoma et al, Pharmazie, 46:331-336 (1991); Morishita et al, Drug
Design and Delivery, 7:309-319 (1991); and Lin et al,
Pharmaceutical Res., 8:919-924 (1991)); each of which is
incorporated by reference herein in its entirety).
[0041] Tablets are made gastroresistent by the addition of, e.g.,
either cellulose acetate phthalate or cellulose acetate
terephthalate.
[0042] Capsules are solid dosage forms in which the peptide
antagonist(s) is enclosed in either a hard or soft, soluble
container or shell of gelatin. The gelatin used in the manufacture
of capsules is obtained from collagenous material by hydrolysis.
There are two types of gelatin. Type A, derived from pork skins by
acid processing, and Type B, obtained from bones and animal skins
by alkaline processing. The use of hard gelatin capsules permit a
choice in prescribing a single peptide antagonist or a combination
thereof at the exact dosage level considered best for the
individual subject. The hard gelatin capsule consists of two
sections, one slipping over the other, thus completely surrounding
the peptide antagonist. These capsules are filled by introducing
the peptide antagonist, or gastroresistent beads containing the
peptide antagonist, into the longer end of the capsule, and then
slipping on the cap. Hard gelatin capsules are made largely from
gelatin, FD&C colorants, and sometimes an opacifying agent,
such as titanium dioxide. The USP permits the gelatin for this
purpose to contain 0.15% (w/v) sulfur dioxide to prevent
decomposition during manufacture.
[0043] In the context of the present invention, oral dosage
compositions for small intestinal delivery also include liquid
compositions which contain aqueous buffering agents that prevent
the peptide antagonist from being significantly inactivated by
gastric fluids in the stomach, thereby allowing the peptide
antagonist to reach the small intestines in an active form.
Examples of such aqueous buffering agents which can be employed in
the present invention include bicarbonate buffer (pH 5.5 to 8.7,
preferably about pH 7.4).
[0044] When the oral dosage composition is a liquid composition, it
is preferable that the composition be prepared just prior to
administration so as to minimize stability problems. In this case,
the liquid composition can be prepared by dissolving lyophilized
peptide antagonist in the aqueous buffering agent.
[0045] The peptide antagonists can be used as to inhibit breakdown
of the blood brain barrier. Thus, the peptide antagonists of the
present invention are useful, e.g., in the treatment of conditions
associated with breakdown of the blood brain barrier. Examples of
such conditions include osmotic injuries, e.g., cerebral ischemia,
stroke or cerebral edema; hypertension; carbon dioxide; convulsive
seizure; chemical toxins; uremia (renal insufficiency); meningitis,
encephalitis, encephalomielitis, e.g., infective (viral (SRV, HIV,
etc.), or bacterial (TB, H. influenzae, meningococcus, etc.) or
allergic; tumors; traumatic brain injuries; radiation brain injury;
immaturity and kernicterus; demyelinating diseases, e.g., multiple
sclerosis or Guillian-Barre syndrome.
[0046] Hence, in another embodiment, the present invention relates
to a method for treatment of conditions associated with breakdown
of the blood brain barrier comprising administering to a subject in
need of such treatment, a pharmaceutically effective amount of a
peptide antagonist of zonulin, wherein said peptide antagonist
comprises amino acid sequence SEQ ID NO:35, wherein said peptide
antagonist binds to ZOT receptor in the brain of said subject, yet
does not physiologically modulate the opening of tight junctions in
said brain.
[0047] To this end, the peptide antagonists can be administered as
intravenous dosage compositions for delivery to the brain. Such
compositions are well-known in the art, and compositions generally
comprise a physiological diluent, e.g., distilled water, or 0.9%
(w/v) NaCl.
[0048] The pharmaceutically effective amount of peptide antagonist
employed is not critical to the present invention and will vary
depending upon the disease or condition being treated, as well as
the age, weight and sex of the subject being treated. Generally,
the amount of peptide antagonist employed in the present invention
to inhibit gastrointestinal inflammation or inhibit breakdown of
the blood brain barrier, e.g., to inhibit zonulin biological
activity, is in the range of about 7.5.times.10.sup.-6 M to
7.5.times.10.sup.-3 M, preferably about 7.5.times.10.sup.-6 M to
7.5.times.10.sup.-4 M. To achieve such a final concentration in,
e.g., the intestines or blood, the amount of peptide antagonist in
a single oral dosage composition of the present invention will
generally be about 1.0 .mu.g to 1000 .mu.g, preferably about 1.0
.mu.g to 100 .mu.g.
[0049] The peptide antagonists can be also be used as an immunogen
to obtain antibodies, either polycolonal or monoclonal, having
binding specificity for zonulin, using techniques well-known in the
art (Abrams, Methods Enzymol., 121:107-119 (1986)). These
antibodies can in turn can be used to assay for zonulin in body
tissue or fluids, or in affinity-purification of zonulin, or
alternatively, to bind to zonulin, and thereby inhibit zonulin
activity, e.g., to inhibit gastrointestinal inflammation or to
inhibit breakdown of the blood brain barrier.
[0050] The following examples are provided for illustrative
purposes only, and are in no way intended to limit the scope of the
present invention.
Example 1
Purification of ZOT
[0051] 5000 ml of the supernatant fraction obtained after culturing
V. cholerae strain CVD110 (Michalski et al, Infect. Immun.,
G1:4462-4468 (1993), which had been transformed with plasmid pZ14,
was concentrated 1000-fold using a lamina flow filter with a MW
cutoff of 10 kDa. The construction of pZ14, which contains the
Vibrio cholera zot gene, is described in detail in, inter alia, WO
96/37196. The resulting supernatant was then subjected to 8.0%
(w/v) SDS-PAGE. Protein bands were detected by Coomassie blue
staining of the SDS-PAGE gel. No protein band corresponding to ZOT
was detectable when compared to control supernatant from strain
CVD110 transformed with plasmid pTTQ181 (Amersham, Arlington
Heights, Ill.), and treated in the same manner. Therefore, even
though the zot gene was placed behind the highly inducible and
strong tac promoter in pZ14, the level of the protein in 1000-fold
concentrated pZ14 supernatant was still not detectable by the
Coomassie stained SDS-PAGE gel.
[0052] A. MBP-ZOT
[0053] To increase the amount of ZOT produced, the zot gene was
fused in frame with the maltose binding protein (hereinafter "MBP")
gene to create a MBP-ZOT fusion protein.
