A Peptide Inhibiting the Collagen Binding Function of
Integrin
2I Domain*
Johanna
Ivaska
,
Jarmo
Käpylä
§,
Olli
Pentikäinen¶
,
Anna-Marja
Hoffrén¶**,
Jorma
Hermonen
,
Pasi
Huttunen
,
Mark S.
Johnson¶
, and
Jyrki
Heino
§§§
From the MediCity Research Laboratory and the Departments of
Medical Biochemistry and

Virology, University of Turku, Finland,
Turku Centre for Biotechnology, University of Turku and Åbo
Akademi University, ¶ Department of Biochemistry, Åbo Akademi
University, and § Department of Biological and Environmental
Science, University of Jyväskylä, Finland
 |
ABSTRACT |
Integrin
2 subunit forms in
the complex with the
1 subunit a cell surface receptor
binding extracellular matrix molecules, such as collagens and
laminin-1. It is a receptor for echovirus-1, as well. Ligands are
recognized by the special "inserted" domain (I domain) in the
integrin
2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the
interaction of platelet
2
1 integrin with
collagen because of the action of a disintegrin/metalloproteinase named
jararhagin. The finding that crude B. jararaca venom could
prevent the binding of human recombinant r
2I domain to
type I collagen led us to study jararhagin further. Synthetic peptides
representing hydrophilic and charged sequences of jararhagin, including
the RSECD sequence replacing the well known RGD motif in the
disintegrin-like domain, were synthesized. Although the
disintegrin-like domain derived peptides failed to inhibit
r
2I domain binding to collagen, a basic peptide from the
metalloproteinase domain proved to be functional. In an in
vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind
strongly to human recombinant
2I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids,
arginine-lysine-lysine (RKK), is essential for r
2I
domain binding, whereas the mutation of the other amino acids in the
peptide had little if any effect on its binding function. Importantly,
the peptide was functional only in the cyclic conformation and its
affinity was strictly dependent on the size of the cysteine-constrained
loop. Furthermore, the peptide could not bind to
2I
domain in the absence of Mg2+, suggesting that the
conformation of the I domain was critical, as well. Cells could attach
to the peptide only if they expressed
2
1
integrin, and the attachment was inhibited by anti-integrin antibodies.
 |
INTRODUCTION |
Integrins
1
1 and
2
1 are the major cellular receptors for
native collagens (for review, see Refs. 1 and 2). Like all integrins
their interaction with ligands is dependent on divalent cations (3).
The
1 and
2 subunits contain an special
inserted domain, the I domain, resembling the A domain found
e.g. in von Willenbrand factor (4). It is evident that
1I and
2I domains are responsible for the
primary recognition of collagen by the corresponding integrins (5, 6).
Two other ligands for
2
1 integrin, namely
laminin-1 and echovirus-1, each bind to
2I domain, as
well. However, echovirus-1 seems to recognize a different site on the
2I domain than the matrix proteins do (7).
The binding sites of
1
1 and
2
1 integrins in collagens have been
localized to the triple helical areas of the molecules (8, 9). One
peptide sequence derived from the collagen
chain has been reported
to block integrin-collagen interaction (10), but in many studies it has
been ineffective and it probably does not represent the actual binding
site in collagen (11-13). More likely, collagen-receptor integrins
recognize amino acid residues in more than one
chain. In type IV
collagen-
1
1 integrin interaction, the
importance of one arginine and two aspartic acid residues, all from
different
chains, has been indicated (8). One model of collagen
binding to
2I domain suggests the interaction of a
glutamate residue on the collagen surface with Mg2+ in
2I domain (14). The recognition of collagen must,
however, include the interaction of several other amino acids. In the
structure-function analysis of other integrins, the use of short
integrin binding peptides have been of great importance. Peptides
binding to
2I domain have so far not been available.
Venoms from several snake species contain disintegrin proteins, which
block platelet integrin function and are responsible for the
anticoagulation effect of the venoms. Many disintegrins harbor the RGD
motif and inhibit the function of platelet
IIb
3 and
V
3
integrins. However some toxins, such as jararhagin and catrocollastatin, have a disintegrin-like domain that differs from the
disintegrin peptides found in crotalid and viperid venoms by the nature
of their different disulfide bond structure and the fact that the RGD
motif is replaced by an XXCD disulfide-bonded cysteinyl
sequence (with X being any amino acid).
Jararhagin (15) is a potent inhibitor of platelet adhesion to collagen
and its effect is based on the inhibition of
2
1 integrin function (16). The exact
mechanism of its action has been unknown. Intact jararhagin is cleaved
in the snake venom-producing jaracetin, the disintegrin-like domain of
jararhagin. The fate of the metalloproteinase domain after this
cleavage is not known. Integrin
2
1 can
also interact with jaracetin, but the interaction seems to be weaker
than with jararhagin (16). Here, we show that a short, cyclic peptide
derived from the metalloproteinase domain of jararhagin binds strongly
to human recombinant
2I domain and is a potent inhibitor
of its interaction with collagens I and IV and laminin-1. Peptides
corresponding to other regions of the jararhagin sequence, including
the RSECD sequence analogous to the RGD region, had no effect. Our
studies reveal a novel integrin binding sequence, RKK. The role of RKK
motif in jararhagin function remains to be shown, but the peptides
create new opportunities for structural studies on
2
1 integrin.
 |
EXPERIMENTAL PROCEDURES |
Molecular Modeling of Jararhagin Metalloproteinase Domain and the
RKK Peptides--
The sequence of human
2I domain was
aligned with the sequences of the available I domain x-ray structures:
the I domain of human integrin
M
2 with
bound Mg2+ at 2.0 Å resolution (17) and the human
L
2
I-domain with bound Mn2+ at 1.8 Å resolution (18); using
the alignment programs MALIGN and MALFORM (19, 20). Based on this
alignment, the
2I domain was modeled using the programs
COMPOSER (Tripos Associates, St. Louis, MO, USA) and MODELLER 4.0 (21)
a kind gift of Andrej Sali, Rockefeller University.
