From the Departament de Ciències Experimentales
i de la Salut, Facultat de Ciències de la Salut i de la Vida,
Universitat Pompeu Fabra, 08003-Barcelona, Spain and the
§ Unitat de Biologia Cel·lular i Molecular, Institut
Municipal d'Investigació Mèdica,
08003-Barcelona, Spain
Received for publication, July 29, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Annexin A2 has been described as an
important receptor for tissue-type plasminogen activator in endothelium
and other cell types. Interaction between tissue-type plasminogen
activator and its cellular receptor is critical for many of the
functions of this protease. The annexin A2 motif that mediates tissue
plasminogen activator interaction has been assigned to the hexapeptide
LCKLSL in the amino-terminal domain of the protein, and it has been
proposed that Cys8 of this sequence is essential for
tPA binding. In an attempt to identify other amino acids critical for
tPA-annexin A2 interaction, we have analyzed a set of peptides
containing several modifications of the original hexapeptide, including
glycine scans, alanine scans, D-amino acid scans,
conservative mutations, cysteine blocking, and enantiomer and
retroenantiomer sequences. Using a non-radioactive competitive binding
assay, we have found that all cysteine-containing peptides,
independently of their sequence, compete the interaction between tPA
and annexin A2. Cysteine-containing peptides also inhibit tPA binding
to the surface of cultured human umbilical vein endothelial cells
(HUVEC). Mass spectrometry demonstrates that the peptides bind through
a disulfide bond to a cysteine residue of annexin A2, the same
mechanism that has been suggested for the inhibition mediated by
homocysteine. These data call for a revision of the role of the
LCKLSL sequence as the sole annexin A2 structural region required to
bind tPA and indicate that further studies are necessary to better
define the annexin A2-tPA interaction.
Tissue-type plasminogen activator
(tPA)1 is a serine protease
that converts the zymogen plasminogen to the active enzyme plasmin, which in turn degrades the fibrin network of thrombi and blood clots
(1, 2). In addition to its important role in thrombolysis, plasmin
participates in the extravascular breakdown of matrix and basement
membrane in events such as cell migration, tissue remodeling, and
invasive growth (3-5). tPA is mainly synthesized in vascular
endothelial cells and secreted into the circulating blood as a
527-residue single-chain glycoprotein that can be further converted
into the two-chain form upon specific cleavage at the Arg275-Ile276 peptide bond (6).
Characterization of specific receptors for components of the
fibrinolytic system has been a crucial point of interest in this area.
In addition to regulating the dynamics of clot lysis, these receptors
may contribute to numerous cellular functions that are dependent upon
cell-surface proteolytic activity (7). Endothelial cell receptors for
tPA and plasminogen are particularly relevant because of the proximity
of these cells to vascular injury and fibrin deposition sites.
Annexin A2 (also termed annexin II, p36, calpactin 1, or lipocortin II)
has been identified as a receptor for tPA and plasminogen (8, 9) in the
surface of endothelial cells. The annexins (for a review see Refs. 10
and 11) are a family of proteins that bind to acidic phospholipids in
the presence of Ca2+. All members of the annexin family
contain four or more units of a conserved structural element of ~70
amino acids, designated the annexin repeat, and a highly variable
amino-terminal domain believed to determine individual annexin
functions. A variety of biological functions have been described for
the annexins, including regulation of membrane traffic (12-14),
transmembrane ion channel (15, 16), inhibition of blood coagulation
(17-20), signal transduction in mitogenesis or differentiation
(21-24), and regulation of cell-matrix or cell-cell interactions
(25-29).
Extracellular AnxA2 has also been described as a membrane-bound
receptor for a number of different molecules, although its interaction
with the plasminogen system elements on the endothelial cell surface is
the best characterized. Simultaneous binding of tPA and plasminogen to
AnxA2 at the endothelial cell surface results in a 60-fold increase in
catalytic efficiency of plasmin generation (9, 30). Previous reports
(31) have mapped the tPA binding site of AnxA2 to the hexapeptide
LCKLSL, corresponding to residues 7-12 of its amino-terminal domain.
