U.S. patent application number 12/545606 was filed with the patent office on 2010-01-21 for heparin binding motif and use thereof.
This patent application is currently assigned to NATIONAL TSING HUA UNIVERSITY. Invention is credited to Margaret Dah-Tsyr Chang, Tan-chi Fan, Shu-Chuan Lin.
Application Number | 20100016239 12/545606 |
Document ID | / |
Family ID | 41109804 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100016239 |
Kind Code |
A1 |
Chang; Margaret Dah-Tsyr ;
et al. |
January 21, 2010 |
HEPARIN BINDING MOTIF AND USE THEREOF
Abstract
A method for reducing cytotoxicity of eosinophil derived toxins
comprising administering to a subject an effective amount of
heparin, heparan sulfate, a potent heparanase inhibitor or a
polypeptide which has sequence as follows: BZBXBX, XBBBXXBX,
XBBXBX, BBXXBBBXXBB, BXBBXB, XBBBXXBBBXXBBX, or TXXBXXTBXXXTBB,
wherein X represents any amino acid, Z represents an aromatic amino
acid, and B represents a basic amino acid and T represents a
turn.
Inventors: |
Chang; Margaret Dah-Tsyr;
(Hsinchu, TW) ; Fan; Tan-chi; (Hsinchu, TW)
; Lin; Shu-Chuan; (Taipei County, TW) |
Correspondence
Address: |
WPAT, PC;INTELLECTUAL PROPERTY ATTORNEYS
2030 MAIN STREET, SUITE 1300
IRVINE
CA
92614
US
|
Assignee: |
NATIONAL TSING HUA
UNIVERSITY
Hsinchu
TW
|
Family ID: |
41109804 |
Appl. No.: |
12/545606 |
Filed: |
August 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12125008 |
May 21, 2008 |
7595374 |
|
|
12545606 |
|
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Current U.S.
Class: |
514/1.1 ;
530/329 |
Current CPC
Class: |
C12N 9/22 20130101; C07K
14/47 20130101; A61P 11/06 20180101 |
Class at
Publication: |
514/14 ; 530/329;
514/17; 514/16; 514/15 |
International
Class: |
A61K 38/08 20060101
A61K038/08; C07K 7/06 20060101 C07K007/06; A61K 38/10 20060101
A61K038/10; A61P 11/06 20060101 A61P011/06 |
Claims
1. A heparin binding motif comprising BZBXBX, wherein X represents
any amino acid, Z represents an aromatic amino acid and B
represents a basic amino acid, provided that the motif excludes SEQ
ID NO. 8.
2. The heparin binding motif of claim 1, which is derived from
eosinophil cationic protein comprises the sequence of claim 1.
3. A method for reducing cytotoxicity of eosinophil derived toxins
comprising administering to a subject an effective amount of
heparin, heparan sulfate, potent heparanase inhibitor or a
polypeptide which has sequence as follows: BZBXBX, XBBBXXBX,
XBBXBX, BBXXBBBXXBB, BXBBXB, XBBBXXBBBXXBBX, or TXXBXXTBXXXTBB,
wherein X represents any amino acid, Z represents an aromatic amino
acid, B represents a basic amino acid and T represents a turn.
4. The method of claim 3, wherein the polypeptide comprising a high
portion of positively charged residues.
5. The method of claim 3, wherein the polypeptide comprises QRRCKN
(SEQ ID NO:7) or RWRCKN (SEQ ID NO:8).
6. The method of claim 3, wherein the polypeptide is SEQ ID NO: 3
(NYRWRCKNQNK) or SEQ ID NO: 4 (NYQRRCKNQNK).
7. The method of claim 3, wherein the heparin comprises low
molecular weight heparin (LMWH), high molecular weight heparin
(HMWH), heparan sulfate proteoglycans (HSPG) or synthetic heparin
oligosaccharides and heparan sulfates.
8. The method of claim 7, wherein the synthetic heparin
oligosaccharides comprises degree of polymerization (dp) 3 to
15.
9. The method of claim 7, wherein the synthetic heparan sulfates
comprises degree of polymerization (dp) 5 to 9.
10. The method of claim 3, wherein the cytotoxicity of eosinophil
derived toxins is reduced by reducing endocytosis of eosinophil
derived toxins.
11. The method of claim 10, wherein the eosinophil derived toxins
is eosinophil cationic protein (ECP), eosinophil-derived neurotoxin
(EDN), eosinophil peroxidase (EPO, also called EPX), or major basic
protein (MBP).
12. The method of claim 3, wherein the subject is mammalian.
13. The method of claim 3, which further inhibits asthma.
14. The method of claim 13, wherein the asthma is ECP/EDN-induced
asthma, cytotoxic RNase-induced asthma or high pI toxin-induced
asthma.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of the pending U.S. patent
application Ser. No. 12/125,008 filed on May 21, 2009, is hereby
incorporated by reference in its entirety.
[0002] Although incorporated by reference in its entirety, no
arguments or disclaimers made in the parent application apply to
this divisional application. Any disclaimer that may have occurred
during the prosecution of the above-referenced application(s) is
hereby expressly rescinded. Consequently, the Patent Office is
asked to review the new set of claims in view of the entire prior
art of record and any search that the Office deems appropriate.