From the sequence of the metalloprotease domain of Bothrops
jararaca the three-dimensional model was built using the same methods as in the modeling of the
2I domain above. The
model was based on the x-ray structure of Adamalysin II from
Crotalus adamanteus at 2.0 Å resolution (22).
The models were energy minimized in SYBYL 6.3 (Tripos Associates, St
Louis, MO, USA) using the TRIPOS force field. In the first application
of energy minimization, the backbone was kept rigid and only the side
chains were allowed to move. In the second step, all atoms were allowed
to move. Energy minimization was performed until all short contacts and
inconsistencies in geometry were rectified. The electrostatic term was
not included, as the main purpose was to remove sterical hindrances and
to correct bad geometry.
The conformational flexibility of the original cyclic peptide in the
cyclic form was assessed using molecular dynamics simulations. The
starting conformation of the peptide was taken from the
metalloproteinase model structure, cysteines were added to each end,
and a disulfide bond was created between them. Peptides derived from
the metalloproteinase were first minimized to remove atom-atom clashes
and further refined by molecular dynamics simulations. Simulations were
performed in vacuum at 300,000 and consisted of 20 pS equilibration
followed by a 200 pS production run. The SHAKE algorithm was applied to constrain the lengths of all bonds between heavy atoms and hydrogen atoms to allow longer 1 femtosecond time step to be used.
Electrostatics were excluded, because small peptides tend to form
intramolecular hydrogen bonds to make the structure globular,
especially when many charged residues are present within a peptide. All
calculations were made using SYBYL 6.3 and the TRIPOS force field using
an Onyx II workstation.
Generation of Human Recombinant Integrin
2I
Domain--
DNA encoding the
2I domain was generated by
polymerase chain reaction using human integrin
2
cDNA as a template (integrin
2 cDNA was a gift
from Dr. M. Hemler, Dana-Farber, Boston; Ref. 27). The forward primer
was 5'-CACAGGGATCCCCTGATTTTCAGCTC-3' and the reverse primer was
5'-GTGGCTGAATTCAACAGTACCTTCAATG-3'. Primers were designed to introduce
two restriction sites into the product: a BamHI site at the
5'-end and an EcoRI site at the 3'-end. Polymerase chain
reaction product and pGEX2T (Amersham Pharmacia Biotech) were digested
with BamHI and EcoRI, ligated, and transformed
into Escherichia coli DH
5F' cells. Plasmid having the
2I domain insert (pJK
2I) was then
sequenced and transformed into E. coli BL21 for production
of recombinant protein r
2I. Production and purification
of glutathione S-transferase-r
2I fusion
protein were carried out as follows: typically, 400 ml Luria Bertaini
medium (carbenicillin, 50 µg/ml) was inoculated with 40 ml of
overnight culture of BL21/pJK
2I and the culture was
grown for 1 h at 37 °C. Then an inducer,
isopropyl-
-D-thiogalactopyranoside (final concentration
0.1 mM), was added for 4 h. Cells were harvested by
centrifugation and pellets were resuspended in phosphate-buffered saline (PBS)1 pH 7.4. Suspensions were sonicated, centrifuged, and the supernatant was
retained. Pellets were resuspended in PBS, sonicated and centrifuged a
further two times, and the supernatants were pooled.
Glutathione-Sepharose (Amersham Pharmacia Biotech) was added to the
resulting lysate and incubated at room temperature for 30 min by gently
agitating. The lysate was centrifuged, the supernatant was removed, and
glutathione-Sepharose with bound fusion protein was transferred onto
the suitable column. The column was then washed with 10 volumes of PBS,
and the fusion protein was eluted with glutathione eluting buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH
8.0) (Amersham Pharmacia Biotech). The fusion protein was cleaved with
the protease thrombin (Pharmacia) (10 units) for at least 2 h at
room temperature and dialyzed against PBS to remove glutathione. The
cleavage mixture was passed down the glutathione-Sepharose column a
second time to remove glutathione S-transferase.
r
2I was collected from the flowthrough. It was necessary
to treat the recombinant protein with 5 mM dithiothreitol to allow proper folding, because extra bands were seen without the
treatment when analyzed by native polyacrylamide gel electrophoresis (data not shown). The recombinant
2I domain produced was
223 amino acids long having two nonintegrin amino acids at the
amino-terminal (GS), amino acids corresponding to
2
integrin sequence 124-339 (PDFQ ... IEGTV) and six nonintegrin
amino acids at the carboxyl-terminal (EFIVTD).
Labeling of r
2I with europium was carried out as
follows:
volume 1 M NaHCO3 (pH 8.5)
was added to the purified r
2I to elevate the pH for
labeling with isothiocyanate. The europium-labeling reagent (Wallac)
was added at a 100-fold molar excess and incubated overnight at
4 °C. The unbound label was removed by gel filtration on a Sephadex
G50/Sepharose 6B column (Pharmacia), and fractions containing the
labeled protein were pooled.