These results were obtained using a competitive solid phase radioligand
binding assay with synthetic peptides corresponding to the AnxA2
amino-terminal domain sequence as competitors of either purified or
recombinant AnxA2. Results were further confirmed in vivo
using primary cultures of human umbilical vein endothelial cells
(HUVEC). The LCKLSL sequence contains a free thiol group
(Cys8) (32) that is crucial for tPA interaction. Evidence
for this conclusion included i) mutation of AnxA2 Cys8 to
Gly resulted in loss of tPA binding, whereas mutations in the other Cys
residues of the protein (C133G, C262G, and C335G) did not (31), and ii)
homocysteine (hC), an amino acid with prothrombotic properties,
competed the binding of tPA to AnxA2 by forming a disulfide bond with
Cys8 (31).
AnxA2 is expressed not only in endothelial cells but also in other cell
types (33, 34), including tumor cells (35). Interestingly, pancreas
cancer cells overexpress AnxA2 (36, 37). Previous results from our
group have demonstrated that tPA is also overexpressed in human
pancreas tumors, and its inhibition using neutralizing antibodies or
chemicals results in decreased invasiveness and tumorigenicity (37,
38). Therefore, it is important to identify tPA receptors in pancreatic
cells and their involvement in tumorigenesis. The fact that AnxA2 has
been shown to be overexpressed in pancreatic tumors supports the
hypothesis that it could act as a tPA receptor. Recent
work2 indicates that AnxA2
may mediate, at least in part, the effects of tPA on pancreas cancer
cells and therefore supports the notion that this protein could be used
as a potential therapeutic target. To further probe this hypothesis, we
evaluated a panel of synthetic peptides as competitors of the tPA-AnxA2
interaction. On the basis of the original LCKLSL motif, we generated a
small library of peptides with alterations in sequence, chain length,
configuration/conformation, and/or availability of the Cys-free thiol
group. Our results demonstrate that all Cys-containing peptides,
regardless of their sequence, are able to compete the tPA-AnxA2
interaction. MALDI-TOF mass spectrometry was used to determine the
nature of the interaction between recombinant AnxA2 and Cys (LCKLSL) or
homocysteine (L(hC)KLSL)-containing peptides and a Cys-lacking peptide
of similar sequence (LAKLSL). In the first two instances, an increase
in molecular weight compatible with a Cys and HC residue, respectively,
strongly argued for the formation of the disulfide bond, whereas
incubation of recombinant AnxA2 (rAnxA2) with the peptide lacking a
thiol group had no effect. These results suggest that the LCKLSL
hexapeptide, which has been assumed to be a key feature in tPA-AnxA2
binding, plays a distinctive, though probably not exclusive, role in
such interaction, to the extent that blocking of the Cys residue will
preclude the association of both proteins. Further research thus
appears to be necessary to identify additional domains of AnxA2
involved in binding to tPA.
Materials--
All reagents were purchased from Sigma unless
otherwise indicated. rAnxA2 was prepared from BL21
Escherichia coli transformed with the pET21b(+) vector
containing the human AnxA2 cDNA, kindly provided by Dr. K. A.
Hajjar (Cornell University Medical College, New York, NY), as
previously described (40). Purification was performed using a nickel
nitrilotriacetic acid-agarose column after elution using a pH gradient
from pH 8 to 4.8. Two elution peaks were collected, one at pH 5.2, corresponding to the monomeric form of the protein, and another one at
pH 4.8, corresponding to the dimer. Both peaks were pooled and stored
at Peptides--
Hexapeptides designed on the basis of the LCKLSL
sequence (Table I) were synthesized on
p-methylbenzhydrylamine resin as C-terminal carboxamides by
solid phase methods using Boc chemistry. After HF cleavage, the
peptides were purified to homogeneity ( Cell Culture--
Human umbilical vein endothelial cells (HUVEC,
passage 2-6) were cultured in M199 medium with 10% fetal calf serum
supplemented with heparin and endothelial cell growth factors as
previously described (42).
tPA Biotinylation--
Recombinant tPA (actilyse, Roche
Molecular Biochemicals) (2 mg) was biotinylated using a 20-fold
molar excess of Sulfo-NHS-LC-biotin (Pierce, Rockford, IL) for 2 h
at 25 °C, according to the manufacturer's instructions. Excess
unreacted biotin was removed by gel filtration using Sephadex G25. The
integrity of biotinylated tPA was examined by SDS-PAGE (43), and
protein concentration was determined by the Bradford method (44) using
the Bio-Rad protein assay (Bio-Rad).