[0054] The MBP vector pMAL-c2 (Biolab) was used to express and
purify ZOT by fusing the zot gene to the malE gene of E. coli. This
construct uses the strong, inducible tac promoter, and the malE
translation initiation signals to give high level expression of the
cloned zot gene. The vector pMAL-c2 has an exact deletion of the
malE signal sequence, which leads to cytoplasmic expression of the
fusion protein. Affinity chromatography purification for MBP was
used to facilitate isolation of the fusion protein (Biolab).
[0055] More specifically, vector pMAL-c2 was linearized with EcoRI
(that cuts at the 3' end of the malE gene), filled in with Klenow
fragment, and digested with XbaI (that has a single site in pMAL-c2
polylinker). The orf encoding ZOT was subcloned from plasmid pBB241
(Baudry et al, Infect. Immun., 60:428-434 (1992)). Plasmid pBB241
was digested with BssHII, filled in with Klenow fragment, and
digested with XbaI. Then, the blunt-XbaI fragment was subcloned
into pMAL-c2 to give plasmid pLC10-c. Since both the insert, and
the vector had blunt and sticky ends, the correct orientation was
obtained with the 3' end of malE fused with the 5' terminus of the
insert. pLC10-c was then electroporated into E. coli strain
DH5.alpha.. In pBB241, the BssHII restriction site is within the
zot orf. Thus, amino acids 1-8 of ZOT are missing in the MBP-ZOT
fusion protein.
[0056] In order to purify the MBP-ZOT fusion protein, 10 ml of
Luria Bertani broth containing 0.2% (w/v) glucose and 100 .mu.g/ml
ampicillin were inoculated with a single colony containing pLC10-c,
and incubated overnight at 37.degree. C. with shaking. The culture
was diluted 1:100 in 1.0 ml of the same fresh medium, and grown at
37.degree. C. while shaking, to about 1.0.times.10.sup.6 cells/ml.
0.2 mM IPTG was then added to induce the MBP-ZOT expression, and
the culture was incubated at 37.degree. C. for additional 3 hr. The
bacteria were then pelleted and resuspended in 20 ml of ice cold
"column buffer" comprising 20 mM Tris-HCl, 0.2 M NaCl, 1.0 mM EDTA,
10 mM 2-ME, 1.0 mM NaN.sub.3. The bacterial suspension was lysed by
French press treatment and spun for 30 min at 13,000.times.g at
4.degree. C. The supernatant was collected, diluted 1:5 with column
buffer and loaded into a 1.times.10 column of amylose resin
(Biolabs, MBP-fusion purification system), pre-equilibrated with
column buffer. After washing the column with 5 volumes of column
buffer, the MBP-ZOT fusion protein was eluted by loading 10 ml of
10 mM maltose in column buffer. The typical yield from 1.0 ml of
culture was 2-3 mg of protein.
[0057] The MBP fusion partner of the purified MBP-ZOT fusion
protein was then cleaved off using 1.0 .mu.g of Factor Xa protease
(Biolabs) per 20 .mu.g of MBP-ZOT. Factor Xa protease cleaves just
before the amino terminus of ZOT. The ZOT protein so obtained was
run on a 8.0% (w/v) SDS-PAGE gel, and electroeluted from the gel
using an electroseparation chamber (Schleicher & Schuell,
Keene, N.H.).
[0058] When tested in Ussing chambers, the resulting purified ZOT
induced a dose-dependent decrease of Rt, with an ED.sub.50 of
7.5.times.10.sup.-8 M.
[0059] B. 6.times.His-ZOT
[0060] The zot gene was amplified by PCR with Deep Vent polymerase
(New England Biolabs), using pBB241 plasmid (Baudry et al, supra)
DNA as a template. The forward and reverse primers used were:
5'-CGGGATCCCGTATGAGTATCTTT-3' (SEQ ID NO:39); and
5'-CCCAAGCTTGGGTCAAAATATACT-3' (SEQ ID NO:40), respectively. The 5'
tails of these oligonucleotides contain a BamHI and a HindIII
restriction site, respectively. The resulting amplicon (1.2 kb) was
analyzed by 8.0% (w/v) agarose gel electrophoresis, and purified
from salts and free nucleotides using an Xtreme spin column
(Pierce). The above-noted two restriction enzymes were then used to
digest the amplicon, and the resulting digested-amplicon was then
inserted in the vector pQE30 (Quiagen), which had been previously
digested with BamHI and HindIII, so as to obtain plasmid pSU113.
pQE30 is an expression vector that provides high level expression
of a recombinant protein with a 6 poly-histidine tag (6.times.His).
The expression product of plasmid pSU113 is therefore a
6.times.His-ZOT fusion protein. pSU113 was then transformed into E.
coli DH5.alpha..
[0061] In order to purify the 6.times.His-ZOT fusion protein, the
resulting transformed E. coli were grown overnight at 37.degree. C.
in 150 ml of Luria Bertani broth containing 2.0% (w/v) glucose, 25
.mu.g/ml of kanamycin and 200 .mu.g/ml of ampicillin until the
A.sub.600 was about 1.10. Next, 75 ml of the overnight cultures
were added to 1000 ml of Luria Bertani broth containing 2.0% (w/v)
glucose, 25 .mu.g/ml of kanamycin and 200 .mu.g/ml of ampicillin,
incubated for about 3 hrs at 37.degree. C., with vigorous shaking,
until the A.sub.600 was about 0.7-0.9. Then, IPTG was added to a
final concentration of 2.0 mM, and growth was allowed to continue
for 5 hrs at 37.degree. C. Next, the cells were harvested by
centrifugation at 4000.times.g for 20 min, the cells resuspend in
5.0 ml/g wet weight of buffer A comprising 6.0 M GuHCl, 0.1 M
sodium phosphate, and 0.01 M Tris-HCl (pH 8.0), and stirred for 1
hr at room temperature. Then, the mixture was centrifuged at
10,000.times.g for 30 min at 4.degree. C., and to the resulting
supernatant was added 4.0-5.0 ml/g wet weight of a 50% slurry of
SUPERFLOW resin (QIAGEN), and stirring was carried out for 1 hr at
room temperature. The resulting resin was loaded into a
1.6.times.8.0 column, which was then washed sequentially with
buffer A, buffer B comprising 8.0 M urea, 0.1 M sodium phosphate,
and 0.01 M Tris-HCl (pH 8.0) and buffer C comprising 8.0 M urea,
0.1 M sodium phosphate, and 0.01 M Tris-HCl (pH 6.3). Each wash was
carried out until the A.sub.600 of the flow-through was less than
0.01. The 6.times.His-ZOT fusion protein was eluted from the column
using 20 ml of buffer C containing 250 mM imidazole. Then, the
fractions containing with the 6.times.His-ZOT fusion protein were
checked by SDS-PAGE using the procedure described by Davis, Ann.
