From the Department of Physiology, Sol Sherry
Thrombosis Research Center, Fels Cancer Research Institute, Temple
University, School of Medicine, Philadelphia, Pennsylvania 19140, ¶ Institute of Peptide Research, Hannover, Germany 30559, ** Biogen
Inc., Cambridge Massachusetts 02142, § Instituto de
Biomedicina, Consejo Superior de Investigaciones Científicas,
Valencia, Spain E 46010, and
Kimmel Cancer Institute, Jefferson
Medical College, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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EC3, a heterodimeric disintegrin
(Mr = 14,762) isolated from Echis
carinatus venom is a potent antagonist of Integrins are a family of cell surface proteins that mediate
cell-cell interactions and the adhesion of cells to extracellular matrix proteins and other ligands. Integrins are heterodimeric structures composed of noncovalently bound The Over the last decade a number of investigators have sought naturally
occurring or synthetic peptides that may selectively inhibit
integrin-ligand interactions. Research on disintegrins, low molecular
weight, cysteine-rich, RGD-containing peptides isolated from viper
venoms was stimulated by this long term objective. The first
disintegrin described in the literature, trigramin, was identified and
characterized on the basis of its ability to block platelet aggregation
and inhibit fibrinogen binding to It is known that disintegrin-like and cysteine-rich domains occur in
larger venom proteins containing a metalloproteinase domain and that
the RGD sequence in these proteins is substituted with other amino
acids (15). We considered the possibility that viper venoms may contain
low molecular weight disintegrins with anti-adhesive properties
mediated by epitopes other than RGD. We fractionated E. carinatus venom on HPLC reverse-phase column, and we tested each
fraction for its ability to bind to Jurkat cells, which express
Materials--
Monoclonal antibodies (mAb) HP2/1 (anti- Cell Lines--
A5 and VNRC3 cells, Chinese hamster ovary (CHO)
cells transfected with human Purification of EC3--
Lyophilized E. carinatus
suchoreki venom obtained from Latoxan (Rosans, France) was
dissolved in 0.1% trifluoroacetic acid (30 mg/ml). The solution was
centrifuged for 5 min at 5000 rpm to remove the insoluble proteins. The
pellet was discarded, and the supernatant was applied to a C-18 HPLC
column. The column was eluted with an acetonitrile linear gradient of
0-80% over 45 min. The venom was separated into 17 fractions. EC3
fraction, eluting at approximately 40% of acetonitrile, was collected,
lyophilized, and then dissolved in water. This solution was re-injected
into the same HPLC column. However, a "flatter" gradient of
acetonitrile was applied (0-60% over 45 min). The main peak, which
contained EC3, was collected and lyophilized. Purity of EC3 was tested
by SDS-polyacrylamide gel electrophoresis and mass spectrometry. The
yield of EC3 was about 4 mg/1 g of crude venom.
Separation of Reduced and Ethylpyridylethylated EC3
Subunits--
Reduction and alkylation of EC3 were performed according
to procedures used before for trigramin (6). Briefly, 100 µg of EC3
was incubated in 200 µl of 0.1 M Tris-HCl, pH 8.5, buffer containing 6 M guanidine hydrochloride, 4 mM
EDTA, 3.2 mM dithiothreitol together with 2 µl of
4-vinylpyridine for 2 h in the dark at room temperature. Modified
subunits epEC3A and epEC3B were isolated by reverse-phase HPLC on a
C-18 column with an acetonitrile gradient of 0-80% over 45 min. In
some experiments, EC3 subunits were reduced and carboxymethylated
with iodoacetate acid before HPLC separation.