Non-radiaoactive Binding Assays--
Binding experiments were
performed essentially as previously described (31, 40) except that
biotinylated tPA (btntPA) was used instead of
125I-tPA.
rAnxA2 Binding Assays--
Ninety-six well Nunc Maxisorp plates
(Nunc, Naperville, IL) were incubated with rAnxA2 (50 µl/well at 10 µg/ml) overnight at 4 °C. After three washes, plates were
equilibrated (2 h, 37 °C) with incubation buffer (11 mM
Hepes, 137 mM NaCl, 4 mM KCl, 3 mM
CaCl2, 2 mM MgCl2, 1 mM
glucose, 0.5% bovine serum albumin, pH 7.2) and btntPA
(100 nM, 2 h, 37 °C) was then added in the presence
or in the absence of peptides. Alternatively, peptides were directly added to rAnxA2, either directly or after washing, and
btntPA was subsequently added. Plates were washed 3 times
with 0.02% Tween 20, 0.5% bovine serum albumin in phosphate-buffered
saline and then incubated for 1 h at 37 °C with alkaline
phosphatase-coupled streptavidin (Zymed Laboratories
Inc., San Francisco, CA). Enzymatic activity was measured using
4-methylumbelliferyl phosphate (1 mg/ml in triethanolamine buffer, pH
9.5) for 20 min at room temperature. Quantification was performed using
a Cytofluor 235 instrument (Millipore, Bedford, MA).
HUVEC Binding Assays--
Confluent HUVEC, cultured in 96-well
Nunclon plates (Nunc, Naperville, IL), were equilibrated for 1 h
at 37 °C with incubation buffer (same as above). 100 nM
btntPA was then added for 1 h at 37 °C in the
absence or presence of selected peptides (10-1000 µM).
After 3 washes with incubation buffer, cells were fixed with methanol
(5 min at Mass Spectrometry--
rAnxA2 (450 µM in
phosphate-buffered saline) was incubated with a 5-mM
solution of peptide (LCKLSL, L(hC)KLSL or LAKLSL) and with 5 mM free homocysteine (hC) for 3 h at 37 °C. The pH
was adjusted to 7.4 with NH4CO3 before the
incubation. The reaction was stopped with 1 µl of formic acid and
diluted 1:5 with 50% methanol, 0.1% trifluoroacetic acid. One
microliter of the resulting solution was mixed with sinapinic acid
(1:1) and analyzed by MALDI-TOF mass spectrometry (Voyager DE-STR,
Applied Biosystem). The direct binding of LCKLSL, L(hC)KLSL, and LAKLSL
peptides to rtPA was analyzed under the same experimental conditions.
Amino Acid Modifications of the LCKLSL AnxA2
Sequence--
Previous work led to the identification of the
hexapeptide LCKLSL (residues 7-12) from the AnxA2 amino-terminal
domain as the minimum sequence required for interaction between AnxA2
and tPA (31). Replacement of the Cys8 of this sequence by
Gly abolished the interaction with tPA, indicating that this amino acid
is critical for such interaction. To determine whether additional amino
acids of this hexapeptide are required for the tPA-AnxA2 interaction
and to identify peptides with improved blocking properties, we designed
a small hexapeptide library with the modifications summarized on
Table I. These modifications include
glycine scans (peptides 2-7), alanine scans (peptides 8-13),
D-amino acid scans (peptides 14-19), conservative
replacements (Leu/Ile, peptides 20-22; Leu/Val, peptides 23-25;
Lys/Arg, peptide 26; Ser/Thr, peptide 27), Cys protection (peptide 28),
and enantiomer and retroenantiomer sequences (peptides 29 and 30). The
competitive properties of the peptides were then evaluated in the
tPA-AnxA2 interaction assays described below.