N.Y. Acad. Sci., 121:404 (1964), and the gel stained with Comassie
blue. The fractions containing 6.times.His-ZOT fusion protein were
dialyzed against 8.0 M urea, combined, and then diluted 100 times
in PBS. Next, 4.0 ml of a 50% slurry of SUPERFLOW resin was added,
stirring was carried out for 2 hrs at room temperature, and the
resulting resin loaded into a 1.6.times.8.0 column, which was then
washed with 50 ml of PBS. The 6.times.His-ZOT fusion protein was
eluted from the column with 10 ml of PBS containing 250 mM
imidazole. The resulting eluant was dialyzed against PBS, and the
6.times.His-ZOT fusion protein was checked by SDS-PAGE, as
described above.
Example 2
Production of Affinity-Purified Anti-ZOT Antibodies
[0062] To obtain specific antiserum, a chimeric glutathione
S-transferase (GST)-ZOT protein was expressed and purified.
[0063] More specifically, oligonucleotide primers were used to
amplify the zot orf by polymerase chain reaction (PCR) using
plasmid pBB241 (Baudry et al, supra) as template DNA. The forward
primer (TCATCACGGC GCGCCAGG, SEQ ID NO:25) corresponded to
nucleotides 15-32 of zot orf, and the reverse primer (GGAGGTCTAG
AATCTGCCCG AT, SEQ ID NO:26) corresponded to the 5' end of ctxA
orf. Therefore, amino acids 1-5 of ZOT were missing in the
resulting fusion protein. The amplification product was inserted
into the polylinker (SmaI site) located at the end of the GST gene
in pGEX-2T (Pharmacia, Milwaukee, Wis.). pGEX-2T is a
fusion-protein expression vector that expresses a cloned gene as a
fusion protein with GST of Schistosoma japonicum. The fusion gene
is under the control of the tac promoter. Upon induction with IPTG,
derepression occurs and GST fusion protein is expressed.
[0064] The resulting recombinant plasmid, named pLC11, was
electroporated in E. coli DH5.alpha.. In order to purify GST-ZOT
fusion protein, 10 ml of Luria Bertani broth containing 100
.mu.g/ml ampicillin were inoculated with a single colony containing
pLC11, and incubated overnight at 37.degree. C. with shaking. The
culture was diluted 1:100 in 1.0 ml of the same fresh medium and
grown at 37.degree. C. while shaking, to about 1.0.times.10.sup.6
cells/ml. 0.2 mM IPTG was then added to induce the GST-ZOT
expression, and the culture was incubated at 37.degree. C. for
additional 3 hr. The bacteria were then pelleted, resuspended in 20
ml of ice cold PBS (pH 7.4), and lysed by the French press method.
The GST-ZOT fusion protein was not soluble under these conditions
as it sedimented with the bacterial pellet fraction. Therefore, the
pellet was resuspended in Laemli lysis buffer comprising 0.00625 M
Tris-HCl (pH 6.8), 0.2 M 2-ME, 2.0% (w/v) SDS, 0.025% (w/v)
bromophenol blue and 10% (v/v) glycerol, and subjected to
electrophoresis on a 8.0% (w/v) PAGE-SDS gel, and stained with
Coomassie brilliant blue. A band of about 70 kDa (26 kDa of GST+44
kDA of ZOT), corresponding to the fusion protein, was electroeluted
from the gel using an electroseparation chamber (Schleicher &
Schuell, Keene, N.H.).
[0065] 10 .mu.g of the resulting eluted protein (10-20 .mu.g) was
injected into a rabbit mixed with an equal volume of Freund's
complete adjuvant. Two booster doses were administered with
Freund's incomplete adjuvant four and eight weeks later. One month
later the rabbit was bled.
[0066] To determine the production of specific antibodies,
10.sup.-10 M of ZOT, along with the two fusion proteins MBP-ZOT and
GST-ZOT, was transferred onto a nylon membrane and incubated with a
1:5000 dilution of the rabbit antiserum overnight at 4.degree. C.
with moderate shaking. The filter was then washed 15 min 4 times
with PBS containing 0.05% (v/v) Tween 20 (hereinafter "PBS-T"), and
incubated with a 1:30,000 dilution of goat anti-rabbit IgG
conjugated to horseradish peroxidase for 2 hr at room temperature.
The filter was washed again for 15 min 4 times with PBS containing
0.1% (v/v) Tween, and immunoreactive bands were detected using
enhanced chemiluminescence (Amersham).
[0067] On immunoblot, the rabbit antiserum was found to recognize
ZOT, as well as MBP-ZOT and GST-ZOT fusion proteins, but not the
MBP negative control.
[0068] Moreover, to confirm the production of appropriate anti-ZOT
antibodies, neutralization experiments were conducted in Ussing
chambers. When pre-incubated with pZ14 supernatant at 37.degree. C.
for 60 min, the ZOT-specific antiserum (1:100 dilution), was able
to completely neutralize the decrease in Rt induced by ZOT on
rabbit ileum mounted in Ussing chambers.
[0069] Next, the anti-ZOT antibodies were affinity-purified using
an MBP-ZOT affinity column. More specifically, a MBP-ZOT affinity
column was prepared by immobilizing, overnight at room temperature,
1.0 mg of purified MBP-ZOT, obtained as described in Example 1
above, to a pre-activated gel (Aminolink, Pierce). The column was
washed with PBS, and then loaded with 2.0 ml of anti-ZOT rabbit
antiserum. After a 90 min incubation at room temperature, the
column was washed with 14 ml of PBS, and the specific anti-ZOT
antibodies were eluted from the column with 4.0 ml of a solution
comprising 50 mM glycine (pH 2.5), 150 mM NaCl, and 0.1% (v/v)
Triton X-100. The pH of the 1.0 ml eluted fractions was immediately
neutralized with 1.0 N NaOH.
Example 3
Purification of Zonulin
[0070] Based upon the observation in U.S. patent application Ser.