Binding Assays for Europium-labeled
2I Domain and
Radioactively Labeled EV1--
A sensitive r
2I binding
assay based on the use of europium-labeled r
2I was
developed. The coating of a 96-well immunoplate (Maxisorp, Nunc) was
done by exposure to 0.1 ml of PBS containing 150 µg/ml (5 µg/cm2) type I collagen (bovine dermal, Cellon), type IV
collagen (Sigma), laminin-1 (purified from basement membranes of the
Engelbreth-Holm-Swarm mouse tumor, Collaborative Research), fibronectin
(human plasma fibronectin, Boehringer Mannheim), or 3.3 µg/ml
echovirus-1 or echovirus-7 for 12 h at 4 °C. Alternatively,
peptides and B. jararaca venom (Sigma) or purified
jararhagin or jaracetin (a kind gift to us from Dr. Berndt, Baker
Medical Research Institute, Australia) were coated at various
concentrations on 96-well amine binding plates (Costar) according to
the manufacturer's instructions. Residual protein absorption sites on
all wells were blocked with 0.1% heat-inactivated bovine serum albumin
in PBS for 1 h at 37 °C. Echovirus-1 (Farouk strain) and -7 (Wallace) were obtained from the ATCC. They were propagated in LLC
M
2 cells and purified using the method described by
Abraham and Colonno (23). The purified viruses were diluted in PBS
containing 0.5 mM MgCl2 and stored at
70 °C until used. Europium-labeled r
2I was added at a concentration of 500 ng/ml in PBS, 2 mM
MgCl2, and 1 mg/ml BSA to the coated wells and incubated
for 3 h at 37 °C. Wells were then washed three times with PBS,
2 mM MgCl2. Delfia enhancement solution (0.1 ml) (Wallac) was added to each well, and the europium signal was
measured by fluorometry (Model 1232 Delfia, Wallac). In some
experiments, anti-
2 integrin antibody 12F1 (24) was used. For the virus binding assays, EV1 was metabolically labeled with
culture medium containing [35S]methionine (Amersham
Pharmacia Biotech). Radioactively labeled virus (20,000 cpm) was
allowed to bind 3 h in PBS, 2 mM MgCl2. The wells were washed three times, and the bound radioactivity was
measured in a liquid scintillation counter (Wallac).
When peptides were added endogenously, the lyophilized peptides were
solubilized directly with europium-labeled r
2I, 500 ng/ml in PBS, 2 mM MgCl2, 1 mg/ml BSA, and then
added to the wells. When EDTA was used instead of MgCl2,
europium-labeled r
2I was diluted with PBS, 2 mM EDTA, and subsequent washes were performed with this buffer.
Peptides and Binding Assay Using Biotinylated 229ox--
The
jararhagin-derived peptides were designed based on the secondary
structure prediction of the jararhagin amino acid sequence. Secondary
structure prediction was performed using the PeptideStructure program
from the Genetics Computer Group (GCG) Software package (Madison, WI).
Surface probability according to the Emini method (25) and
hydrophilicity according to the Kyte-Doolittle method (26) were taken
into account.
The peptides were synthesized on an automated peptide synthesizer
(Applied Biosystems 431A) using
N-(9-fluorenyl)methoxycarbonyl chemistry. The peptides for
the alanine substitution series were purchased from Research Genetics
(Huntsville, AL). After synthesis, the peptides were oxidized to form
disulfide bridges. The peptides were solubilized at 1 mg/ml
concentration with 0.1 M ammonium carbonate buffer and
incubated for 16-24 h at 4 °C. The oxidation was checked with
reverse-phase high pressure liquid chromatography, and the oxidized
peptides were lyophilized. In the 261ox peptide, the carboxyl-terminal
cysteinyl residue was protected with an acetoamidomethyl group, and the
cysteinyl residues at positions 1 and 8 were protected with trityl
groups that were removed during cleavage of the peptide from the resin.
The isoelectric points (pI) of the various peptides were also
determined from the primary sequence using the isoelectric program from
the Genetics Computer Group software package. Peptide 225ox was found
to have a similar isoelectric point as 229ox and was therefore chosen
to be the control peptide in some experiments.
Biotinylation of 229ox was carried out as follows: lyophilized 229ox
peptide was solubilized in PBS and
volume 0.1 M
NaHCO3, 0.5 M NaCl (pH 8.0) was added to
elevate the pH for biotinylation. Sulfobiotin-NHS (Calbiochem) was
added 1:2 (w/w) 229ox:biotin and incubated for 2 h at room
temperature.
volume 0.5 M Tris-HCl (pH 8.0) was
added to stop the biotinylation reaction.
For the binding assays using biotinylated 229ox peptide, 96-well amine
binding plates (Costar) were coated with various concentrations of
r
2I domain- or r
2I domain-derived
peptides according to the manufacturer's instructions. Residual
protein absorption sites on all wells were blocked with 0.1%
heat-inactivated bovine serum albumin in PBS for 1 h at 37 °C.
100 µM biotinylated 229ox in PBS, 2 mM
MgCl2 and 1 mg/ml BSA was added to the coated wells and
incubated for 3 h at 37 °C. Wells were then washed three times with PBS, 2 mM MgCl2 and europium-labeled
streptavidin (Wallac) was added at a concentration of 500 ng/ml in PBS,
2 mM MgCl2, 1 mg/ml BSA for 30 min at room
temperature. Wells were again washed three times. 0.1 ml of Delfia
enhancement solution (Wallac) was added to each well, and the europium
signal was measured by fluorometry (Model 1232 Delfia, Wallac). When
EDTA was used instead of MgCl2, europium-labeled
r
2I was diluted with PBS, 2 mM EDTA, and
subsequent washes were performed with this buffer.