Receptor Binding Properties of Biotinylated tPA--
We employed a
non-radioactive modification of the competitive binding experiments
already described (31) using tPA labeled with biotin (see
"Experimental Procedures" for details). Fig. 1 shows that soluble
btntPA interacts in a dose-dependent fashion
with immobilized rAnxA2, showing half-maximal binding at a 200 nM concentration. This binding profile is in the same range
as that already described for 125I-tPA (8), indicating the
comparability of both assays. Moreover, an excess of unlabeled tPA
completely inhibited the binding of btntPA to AnxA2 (not
shown).
In addition, cell binding studies were performed by assaying the
binding of btntPA to HUVEC cells because AnxA2 is the main
receptor for tPA in endothelial cells. As shown in Fig.
2, btntPA binds to the
surface of HUVEC in a selective and dose-dependent manner,
indicating that the labeled protein also recognizes native AnxA2 on the
cell membrane.
Competitive tPA-AnxA2 Binding Properties of LCKLSL and Modified
Hexapeptides in Vitro and in Vivo--
The peptides described in Table
I were tested for their ability to compete with the interaction between
btntPA and rAnxA2. Fig. 3
shows the results obtained with several representative peptides. As
expected, peptides lacking Cys8 (Fig. 3, peptide 9, LAKLSL)
did not compete for tPA binding; the Ala substitution is shown, but
similar results were obtained with the Gly substitution and when the
Cys was protected, confirming the crucial role of this amino acid in
the interaction. Surprisingly, the enantiomer (Fig. 3, peptide 29, lcklsl) and the retroenantiomer (Fig. 3, peptide 30, lslkcl) competed
the interaction in a manner similar to that of the canonical
hexapeptide (Fig. 3, peptide 1, LCKLSL), indicating that
configurational/conformational characteristics are not determinant for
the binding. Moreover, all other amino acid modifications resulting
from the Ala and Gly scans showed the same competition activity as the
LCKLSL sequence (Table I).
We next assayed the inhibitory effect of the modified hexapeptides on
tPA binding to the surface of endothelial cells. Fig. 4 shows that the canonical hexapeptide
(peptide 1, LCKLSL) and its enantiomer (peptide 29, lcklsl) cause a
40-60% reduction of the binding of btntPA to HUVEC.
Peptides with mutations in Cys had no effect (peptide 9, LAKLSL, is
shown in Fig. 4).
All Cysteine-containing Peptides Compete the Binding of
tPA-AnxA2--
The results described above strongly suggested that the
presence of a Cys residue in the sequence was sufficient to compete the
tPA-AnxA2 interaction. To test this hypothesis, a completely unrelated
peptide containing one Cys residue (peptide 31, SFQTTTTYPTPSHPQTTLPC), a randomized version of the LCKLSL sequence (peptide 32, KLLCLS), and a
four-amino acid peptide fragment of the canonical hexapeptide (peptide
33, LCKL) were next assayed. As shown in Fig.
5, all these peptides display the same
competitive activity as LCKLSL, indicating that the presence of a Cys
residue in the competing peptide is sufficient to disrupt the
interaction.
To determine whether binding of peptides to tPA, rather than to AnxA2,
was required for the inhibition, peptides were directly added to
rAnxA2, plates were washed, and btntPA was then added. The
results were similar to those described above, strongly suggesting the
direct interaction of AnxA2 with the peptides (data not shown).
Homocysteine Inhibits the Binding of Biotinylated tPA to
AnxA2--
The specific interaction of tPA with endothelial cells is
inhibited by the thiol-containing amino acid hC, a fact that has been
postulated as an explanation for the thrombotic events related to
homocysteinemia (45-47). The molecular mechanism by which hC impairs
tPA-endothelium interaction has been proposed to be based on the direct
blockade of the tPA binding domain of AnxA2 through formation of a
disulfide link with Cys8 (31). We have compared the effects
of hC and the peptides used in this study on the binding of tPA to
AnxA2 under the experimental conditions described above. Fig.