No. 08/803,364, filed Feb. 20, 1997, that ZOT interacts with a
specific epithelial surface receptor, with subsequent activation of
a complex intracellular cascade of events that regulate tj
permeability, it was postulated in the present invention that ZOT
may mimic the effect of a physiological modulator of mammalian tj.
It was postulated in U.S. patent application Ser. No. 08/859,931,
filed May 21, 1997, that ZOT, and its physiological analog
(zonulin), would be functionally and immunologically related.
Therefore, as described therein, affinity-purified anti-ZOT
antibodies and the Ussing chamber assay were used in combination to
search for zonulin in various rabbit and human tissues.
[0071] A. Rabbit Tissues
[0072] Initially, zonulin was purified from rabbit intestine. The
tissue was disrupted by homogenization in PBS. The resulting cell
preparations were than centrifuged at 40,000 rpm for 30 min, the
supernatant collected and lyophilized. The resulting lyophilized
product was subsequently reconstituted in PBS (10:1 (v/v)),
filtered through a 0.45 mm membrane filter, loaded onto a Sephadex
G-50 chromatographic column, and eluted with PBS. Then, 2.0 ml
fractions obtained from the column were subjected to standard
Western immunoblotting using the affinity-purified anti-ZOT
antibodies obtained as described in Example 2 above.
[0073] Positive fractions, i.e., those to which the anti-ZOT
antibodies bound, were combined, lyophilized, reconstituted in PBS
(1:1 (v/v)), and subjected to salt gradient chromatography through
a Q-Sepharose column. The salt gradient was 0-100% (w/v) NaCl in 50
mM Tris buffer (pH 8.0). Five 20 ml fractions were collected, and
subjected to standard Western immunoblotting using the
affinity-purified anti-ZOT antibodies obtained as described in
Example 2 above. Fraction 1 (20% (w/v) NaCl) was the only fraction
that was found to be positive in the Western immunoblot assay.
[0074] The fractions obtained from the Q-Sepharose column were then
tested for their tissue resistance effects on both CaCo2
monolayers, and rabbit small intestine in Ussing chambers.
[0075] More specifically, CaCo2 cells were grown in cell-culture
flasks (Falcon) under humidified atmosphere of 95% O.sub.2/5% CO,
at 37.degree. C. in Dulbecco's modified Eagle's medium containing
10% (v/v) fetal-calf serum, 40 .mu.g/l penicillin and 90 .mu.g/l
streptomycin. The cells were subcultured at a surface ratio of 1:5
after trypsin treatment every 5 days, when they had reached 70-80%
confluence. The passage number of the cells used in the this study
varied between 15 and 30.
[0076] The CaCo2 monolayers were grown to confluence (12-14 days
after plating at a 1:2.5 surface ratio) on tissue-culture-treated
polycarbonate filters firmly attached to a polystyrene ring (6.4 mm
diameter, Transwell Costar). The filters were placed in a tightly
fitting insert separating the serosal and mucosal compartment of a
modified Ussing chamber, and the experiments were carried out as
described by Fasano et al, Proc. Natl. Acad. Sci., USA, 8:5242-5246
(1991), for rabbit intestines in Ussing chambers. The results are
shown in FIG. 1.
[0077] As shown in FIG. 1, the zonulin-containing fraction induced
a significant reduction of CaCo2 monolayers' resistance, as
compared to zonulin-negative fractions.
[0078] Next, Ussing chamber assays were carried out using ileum
from 2-3 kg adult male New Zealand white rabbits, which were
sacrificed by cervical dislocation. A 20 cm segment of ileum was
removed, rinsed free of the intestinal content, opened along the
mesenteric border, and stripped of muscular and serosal layers.
Eight sheets of mucosa so prepared were then mounted in lucite
Ussing chambers (1.12 cm.sup.2 opening), connected to a voltage
clamp apparatus (EVC 4000 WPI, Saratosa, Fla.), and bathed with
freshly prepared Ringer's solution comprising 53 mM NaCl, 5.0 mM
KCl, 30.5 mM mannitol, 1.69 mM Na.sub.2HPO.sub.4, 0.3 mM
NaH.sub.2PO.sub.4, 1.25 mM CaCl.sub.2, 1.1 mM MgCl.sub.2, and 25 mM
NaHCO.sub.3. The bathing solution was maintained at 37.degree. C.
with water-jacketed reservoirs connected to a constant-temperature
circulating pump and gassed with 95% O.sub.2/5% CO.sub.2.
[0079] 100 .mu.l of zonulin purified from rabbit intestine was
added to the mucosal side. The potential difference (PD) was
measured every 10 min, and the short-circuit current (Isc) and
tissue resistance (Rt) were calculated as described by Fasano et
al, supra. Because of tissue variability, data were calculated as
.DELTA.Rt (Rt at time x)-(Rt at time 0). The results are shown in
FIG. 2.
[0080] As shown in FIG. 2, the zonulin-containing fraction induced
a significant reduction in rabbit small intestinal resistance, as
compared to a zonulin-negative fraction. This effect was completely
reversible once zonulin was withdrawn from the reservoir.
[0081] The zonulin-positive fraction was also subjected to 8.0%
(w/v) SDS-PAGE, followed by Western immunoblotting using the
anti-ZOT antibodies. The protein bands separated by SDS-PAGE were
then transferred onto PVDF filter (Millipore) using CAPS buffer
comprising 100 ml of (3-[cyclohexylamino]-1 propanesulfonic acid)
10.times., 100 ml of methanol, 800 ml of distilled water. The
protein that aligned to a single band that was detected by Western
immunoblotting had an apparent molecular weight of about 47 kDa.
This band was cut out from the PVDF filter, and subjected to
N-terminal sequencing as described by Hunkapiller, In: Methods of
Protein Microcharacterization, Ed. Shibley, Chapters 11-12, Humana
Press, pages 315-334 (1985), using a Perkin-Elmer Applied
Biosystems Apparatus Model 494. The N-terminal sequence of zonulin
purified from rabbit intestine is shown in SEQ ID NO:27.
[0082] The rabbit zonulin N-terminal sequence was compared to other
protein sequences by BLAST search analysis. The result of this
analysis revealed that the N-terminal sequence of rabbit zonulin is
85% identical, and 100% similar, to the N-terminal sequence of tau
protein from Homo sapiens.