Platelet Aggregation Assay--
Human blood was obtained from
healthy donors who had not taken any medication within the previous ten
days. Blood samples were drawn into a Becton Dickinson Vacutainer 228 containing 0.129 M sodium citrate. The tube was then
centrifuged 250 × g for 10 min and the platelet-rich
plasma was transferred to a clean tube. Platelet aggregation was
conducted at 37 °C in an aggregometer (Payton, CO). 450 µl of
platelet-rich plasma was preincubated 3 min at 37 °C, and platelet
aggregation was quantified by measuring the total amplitude of
aggregation at a predetermined time interval following the addition of
50 µl of 2 mg/ml collagen (Sigma). To assay the ability of the
synthetic peptides to inhibit platelet aggregation, the antagonists
were dissolved in phosphate-buffered saline at pH 7.4, 20 mM MgCl2 immediately before use. The antagonist solution was preincubated with platelet-rich plasma for 4 min at
37 °C before collagen stimulation. The extent of inhibition of
platelet aggregation was assessed by comparison with the maximal aggregation induced by collagen and then expressed as a precentage.
Cell Lines and Construction of the
2 Integrin
Expression Plasmid--
Human osteogenic cell line SAOS-2 (American
Type Culture Collection) were used. HACAT cells are immortalized human
keratinocytes originally obtained from Dr. N. E. Fusenig (DKFZ,
Heidelberg). Cells were maintained in Dulbecco's modification of
Eagle's medium supplemented with 10% fetal calf serum. Integrin
2 cDNA corresponding to nucleotides 1-4559 in the
published sequence (27) was kindly provided by Dr. M. Hemler
(Dana-Farber Cancer Institute, MA). cDNA was ligated into the
pAWneo2 expression vector (a kind gift from Dr. A. Weiss, University of
California San Francisco; Ref. 28), which carries the spleen
focus-forming virus long terminal repeat promoter and a
neomycin-resistant gene. Stable transfections were carried out using
Lipofectin reagent (Life Technologies Inc.) according to the
manufacturer's recommendations. 400 µg/ml neomycin analogue G418 was
added to the culture media. After 2-3 weeks of selection, the
nontransfected control cells were dead, and G418 resistant clones were
isolated and analyzed for their expression of
2 integrin
mRNA and protein.
Cell Adhesion Experiments--
The coating of a 96-well amine
binding plate (Costar) with 5 µg/cm2 (150 µg/ml) type I
collagen (bovine dermal, Cellon) or the various peptides at the
concentrations indicated was done according to the manufacturer's
instructions. Residual protein absorption sites on all wells were
blocked with 0.1% heat-inactivated bovine serum albumin in PBS for
1 h at 37 °C. Cells were detached by using 0.01% trypsin and
0.02% EDTA. Trypsin activity was inhibited by washing the cells with 1 mg/ml soybean trypsin inhibitor (Sigma). Cells were suspended in
Dulbecco's modification of Eagle's medium (Life Technologies, Inc.)
containing 0.1% glycine. When antibodies (anti-
1
(Mab13, Becton Dickinson), anti-
2 (5E8 a kind gift from Dr. Bankert, Roswell Park Cancer Institute)) or peptides were added the
cells were preincubated for 15 min at room temperature before transfer
to the wells. 10,000 cells were transferred to each well and incubated
at 37 °C for 1 h. Nonadherent cells were removed by washing the
wells once with PBS. Adherent cells were fixed with 2%
paraformaldehyde, stained with 0.5% crystal violet in 20% ethanol,
and washed with distilled water. The bound stain was dissolved in 10%
acetic acid and measured spectrophotometrically at 600 nm.
 |
RESULTS |
Ligand Binding Properties of Europium-labeled Human Recombinant
Integrin
2I Domain--
The human recombinant integrin
2I domain (r
2I) was produced in E. coli and purified. The recombinant protein was at least 90% pure
by SDS-polyacrylamide gel electrophoresis and only a single band was
observed by native polyacrylamide gel electrophoresis (not shown). To
further characterize the recombinant protein, the amino-terminal
sequence of the purified product was determined and found to be correct
(GSPDF). r
2I was labeled with europium and used in
solid-phase ligand binding assays. r
2I bound type I
collagen, type IV collagen, and laminin-1, although its binding to
fibronectin and albumin was not considered significant (Fig. 1A). r
2I bound
type I collagen in a Mg2+-dependent manner and
the addition of 2 mM EDTA abolished binding completely
(Fig. 1B). This is consistent with the fact that
2
1 integrin interacts with collagen only
in the presence of divalent cations (3). The binding of
r
2I domain to echovirus-1 was much weaker than to the
matrix molecules. However, the binding was about 5-fold higher than to
echovirus-7, a negative control virus known not to use
2
1 integrin as its cellular receptor (not
shown). Furthermore, monoclonal antibody 12F1 could block r
2I domain binding to echovirus-1 but not to collagen
(not shown), in agreement with the previous observation that matrix
molecules and echovirus-1 recognize distinct motifs on the
2I domain (7).

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Fig. 1.
Interaction of europium-labeled
r 2I with its ligands. Microtiter plate wells were
precoated with type I collagen, type IV collagen, laminin-1, and
fibronectin each at a concentration of 5 µg/cm2 (150 µg/ml). Europium-labeled r 2I was allowed to attach for
3 h in the presence of 2 mM MgCl2; wells
were washed three times and the europium signal was measured
(A). Europium-labeled r 2I was allowed to
attach to precoated type I collagen for 3 h in the presence of 2 mM Mg2+ or EDTA (B) or 1 µg/ml to
1,000 µg/ml B. jararraca venom (C). The data
shown are the mean values (±S.D.) of a representative experiment done
in triplicate.
|
|
Previous studies have shown that jararhagin, a large hemorrhagic
disintegrin/metalloproteinase from B. jararaca venom
inhibits the interaction of platelet
2
1
integrin with collagen (16). To investigate whether proteins found in
the crude B. jararaca venom could interact directly with the
r
2I domain, indicating a possible interaction between
jararhagin and
2I domain, two solid-phase ligand binding
assays were performed. First, europium-labeled r
2I
domain was allowed to attach to collagen I substratum in the presence
of 2 mM Mg2+, and the amount of bound
r
2I was then determined. The effect of the venom was
tested with concentrations ranging from 1 µg/ml to 1,000 µg/ml. The
venom inhibited the r
2I domain-collagen interaction efficiently and in a concentration dependent manner (Fig.