FIELD OF THE INVENTION
[0003] The present invention relates to a heparin binding motif of
eosinophil toxins and use thereof.
BACKGROUND OF THE INVENTION
[0004] Eosinophil cationic protein (ECP), a member of the
ribonuclease A (RNase A) superfamily, is found in the specific
granules of eosinophilic leukocytes. It is a single polypeptide
with a molecular mass ranging from 16 to 21.4 kDa due to varying
degrees of glycosylation. It shows a 67% amino acid sequence
identity with eosinophil-derived neurotoxin (EDN), another
eosinophil-secreted RNase. Although ECP shares the overall
three-dimensional structure of RNase A, it has relatively lower
RNase activity (Boix, E., et al. (1999) Journal of Biological
Chemistry 274, 15605-15614). ECP released by activated eosinophils
contributes to the toxicity against helminth parasites, bacteria,
and single-strand RNA viruses (Lehrer, R., et al. (1989) Journal of
Immunology 142, 4428-4434; Domachowske, J. B., et al. (1998)
Nucleic acids research 26, 3358-3363). Together with other proteins
secreted from eosinophils such as EDN, eosinophil peroxidase (EPO;
also EPX), and major basic protein (MBP), ECP is thought to cause
damage to epithelial cells, a common feature of airway inflammation
in asthma (Gleich, G. J. (2000) Journal of Allergy and Clinical
Immunology 105, 651-663).
[0005] The mechanism underlying the cytotoxic property of ECP is
unclear. It has been hypothesized that ECP cytotoxicity is due to
destabilization of lipid membranes of target cells (Young, J., et
al. (1986) Nature 321, 613-616), and the degree of cytotoxicity is
dependent on the cellular concentration (Carreras, E., et al.
(2005) Molecular and Cellular Biochemistry 272, 1-7). The binding
of ECP to target cells has been attributed to its high arginine
content (estimated pI=10.8), which facilitates the interaction
between ECP and negatively charged molecules on the cell surface
(Carreras, E., et al. (2005) Molecular and Cellular Biochemistry
272, 1-7; Carreras, E., et al. (2003) Biochemistry 42, 6636-6644).
Recently, we found that binding and endocytosis of ECP into
bronchial epithelial cells were greatly dependent on the cell
surface glycosaminoglycan (GAG), specifically heparan sulfate
proteoglycans (HSPG) (Fan, T. C., et al. (2007) Traffic 8,
1778-1795). The cytotoxicity of ECP was severely reduced toward
cell lines with heparan sulfate (HS) deficiency.
[0006] Heparin and HS are complex polysaccharides composed of
alternating units of hexuronic acid and glucosamine. The uronic
acid residues of heparin typically consist of 90%
L-idopyranosyluronic acid and 10% D-glucopyranosyluronic acid
(Capila, I. and Linhardt, R. J. (2002) Angewandte Chemie
International Edition 41, 391-412). The N position of glucosamine
may be substituted with an acetyl or sulfate group. The 3-O and 6-O
positions of glucosamine and the 2-O of uronic acid may be
sulfated. Through the combination of different negatively charged
moieties, heparin and HS have been demonstrated to bind a variety
of proteins with diverse functions, including growth factors,
thrombin, chemokines and viral proteins. The HS chains contain
domains with a high level of sulfation and epimerization
(S-domains), regions with mixed N-acetylation and N-sulfation
(NA/S-domains), and unmodified domains that are mostly N-acetylated
and contain little sulfate (Tumova, S., et al. (2000) The
international journal of biochemistry & cell biology 32,
269-288). Because HS chains contain heparin regions, heparin and
its mimetics can be used to study interactions between proteins and
polysaccharides.
[0007] The structure of ECP has been determined and refined to a
resolution up to 1.75 .ANG., displaying a folding topology that
involves three .alpha. helices and five .beta. strands
(Mallorqui-Fernandez, G., et al. (2000) Journal of Molecular
Biology 300, 1297-1307). The most interesting feature is the 19
surface-oriented arginine residues, conferring a strong basic
character to ECP. However, the heparin binding site in ECP has not
been identified. Heparin binding domains within proteins usually
contain a high proportion of positively charged residues, which
bind to the acidic groups of heparin through electrostatic
interactions. It has been proposed that the three-dimensional
structure of the HS chain is critical for protein binding (Hileman,
R. E., et al. (1998) BioEssays 20, 156-167). However, not much is
known about the three-dimensional structure of HS. After examining
a series of heparin-binding protein sequences, Cardin and Weintraub
proposed that the pattern XBBBXXBX or XBBXBX (where X represents
hydrophobic or uncharged amino acids, and B represents basic amino
acids) is responsible for HS binding to other proteins (Cardin, A.
D. and Weintraub, H. J. (1989) Arteriosclerosis (Dallas, Tex. 9,
21-32). In addition, the following sequences have also been
reported to serve as heparin binding motifs.
[0008] BBXXBBBXXBB (where B is a positively charge residue
(arginine, lysine or hystidine) and X is any residue) (Olenina, L.
V., et al. (2005) J Viral Hepat 12, 584-593).
[0009] BXXBBXB (where B is a basic residue and X is any residue)
(Wu, H. F., et al. (1995) Blood 85, 421-428).