[0083] As a result, to determine whether rabbit zonulin and tau are
the same moiety, cross-neutralization experiments were conducted in
Ussing chambers. More specifically, 10 .mu.l/ml of rabbit zonulin
was added to the mucosal side of rabbit ileum either untreated or
pre-incubated for 60 min at 37.degree. C. with anti-tau antibodies
(dilution 1:10) (Sigma). Both 10 .mu.l/ml of rabbit zonulin
pre-incubated with anti-ZOT antibodies (dilution 1:10) (Example 2);
and 0.4 .mu.g/ml of purified tau (Sigma), were used as controls.
The results are shown in FIG. 3.
[0084] As shown in FIG. 3, rabbit zonulin induced the typical
decrease of tissue resistance that was readily reversible once the
protein was withdrawn from the Ussing chambers. This activity was
completely neutralized by pre-treatment with anti-ZOT antibodies,
but not by pre-treatment with anti-tau antibodies. On the other
hand, there was no significant effect on tissue resistance in
tissues exposed to tau protein.
[0085] Rabbit zonulin was also detected in various other rabbit
tissues, i.e., rabbit heart, brain, muscle, stomach, spleen, lung,
kidney, as well as various portions of rabbits intestines, i.e.,
distal jejunum, proximal jejunum, ileum, caecum and colon. That is,
when these rabbit tissues were processed in the same manner as the
rabbit intestine, discussed above, and subjected to 8.0% (w/v)
SDS-PAGE, followed by Western immunoblotting using
affinity-purified anti-ZOT antibodies obtained as described in
Example 2 above, a single band of approximately 47 kDa in size was
detected in all of the tissues tested.
[0086] B. Human Tissues
[0087] Zonulin was also purified from several human tissues,
including intestine, heart, and brain. Both fetal and adult tissues
were used. The tissues were disrupted by homogenization in PBS. The
resulting cell preparations were than centrifuged at 40,000 rpm for
30 min, the supernatant collected and lyophilized. The resulting
lyophilized product was subsequently reconstituted in PBS (10:1
(v/v)), filtered through a 0.45 mm membrane filter, loaded onto a
Sephadex G-50 chromatographic column, and eluted with PBS. Then,
2.0 ml fractions obtained from the column were subjected to
standard Western immunoblotting using the affinity-purified
anti-ZOT antibodies obtained as described in Example 2 above.
[0088] Positive fractions, i.e., those to which the anti-ZOT
antibodies bound, were combined, lyophilized, reconstituted in PBS
(1:1 (v/v)), and subjected to salt gradient chromatography through
a Q-Sepharose column. The salt gradient was 0-100% (w/v) NaCl in 50
mM Tris buffer (pH 7.4). Five 20 ml fractions were collected, and
subjected to standard Western immunoblotting using the
affinity-purified anti-ZOT antibodies obtained as described in
Example 2 above. Fraction 1 (20% (w/v) NaCl) showed a single band
of 47 kDa in size in the Western immunoblot assay. Fraction 2 (40%
(w/v) NaCl) showed two additional bands of 35 kDa and 15 kDa in
size in the Western immunoblot assay. Fraction 3 (60% (w/v) NaCl)
and Fraction 4 (80% (w/v) showed only the 35 kDa and 15 kDa bands.
These results suggest that zonulin may be subjected to degradation
by proteases, probably present in the human tissues used, and that
the breakdown products elute from the column at higher salt
concentrations as compared to the holoprotein.
[0089] Fraction 1 (from human heart, intestine and brain tissues)
and Fraction 4 (from heart tissue) obtained from the Q-Sepharose
column were then tested for their tissue resistance effects on both
rabbit intestine and Rhesus monkey intestine in Ussing
chambers.
[0090] Ussing chamber assays were carried out using different
tracts of intestine, including jejunum, ileum, or colon from either
2-3 kg adult male New Zealand white rabbits, or 5-6 kg adult male
Rhesus monkeys. After the animals were sacrificed, different
segments of intestine, including jejunum, ileum, and colon, were
removed, rinsed free of the intestinal content, opened along the
mesenteric border, and stripped of muscular and serosal layers.
Eight sheets of mucosa so prepared (three jejunum, three ileum, and
two colon) were then mounted in lucite Ussing chambers (1.12
cm.sup.2 opening), connected to a voltage clamp apparatus (EVC 4000
WPI, Saratosa, Fla.), and bathed with freshly prepared Ringer's
solution comprising 53 mM NaCl, 5.0 mM KCl, 30.5 mM mannitol, 1.69
mM Na.sub.2HPO.sub.4, 0.3 mM NaH.sub.2PO.sub.4, 1.25 mM CaCl.sub.2,
1.1 mM MgCl.sub.2, and 25 mM NaHCO.sub.3. The bathing solution was
maintained at 37.degree. C. with water-jacketed reservoirs
connected to a constant-temperature circulating pump and gassed
with 95% O.sub.2/5% CO.sub.2.
[0091] 100 .mu.l of Fraction 1 of zonulin purified from human heart
or Fraction 1 of zonulin purified from human brain, or Fraction 1
of zonulin purified from human intestine, or Fraction 4 purified
from human heart, was added to the mucosal side. The potential
difference (PD) was measured every 10 min, and the short-circuit
current (Isc) and tissue resistance (Rt) were calculated as
described by Fasano et al, supra. Data were calculated as Rt for
FIGS. 4A and 4B; but because of tissue variability, data were
calculated as .DELTA.Rt (Rt at time x)-(Rt at time 0) for FIGS. 5A
and 5B. The results are shown in FIGS. 4A and 4B (monkey intestine)
and FIGS. 5A and 5B (rabbit intestine).
[0092] As shown in FIGS. 4A and 4B, zonulin purified from human
heart and intestine (Fraction 1) induced a significant reduction in
monkey intestinal resistance (both jejunum (FIG. 4A) and ileum
(FIG. 4B), as compared to the PBS negative control. No significant
changes were observed when zonulin purified from either human heart
or human intestine were tested in the colon. FIGS. 4A and 4B also
show that no significant effect on both monkey jejunum (FIG. 4A)
and monkey ileum (FIG. 4B) was observed when zonulin purified from
human brain (Fraction 1) was tested. Fraction 4 of zonulin purified
from human heart also induced a significant decrease in monkey
small intestinal tissue resistance.