6 shows that hC competed less efficiently
than the peptides. In contrast, when an analogue of the canonical
hexapeptide containing hC instead of Cys (peptide 34, L(hC)KLSL)
was assayed, it competed as efficiently as the consensus LCKLSL
peptide. The lower effect of free versus
peptide-incorporated hC may be explained by differences in stability of
the reduced state.
Cysteine-containing Peptides Form a Disulfide Linkage
with rAnxA2--
As mentioned above, the mechanism proposed to explain
the blocking effect of hC on the tPA-AnxA2 interaction is direct
binding between hC and Cys8 through a disulfide bond (31).
Our previous results suggest that any Cys-containing sequence can
compete the binding between tPA and rAnxA2 in a similar fashion. To
account for this fact we have considered two possible explanations: i)
a low affinity interaction (disrupted by high concentrations of
thiol-containing peptides) exists between tPA and AnxA2, which
implicates Cys8 of AnxA2, or ii) by forming a disulfide
bond with Cys8 of AnxA2 (as does homocysteine),
Cys-containing peptides render this amino acid unavailable for further
interaction, such as tPA binding. To test these hypotheses, we have
studied the binding of Cys-containing peptides to rAnxA2 by mass
spectrometry. Fig. 7 shows the MALDI-TOF
spectra of AnxA2 incubated, or not, with the hexapeptides LCKLSL
(panel A) and L(hC)KLSL (panel B). Both peptides
bound to rAnxA2, as reflected by the increase of molecular mass from
41,043 Da (free rAnxA2) to 41,619 Da (rAnxA2 incubated with LCKLSL) or
41,598 Da (rAnxA2 incubated with L(hC)KLSL). These increments in 576 and 555 Da, respectively, fit well with the molecular mass of the
hexapeptides and suggest the formation of a complex with a 1:1
stoichiometry. In contrast, incubation of rAnxA2 with the Cys-lacking
hexapeptide LAKLSL had no effect on the mass spectrum (Fig. 7,
panel C). These results indicate that peptide competition is
mediated by direct disulfide bond formation with rAnxA2. This
interaction is likely to be mediated by Cys8, although the
participation of other Cys residues cannot be completely ruled out.
To exclude the possibility that the peptides bound promiscuously to any
Cys-containing protein, and more specifically to tPA, LCKLSL,
L(hC)KLSL, and LAKLSL peptides were incubated with tPA, and the
reaction products were analyzed by MALDI-TOF. The spectra of tPA were
not significantly changed upon incubation with any of the peptides
analyzed (data not shown).
The dramatic increase in tPA catalytic activity resulting from its
binding to cellular membranes provides an interesting target for the
modulation of its biological functions related to the proteolytic
degradation of fibrin as well as other substrates. We have recently
shown that tPA is overexpressed in pancreas cancer cells and the
blockade of tPA is associated with reduced in vitro invasiveness and tumorigenicity (37, 38). Therefore, the tPA system
constitutes an attractive target for the development of novel
therapies. Because cell surface receptors allow focalization of the
proteolytic activity at the cell membrane and may also participate in
signal transduction, their identification is of major importance.
AnxA2 has been shown to be a major tPA receptor in endothelial cells,
among others, and preliminary data indicate that this protein is also
overexpressed in pancreas cancers (36, 37).2 On the basis
of these findings, it can be postulated that AnxA2 might play a role in
stimulating tPA activity and plasmin generation at the membrane of
cancer cells, potentially leading to increased tumorigenic properties.
Nevertheless, our data do not exclude the possibility that some of the
effects of tPA and it(s) receptor(s) take place independently of its
proteolytic activity and/or that of plasmin, as is the case of some
effects mediated by the receptors for urokinase-type plasminogen
activator (48). The blockade of the interaction between tPA and AnxA2
might provide clues about the precise role of this molecule as a tPA
cell surface receptor in cancer cells.