[0010] XBBBXXBBBXXBBX (where B is a basic residue and X is any
residue) (Andersson, E., et al. (2004) Eur J Biochem 271,
1219-1226; Sobel, M., et al. (1992) The Journal of biological
chemistry 267, 8857-8862).
[0011] TXXBXXTBXXXTBB (where B is a basic residue, X is any
residue, and T defines a turn) (Capila, I. and Linhardt, R. J.
(2002) Angewandte Chemie International Edition 41, 391-412;
Hileman, R. E., et al. (1998) BioEssays 20, 156-167).
SUMMARY OF THE INVENTION
[0012] The present invention provides a heparin binding motif
comprising BZBXBX, wherein X represents any amino acid, Z
represents an aromatic amino acid and B represents a basic amino
acid.
of claim 1.
[0013] The present invention further provides a method for reducing
cytotoxicity of eosinophil derived toxins comprising administering
to a subject an effective amount of heparin, heparan sulfate,
potent heparanase inhibitor or a polypeptide which has sequence as
follows: BZBXBX, XBBBXXBX, XBBXBX, BBXXBBBXXBB, BXBBXB,
XBBBXXBBBXXBBX, or TXXBXXTBXXXTBB, wherein X represents any amino
acid, Z represents an aromatic amino acid, B represents a basic
amino acid and T represents a turn
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. The structure of synthetic heparin oligosaccharides
used in the present invention. n=1-4 (n=1, dp3; n=2, dp5; n=3, dp7;
n=4, dp9).
[0015] FIG. 2. FACE analysis to evaluate binding of ECP to various
sugar-containing substrates. A, AMAC-labeled LMWH was incubated
with or without ECP in PBS for 10 min at 25.degree. C., and the
binding reaction products were separated on a 1% agarose gel. The
numbers above the gel indicate the molar ratio of ECP to LMWH. B,
Representative oligosaccharide binding pattern of ECP. AMAC-labeled
heparin dp3 (10.8 nmol), dp5 (3.3 nmol), dp7 (1.4 nmol), dp9 (1.1
nmol), LMWH (0.03 nmol) and PI-88 (0.03 nmol) were incubated with
or without 10-fold molar excess of ECP, and the binding reaction
products separated on a 1% agarose gel. C, Representative
oligosaccharide binding pattern of EDN. AMAC-labeled heparin dp3
(10.8 nmol), dp5 (3.3 nmol), dp7 (1.4 nmol), dp9 (1.1 nmol), LMWH
(0.03 nmol) and PI-88 (0.03 nmol) were incubated with or without
10-fold molar excess of ECP, and the binding reaction products
separated on a 1% agarose gel.
[0016] FIG. 3. Heparin oligosaccharides inhibit ECP binding to
cells. Beas-2B cells were preincubated with heparin of different
sizes in RPMI 1640 medium for 30 min at 4.degree. C. before
incubation with MBP-ECP for an additional 1 h. After treatment, the
cells were washed with PBS and fixed with PFA. The level of bound
MBP-ECP was assessed by ELISA. The amount of MBP-ECP bound to cells
without GAG treatment was set to 100%. C, control (cells were
incubated with MBP.) The data shown are the means of triplicate
experiments.
[0017] FIG. 4. Identification of the heparin binding site in ECP.
A, Alignment of human and bovine RNases. The sequences between
.alpha.2 and .beta.1 of the RNases are aligned. Regions in other
RNases that are highly similar to the putative heparin binding site
of ECP are boxed. Positively charged amino acids are indicated in
bold. B, Beas-2B cells were incubated with MBP-ECP for 1 h at
4.degree. C. The amount of MBP-ECP bound to cells was assessed as
for FIG. 3.
[0018] FIG. 5. ITFE profiles of ECP wt and mt1 with dp5. Tryptophan
titration emission spectra of 0.2 mM ECP wt or mt1 bound with dp5.
The emission spectra at 340 nm were recorded. The resulting
isotherms were fitted by nonlinear regression least-squares
computer fit using the KaleidaGraph Synergy Software. AF, the
relative fluorescence change, equals F.sub.0-F.sub.obs, where
F.sub.0 and F.sub.obs represent the initial and observed
fluorescence values, respectively.
[0019] FIG. 6. CD spectra of ECP wt and mt1. The CD spectra were
scanned from 200 to 260 nm.
[0020] FIG. 7. Synthetic peptides inhibit ECP binding to cells. A,
Beas-2B cells were preincubated with peptides in RPMI 1640 medium
for 30 min at 4.degree. C. before incubation with MBP-ECP for an
additional 1 h. After treatment, the cells were washed with PBS and
fixed with PFA. The level of bound MBP-ECP was assessed by ELISA.
The amount of MBP-ECP bound to cells without peptide treatment was
set to 100%. The data shown are the means of triplicate
experiments. B, Beas2-B cells were incubated with biotinylated
peptides for 1 h at 4.degree. C. The amount of peptides bound to
cells was assessed as for FIG. 3.
[0021] FIG. 8. Heparin-binding synthetic peptides. Human Beas-2B
cells were incubated with synthetic peptide (A, TAT; B, D1) at
4.degree. C. for 30 min and then washed, fixed and analyzed.
Synthetic peptide was identified with mouse anti-biotin monoclonal
antibody and FITC-conjugated goat anti-mouse antibody. The
distribution of synthetic peptide was examined by confocal
microscopy. Scale bar, 10 mm.