Structural Characterization of EC3A and EC3B--
Determination
of the molecular mass of native EC3 or reduced and alkylated EC3
subunits was done by electrospray ionization mass spectrometry using a
Sciex API-III triple quadrupole instrument. The sequences of native EC3
electroblotted onto a polyvinylidene difluoride membrane (19), and
residues 1-40 of epEC3A and epEC3B were determined by N-terminal
sequence analysis using an Applied Biosystems Procise instrument. The
primary structures of EC3A and EC3B were deduced from Edman degradation
of overlapping peptides obtained by digestion with endoproteinase Lys-C
(Roche Molecular Biochemicals) (2 mg/ml protein in 100 mM
ammonium bicarbonate, pH 8.3, for 18 h at 37 °C using an
enzyme:substrate ratio of 1:100 (w/w)) and CNBr (10 mg/ml protein and
100 mg/ml CNBr in 70% formic acid for 6 h under N2
atmosphere and in the dark). Peptides were separated by reverse-phase
of HPLC using a 0.4 × 25-cm Lichrospher RP100 C-18 (5-µm
particle size) column (Merck) eluting at 1 ml/min with acetonitrile
gradient. For determination of sulfhydryl groups (free cysteines),
native EC3 (2 mg/ml in 100 mM ammonium bicarbonate, pH 8.3, containing 6 M guanidine hydrochloride) was treated for 2 h at room temperature with a 100-fold molar excess of
iodoacetamide, dialyzed against distilled water, lyophilized, and
subjected to amino acid analysis (after sample hydrolysis with 6 N HCl for 18 h at 110 °C) using a Amersham
Pharmacia Biotech AlphaPlus amino acid analyzer.
Peptide Synthesis--
The peptides were prepared by solid phase
synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl)
strategy on a 430A peptide synthesizer (Applied Biosystems, Foster
City, CA) and a 9050 Pepsynthesizer Plus (Perseptive Biosystems,
Cambidge, MA), as described previously (20).
Adhesion Studies--
Adhesion of cultured cells labeled with
5-chloromethylfluorescein diacetate was performed as described
previously (21). Briefly, ligands EC3, fibrinogen, vitronectin,
fibronectin, or VCAM-1 were immobilized on 96-well microtiter plates
(Falcon, Pittsburgh, PA) in phosphate-buffered saline overnight at
4 °C. Wells were blocked with 1% bovine serum albumin in Hanks'
balanced salt solution. Cells were labeled by incubation with 12.5 µM 5-chloromethylfluorescein diacetate in Hanks'
balanced salt solution buffer containing 1% bovine serum albumin at
37 °C for 15 min. Unbound label was removed by washing with the same
buffer. Labeled cells (1 × 105/sample) were added to
the well in the presence or absence of inhibitors and incubated at
37 °C for 30 min. Unbound cells were removed by washing the wells,
and bound cells were lysed by the addition of 0.5% Triton X-100. In
parallel, a standard curve was prepared in the same plate using known
concentrations of labeled cells. The plates were read using a Cytofluor
2350 fluorescence plate reader (Millipore, Bedford, MA) with a 485-nm
excitation filter and a 530-nm emission filter.
Flow Cytometry Analysis--
Samples for flow cytometry analysis
were prepared as described (22) and analyzed in a Coulter Epics flow
cytometer (Miami, FL).
Direct Binding Assay--
Direct binding assay alkaline
phosphatase conjugated with VCAM-1Ig was performed using Jurkat
( Amino Acid Sequence and Subunit Composition of EC3--
Analysis
of the nonreduced EC3 band excised from the Immobilon-P membrane
revealed a single amino acid sequence:
NSVHPXXDPV(K/T)XEPREGEHXISGP. The
complete amino acid sequences of EC3A and EC3B were determined by
N-terminal sequence analysis of reverse phase HPLC-isolated peptides
derived by degradation of each subunit with endoproteinase Lys-C and
CNBr. Both EC3A and EC3B are cysteine-rich proteins of 67 amino acids.