Hajjar et al. (31) have recently reported on the critical
role of a Cys residue in the LCKLSL (residues 7-12) sequence at the N
terminus of AnxA2 for tPA and hC binding and have proposed that the
interaction between the latter and AnxA2 leads to a reduction of tPA
binding, thus providing an explanation for the prothrombotic effects of
hC (45-47). This conclusion was based on the fact that: 1) an AnxA2
33-kDa chymotryptic peptide lacking the N-terminal peptide did not
compete tPA-AnxA2 binding, 2) the hexapeptide abolished the binding of
125I-tPA to immobilized purified AnxA2 and to HUVEC cells,
3) synthetic peptides lacking Cys8 did not inhibit binding,
and 4) a systematic mutation of all Cys residues of AnxA2 showed that
only Cys8 was required for the interaction. Therefore, we
initiated a search for variant peptides that were able to better
compete AnxA2-tPA binding and had more suitable pharmacological
properties. These peptides were tested using the experimental
procedures previously described (31) except that tPA was labeled with
biotin instead of 125I. Our findings confirm the
requirement of Cys in the LCKLSL sequence but also suggest that other
regions of the molecule must be necessary in order to confer
specificity to the interaction.
We found that all peptides containing a Cys residue, regardless of the
rest of their sequence and of the position in which the Cys was
located, were able to compete the interaction between tPA and AnxA2
because of their capacity to bind AnxA2. The fact that the consensus
peptide, as well as its enantiomer and retroenantiomer versions, showed
similar properties in these assays and the small size of the peptides
used argue in favor of mechanisms independent of a strong secondary
structure. Because hC has been shown to block AnxA2-tPA binding through
the formation of a disulfide bond with the thiol group of
Cys8, we considered the possibility that the effects of
synthetic peptides used in the assays performed here and in the work of Hajjar et al. might act through similar mechanisms. Our
results indicate that the peptides compete with AnxA2-tPA binding more efficiently than hC and that peptide LCKLSL and its enantiomer bind
covalently to AnxA2 and induce an increase in molecular mass, as
determined by mass spectrometry, whereas a peptide with a Cys-Ala substitution does not bind. Although we cannot exclude the covalent binding of the peptides to other Cys residues, all available evidence points to an interaction with Cys8, because it has been
shown that it is the only residue to which hC binds (31). Similar
analyses using mass spectrometry showed that the same peptides do not
bind tPA.
AnxA2 has been described (49) to exist as at least three different
forms, monomer, heterodimer (composed of one molecule of AnxA2 and one
molecule of 3-phosphoglycerate kinase), and heterotetramer (two AnxA2
subunits and two 11-kDa regulatory subunits called p11 or S100A10).
These different forms seem to be present in distinct subcellular
compartments; AnxA2 monomer is mainly cytosolic (49-51), whereas the
heterodimer has been described in the nucleus (52-54) and the
heterotetramer is associated with the plasma membrane (55). The AnxA2
heterotetramer is the most abundant form of the protein, representing
90-95% of the total AnxA2 in endothelial, epithelial, and Madin-Darby
canine kidney cells (34, 56). The p11 light chain regulates many of the
activities of AnxA2 and confers to the heterotetramer biochemical
properties distinct from those of the monomer (49, 57, 58). In
particular, the tetramer is an extremely potent activator of
plasminogen, stimulating the rate of activation of (Glu) plasminogen
about 341-fold, compared with an approximate 6-fold stimulation by the
monomer, and inducing a 90-fold increase in the catalytic efficiency of
tPA for (Glu) plasminogen (59). Binding of p11 to AnxA2 also decreases
the Kd (Ca2+) for the binding of AnxA2
to biological membranes. In addition, the monomer bundles F-actin to a
much lesser extent than the tetramer, and actin binding is important
for regulation of intracellular AnxA2-membrane-mediated functions.