[0022] FIG. 9. FACE analysis of synthetic peptides. AMAC-labeled
LMWH were incubated without or with increasing concentrations of
C1, TAT, D1 and R1 peptides at room temperature for 15 min. Samples
were loaded and separated by agarose gel electrophoresis as
described for FIG. 2.
[0023] FIG. 10. Titration profiles of synthetic peptides by
synthetic heparin oligosaccharides. Titrations were carried out on
1 .mu.M peptides in the presence of heparin oligosaccharides. The
K.sub.d of each peptide-oligosaccharide interaction was determined
by measuring the fluorescence change of intrinsic tryptophan at 340
nm. Data were fitted to a single-binding-site curve using nonlinear
least-squares analysis.
[0024] FIG. 11. Isothermal titration calorimetry of heparin-peptide
interaction. The top panels show the differential power time
course. Raw data for sequential 6-.mu.l injections of each
synthetic peptide in PBS buffer at 25.degree. C., TAT (positive
control), B, R1 (negative control), C, C1, and D, D1 into the
sample cell containing 1.4 ml HMWH solution (10-20 .mu.M). The
total heat released in each injection is proportional to the area
under the corresponding peak. The lower panels show a fit of the
integrated areas based on peptide binding. The solid line
represents a non-linear least squares of the reaction heat for the
injection.
[0025] FIG. 12. Cytotoxicity of wild-type and mutant ECP. Beas-2B
cells were incubated with increasing concentrations of wild-type
and mt1 ECP at 37.degree. C. for 48 h, followed by the MTT assay.
The error bars show standard deviation among triplicate
experiments.
[0026] FIG. 13. Immunohistochemical localization of ECP using the
Super Sensitive Non-Biotin HRP Detection System. Representative
immunohistochemical staining patterns. ECP (red color) was detected
in tracheo-epithelial cells (arrow) and cartilage cells 1 hr
post-iv injection (A, B). Decreased ECP signal was observed in
tracheo-epithelial cells when ECP was co-injected with heparin (C).
A control section of lung tissue subjected to MBP injection was
used as a negative control. Magnification: A, 200.times.; B, C, and
D, 400.times.. Scale bars: 20 .mu.m.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The term "eosinophil derived toxins" used herein means the
toxins derived from eosinophil and damage cells when the toxins are
entered into the cells. The species of eosinophil derived toxins
include but is not limited to eosinophil cationic protein (ECP),
eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO,
also called EPX), and major basic protein (MBP).
[0028] In the present invention, a linear heparin binding site on
ECP and the shortest ECP-binding heparin oligosaccharide unit have
been identified. Loop L3 of ECP mediates the interaction between
ECP and cell surface HSPG, contributing to ECP cytotoxicity.
Furthermore, the dissociation constants between oligosaccharides
and ECP were determined by tryptophan emission titration.
[0029] Accordingly, the present invention provides a heparin
binding motif comprising BZBXBX, wherein X represents any amino
acid, Z represents an aromatic amino acid and B represents a basic
amino acid. The present invention also provides a heparin binding
motif of eosinophil cationic protein comprising the above sequence.
In the more embodiment of the present invention, the said motif is
SEQ ID NO: 8.
[0030] The present invention further provides a method for reducing
cytotoxicity of eosinophil derived toxins comprising administering
to a subject an effective amount of heparin, heparan sulfate,
potent heparanase inhibitor or a polypeptide which has sequence as
follows: BZBXBX, XBBBXXBX, XBBXBX, BBXXBBBXXBB, BXBBXB,
XBBBXXBBBXXBBX, or TXXBXXTBXXXTBB, wherein X represents any amino
acid, Z represents an aromatic amino acid, B represents a basic
amino acid and T represents a turn. The said subject is mammalian.
The said polypeptide comprises a high portion of positively charged
residues. In the embodiment of the present invention, the
polypeptide comprises QRRCKN (SEQ ID NO:7) or RWRCKN (SEQ ID NO:8).
In the more embodiment of the present invention, the polypeptide is
SEQ ID NO: 3 (NYRWRCKNQNK) or SEQ ID NO: 4 (NYQRRCKNQNK). The said
heparin comprises low molecular weight heparin (LMWH,
Sigma-Aldrish, average MW 3,000), high molecular weight heparin
(HMWH, Sigma-Aldrish, average MW 16,000), heparan sulfate
proteoglycans (HSPG) or synthetic heparin oligosaccharides and
heparan sulfates from the degree of polymerization (dp) of 3 to 15.
In the embodiment of the present invention, the synthetic heparan
sulfates comprises degree of polymerization (dp) 5 to 9 (dp 3 to dp
9: MW 926-2,642; Molecular formula: P+nQ where P is a 2
methoxy-glucosamine, Q represents disaccharide units consisted of
hexuronic acid and glucosamine, and n is an integral. The molecular
weight of X is 349 and that of Y ranges from 333 to 573.
Furthermore, the glucosamine of P could be substituted by other
protecting group).
[0031] The cytotoxicity of eosinophil derived toxins is reduced by
reducing endocytosis of eosinophil derived toxins such as ECP, EDN,
EPO, and MBP. The method can further inhibit asthma. The said
asthma includes ECP/EDN-induced asthma, cytotoxic RNase-induced
asthma and high pI toxin-induced asthma.