They display amino acid sequence heterogeneity at several positions,
indicating the existence of isoforms. The isotope-averaged molecular
masses calculated for the reduced EC3A isoforms (1-67: N33 I37 G64
E66), (1-67: R33, V37, G64, E66), and (1-66: N33, I37, D64, D66) are
7412 Da, 7440 Da, and 7341 Da, respectively, corresponding to major and
minor ions of reduced EC3 mass spectrum. The major EC3A isoforms might
be the one with molecular weight 7412, which yields a mass of 8478 after reduction and ethylpyridylethylation. On the other hand, reduced
EC3B isoforms (1-67: T11, K40, S55), (1-67: K11. R40, T55), and
(1-67: T11, R40, T55) have calculated masses of 7370 Da, 7439 Da, and
7412 Da, respectively. The major EC3B isoform, i.e. the one
that would have a molecular mass of 7950 after reduction and
carboxymethylation, is the 7370-Da isoform. The possibility of a number
of dimers involving combinations of various EC3A and EC3B isoforms
should be considered. However, we propose that EC3A-EC3B heterodimers may represent the major species because homodimers would not yield separated subunits displaying the distinct biological activities demonstrated for the HPLC-purified EC3A and EC3B fractions, and a
mixture of EC3A and EC3B homodimers would display a more complex HPLC
separation profile.
EC3A and EC3B showed a high degree of sequence similarity to each other
and to eristostatin, echistatin, flavoridin, and kistrin, including the
alignment of conserved cysteines identified in each subunit. The EC3A
amino acid sequence had high homology with the disintegrin domain of
Le3, a metalloproteinase-disintegrin identified in Vipera
lebetina (24) (Fig. 1).
Surprisingly, neither EC3 subunit contained an RGD sequence. The
hairpin loop sequence of echistatin, KRARGDDMDDY, was
substituted in EC3A and EC3B with KRAVGDDVDDY and
KRAMLDGLNDY, respectively (Fig. 1).
Biological Activities of EC3--
The biological activities of EC3
and the RGD-containing disintegrin echistatin were compared in a panel
of integrin assays (Table I). As
expected, echistatin at concentrations of 20-130 nM
potently inhibited
We also evaluated the biological activity of the EC3A and EC3B subunits
after reduction and ethylpyridylethylation. Although residual activity
of both subunits was significant, it was decreased by approximately
200-fold. It has been previously reported that reduction and
ethylpyridylethylation of flavoridin and albolabrin decreased their
ability to inhibit ADP-induced platelet aggregation by approximately
40-fold (25). epEC3B inhibited adhesion of Jurkat cells to immobilized
VCAM-1 (IC50 = 6 µM), whereas epEC3A was
inactive in this system. However, epEC3A and epEC3B both inhibited adhesion of K562 cells to fibronectin (IC50 = 30 µM and 6 µM, respectively) (Fig.
2). This experiment suggests that the
specificity of EC3 for
Further experiments showed that EC3 competes with mAb HP2/1 for binding
to The experimental data described in this paper identify a novel,
heterodimeric disintegrin in the venom of E. carinatus. This disintegrin, named EC3, is a potent and relatively selective antagonist of EC3 is a new naturally occurring ligand for Because EC3A, EC3B, kistrin, and flavoridin show identical alignment of
cysteines (Fig. 1) and because the pattern of S-S bonds in kistrin and
flavoridin is well established (26, 33-35), it is possible to deduce a
hypothetical structure of EC3. Assuming that both subunits of EC3 may
have the S-S pattern of kistrin/flavoridin, we propose that Cys-7 and
Cys-12 may be involved in two intermolecular bridges. The
intramolecular disulfides are likely formed between Cys-6-Cys-29,
Cys-20 EC3 inhibits quite selectively adhesion of It should be noted that EC3 weakly inhibited ADP-induced platelet
aggregation and the binding of CHO cells transfected with Trikha et al. (39) and Clark et al. (40) isolated
a homodimeric, RGD-containing protein, contortrostatin, from the venom of Agkistrodon contortrix contortrix. The amino acid
sequences of this protein, which appears to be a disintegrin, have not
been reported. As determined by mass spectrometry, molecular mass of nonreduced contortrostatin is 13,505 Da, and the molecular mass of