1C). Second, we coated microtiter wells with the venom
proteins and tested r
2I binding to this substratum.
r
2I was found to bind the venom directly in a
concentration-dependent manner (not shown).
A Short, Cyclic Jararhagin-derived Peptide Directly Interacts with
Integrin r
2I Domain and Inhibits the Ligand
Binding--
To study if the jararhagin sequence could be used to find
a peptide that would interact with
2I domain, we used a
series of short cyclic peptides corresponding to regions along the
protein. The most obvious target was to design a peptide corresponding to the RGD-analogous region in the disintegrin-like domain. The other
tested regions were selected based on the following facts: (i) many of
the integrin binding motifs in matrix proteins and in snake venom
disintegrins are found in loop structures (29, 30); (ii) many of the
known integrin binding motifs contain an aspartic acid residue (29,
31-35); and (iii) the published models of integrin-collagen
interactions emphasize the role of arginine and glutamic acid residues
in addition to aspartic acid residue (8). Peptides corresponding to
some of the more promising charged sequences were synthesized (Table
I). To investigate whether any of these
peptides could directly interact with
2I domain, the
jararhagin peptides along with cyclic RGD peptide, type I collagen,
type IV collagen, and fibronectin were coated on microtiter wells and
r
2I-europium was added. The results show that one of the
jararhagin peptides, 229ox, bound to r
2I domain efficiently, whereas other peptides, including 261ox, which is analogous to the atrolysin A functional motif (36), showed no effect
(Fig. 2A). The peptides were
then tested for their ability to influence r
2I binding
to type I collagen at a concentration of 500 µM. Again,
only peptide 229ox had any significant effect; it almost completely
inhibited the interaction between r
2I domain and
collagen (Fig. 2B).
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Table I
Sequences of the synthetic peptides used in this study
The name and the position of the peptides synthesized based on the
primary sequence of jararhagin (15) is shown. *, disulfide-bonded
residue; Acm, acetoamidomethylated
cysteine.
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Fig. 2.
Binding of europium-labeled
r 2I to adhesion proteins and jararhagin-derived peptides
in a solid-phase assay. Amine binding microtiter plate wells were
precoated with various peptides, type I collagen, type IV collagen and
fibronectin. The data are means ± S.D. from a representative
experiment done in triplicate showing r 2I binding to
different substrata in the presence of 2 mM
MgCl2 (A). Microtiter plate wells were precoated
with BSA and type I collagen, and the europium-labeled
r 2I was allowed to attach for 3 h in the presence
of 2 mM MgCl2 and 500 µM peptide
(B).
|
|
The Sequence of Three Amino Acids, RKK, As Well As the Proper
Cyclic Conformation Is Critical for Binding to r
2I
Domain--
To reveal which of the amino acid residues of the 229ox
peptide are critical for its function, we tested a series of new
peptides where amino acids in peptide 229 were replaced one at a time
with alanine residues. The peptides were bound to the solid phase and tested for their ability to bind r
2I. As predicted based
on molecular modeling, the three positively charged amino acids
arginine-lysine-lysine (RKK) were found to be essential. In addition,
the adjacent histidine showed some effect. The substitution of the
aspartic acid or the asparagine residues had no effect (Fig.
3A). Consistent with this, r
2I binding to type I collagen was poorly inhibited with
the peptides containing alanine substitutions of the RKK sequence, whereas substitution of the aspartic acid or the asparagine residues did not impair this function (Fig. 3B).

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Fig. 3.
Inhibition of the binding of
r 2I to type I collagen by alanine-substituted 229ox
peptides. The peptides were precoated to amine binding microtiter
plate wells and the europium-labeled r 2I was allowed to
bind for 3 h. Wells were washed three times, and the europium
signal was measured (A). Alternatively, microtiter plate
wells were precoated with type I collagen and labeled
r 2I was added in the presence of 500 µM
peptide (B). The data shown are the mean values (±S.D.) of
a representative experiment done in triplicate.
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|
To exclude the possibility that the binding of 229ox to
2I domain was because of nonspecific interactions
between positively and negatively charged amino acid side chains, we
used both oxidized and linear p229 peptide in our solid-phase binding
assay. Both peptides were of identical sequence, but measurements with
the non-oxidized 229 were done in the presence of 5 mM
dithiothreitol to prevent the formation of a disulfide bond. The cyclic
229ox showed the ability to inhibit r
2I adhesion to type
I collagen, whereas the linear form of the peptide had only a small
effect (Fig. 4). Thus, the function of
the peptide was dependent on the cyclic conformation. Optimization of
the loop size of the peptide was done with the help of molecular
modeling and dynamic simulations. Predicted three-dimensional
structures of RKK-containing peptides with varying numbers of residues
were compared with the predicted structure of the RKK-loop found in the
molecular model of the jararhagin metalloproteinase domain.
Computer-based simulations on the dynamic movements available to the
cyclic peptides suggested that 248ox could maintain conformation very
similar to the corresponding loop region of the jararhagin
metalloproteinase model structure. The 248ox is more rigid than the
two-residue longer 229ox peptide, but both peptides have similar
conformations, and the side chains are similarly positioned. In
contrast, the eight-residue-long peptide (252ox) did not mimic the loop
in the jararhagin model but after extended molecular dynamics
simulations was seen to be kinked.

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Fig. 4.