EXAMPLE
Example 1
Materials
[0032] Mouse anti-MBP (maltose binding protein) was obtained from
Santa Cruz Biotechnology (Santa Cruz, Calif.). RNase A was
purchased from Promega (Madison, Wis.). Chemicals were purchased
from Sigma-Aldrich (St. Louis, Mo.) unless otherwise specified.
Human ECP peptide NYRWRCKNQNK-biotin (C1, SEQ ID NO: 3), EDN
peptide NYQRRCKNQNK-biotin (D1, SEQ ID NO: 4), RNase1 peptide
MTQGRCKPVNK-biotin (R1, SEQ ID NO: 5), and HIV-TAT peptide
YGRKKRRQRRRK-biotin (TAT, SEQ ID NO: 6) were purchased from Genemed
Synthesis (South San Francisco, Calif.).
Example 2
Oligosaccharide Length-Dependence of ECP-Heparin Interaction
[0033] Recombinant hECP containing a C-terminal His.sub.6 tag was
expressed in E. coli BL21-CodonPlus(DE3) (Novagen, Madison, Wis.),
purified by chromatography, and refolded as previously described
(Boix, E., et al. (1999) Journal of Biological Chemistry 274,
15605-15614). MBP-ECP was purified using amylase affinity
chromatography.
[0034] The prior art demonstrated that binding and endocytosis of
ECP are HSPG dependent (Fan, T. C., et al. (2007) Traffic 8,
1778-1795). An early study demonstrated ECP purification using
heparin-Sepharose chromatography (Gleich, G. J., et al. (1986) Proc
Natl Acad Sci USA 83, 3146-3150). Thus, the minimal heparin length
(or disaccharide unit) required to interact with ECP was
investigated by FACE (Fluorescence-assisted carbohydrate
electrophoresis).
[0035] Synthetic heparin oligosaccharide (FIG. 1) (Lee, C. J., et
al. (2004) J Am Chem Soc 126, 476-477), low molecular weight
heparin (LMWH) and PI-88 (kindly provided by Progen Inc.,
Australia) were labeled with 2-aminoacridone (AMAC) as described
(Calabro, A., et al. (2000) Glycobiology 10, 273-281).
AMAC-oligosaccharide and proteins were incubated at room
temperature for 15 min, loaded onto 1% gels and electrophoresed as
described (Holmes, O., et al. (2007) J Mol Biol 367, 395-408).
[0036] Initially, ECP was co-incubated with LMWH at various molar
ratios, and the binding products were analyzed by gel
electrophoresis. The decrease in the free LMWH signal was monitored
as ECP concentration was increased (FIG. 2A). Subsequently, the
dependence of ECP binding on heparin oligosaccharide size was
examined in the presence of synthetic heparin oligosaccharides from
the degree of polymerization (dp) 3 to 9 using LMWH as a positive
control. It was apparent that dp5 served as the shortest heparin
fragment that retained the ability to bind ECP (FIG. 2B).
Furthermore, we tested a potent heparanase inhibitor (PI-88),
undergoing clinical trials for its anti-angiogenic and
anti-metastatic effects (Joyce, J. A., et al. (2005) Oncogene 24,
4037-4051; Parish, C. R., et al. (1999) Cancer research 59,
3433-3441), that contains a mixture of highly sulfated
mannose-containing di- to hexasaccharides (Yu, G., et al. (2002)
European journal of medicinal chemistry 37, 783-791; Ferro, V, Li,
C., et al. (2002) Carbohydrate research 337, 139-146).
Interestingly, this heparin mimetic could also bind ECP (FIG. 2B).
In addition, similar results were observed for EDN (FIG. 2C).
Example 3
Competitive Inhibition of ECP Binding to Cells by Synthetic
Oligosaccharides
[0037] Beas-2B, a human bronchial epithelial cell line, was
cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with
heat-inactivated 10% fetal bovine serum (Gibco/Invitrogen,
Carlsbad, Calif.).
[0038] The ability of MBP-ECP to bind cells in the presence of
serial dilutions of oligosaccharides or peptides was determined as
described (Fan, T. C., et al. (2007) Traffic 8, 1778-1795). At the
present invention, synthetic heparins (dp3-9) were tested for their
ability to interfere with ECP binding to Beas-2B cells. Briefly,
confluent monolayers of Beas-2B cells in 96-well plates were
pretreated with various concentrations of oligosaccharides or
peptides in serum-free RPMI 1640 medium at 4.degree. C. for 30 min
before incubation with 5 .mu.g/ml MBP-ECP at 4.degree. C. for 1 h.
The cells were then washed with ice-cold PBS and fixed with 2% PFA
at room temperature for 15 min prior to blocking with 2% BSA/PBS at
room temperature for 90 min. The level of bound MBP-ECP was
quantified by ELISA analysis. MBP-ECP was detected using mouse
monoclonal anti-MBP and goat anti-mouse horseradish peroxidase
(HRP)-conjugated secondary antibody, followed by the enhanced
chemiluminescence detection system. The amount of MBP-ECP bound to
cells without oligosaccharide or peptide treatment was set to
100%.
[0039] Beas-2B cells were preincubated with oligosaccharides, and
bound ECP was detected essentially as described (Fan, T. C., et al.