reduced and pyridylethylated contortrostatin is 8,000 Da. This bivalent
protein is a potent inhibitor of platelet aggregation, and in contrast
to monomeric disintegrins, it induces tyrosine phosphorylation of
platelet proteins. In addition, contortrostatin is a potent inhibitor
of In conclusion, we describe a novel dimeric disintegrin, EC3, that is a
potent inhibitor of 4 integrins. Two
subunits called EC3A and EC3B were isolated from reduced and alkylated
EC3 by reverse-phase high performance liquid chromatography. Each
subunit contained 67 residues, including 10 cysteines, and displayed a
high degree of homology to each other and to other disintegrins. EC3
inhibited adhesion of cells expressing
4
1 and
4
7 integrins
to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and
mucosal addressin cell adhesion molecule 1 (MadCAM-1) with
IC50 = 6-30 nM, adhesion of K562 cells
(
5
1) to fibronectin with IC50 = 150 nM,
and adhesion of
IIb
3 Chinese hamster ovary cells to fibrinogen
with IC50 = 500 nM; it did not inhibit adhesion of
v
3 Chinese hamster ovary cells to vitronectin.
Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to
immobilized VCAM-1 (IC50 = 6 µM), whereas
EC3A was inactive in this system. The MLDG motif appeared to be
essential for activity of EC3B. Linear MLDG peptide inhibited the
adhesion of Jurkat to VCAM-1 in a dose-dependent manner
(IC50 = 4 mM), whereas RGDS peptide was not
active at the same concentration. MLDG partially inhibited adhesion of
K562 cells to fibronectin (5-10 mM) in contrast to RGDS
peptide (IC50 = 3 mM), inhibiting completely at
10 mM.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits (1, 2).
In humans there are at least 15 different
subunits and 8 different
subunits, and they can combine to form proteins with diverse ligand
specificities and biological activities. The integrins play important
roles in many diverse biological processes including platelet
aggregation, tissue repair, angiogenesis, bone destruction, tumor
invasion, and inflammatory and immune reactions. Integrin
IIb
3
(glycoprotein IIb/IIIa complex) binds fibrinogen on the platelet
surface and mediates platelet aggregation. Integrin
v
3 is
predominantly expressed on endothelial cells and plays an important
role in angiogenesis. It is also expressed on osteoclasts and
participates in bone destruction. Integrin
5
1 is widely distributed on a variety of cells; it plays a critical role in cell
adhesion to extracellular matrix as well as in the formation of tissues
and organs during embryonic development (3). All three integrins,
IIb
3,
v
3, and
5
1, recognize RGD sequence in the
adhesive ligands (1, 2).
4 integrins
4
1 and
4
7 are widely expressed on
leukocytes and lymphoid cells and play a major role in inflammation and
autoimmune diseases (4). The
4
1 integrin (also called VLA-4, very
late antigen-4) mediates cell adhesion to vascular cell adhesion
molecule 1 (VCAM-1),1 an
adhesive molecule belonging to the immunoglobulin (Ig) superfamily that
is expressed on endothelial cells at sites of inflammation.
4
1
also binds to alternatively spliced variants of fibronectin that
contain connecting segment 1 (CS-1). The
4
7 integrin binds to
mucosal addressin cell adhesion molecule 1 (MadCAM-1) and to a lesser
extent to VCAM-1 and CS-1. Interaction of these integrins with VCAM-1
or MadCAM-1 (which are up-regulated by cytokines) on endothelium
mediates leukocyte infiltration, which can lead to tissue and organ
destruction (4). Selectins and
2 integrins (expressed on neutrophils
and monocytes) also contribute to this process. Leukocyte engagement
via
4 integrins is believed to play a significant role in the
progression of many diseases including insulin-dependent
diabetes, multiple sclerosis, rheumatoid arthritis, ulcerative colitis,
arteriosclerosis, asthma, allergy, and re-stenosis of arteries after
surgery and angioplasty (4, 5).
IIb
3 (6). Subsequently a number
of laboratories have isolated several other RGD containing viper venom
disintegrins of similar size, including kistrin (rhodostomin) (7),
applagin (8), and flavoridin (triflavin) (9, 10). Two short (49 amino
acids) RGD disintegrins, echistatin (11) and eristostatin (12, 13),
have been isolated from the venoms of Echis carinatus and
Eristocophis macmahoni, respectively. A number of NMR
studies on kistrin, echistatin, and flavoridin showed that their RGD
sequences are located in a mobile loop joining two strands of
sheet
protruding from the protein core (reviewed in Ref. 14). The disulfide
bonds around the RGD sequence in disintegrins maintain the hairpin loop
conformation in each peptide, which is important for their potency and selectivity.