Overall, the data suggest that p11 acts as a modulator of properties
displayed by the p36 core protein. Interestingly, the interaction
domain for p11 has been located, using fluorescence spectroscopy,
within the first 9 amino acids of the AnxA2 tail (STVHEILCK) (32). This
sequence partially overlaps with the LCKLSL peptide, suggesting that
both p11 and tPA could compete for binding to AnxA2. However, the fact
that tPA can bind to the tetramer, thereby leading to enhanced plasmin generation activity, supports the notion that non-overlapping domains
must also be involved in binding of AnxA2 to p11 and to tPA. Our
studies do not modify the interpretation of the mechanism of
interaction of AnxA2 with p11, because the latter has been shown with a
fluorophore used in fluorescence spectroscopy studies to be dependent
on the N-terminal acetyl group of Ser1 (60, 61) and
unaffected by substitution of Cys8 (32). Because the AnxA2
tetramer is overexpressed at the extracellular side of the membrane in
tumor cells (35) and its expression has been associated with
cellular transformation and metastasis (35, 28), elucidating the
precise structure of the complex remains an important task.
Regarding the binding site in the tPA molecule, Beebe et al.
(62) have proposed that residues 7-17 of its finger domain (RDEKTQMIYQQ) are involved in binding to AnxA2. This sequence mimics
partially the sequence of p11 responsible for AnxA2 binding (CRDGK, residues 61-65), suggesting again the possibility
of competition between both molecules for AnxA2 binding. However, the
high concentrations (mM) of tPA-derived peptide required
for HUVEC binding suggest that other regions of the molecule may also
play a role. For instance, tPA deletion mutants lacking the finger
domain or both the finger and growth factor domains are still able,
although to a much lesser extent, to bind to endothelial cells (63) and
stimulate plasminogen activation (39). However, in these assays the
cellular receptors involved in the interaction with tPA or tPA-derived
fragments were not molecularly characterized. Altogether, the available evidence indicates that a reassessment of the domains involved in the
binding of AnxA2, tPA, and p11 is necessary and that it is important to
take into account that different domains may be involved in different
cellular processes, given the broad range of functions ascribed to the
molecules involved in this complex.
The precise mechanisms through which the overexpression of tPA and
AnxA2 may contribute to tumor progression in pancreas cancer, as well
as in other tumor types, are not clear and may be severalfold. To
tackle more effectively such processes and to develop therapeutic strategies, a better understanding of the molecular interactions between these two proteins is necessary.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
90% by analytical HPLC) by
preparative reverse phase HPLC (41). Peptides were satisfactorily
characterized by amino acid analysis and by MALDI-TOF mass spectrometry
(Voyager DE-STR, Applied Biosystems, Foster City, CA), which was also
used to confirm their oxidative state (free thiol).
20 °C), washed 3 times with 0.02% Tween 20 in
phosphate-buffered saline, and incubated with alkaline
phosphatase-coupled streptavidin (1 h, 37 °C). Reactions were
developed and quantified as described above. To determine specific
binding, a 50-fold excess of unlabeled tPA was added during the
incubation with btntPA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Synthetic peptides used to compete the binding of tPA to AnxA2
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Fig. 1.
Binding of btntPA to recombinant
AnxA2. Wells of a 96-well plate were coated with rAnxA2 as
described under "Experimental Procedures" and incubated with
increasing concentrations of btntPA. Results of one
representative experiment of two independent assays performed are
shown. Error bars indicate S.E. (n = 3).
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Fig. 2.
Binding of btntPA to HUVEC.
Confluent HUVEC cultured in 96-well plates were incubated with
increasing concentrations of btntPA. Results of one
representative experiment of two independent assays performed are
shown. Error bars indicate S.E. (n = 3).
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Fig. 3.
Effect of synthetic LCKLSL-derived
hexapeptides on btntPA binding to rAnxA2. Wells of a
96-well plate were coated with rAnxA2 as described under
"Experimental Procedures." Peptides, at the indicated
concentrations, and btntPA (100 nM) were
simultaneously added to rAnxA2 and incubated at 37 °C for 2 h.