(2007) Traffic 8, 1778-1795). The degree of inhibition increased
with increasing oligosaccharide length (FIG. 3). Fifty micrograms
per milliliter of heparin dp5 inhibited 50% of ECP binding to
cells, and the same amount of dp7 and dp9 was capable of inhibiting
over 70% and 80% of ECP binding, respectively. These data revealed
that pentasaccharide was the minimal length sufficient to interfere
with ECP binding to Beas-2B cells. In addition, the concentration
dependence of PI-88 against cellular binding of ECP was similar to
that of dp7 (FIG. 3).
Example 4
Identification of Heparin-Binding Sequence in ECP
[0040] A consensus sequence of the heparin binding site (e.g.,
XBBXBX or XBBBXXBX) has been found in many GAG-binding proteins or
peptides (Hileman, R. E., et al. (1998) BioEssays 20, 156-167;
Cardin, A. D. and Weintraub, H. J. (1989) Arteriosclerosis Dallas,
Tex. 9, 21-32). Inspection of the sequences of human RNase A family
members revealed a surface loop L3 region, .sup.34QRRCKN (SEQ ID
NO: 7), in EDN that exactly matches the XBBXBX motif (FIG. 4A), but
no consensus heparin-binding motif was found in ECP. Therefore, it
was speculated that residues 34-39 (.sup.34RWRCKN, SEQ ID NO: 8) in
ECP that correspond to the consensus motif in EDN might also bind
heparin. To determine whether this region contributes to cellular
binding, cell ELISA analysis was conducted using MBP-ECP and
MBP-ECP mt1 containing the mutations R34A/W35A/R36A/K38A.
[0041] Amino acid residues R34, W35, R36, and K38 of ECP were
simultaneously substituted to alanine using QuickChange
site-directed mutagenesis (Stratagene, La Jolla, Calif.) and the
resultant mutant was named ECP mt1. The primers used were as
follows: mt1 forward, 5'-TATGCAGCGGCTTGCGCAAACCAAAAT-3' (SEQ ID
NO:1), and mt1 reverse, 5'-TTTGCGCAAGCCGCTGCATAATTGTTA-3' (SEQ ID
NO: 2). E. coli BL21-CodonPlus(DE3) cells were used to transform
various plasmids. This mutant had only 50% of the cell-binding
activity, indicating the importance of the .sup.34RWRCKN motif in
ECP for cellular HS binding (FIG. 4B).
Example 5
Characterization of Association Between ECP and Heparin
Oligosaccharides
[0042] The binding affinities of wild-type and mutant ECP for
heparin oligosaccharides were subsequently monitored by intrinsic
tryptophan fluorescence titration (Lau, E. K., et al. (2004) The
Journal of biological chemistry 279, 22294-22305).
[0043] Binding of ECP to heparin was monitored by changes in
intrinsic tryptophan fluorescence emission (IFTE). ECP (200 nM) in
PBS at 25.degree. C. was titrated with small aliquots of a high
concentration of pentasaccharides with minimal dilution (<2%).
Protein fluorescence measurements were recorded 2 min after each
addition on a Hitachi 8000 spectrofluorimeter at emission
wavelength of 340 nm using an excitation wavelength of 280 nm. AF,
the relative fluorescence change, equals F.sub.0-F.sub.obs, where
F.sub.0 and F.sub.obs represent the initial and observed
fluorescence values, respectively. Binding constants were estimated
from the titration data using a nonlinear least-squares computer
fit to the equation based on 1:1 binding stoichiometry (Venge, P.
and Bystrom, J. (1998) The international journal of biochemistry
& cell biology 30, 433-437):
[0044]
.DELTA.F=.DELTA.F.sub.max.times.([P]+[H]+K.sub.d-(([P]+[H]+K.sub.d)-
.sup.2-4.times.[P].times.[H]).sup.1/2)/(2.times.[P]) (Eq. 1) where
.DELTA.F.sub.max is the maximum relative fluorescence change, P is
the total concentration of ECP, H is the concentration of heparin,
and K.sub.d is the dissociation constant for the ECP-heparin
interaction.
[0045] The change in tryptophan fluorescence revealed that
wild-type ECP bound to dp5 with high affinity, and the
corresponding K.sub.d was 139.6 nM (FIG. 5). As expected, a 4- to
5-fold increase in K.sub.d to 568.1 nM was observed for ECP mt1
(R34A/W35A/R36A/K38A) (Table I), indicating decreased heparin
binding activity for mutant ECP lacking the key heparin binding
motif in the L3 region.
TABLE-US-00001 TABLE I Dissociation constant (K.sub.d)
determination for wild-type ECP and mt1 with heparin
oligosaccharides. ECP wt ECP mt1 Oligosaccharide K.sub.d (nM)
K.sub.d (nM) dp9 62.6 400.3 dp5 139.6 568.1 K.sub.d was measured in
PBS at 25.degree. C. The change in intrinsic tryptophan
fluorescence was fit by the equation 1 to obtain K.sub.d.