4
1 and
5
1 integrins but do not express
3 integrins. We
isolated and characterized a new protein, referred as EC3, that is
selective and a highly potent inhibitor of
4 integrins and shows a
low level of interaction with
3 integrins. EC3 is the member of a
new protein family called heterodimeric disintegrins, which is first
reported in this paper.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
subunit of VLA-4) and SAM-1 (anti-
5 subunit of VLA-5) were purchased
from Immunotech, Inc. (Westbrook, ME). HP2/4 (anti-
4 subunit of
VLA-4) was a gift from Dr. F. Sanchez-Madrid (Madrid, Spain). Because
the biological effects of HP2/1 and HP2/4 were identical, only data
with HP2/1 are shown. Highly purified human fibrinogen was a gift from
Dr. A. Budzynski (Temple University, Philadelphia, PA). Recombinant human VCAM-1 (16) was a gift from Dr. M. Renz (Genentech, San Francisco, CA). Human vitronectin and fibronectin were purchased from
Calbiochem and Sigma, respectively. GRGDSP and GRGESP peptides were
purchased from Bachem (Torrance, CA). RGDS was purchased from Sigma.
Echistatin was isolated from E. carinatus suchoreki venom as
described previously (13). Fluorescein isothiocyanate-conjugated goat
anti-mouse IgG for flow cytometry was purchased from Jackson Immune
Research (West Grove, PA).
IIb
3 and
v
3 integrins,
respectively (17), were kindly provided by Dr. M. Ginsberg (Scripps
Research Institute, La Jolla, CA). CHO cells with deleted
5 integrin
(B2 cells) were kindly provided by Dr. R. Juliano (University of North
Carolina, Chapel Hill, NC). CHO cells transfected with human
4 or
its G190A mutant (18) and B2 cells transfected with human
4 (
4B2)
were kindly provided by Dr. Y. Takada (Scripps Research Institute). JY
cells expressing
4
7 were a gift from Dr. S. Burakoff (Dana-Farber Cancer Institute, Boston MA), and RPMI8866 cells were from Dr. A. Garcia-Padro, Madrid, Spain. K562 cells transfected with
6 and
2
integrin were gifts of Dr. A. Sonnenberg (Netherlands Cancer Institute,
Amsterdam, Holland) and M. E. Hemler (Dana Farber, Boston, MA),
respectively. Jurkat cells, K562 cells, and nontransfected CHO K1 cells
were purchased from ATCC (Manassas, VA).
4
1-expressing) and JY (
4
7-expressing) cells according to
procedures described previously (23).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparison of amino acid sequences of EC3A
and EC3B with other disintegrins. Eristostatin (13) and echistatin
(12) represent short disintegrins; kistrin (7) and flavoridin (9)
represent medium size disintegrins. Le3 is a metalloproteinase from
V. lebetina venom with a disintegrin domain (24). The
cysteines are boxed. EC3A and EC3B now have Swiss-Prot entry
codes P81630 and P81631.