[0093] As shown in FIGS. 5A and 5B, similar results were obtained
when rabbit intestine was used. That is, zonulin purified from
human heart (Fraction 1) showed a significant effect on tissue
resistance both in the rabbit jejunum (FIG. 5A) and rabbit ileum
(FIG. 5B), but not in the colon. FIGS. 5A and 5B also show that no
significant effect on both rabbit jejunum (FIG. 5A) and rabbit
ileum (FIG. 5B) was observed when zonulin purified from human brain
(Fraction 1) was tested.
[0094] To establish whether zonulin increases the oral delivery of
insulin, in vitro model experiments using rabbit intestine were
performed. Briefly, adult male New Zealand white rabbits (2-3 kg)
were sacrificed by cervical dislocation. Segments of rabbit small
intestine (either jejunum or ileum) were removed, rinsed free of
the intestinal content, opened along the mesenteric border, and
stripped of muscular and serosal layers. Eight sheets of mucosa so
prepared were then mounted in Lucite Ussing chambers (1.12 cm.sup.2
opening), connected to a voltage clamp apparatus (EVC 4000 WPI,
Sarasota, Fla.), and bathed with freshly prepared buffer containing
53 mM NaCl, 5.0 mM KCl, 30.5 mM mannitol, 1.69 mM
Na.sub.2HPO.sub.4, 0.3 mM NaH.sub.2PO.sub.4, 1.25 mM CaCl.sub.2,
1.1 mM MgCl.sub.2, and 25 mM NaHCO.sub.3. The bathing solution was
maintained at 37.degree. C. with water-jacketed reservoirs
connected to a constant-temperature circulating pump and gassed
with 95% O.sub.2/5% CO.sub.2. Potential difference (PD) was
measured, and short-circuit current (Isc) and tissue resistance
(Rt) were calculated. Once the tissues reached a steady state
condition, paired tissues, matched on the basis of their
resistance, were exposed luminally to 10.sup.-11 M
.sup.125I-insulin (Amersham, Arlington Heights, Ill.; 2.0
.mu.Ci=10.sup.-12 M), alone or in the presence of 100 .mu.l of
heart zonulin from Fraction 1. A 1.0 ml aliquot from the serosal
side and a 50 .mu.l aliquot from the mucosal side were immediately
obtained to establish baseline values. Samples from the serosal
side were then collected at 20 min intervals for the following 100
min.
[0095] It was found that heart zonulin increased the intestinal
absorption of insulin both in the jejunum (0.058.+-.0.003
fmol/cm.sup.2min vs. 0.12.+-.0.005 fmol/cm.sup.2min, untreated vs.
zonulin-treated tissues, respectively, p=0.001), and in the ileum
(0.006.+-.0.0002 fmol/cm.sup.2min vs. 0.018.+-.0.005
fmol/cm.sup.2min, untreated vs. zonulin-treated tissues,
respectively, p=0.05) in a time-dependent manner.
[0096] Fraction 1 of zonulin purified from human heart, Fraction 1
of zonulin purified from human intestine, and Fraction 1 of zonulin
purified from human brain were also subjected to 8.0% (w/v)
SDS-PAGE, followed by Western immunoblotting using the anti-ZOT
antibodies obtained as described in Example 2 above. The protein
bands separated by SDS-PAGE were then transferred onto PVDF filter
using CAPS buffer comprising 100 ml of (3-[cyclohexylamino]-1
propanesulfonic acid) 10.times., 100 ml of methanol, 800 ml of
distilled water. The protein that aligned to a single band that was
detected by Western immunoblotting had an apparent molecular weight
of about 47 kDa. This band was cut out from the PDVF filter, and
subjected to N-terminal sequencing as described by Hunkapiller, In:
Methods of Protein Microcharacterization, Ed. Shibley, Chapters
11-12, Humana Press, pages 315-334 (1985), using a Perkin-Elmer
Applied Biosystems Apparatus Model 494. The N-terminal sequence of
zonulin purified from adult human heart is shown in SEQ ID NO:28,
the N-terminal sequence of zonulin purified from adult human brain
is shown in SEQ ID NO:29, and the N-terminal sequence of zonulin
purified from adult fetal brain is shown in SEQ ID NO:36.
[0097] The first nine amino acids from the N-terminal sequence of
zonulin purified from adult human intestine (SEQ ID NO:31) were
also sequenced, and found to be identical to the first nine amino
acids of zonulin purified from human heart shown in SEQ ID NO:28
(see FIG. 6). The first twenty amino acids from the N-terminal
sequence of zonulin purified from human fetal intestine were also
sequenced: Met Leu Gln Lys Ala Glu Ser Gly Gly Val Leu Val Gln Pro
Gly Xaa Ser Asn Arg Leu (SEQ ID NO:30), and found to be almost
identical to the amino acid sequence of zonulin purified from human
heart shown in SEQ ID NO:28 (see FIG. 6).
[0098] The N-terminal sequence of zonulin purified from adult human
brain (SEQ ID NO:29) and fetal human brain (SEQ ID NO:36) was
completely different than the N-terminal of zonulin purified from
each of heart (SEQ ID NO:28), fetal intestine (SEQ ID NO:30) and
adult intestine (SEQ ID NO:31) (see FIGS. 6-7). This difference is
believed to explain the tissue-specificity of zonulin in
determining the permeability of tissues, such as the intestine,
demonstrated above.
[0099] The N-terminal sequences of human zonulin purified from
heart, intestine, and brain, all differ from the N-terminal
sequence of zonulin purified from rabbit intestine (FIG. 6). To
establish whether these proteins represent different isoforms of a
tau-related family of proteins, tissues from both rabbit and human
were subjected to 8.0% (w/v) SDS-PAGE, followed by Western
immunoblotting using either anti-ZOT or anti-tau antibodies. The 47
kDa zonulin bands purified from both rabbit and human tissues
(including brain, intestine, and heart) which were found to be
recognized by the anti-ZOT antibodies, were also found to
cross-react with anti-tau antibodies. The different fractions of
zonulin purified from human brain obtained by salt chromatography
were also subjected to Western immunoblotting using either anti-ZOT
antibodies or anti-tau antibodies. While anti-ZOT antibodies
recognized the intact 47 kDa protein and both of the 35 kDa and 15
kDa breakdown fragments, the anti-tau antibodies only recognized
the intact 47 kDa protein and the 35 kDa fragment, while the
anti-tau antibodies did not recognize the 15 kDa fragment. To
establish whether the 35 kDa fragment includes the N-terminus or
the C-terminus of zonulin, the N-terminal sequence of the 35 kDa
band was obtained and found to be: Xaa Xaa Asp Gly Thr Gly Lys Val
Gly Asp Leu (SEQ ID NO:32). This sequence is different from the
N-terminal sequence of the intact human brain zonulin (SEQ ID
NO:29). These results suggest that the 15 kDa fragment represents
the N-terminal portion of zonulin, while the 35 kDa fragment
represents the C-terminal portion of zonulin.