Peptide 1 (LCKLSL), . Peptide 15 (LAKLSL),
. Peptide 29 (lcklsl),
. Peptide 30 (lslkcl),
. Results of one representative experiment
of two independent assays performed are shown. Error bars
indicate S.E. (n = 3).
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Fig. 4.
Effect of synthetic LCKLSL-derived
hexapeptides on btntPA binding to HUVEC. HUVEC were
cultured in 96-well plates and grown to confluence as described under
"Experimental Procedures." btntPA (100 nM)
was added in presence of peptide 1 (LCKLSL), , peptide 15 (LAKLSL),
, and peptide 29 (lcklsl),
. Results of one representative
experiment of three performed are shown.
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Fig. 5.
Effect of Cys-containing synthetic peptides
on btntPA binding to rAnxA2. Wells of a 96-well plate
were coated with rAnxA2 as described under "Experimental
Procedures." Peptides, at the indicated concentrations, and
btntPA (100 nM) were added to rAnxA2 and
incubated at 37 °C for 2 h. Peptide 1 (LCKLSL), . Peptide 32 (KLLCLS),
. Peptide 33 (LCKL),
. Peptide 31 (SFGTTTTYPTPSHPQTTLPC),
. Results of one representative experiment
of three independent assays performed are shown. Error bars
indicate S.E. (n = 3).
View larger version (13K):
[in a new window]
Fig. 6.
Effect of homocysteine and hC-containing
synthetic peptide on btntPA binding to rAnxA2. Wells
of a 96-well plate were coated with rAnxA2 as described under
"Experimental Procedures." Peptides, at the indicated
concentrations, and btntPA (100 nM) were added
to rAnxA2 and incubated at 37 °C for 2 h. Peptide 1 (LCKLSL),
. Homocysteine,
. Peptide 34 [L(hC)KLSL],
. Results of one
representative experiment of two performed are shown. Error
bars indicate S.E. (n = 3).
View larger version (22K):
[in a new window]
Fig. 7.
Mass spectrometry analysis of untreated and
peptide-treated rAnxA2. rAnxA2 was dissolved in phosphate-buffered
saline, incubated with different peptides for 3 h at 37 °C, and
analyzed by MALDI-TOF mass spectrometry as described under
"Experimental Procedures." Analyses of untreated rAnxA2
(panels A, B, and C, left
graph) as well as rAnxA2 treated with peptide 1 (LCKLSL)
(panel A, right graph), peptide 34 (L(hC)KLSL) (panel B, right graph), or
peptide 15 (LAKLSL) (panel C, right graph).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. C. Castellernau and M. G. Lampugnani for kindly providing the HUVEC cells and Drs. A. García de Herreros and J. Domínguez for helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from Instituto de Salud Carlos III (00/0462), Biomed Program (BMH4-CT98.3085), Dirección General de Enseñanza Superior e Investigación Científica (PM97-0077), and Comissió Interdepartamental de Recerca i Innovació Tecnològica (Generalitat de Catalunya) (SGR-00245 and SGR-00410).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.
¶ Supported by a fellowship from the Ministerio de Educación, Cultura y Deporte.
Both authors contributed equally to this work.
** To whom correspondence should be addressed: Unitat de Biologia Cel·lular i Molecular, Institut Municipal d'Investigació Mèdica, Dr. Aiguader, 80, 08003-Barcelona, Spain. Tel.: 34-93-2211009; Fax: 34-93-2213237; E-mail: pnavarro@imim.es.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M207605200
2 S. Peiró, S. Aguilar, J. M. Corominas, F. X. Real, and P. Navarro, unpublished work.
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ABBREVIATIONS |
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The abbreviations used are: tPA, tissue-type plasminogen activator; btntPA, biotinylated tPA; AnxA2, annexin A2; rAnxA2, recombinant annexin A2; hC, homocysteine; p11, p11 light chain of annexin A2 tetramer or S100A10; HUVEC, human umbilical vein endothelial cells; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight.
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