IIb
3-dependent platelet
aggregation and
IIb
3-,
v
3-, and
5
1-dependent cell adhesion (Table I). In contrast,
EC3 only weakly inhibited
IIb
3-dependent interactions (IC50 = 1 µM for platelet aggregation and
IC50 = 500 nM for A5 cell adhesion to
fibrinogen) and showed no inhibition of
v
3-dependent adhesion up to 10 µM, although inhibition of
5
1-dependent adhesion was observed at an
IC50 of 150 nM (Table I). When the two
disintegrins were evaluated in a panel of
4 integrin-mediated cell
adhesion assays, the specificities were reversed. At concentrations of 25-100 nM, EC3 was a highly potent inhibitor of the
interaction of both anchorage-dependent and -independent
cells expressing
4
1 with either VCAM-1 or the CS-1 fragment of
fibronectin, whereas echistatin showed no detectable activity at 10 µM (Table I). EC3 inhibited to the same extent adhesion
of A2 (CHO
4+
5+) cells and
4B2 (CHO
4+
5
) cells to
immobilized VCAM-1, confirming direct inhibition of binding to
4
1
integrin. To further extend the data on
4 integrins, the potency of
EC3 in assays measuring VCAM-Ig binding directly to either
4
1 on
Jurkat cells or
4
7 on JY cells was evaluated. EC3 potently
inhibited
4
1 and
4
7 binding at concentrations of 28 nM and 6 nM, respectively. Moreover, adhesion
of RPMI 8866 cells was inhibited by EC3 with IC50 = 17 nM, whereas echistatin was not inhibitory. Cell adhesion
assays and direct binding assays yielded similar results. Neither
echistatin nor EC3 inhibited adhesion of
6
1-transfected cells to
laminin and adhesion of
2
1 cells to collagen (Table I). We also
studied biological function of both disintegrins in direct binding
assay, confirming the specificity of EC3 for
4
1 and
4
7
integrin.
Comparison of the inhibitory effects of echistatin and EC3 on various
integrins
4 integrins likely resides in the MLD
sequence in the EC3B subunit, whereas the ability if EC3 to inhibit
5
1 likely resides in both subunits. Obviously, the MLDG sequence
in EC3B is replacing the RGDX motif in monomeric
disintegrins. Both RGDX and MLDG motifs appear to represent
integrin binding sites. Fig. 3 shows that
MLDG peptide inhibited adhesion of Jurkat cells to immobilized VCAM-1
in a dose-dependent manner approaching saturation at 5-10
mM. Adhesion of K562 cells to immobilized fibronectin showed a similar pattern of inhibition by RGDS. On the other hand RGDS
did not cause any significant inhibition of Jurkat cell adhesion to
immobilized VCAM-1. Inhibition of K562 to fibronectin by MLDG was only
partial at 10 mM. It should be noted that the inhibitory effect on Jurkat cell adhesion to VCAM-1 was increased when longer MLDG-containing peptides were used. For instance CKRAMLDGLNDYC inhibited Jurkat cell adhesion with IC50 of 800 µM, whereas the peptide CRAMLDGLNDYCTGKSSD caused 50%
inhibition at 50 µM (not shown).
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Fig. 2.
Effect of reduced and ethylpyridylethylated
EC3A and EC3B on adhesion of Jurkat cells to immobilized VCAM-1
(A) and K562 cells to immobilized fibronectin
(B). Recombinant VCAM-1 (0.5 mg/well) or
fibronectin (0.5 mg/well) were immobilized overnight at 4 °C on a
96-well plate in phosphate-buffered saline buffer. After blocking, the
5-chloromethylfluorescein diacetate-labeled cells were added to each
well in the presence or absence EC3 subunits. The adhesion was
performed as described under "Experimental Procedures." Open
circles and closed circles indicate different
concentrations of EC3A and EC3B, respectively. Error bars
indicate S.D. from three independent experiments.
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Fig. 3.
Effect of MLDG and RGDS peptides on the
adhesion of Jurkat cells to immobilized VCAM-1 (A) and
on the K562 cells adhesion to immobilized fibronectin
(B). The experiment was performed as described in
the legend to Fig. 2. The inhibitory effects of MLDG peptide
(open circles) and RGDS peptide (closed circles)
are shown. Error bars indicate S.D. from three independent
experiments.
4 integrin. HP 2/1 at a concentration of 1 µg/sample blocked
adhesion of Jurkat cells to immobilized EC3, whereas at the
concentration of 1 mM, neither the hexapeptide GRGDSP nor a
control peptide GRGESP had any effect (Fig.
4A). Competition between EC3
and HP2/1 was also confirmed using fluorescence-activated cell sorter
analysis. Fig. 4B shows EC3-mediated inhibition of HP2/1
binding to
4B2 (CHO
4+
5
) cells. The inactive G190A
4 mutant did not interact with EC3 (data not shown). In addition, the
ability to directly inhibit binding to
5
1 was confirmed using mAb
SAM-1, which blocked adhesion of K562 cells to immobilized EC3 (data
not shown).