[0046] Circular dichroism spectroscopy (CD spectroscopy) was used
to compare the conformations of wild-type ECP and ECP mt1. CD
spectra were recorded using an Aviv model 202 CD Spectrometer
equipped with a 450-watt Xenon arc lamp. Far-UV spectra were
recorded at 25.degree. C. from 200 to 250 nm using a 0.1-cm cuvette
containing 10 .mu.M protein in PBS. The CD spectra were recorded at
1.5-min intervals with a bandwidth of 1 nm. Each spectrum is an
average of three consecutive scans and was corrected by subtracting
the buffer spectrum. The results showed that the secondary
structure of ECP mt1 was very similar to that of wild-type ECP
(FIG. 6). These results strongly indicated that the decrease in
heparin binding affinity resulted from a loss of the specific
recognition sequence motif, and not a conformational change in ECP
mt1.
Example 6
Competitive Inhibition of ECP Binding to Cells by Synthetic
Peptides
[0047] To investigate whether the RWRCK (SEQ ID NO: 8) motif is
directly responsible for heparin binding, cell ELISA was conducted
using a synthetic peptide, C1 (SEQ ID NO: 3), along with a positive
control peptide derived from HIV-TAT, TAT (SEQ ID NO: 6) (Vives,
E., et al. (1997) The Journal of biological chemistry 272,
16010-16017; Ziegler, A. and Seelig, J. (2004) Biophysical journal
86, 254-263). The dose-dependent competition of these peptides is
shown in FIG. 7A. Binding of MBP-ECP to cell-surface HS was
significantly reduced in the presence of both C1 (SEQ ID NO: 3) and
TAT (SEQ ID NO: 6) peptides. Therefore, synthetic peptides with
heparin binding activity may compete with ECP for cellular binding.
Furthermore, the cell surface binding ability of peptides was
tested using cell ELISA assay. As expected, similar to the TAT (SEQ
ID NO: 6), C1 (SEQ ID NO: 3), and D1 (SEQ ID NO: 4) peptides bound
to the cell surface, whereas R1 (SEQ ID NO: 5) peptide was devoid
of such function (FIG. 7B).
Example 7
Interaction Between Synthetic Heparin Binding Peptide and Cell
Surface
[0048] To investigate whether the heparin binding QRRCK motif on
EDN, corresponding to the "RWRCK" motif on ECP (SEQ ID NO: 8), was
directly responsible for heparan sulfate binding on cell surface,
Beas-2B cell-peptide binding was conducted using a synthetic
peptide, D1 (SEQ ID NO: 4), and a positive control, TAT (SEQ ID NO:
6) purchased from Genemed Synthesis (South San Francisco, Calif.).
The data demonstrated that synthetic peptide D1 (SEQ ID NO: 4)
bound to Beas-2B cell surface heparan sulfate at low temperature,
in consistent with the results obtained from cell-ELISA competitive
inhibition experiments shown in FIG. 8.
Example 8
Interaction of Synthetic Peptides with LMWH
[0049] The ability of C1 (SEQ ID NO: 3) to directly interact with
LMWH was further investigated by FACE analysis. As expected, a
decreased amount of free AMAC-LMWH signal was observed with
increasing concentration of C1 (SEQ ID NO: 3) peptide or TAT (SEQ
ID NO: 6) peptide (FIG. 9). In addition, because EDN contains a
conventional heparin binding sequence, the corresponding peptide,
D1 (SEQ ID NO: 4), was also synthesized and tested. As shown in
FIG. 8, D1 (SEQ ID NO: 4) bound heparin as tightly as C1 (SEQ ID
NO: 3). Interestingly, although the peptide segment in the
corresponding location of human RNase 1 (R1, SEQ ID NO: 5) also
contains several positively charged residues, it did not bind
heparin. Taken together, these results indicate that the RWRCK (SEQ
ID NO: 8) motif within loop L3 of ECP serves as a specific heparin
binding site.
Example 9
Characterization of Association Between C1 and Heparin
Oligosaccharides
[0050] The definitive heparin binding activity of the C1 (SEQ ID
NO: 3) peptide was determined by ITFE, which clearly demonstrated
that C1 has high affinity for heparin (FIG. 10). The K.sub.d values
obtained for LMWH, dp9, and dp5 binding to the C1 (SEQ ID NO: 3)
peptide were 192.0, 229.1, and 274.8 nM, respectively (Table II),
indicating that C1 (SEQ ID NO: 3) bound tighter to longer heparin
oligosaccharides.
TABLE-US-00002 TABLE II Determination of dissociation constants
(K.sub.d) for C1 peptide with heparin oligosaccharides.
Oligosaccharide K.sub.d (nM) LMWH 192.0 dp9 229.1 dp5 274.8
Example 10
Characterization of Association Between Synthetic Heparin Binding
Peptides and HMWH
[0051] In general exothermal reaction takes place upon molecular
binding between synthetic peptide and HMWH. As expected, the
positive control TAT (SEQ ID NO: 6) peptide showed significant
exothermal reaction mode employing ITC (FIG. 11A), whereas the
negative control R1 (SEQ ID NO: 5) peptide revealed no heat release
(FIG. 11B), strongly suggesting that no binding occurred between
the test molecules. As for the synthetic C1 (SEQ ID NO: 3) and D1
(SEQ ID NO: 4) peptides, exothermal reaction modes were clearly
observed (FIGS. 11C-D). These data further proved that peptide
motifs C1 (SEQ ID NO: 3) and D1 (SEQ ID NO: 4) acted as crucial in
vitro HMWH binding sites residing in human eosinophil RNases.