[0100] Combined together, these results suggest that the zonulin
domain recognized by the anti-tau antibodies is toward the
C-terminus of the protein, is common to the different isoforms of
zonulin from either human or rabbit tissues (while the N-terminal
portion may vary), and is probably involved in the permeabilizing
effect of the protein (based on the observation that tau binds to
.beta.-tubulin with subsequent rearrangement of the cell
cytoskeleton, and the effect of Fraction 4 on monkey small
intestinal tissue resistance).
[0101] The N-terminal sequence of human zonulin purified from both
the heart and intestine was compared to other protein sequences by
BLAST search analysis. The result of this analysis revealed that
the N-terminal sequence of human zonulin is 95% identical, to the
N-terminal sequence of the heavy variable chain of IgM from Homo
sapiens (SEQ ID NO:37).
[0102] As a result, to determine whether human zonulin purified
from heart and human IgM are the same moiety, a partial digestion
of the human zonulin was performed to obtain an internal fragment,
which was then sequenced.
[0103] More specifically, 1.0 mm of the PVDF filter containing
zonulin purified from human heart was placed in a plastic tube
previously washed with 0.1% (w/v) trifluoracetic acid (TFA), and
rinsed with methanol. 75 .mu.l of a buffer solution comprising 100
mM Tris (pH 8.2), 10% (v/v) CH.sub.3CN, and 1.0% (v/v)
dehydrogenated Triton X-100 was added, and incubated with the
membrane at 37.degree. C. for 60 min. 150 ng of trypsin was then
added, and an additional 24 hr incubation period at 37.degree. C.
was carried out. The resulting solution was sonicated for 10 min,
and the supernatant decanted. 75 .mu.l of 0.1% (w/v) TFA was then
added, the solution was sonicated for additional 10 min, and the
supernatant decanted. Both aliquots were loaded on a 0.5
mm.times.250 mm C.sub.18 column, 5.0 .mu.m particle size, 300 .ANG.
pore size. A gradient from 0.1% (w/v) TFA to 45% CH.sub.3CN
water+0.1% (w/v) TFA was developed for 2 hr and 15 min. The peaks
were finally collected and sequenced.
[0104] The internal sequence of human zonulin purified from adult
human heart was found to be: Leu Ser Glu Val Thr Ala Val Pro Ser
Leu Asn Gly Gly (SEQ ID NO:33).
[0105] The human zonulin internal sequence was compared to other
protein sequences by BLAST search analysis. The result of this
analysis revealed that the internal sequence of human zonulin has
0% identity to any internal sequence of the heavy variable chain of
IgM from Homo sapiens.
[0106] The results in Example 3 above demonstrate that (1) zonulin
represents the physiological modulator of the paracellular pathway;
(2) the N-terminal sequence of rabbit zonulin is highly homologous
to the N-terminal sequence of the tau protein; (3) zonulin and tau
are two distinct moieties that are immunologically related, yet
functionally different; (4) the N-terminal sequence of human
zonulin obtained from heart and intestine is highly homologous to
the N-terminal sequence of the heavy chain of the variable region
of IgM; (5) human zonulin and IgM are two distinct moieties that
are structurally related, yet functionally different; and (6)
zonulin represents a family of tau-related proteins with common,
active C-terminal sequences, and variable N-terminal sequences.
Example 4
Peptide Antagonists of Zonulin
[0107] Given that ZOT, human intestinal zonulin (zonulin.sub.i) and
human heart zonulin (zonulin.sub.h) all act on intestinal (Fasano
et al, Gastroenterology, 112:839 (1997); Fasano et al, J. Clin.
Invest., 96:710 (1995); and FIGS. 1-5) and endothelial tj and that
all three have a similar regional effect (Fasano et al (1997),
supra; and FIGS. 1-5) that coincides with the ZOT receptor
distribution within the intestine (Fasano et al (1997), supra; and
Fasano et al (1995), supra), it was postulated in the present
invention that these three molecules interact with the same
receptor binding site. A comparison of the primary amino acid
structure of ZOT and the human zonulins was thus carried out to
provide insights as to the absolute structural requirements of the
receptor-ligand interaction involved in the regulation of
intestinal tj. The analysis of the N-termini of these molecules
revealed the following common motif (amino acid residues 8-15 boxed
in FIG. 7): non-polar (Gly for intestine, Val for brain), variable,
non-polar, variable, non-polar, polar, variable, polar (Gly). Gly
in position 8, Val in position 12 and Gln in position 13, all are
highly conserved in ZOT, zonulin.sub.i and zonulin.sub.h (see FIG.
7), which is believed to be critical for receptor binding function
within the intestine. To verify the same, the synthetic octapeptide
Gly Gly Val Leu Val Gln Pro Gly (SEQ ID NO:15) (named FZI/0, and
corresponding to amino acid residues 8-15 of human fetal
zonulin.sub.i) was chemically synthesized.
[0108] Next, rabbit ileum mounted in Ussing chambers as described
above, were exposed to 100 .mu.g of FZI/0 (SEQ ID NO:15), 100 .mu.g
of FZI/1 (SEQ ID NO:34), 1.0 .mu.g of 6.times.His-ZOT (obtained as
described in Example 1), 1.0 .mu.g of zonulin.sub.i (obtained as
described in Example 3), or 1.0 .mu.g of zonulin.sub.h (obtained as
described in Example 3) alone; or pre-exposed for 20 min to 100
.mu.g of FZI/0 or FZI/1, at which time 1.0 .mu.g of
6.times.His-ZOT, 1.0 .mu.g of zonulin.sub.i, or 1.0 .mu.g of
zonulin.sub.h, was added. .DELTA.Rt was then calculated as
described above. The results are shown in FIG. 8.
[0109] As shown in FIG. 8, FZI/0 did not induce any significant
change in Rt (0.5% as compared to the negative control) (see closed
bar). On the contrary, pre-treatment for 20 min with FZI/0
decreased the effect of ZOT, zonulin.sub.i, and zonulin.sub.h on Rt
by 75%, 97%, and 100%, respectively (see open bar). Also as shown
in FIG. 8, this inhibitory effect was completely ablated when a
second synthetic peptide (FZI/1) was chemically synthesized by
changing the Gly in position 8, the Val in position 12, and the Gln
in position 13 (as referred to zonulin.sub.i) with the
correspondent amino acid residues of zonulin.sub.b (Val, Gly, and
Arg, respectively) was used (see shaded bar).