The cyclic conformation of the RKK peptide is
essential for its function. Dose response curves for inhibition of
europium-labeled r 2I binding to precoated type I
collagen in the presence of variants of the RKK peptide.
Europium-labeled r 2I was allowed to attach to precoated
microtiter plate wells for 3 h in PBS, 2 mM
MgCl2 in the presence of varying concentrations of cyclic
or linear 229 peptide (the measurements with the linear 229 peptide
were done in the presence of 5 mM dithiothreitol). Wells
were washed three times, and the europium signal was measured. The data
are means ± S.D. from a representative experiment done in
triplicate showing r 2I binding to type I collagen in the
presence of different soluble ligands and 2 mM
MgCl2.
|
|
Peptides of six (248ox) and eight (252ox) residues (we do not count the
artificially added cysteines) were synthesized and tested for their
ability to inhibit r
2I adhesion to type I collagen. As
shown in Fig. 5, soluble type I collagen
was the most potent inhibitor with an apparent IC50 value
of 0.004 ± 0.002 µM. In agreement with the modeling
data, peptide 248ox with six residues was the most potent peptide
inhibitor of
2I domain adhesion to type I collagen
having an apparent IC50 of 1.3 ± 0.2 µM
compared with the approximate IC50 values of 52 ± 20 µM and greater than 10 mM for peptides 229ox
and 252ox, respectively.

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Fig. 5.
Properties of the RKK-containing
peptide. The conformation and the length of the RKK peptide have
significant effect on the ability of the peptide to inhibit the binding
of r 2I to type I collagen. Dose response curves for
inhibition of europium-labeled r 2I binding to precoated
type I collagen in the presence of type I collagen or variants of the
RKK peptide. Europium-labeled r 2I was allowed to attach
to precoated microtiter plate wells for 3 h in PBS, 2 mM MgCl2 and varying concentrations of soluble
type I collagen and the peptide variants. Wells were washed three
times, and the europium signal was measured. The data are means ± S.D. from a representative experiment done in triplicate showing
r 2I binding to type I collagen in the presence of
different soluble ligands and 2 mM MgCl2.
|
|
The requirement of the correct architecture of the
2I
domain for 229ox binding was shown in binding assays performed in the presence of EDTA instead of Mg2+. Interaction of the two
molecules was tested by having either 229ox or
2I domain
bound to the solid phase. In both cases, EDTA completely prevented the
binding of the molecules to each other (Fig.
6, A and B). Thus,
the molecular recognition of
2I domain by 229ox was
strictly dependent on the proper three-dimensional structure of both
components.

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Fig. 6.
The importance of Mg2+ for the
peptide-r 2I interaction. Europium-labeled
r 2I was allowed to bind to solid-phase bound type I
collagen, 229ox peptide and 225ox control peptide in PBS, 2 mM MgCl2 or 2 mM EDTA for 3 h,
wells were washed 3 times, and the europium signal was measured
(A). r 2I (1 µg/well) was bound to
solid-phase and biotinylated 229ox was added at a concentration of 100 µM for 3 h at 37 °C. The wells were washed three
times, and the europium-labeled streptavidin was added at a
concentration of 500 ng/ml for 30 min at room temperature. Wells were
washed three times, and the europium signal was measured
(B). The data shown are the mean values ± S.D. of a
representative experiment done in triplicate.
|
|
Some of the peptides were also tested for their ability to inhibit
collagen-induced platelet aggregation. With up to 1 mM concentrations, peptides 225ox and 261ox showed no effect, at 100 µM concentration 248ox showed 88% and RGD peptide 62%
of the aggregation measured in the controls. At higher peptide
concentrations, RGD showed complete inhibition of aggregation, whereas
increasing 248ox concentrations were only slightly more effective.
The RKK peptides seem to be potent inhibitors of
2I
domain suggesting a role for the metalloproteinase domain in
2I domain binding. However, the previous hypothesis (37)
has been that the jararhagin disintegrin domain or jaracetin are
responsible for
2I domain binding. To test this,
type I collagen was coated to the microtiter plate wells and
r
2I-europium was added in the presence of 100 µg/ml
jaracetin. No interaction between
2I domain and
jaracetin was detected (not shown). The venom proteins were also bound
to the solid phase along with type I collagen, and r
2I-europium was added. The
2I domain
bound to collagen but showed no binding to jaracetin (not shown). It
was not possible to study the role of the metalloproteinase domain of
jararhagin in
2I domain binding, because the
purification of the domain has not been described, and the
corresponding cDNA was not available for us to use in the
production of a recombinant protein. Our preliminary experiments with
the purified intact jararhagin protein failed to show binding to
2I domain (not shown). We did not have access to
jararhagin protein in amounts allowing optimization of the test
conditions, but the result may indicate that the disintegrin domain
masks the highly charged RKK motif in the metalloproteinase domain.
Expression of
2
1 Integrin on the Cell
Surface Is Essential for the Recognition of the RKK Peptide--
Two
different cell lines were used to study the effect of the RKK peptide
(248ox; CTRKKHNC). HACAT keratinocytes express
2
1 integrin, but
1
1 integrin was not detectable on their
surfaces. Human osteosarcoma SAOS-2 cells express
1
1 but not
2
1 integrin. To test whether cells bind
to the RKK peptide in the absence of
2
1
integrin, we assayed for adhesion with SAOS-2 cells. These cells could
bind to type I collagen, but no cell adhesion occurred on the RKK
peptide, thus showing that
1
1 integrin
does not recognize the RKK motif (Fig.
7A). Cell adhesion on type I
collagen was prevented by anti-
1 integrin antibody. When
2 integrin cDNA was transfected into SAOS-2 cells
and cell clones overexpressing
2
1
integrin were tested, cell adhesion on the RKK peptide and on type I
collagen increased significantly. Anti-
1 integrin
antibody could decrease cell adhesion on both the RKK peptide and on
collagen (Fig. 7A).