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Fig. 4.
Competition of EC3 with RGD peptides and mAb
HP2/1. A, effect of GRGDSP, GRGESP, and HP 2/1 mAb on
the adhesion of Jurkat cells to immobilized EC3. An adhesion study was
performed using 5-chloromethylfluorescein diacetate-labeled Jurkat
cells in the absence or presence of 1 mM GRGDSP, 1 mM GRGESP, or 10 µg/ml HP 2/1. Error bars
represent S.D. from three independent experiments. B, effect
of EC3 on the binding of HP2/1 mAb to 5-deficient CHO cells
transfected with
4 integrin. Cells were incubated with 10 µg/ml HP
2/1 in the absence (
) or presence (
) of 60 nM EC3 for
30 min at room temperature. After washing, 10 µg/ml of fluorescein
isothiocyanate-conjugated goat anti-mouse IgG was added, and the
samples were incubated for another 30 min at room temperature. The
samples were fixed by the addition of 1% paraformaldehyde before
measurement of fluorescence intensity by flow cytometry. The control
binding of mouse IgG is shown in the unfilled trace.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 integrins, which inhibits their interaction with ligands in an
RGD-independent manner. EC3 is composed of two covalently linked
subunits A and B, which show a high degree of homology (including
alignment of conserved cysteines) with other viper venom disintegrins.
It is likely that the integrin binding sites of EC3 are located in two
loops encompassing 13 amino acids (Cys-38 to Cys-50), corresponding to
hairpin loops extending from Cys-20 to Cys-32 in echistatin and from
Cys-45 to Cys-57 in kistrin and flavoridin. It is well known that the
hairpin loops in disintegrins are maintained in appropriate
conformation by S-S bridges (14, 15, 26), and the same appears to be
true regarding EC3. The biological activity of this protein is
decreased by 2 orders of magnitude after reduction and alkylation (Fig.
3). In contrast to all other viper venom disintegrins, EC3 contains
neither RGD nor KGD sequences. In fact, the RGD motif is substituted
with VGD in EC3A and with MLD in EC3B. The activity of EC3 with regard to inhibition of
5
1 binding resides on both subunits. However, only EC3B was active in the inhibition of
4
1/VCAM-1 interactions. This observation suggests, that MLD is the active sequence in EC3
mediating its anti-
4 effects. The experiment with synthetic peptides
(Fig. 3) confirmed this expectation. MLDG linear peptide blocked
adhesion of Jurkat cells to VCAM-1. In contrast, RGDS peptide, which is
a very well known inhibitor of several
1 and
3 integrins (27),
was not significantly active in this system. The MLDG peptide partially
inhibits adhesion of
5
1-expressing cells to fibronectin (Fig. 3).
This is consistent with the dual inhibitory effect of EC3 and of EC3B
subunit containing MLDG (Fig. 2). The inhibitory effects of anti-
4
and anti-
5 inhibitory antibodies are in agreement with this suggestion.
4 integrins. The LD
motif from its B subunit is also present in other ligands of
4
integrins. An ILDV sequence was found in alternatively spliced connective segment I of fibronectin (28, 29), and KLDAPT is present in
the fibronectin type III5 repeat (30). The LDT sequence occurring in
MadCAM (31) appears to be important for its ability to bind to
4
7. Recently Tselepis et al. (32) produced a number of
mutants of recombinant kistrin and demonstrated that ILDV kistrin (kistrin in which PRGD sequence was substituted with ILDV) inhibited binding of the LDV-containing fibronectin fragment to immobilized
4
1, with an IC50 close to 0.1 µM. It is difficult to compare activities of EC3 with
LDV-kistrin and synthetic peptides because preparations have been
tested in different assay systems; however, in our hands LDV-kistrin
was some 50-fold less active than EC3 in the direct binding
assay.2 Until now, no MLDG
motif has been identified and functionally characterized. Most
investigators have achieved better inhibitory effects for tested
4
inhibitors in the presence of Mn2+. However EC3 has almost
the same activity in the presence or absence of Mn2+ (data
not shown).