[0110] The above results demonstrate that there is a region
spanning between residue 8 and 15 of the N-terminal end of ZOT and
the zonulin family that is crucial for the binding to the target
receptor, and that the amino acid residues in position 8, 12, and
13 determine the tissue specificity of this binding.
[0111] While the invention has been described in detail, and with
reference to specific embodiments thereof, it will be apparent to
one of ordinary skill in the art that various changes and
modifications can be made therein without departing from the spirit
and scope thereof.
Sequence CWU 1
1
4018PRTArtificial SequenceZonulin Peptide Antagonist 1Gly Arg Val
Cys Val Gln Pro Gly1 528PRTArtificial SequenceZonulin Peptide
Antagonist 2Gly Arg Val Cys Val Gln Asp Gly1 538PRTArtificial
SequenceZonulin Peptide Antagonist 3Gly Arg Val Leu Val Gln Pro
Gly1 548PRTArtificial SequenceZonulin Peptide Antagonist 4Gly Arg
Val Leu Val Gln Asp Gly1 558PRTArtificial SequenceZonulin Peptide
Antagonist 5Gly Arg Leu Cys Val Gln Pro Gly1 568PRTArtificial
SequenceZonulin Peptide Antagonist 6Gly Arg Leu Cys Val Gln Asp
Gly1 578PRTArtificial SequenceZonulin Peptide Antagonist 7Gly Arg
Leu Leu Val Gln Pro Gly1 588PRTArtificial SequenceZonulin Peptide
Antagonist 8Gly Arg Leu Leu Val Gln Asp Gly1 598PRTArtificial
SequenceZonulin Peptide Antagonist 9Gly Arg Gly Cys Val Gln Pro
Gly1 5108PRTArtificial SequenceZonulin Peptide Antagonist 10Gly Arg
Gly Cys Val Gln Asp Gly1 5118PRTArtificial SequenceZonulin Peptide
Antagonist 11Gly Arg Gly Leu Val Gln Pro Gly1 5128PRTArtificial
SequenceZonulin Peptide Antagonist 12Gly Arg Gly Leu Val Gln Asp
Gly1 5138PRTArtificial SequenceZonulin Peptide Antagonist 13Gly Gly
Val Cys Val Gln Pro Gly1 5148PRTArtificial SequenceZonulin Peptide
Antagonist 14Gly Gly Val Cys Val Gln Asp Gly1 5158PRTArtificial
SequenceSynthetic Octapeptide 15Gly Gly Val Leu Val Gln Pro Gly1
5168PRTArtificial SequenceZonulin Peptide Antagonist 16Gly Gly Val
Leu Val Gln Asp Gly1 5178PRTArtificial SequenceZonulin Peptide
Antagonist 17Gly Gly Leu Cys Val Gln Pro Gly1 5188PRTArtificial
SequenceZonulin Peptide Antagonist 18Gly Gly Leu Cys Val Gln Asp
Gly1 5198PRTArtificial SequenceZonulin Peptide Antagonist 19Gly Gly
Leu Leu Val Gln Pro Gly1 5208PRTArtificial SequenceZonulin Peptide
Antagonist 20Gly Gly Leu Leu Val Gln Asp Gly1 5218PRTArtificial
SequenceZonulin Peptide Antagonist 21Gly Gly Gly Cys Val Gln Pro
Gly1 5228PRTArtificial SequenceZonulin Peptide Antagonist 22Gly Gly
Gly Cys Val Gln Asp Gly1 5238PRTArtificial SequenceZonulin Peptide
Antagonist 23Gly Gly Gly Leu Val Gln Pro Gly1 5248PRTArtificial
SequenceZonulin Peptide Antagonist 24Gly Gly Gly Leu Val Gln Asp
Gly1 52518DNAArtificial SequenceSynthetic Construct 25tcatcacggc
gcgccagg 182622DNAArtificial SequenceSynthetic Construct
26ggaggtctag aatctgcccg at 222718PRTUnknownRabbit intestine 27Asn
Gln Arg Pro Pro Pro Ala Gly Val Thr Ala Tyr Asp Tyr Leu Val1 5 10
15Ile Gln2820PRTHomo sapiens 28Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu 20299PRTHomo
sapiens 29Val Thr Phe Tyr Thr Asp Ala Val Ser1 53020PRTHomo
sapiensmisc_feature(16)..(16)Xaa can be any naturally occurring
amino acid 30Met Leu Gln Leu Ala Glu Ser Gly Gly Val Leu Val Gln
Pro Gly Xaa1 5 10 15Ser Asn Arg Leu 203111PRTHomo
sapiensmisc_feature(10)..(10)Xaa can be any naturally occurring
amino acid 31Glu Val Gln Leu Val Glu Ser Gly Gly Xaa Leu1 5
103211PRTHomo sapiensmisc_feature(1)..(2)Xaa can be any naturally
occurring amino acid 32Xaa Xaa Asp Gly Thr Gly Leu Val Gly Asp Leu1
5 103313PRTHomo sapiens 33Leu Ser Glu Val Thr Ala Val Pro Ser Leu
Asn Gly Gly1 5 10348PRTArtificial SequenceSynthetic octapeptide
34Val Gly Val Leu Gly Arg Pro Gly1 5358PRTArtificial
SequenceZonulin Peptide Antagonist 35Val Asn Gly Phe Gly Arg Ile
Gly1 53622PRTHomo sapiensmisc_feature(1)..(1)Xaa can be any
naturally occurring amino acid 36Xaa Gly Lys Val Lys Val Gly Val
Asn Gly Phe Gly Arg Ile Gly Arg1 5 10 15Ile Gly Arg Leu Val Ile
203720PRTHomo sapiens 37Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu 203814PRTUnknownZonulin
Occludes Toxin 38Phe Cys Ile Gly Arg Leu Cys Val Gln Asp Gly Phe
Val Thr1 5 103923DNAArtificial SequenceSynthetic Construct
39cgggatcccg tatgagtatc ttt 234024DNAArtificial SequenceSynthetic
Construct 40cccaagcttg ggtcaaaata tact 24
* * * * *