Example 11
Growth Inhibitory Effect of ECP
[0052] The inhibition of lymphocyte and mammalian cell growth by
ECP has been reported (Fan, T. C., et al. (2007) Traffic 8,
1778-1795; Maeda, T., et al. (2002) European Journal of
Biochemistry 269, 307-316). As a physiological test, the
cytotoxicity of ECP mt1 was monitored by MTT assay. The effect of
ECP on the cell growth was determined by a calorimetric assay using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(US Biological, MA, USA). Cells were plated in a 96-well plate
(5000 cells/well) and incubated at 37.degree. C. overnight. Each
sample was incubated with the indicated concentration (1-100 .mu.M)
of ECP. Forty-eight hours after treatment with ECP, MTT was added,
and cell growth was monitored at A.sub.570 to measure the
mitochondrial-dependent formation of a colored product.
[0053] As compared with wild-type ECP, ECP mt1
(R34A/W35A/R36A/K38A) exhibited a 2- to 3-fold increased IC.sub.50
value toward the Beas-2B cell line (FIG. 12, Table III). Thus, ECP
mt1 without the key heparin binding sequence motif appears to be
less cytotoxic than wild-type ECP, presumably due to a lesser
interaction with the cell surface, which in turn leads to less
endocytosis.
TABLE-US-00003 TABLE III IC.sub.50 values for cytotoxic ECP.
Protein IC.sub.50 (.mu.M) ECP wt.sup.a 16.05 ECP mt1 38.52
.sup.aData taken from reference. (Fan, T. C., et al. (2007) Traffic
8, 1778-1795)
Example 12
Immunohistochemical Localization of ECP
[0054] To better understand the possible cellular targets of ECP,
recombinant mature ECP was injected into the rat circulation
through the tail vein.
[0055] Adult specific-pathogen-free (SPF) Sprague-Dawley rats
(Narl:SD) with body weight (BW) of 200-300 g, were purchased and
maintained at the National Laboratory Animal Center (NLAC) in
Taiwan. The rats were separated into three groups. In group 1, each
rat was injected with 5 nmol of ECP through the tail vein. In group
2, each rat was co-injected with heparin (FRAGMIN.RTM., average MW
6000, 5000 IU/0.2 ml) and 5 nmol of ECP. In group 3, each rat was
injected with 5 nmol of MBP as the negative control. All animals
were sacrificed using CO.sub.2 narcosis 1 h after the injection of
these agents. The lung and trachea of these rats were taken and
immediately fixed with 10% neutral buffered formaldehyde. The
tissue samples were processed by routine methods to prepare
paraffin wax-embedded block. These blocks were then sectioned into
6-.mu.m slices. All tissue sections were examined using the Super
Sensitive Non-Biotin HRP Detection System (BioGenex Laboratories,
San Ramon, Calif.) as described (Liang, C. T., et al. (2007)
Journal of comparative pathology 136, 57-64). Briefly, the mouse
anti-ECP or anti-MBP monoclonal antibody was used as the primary
antibody. Antigen unmasking was performed by immersion of sections
in 5% Trilogy (Cell Marque, Rocklin, Calif.) antigen unmasking
solution in Milli-Q water and boiled at 121.degree. C. Endogenous
peroxidase activity was quenched with hydrogen peroxide (3%) in
methanol. These sections were then incubated in Power Block
solution, and mouse anti-ECP or anti-MBP at 1/200 dilution was
applied and left for 24 h. The sections were incubated with Super
Enhancer reagent, followed by Polymer-HRP reagent, and then
incubated with 3-amino, 9 ethyl-carbazole chromogen solution. The
sections were finally counterstained with Mayer's hematoxylin and
mounted with Super Mount permanent aqueous mounting media prior to
examination with a light microscope (Zeiss-Axioplan, Germany).
[0056] Immunohistochemical localization showed strong
internalization of ECP in the tracheo- and broncho-epithelial cells
1 h post-injection (FIGS. 13A-B). ECP internalizaiton was reduced
when co-injection with heparin was carried out (FIG. 13C). For the
negative control, no MBP signal was detected in tracheo- and
broncho-epithelial cells, despite the MBP signal that could be
detected in circulating blood (FIG. 13D).
Sequence CWU 1
1
8127DNAArtificialprimer 1tatgcagcgg cttgcgcaaa ccaaaat
27227DNAArtificialprimer 2tttgcgcaag ccgctgcata attgtta
27311PRTArtificialderived from Eosinophil cationic protein (ECP)
peptide with biotin-labeled at 3' end 3Asn Tyr Arg Trp Arg Cys Lys
Asn Gln Asn Lys1 5 10411PRTArtificialderived from
eosinophil-derived neurotoxin(EDN) peptide with biotin labeled at
3' end 4Asn Tyr Gln Arg Arg Cys Lys Asn Gln Asn Lys1 5
10511PRTArtificialderived from RNase1 peptide with biotin-labeled
at 3' end 5Met Thr Gln Gly Arg Cys Lys Pro Val Asn Lys1 5
10612PRTArtificialderived from HIV-TAT peptide with biotin- labeled
at 3' end 6Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys1 5
1075PRTHomo sapiensBINDING(1)..(5) 7Gln Arg Arg Cys Lys1 585PRTHomo
sapiensBINDING(1)..(5) 8Arg Trp Arg Cys Lys1 5
* * * * *