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Fig. 7.
SAOS-2 cell adhesion and 2
transfected SAOS-2 cell adhesion on RKK peptide and type I
collagen in the absence and presence of anti- 1
antibody (1 µg/ml) (A). HACAT cell adhesion on type I
collagen in the presence of 225ox and RKK peptides and 5E8, a
functional anti- 2 1 integrin antibody
(B). Type I collagen (5 µg/cm2; 150 µg/ml),
BSA (1 mg/ml), or RKK peptide (1 mg/ml) was coated on amine binding
immunoplates. In the inhibition assays, the cells were preincubated
with antibodies (1 µg/ml) or peptides (500 µM) for 15 min at room temperature. 10,000 cells in Dulbecco's modification of
Eagle's medium with 0.1% glycine were added to each well and
incubated at 37 °C for 1 h. Nonadherent cells were removed by
washing the wells once with PBS. Adherent cells were fixed with 2%
paraformaldehyde, stained with 0.5% crystal violet in 20% ethanol,
and washed with distilled water. The bound stain was dissolved in 10%
acetic acid and spectrophotometrically measured at 600 nm. The data
shown are the mean values ± S.D. of a representative experiment
done in quadruplicate.
|
|
HACAT keratinocytes were selected for a second set of experiments,
because in these cells anti-
2 integrin antibody (5E8) could completely block cell adhesion to type I collagen (Fig. 7B), suggesting that
2
1
integrin is the only receptor used by these cells in collagen binding.
In these cells, the RKK peptide was almost as effective an inhibitor of
cell adhesion as the 5E8 antibody (Fig. 7B). These
experiments showed that the expression of
2
1 integrin is needed on the cell surface
for the recognition of the RKK peptide and that other integrins,
including the
1
1, cannot replace
2
1 integrin.
Peptide 229ox Activates the Echovirus-1 Recognition Site in
Integrin r
2I Domain--
In addition to type I
collagen, r
2I domain also binds type IV collagen and
laminin-1. The 229ox peptide inhibited the binding of
r
2I to these ligands, whereas the control peptide 225ox
of the same length and conformation together with a similar pI value had no effect (Fig. 8). This suggests
that the
2I domain binds all of these ligands by the
same mechanism, and that 229ox inhibits the binding either by
interacting directly with the ligand recognition site or by altering
the native three-dimensional structure of the
2I domain
to an inactive one.

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Fig. 8.
229ox peptide inhibits r 2I
domain binding to collagens type I and IV and to laminin-1.
Europium-labeled r 2I was allowed to bind to solid-phase
bound type I collagen, type IV collagen, laminin-1, fibronectin, and
BSA in the presence or absence of 229ox peptide or 225ox control
peptide in PBS, 2 mM MgCl2 for 3 h. Wells
were washed 3 times, and the europium signal was measured. The data
shown are the mean values ± S.D. of a representative experiment
done in triplicate.
|
|
Integrin
2
1 also functions as a virus
receptor, mediating cell surface attachment and infection by a human
pathogen, echovirus-1 (7). Matrix proteins and echovirus-1 have been
found to interact with the integrin in a different manner (7), but the
binding site for echovirus-1 is also located on the I domain of the
2 subunit. As described above, the r
2I
domain showed weak binding to the solid-phase bound echovirus-1, but
the addition of RKK peptide increased this binding about 10-fold, and
the control peptide 225ox had no effect (Fig.
9A). Importantly, the RKK
peptides did not directly bind to the virus itself (Fig.
9B). This result indicates that binding of the RKK peptide
to the
2I domain may induce a structural change in the
protein that increases the binding affinity of r
2I to
echovirus-1.

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Fig. 9.
Interaction of 229ox peptide with
echovirus-1. Microtiter plate wells were precoated with type I
collagen and echovirus-1; europium-labeled r 2I was
allowed to bind for 3 h in PBS, and 2 mM
MgCl2, 0.1% BSA in the presence or absence of 500 µM peptide (A). The data shown are the mean
values ± S.D. of a representative experiment done in triplicate.
Type I collagen (5 µg/cm2; 150 µg/ml), BSA (1 mg/ml),
229ox peptide (0.5 mg/ml), control peptide 261ox (0.5 mg/ml), and
r 2I domain (0.5 mg/ml) were coated on amine binding
immunoplates (B). A radioactively labeled virus was allowed
to bind 3 h in PBS, 2 mM MgCl2. The wells
were washed three times, and the bound radioactivity was determined
with a scintillation counter. The data are mean values ± S.D. of
five parallel measurements.
|
|
 |
DISCUSSION |
The identification of the RGD motif (31) started an intensive
search for other similar short recognition sequences in the integrin
binding proteins. Peptides mimicking these sequences have played a
central role in the studies on structure-function relationship of many
integrins. The regions in collagens recognized by
1 and
2 integrin I domains contain three polypeptides forming an
-helical coiled-coil (8, 9), and most likely the collagen binding
integrins recognize epitopes containing amino acids from all three
chains instead of a linear sequence.