Cys-26, Cys-25
Cys-50, and Cys-38
Cys-57. On the other hand,
if one uses the S-S bonding pattern of albolabrin (25), then Cys-6 and
Cys-7 will form intermolecular bridges, and the intramolecular S-S
bonds would correspond to Cys-12
Cys-26, Cys-20
Cys-50,
Cys-25
Cys-29, and Cys-38
Cys-57. Clearly, further structural studies
are needed to establish the S-S pattern of EC3.
4
1-expressing cells to
immobilized VCAM-1. Its effect on
5
1 and on
IIb
3 appears to
be lower by 1 and 2 orders of magnitude, respectively. The effect of
EC3 on
4
1 does not appear to be related to the inhibition of
5
1 integrin, because this disintegrin inhibited to the same extent the adhesion to VCAM-1 of CHO cells transfected with
4 and of
5-deficient CHO cells transfected with
4. EC3 also inhibits adhesion of
4
7-expressing cells to MadCAM. Because mAb HP2/1 competes with EC3 for binding to
4, it appears that EC3 may bind to
the N-terminal domain of
4, where the epitope of this antibody also
resides (18, 36, 37).
IIb
3 to
fibrinogen, although it had no significant effect on
v
3-mediated
adhesion. This is in agreement with other observations that the RGD
motif in disintegrins is not absolutely required for expression of
platelet aggregation inhibitory activity. For instance, Jia et
al. (38) expressed in insect cells the disintegrin/cysteine-rich domain of atrolysin A from Crotalus atrox and demonstrated
that the recombinant protein inhibited collagen and ADP-induced
platelet aggregation. This recombinant protein contained RSEC instead
of the RGD motif.
1 integrin-mediated melanoma cell adhesion in vitro
and lung colonization in vivo. Most recently we isolated three other dimeric disintegrins, EMF10 from E. macmahoni
and CC5 and CC8 from Cerastes cerastes venom, which all seem
to be heterodimeric disintegrins with an molecular mass of 14-15
kDa.3 We are in the process
of determining the amino acid sequences and function of these novel
disintegrins. Further studies are required to establish how the
bivalent structure of dimeric disintegrins affects the biological
properties of the individual subunits.
4 integrin binding to VCAM-1 and moderately
inhibits
5
1 integrins. We propose that the activity of EC3 is
associated with the MLDG sequence in the putative hairpin loop of this disintegrin.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Renz for the generous gift of
recombinant VCAM-1, Dr. Y. Takada for providing CHO cells transfected
with 4 integrin and its mutants, Drs. M. Ginsberg and J. Loftus for
cells transfected with
IIb
3 and
v
3 integrins, Dr. L. Rosenthal for the critical review of the manuscript, and Diane Leone,
Andrew Sprague, and William Yangt for help with assays.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO3 DE11844, grants from the American Diabetes Association and Barra Foundation, Ardmore Pennsylvania (to S. N.), an Initial Investigatorship American Heart Association (to C. M.), and a grant-in-aid from the American Heart Association, Southeastern Pennsylvania Chapter (to S. N).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed: Dept. of
Physiology, Temple University School of Medicine, 3400 North Broad St.,
Philadelphia, PA 19140. Tel.: 215-707-4408; Fax: 215-707-4003; E-mail:
stni{at}astro.ocis.templ.edu.
2 Lobb, R., Humpries, M., Tselpis, V., unpublished observation.
3 C. Marcinkiewicz, J. J. Calvete, M. M. Marcinkiewicz, M. Raida, S. Vijay-Kumar, R. R. Lobb, and S. Niewiarowski, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: VCAM-1, vascular cell adhesion molecule 1; ep, ethylpyridylethylated; MadCAM-1, mucosal addressin cell adhesion molecule 1; VLA-4, very late antigen-4; HPLC, high performance liquid chromatography; CHO, Chinese hamster ovary cells; CS-1, connecting segment 1.
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REFERENCES |
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