The aim of this study was to find a single amino acid chain ligand
molecule interacting with the
2I domain and to use this to design a peptide that could inhibit the function of the
collagen-binding
2
1 integrin. Previous
studies have shown that venom from B. jararaca can inhibit
platelet adhesion to collagen by blocking the function of
2
1 integrin (16). Here, the crude
B. jararaca venom could directly interact with the
recombinant
2I domain. This led us to synthesize and
test a number of jararhagin-derived peptides. We tested a peptide from
the disintegrin-like region in jararhagin corresponding to the RGD loop
found in other disintegrins and also other hydrophilic and charged
regions along the molecule. In a solid-phase assay, the peptide derived
from the disintegrin-like domain showed no interaction with the
2I domain, whereas a peptide, CTRKKHDNAQC, containing
amino acids 241-249 from the metalloproteinase domain bound tightly to
recombinant
2I domain inhibiting its interaction with
other ligands. None of the other peptides tested showed any binding to
2I domain. Mutational analysis of the peptide sequence
revealed a novel integrin binding motif, RKK or RKKH, because
histidine, the fourth amino acid in the sequence, may also be important
for the full function. The cyclic conformation, the length of the
cyclic peptide and presence of Mg2+ was critical for the binding.
Another snake venom protein catrocollastatin from Crotalus
atrox, also an inhibitor of
2
1
integrin function (38), has an identical RKK motif in its
metalloproteinase domain. However, the role of the RKK motif in the
function of the metalloproteinase domains of jararhagin and
catrocollastatin remains to be shown. On the basis of the present
knowledge, it is not possible to name the structural basis of
jararhagin binding to
2
1 integrin. A recent paper presents a hypothesis that jararhagin binds to
2I domain by its disintegrin domain and then degrades
the
1 subunits by its metalloproteinase domain (37).
However, no direct interaction between
2I domain and
jararhagin disintegrin domain has been shown, and also our experiments
failed to show it. In agreement with this, a peptide representing the
jararhagin sequence corresponding to the collagen-induced platelet
aggregation blocking disintegrin-like motif in atrolysin (36) was
nonfunctional. Thus an alternative model for jararhagin action must be
considered. One possibility is that the disintegrin domain and the
metalloproteinase domain, present also as separate peptides in snake
venom, have independent functions. Our data support the idea that the
metalloproteinase domain can block the function of
2I
domain. The metalloproteinase domain of jararhagin as such could not be
tested because the cDNA was not available to us for the production
of the recombinant protein, and the cleavage product of jararhagin
containing the metalloproteinase domain has not been purified. The fact
that intact jararhagin did not interact with the
2I
domain, in our experiments, can mean that the disintegrin-like domain
adjacent to the metalloproteinase domain folds in a way that it masks
the highly charged RKK loop and therefore inhibits the function of the
RKK motif. Finally, the disintegrin domain of jararhagin (jaracetin) cannot explain the fact that the crude venom can block the function of
2I domain. We cannot exclude the possibility that the
snake venom also contains another disintegrin. However, its binding mechanism to
2I domain should be very similar to what we
propose for jararhagin metalloproteinase domain because the RKK peptide could inhibit r
2I domain binding to the crude B. jararaca venom in a specific
manner.2
We studied the effects and specificity of the RKK peptide at the
cellular level. The RKK peptide can slightly inhibit
collagen-induced platelet aggregation. Receptors other than
2
1 integrin participate in
collagen-dependent platelet aggregation and the number of
different integrins expressed on platelets is limited. Therefore, we
chose two different cell lines that differ in their expression of
1
1 and
2
1
integrins. In addition, we used function-blocking antibodies specific
to
1 and
2 integrin subunits. Results
support the conclusion that the RKK peptide is specific for
2
1 integrin. Most importantly, human
osteosarcoma cell line SAOS-2, which lacks
2
1 but has several other integrins,
including
1
1,
3
1,
4
1,
5
1,
V
3, and
V
5,3
could not bind to the peptide. The same cell line was transfected with
2 integrin cDNA and forced to express
2
1 on the cell surface. These cells
showed
1 integrin mediated binding to the RKK peptide. Our preliminary results indicate that RKK peptide can still bind to
recombinant
1I domain, but the peptide inhibits only
slightly the binding of
1I domain to
collagen.4
The fact that the RKK motif can bind to
2I domain and
inhibit its collagen recognition function opens new possibilities to study the structure-function relationship of
2I domain.
We have docked the jararhagin metalloproteinase domain structure
manually onto the surface of the
2I domain. The side
chains of the RKK motif consist of three positively charged residues
that are oriented roughly with the positively charged groups occupying
the corners of a triangle. We found a region on the
2I
domain surface that provided a complimentary set of acidic residues.
Mutation of one of these amino acids, Asp-219, generated an
2I domain that bound to collagen, but its function could
not be inhibited by the RKK peptides.4 Asp-219 is located
near the predicted collagen binding site close to metal ion binding
MIDAS site. On the basis of the data presented here, it is evident that
the RKK peptides also altered the interaction between
2I
domain and echovirus-1. This was seen as the activation of the
echovirus-1 binding function. This phenomenon can be used in future
studies to unveil the molecular details of the
2I
domain-virus interaction.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. Hemler for cDNA, Drs. V. Woods and R. Bankert for the antibodies, and Dr. Berndt for the
purified jararhagin and jaracetin proteins. We are grateful for
technical assistance from M. Potila.
 |
FOOTNOTES |
*
This study was supported by grants from the Academy of
Finland, the Technology Development Centre in Finland (TEKES), the Sigrid Jusélius Foundation, and the Finnish Cancer Association.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.
**
Present address: Juvantia Pharma Ltd., Biocity, Turku, Finland.
§§
To whom correspondence should be addressed: MediCity Research
Laboratory, University of Turku, Tykistökatu 6A, FIN-20520 Turku,
Finland. Tel.: 358-2-333-7005; Fax: 358-2-333-7000; E-mail: jyrki.heino{at}utu.fi.
The abbreviations used are:
PBS, phosphate-buffered saline; BSA, bovine serum albumin; r
2I, recombinant integrin
2I domain.
2
J. Ivaska et al., unpublished results.
3
P. Koistinen et al., unpublished results.
4
J. Käpylä et al.,
unpublished results.
 |
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