(Received for publication, November 7, 1996)
From the Department of Biological Sciences, Tokyo
Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama
226, Japan, the § Pharmaceutical Research Department,
Sumitomo Metal Industries, Ltd., Kyoto 619-02, Japan, the
¶ Institute for Protein Chemistry, Osaka University, Osaka 565, Japan, and the
Department of Vascular Biology, The Scripps
Research Institute, La Jolla, California 92037
We have previously reported that propolypeptide
of von Willebrand factor (pp-vWF) promotes melanoma cell adhesion in a
1 integrin-dependent manner. In this report, we
identified the
subunit of the cell adhesion receptor for pp-vWF as
4. Human leukemia cell lines that express
4
1 integrin (very
late antigen-4, VLA-4), but not cell lines which lack VLA-4, attached
well to pp-vWF substrate and these adhesions were completely inhibited by anti-
4 integrin monoclonal antibody HP2/1. Adhesion of mouse melanoma expressing
4 integrin was also inhibited by anti-mouse
4
mAb PS/2. Furthermore, transfection of human
4 cDNA into
4
Chinese hamster ovary cells resulted in an
acquisition of adhesive activity to pp-vWF, indicating that pp-vWF is a
ligand for VLA-4 integrin. Using a recombinant fragment of pp-vWF, the
cell attachment site was shown to be located within amino acid residues
376-455 of pp-vWF. A series of synthetic peptides covering this region were tested for the ability to promote cell attachment and a 15-residue peptide designated T2-15 (DCQDHSFSIVIETVQ, residues numbered 395-409) promoted VLA-4 dependent cell adhesion. The peptide was also capable of
inhibiting cell adhesion to pp-vWF, suggesting that this sequence represents the cell attachment site. By affinity chromatography using
peptide T2-15-Sepharose, it was found that
4
1 integrin complex
from extracts of surface iodinated B16 cells specifically bound to the
peptide. These results strongly suggest that pp-vWF is a novel
physiological ligand for VLA-4.
Propolypeptide of von Willebrand factor
(pp-vWF),1 which is also called von
Willebrand antigen II (1), is an unusually large propolypeptide (~100
kDa) produced only in endothelial cells and megakaryocytes together
with blood coagulation protein von Willebrand factor (2). It is
processed from a large precursor of vWF (prepro-vWF) during
biosynthesis and stored in the granule of both endothelial cells and
platelets independent from mature vWF (3, 4). We have been
investigating the biological functions of pp-vWF and found that pp-vWF
bound to collagen and inhibited collagen-induced platelet aggregation
in contrast to the mature vWF (5, 6). Furthermore, we have found that
pp-vWF serves as a substrate for transglutaminase and is specifically
cross-linked to laminin (7), suggesting a possibility that it acts as
transient matrix protein upon secretion from platelets and endothelial
cells at the site of vascular injury. In a previous paper (8), we
reported that pp-vWF promoted the attachment and spreading of melanoma
cells. The receptor responsible for this adhesion was the 1 class of integrin but the corresponding
subunit could not be identified.
Integrins are heterodimeric transmembrane proteins consisting of and
subunits and mediate cell adhesion to extracellular matrix
proteins as well as cell-cell interactions (9-12). To date more than
15
subunits and 8
subunits have been identified and combination
of
and
subunits determines the ligand specificity of individual
integrins. Integrin-mediated cell adhesion plays crucial roles in
regulating the morphology, proliferation, migration, and
differentiation of cells. Ligands for integrins are quite diverse and
include many extracellular matrix proteins as well as cell surface
molecules. VLA-4 (
4
1) is an integrin complex that recognizes both
alternatively spliced segment III (CS1) of fibronectin (13), and
vascular cell adhesion molecule-1 (VCAM-1) (14). Lymphocyte adhesion to
endothelial cells is primarily mediated by interaction of lymphocyte
VLA-4 with VCAM-1 expressed on cytokine-activated endothelial cells and
this pathway is thought to be central to the lymphocyte recruitment to
the site of inflammation (15). Moreover, interactions of VLA-4 and its
counter-receptors have also been implicated in a number of physiologic
and pathogenic processes including CD3-dependent T cell
activation (16), lymphohemopoiesis (17-19), myogenesis (20), and
melanoma cell metastasis (21, 22). Therefore, VLA-4 is attracting broad
attention from those who are investigating the molecular mechanisms
underlining these processes.
In the present paper, we found that pp-vWF is the novel ligand for the
VLA-4. Furthermore, we identified the cell attachment site as a
15-residue linear sequence present in the midregion of pp-vWF molecule.
A synthetic peptide with this sequence could promote cell attachment in
an 4
1 integrin-dependent manner and directly bound to
VLA-4 complex.
Monoclonal antibody (mAb) 4B4 recognizing human
1 integrin was a gift from Dr. C. Morimoto, Dana-Farber Cancer
Institute, Boston, MA. MAbs A1A5 and TS2/16 (both anti-human
1) were
obtained from Dr. M. E. Hemler (Dana-Farber Cancer Institute, Boston,
MA). Mouse mAb 6F1 (anti-
2) was from Dr. B. S. Coller (State
University of New York, Stony Brook, NY), BIIG2 (anti-
5) was from
Dr. C. Damsky (University of California San Francisco, San Francisco, CA). Mouse mAbs P1B5 (anti-
3), HP2/1 (anti-
4), and
LM609(anti-
v
3) were purchased from Terios Pharmaceutical Co. (La
Jolla, CA), Cosmo Bio Co., Ltd (Tokyo Japan), and Chemicon
International Inc. (Temecula, CA), respectively. A rat anti-
6
integrin mAb GoH3 was a gift from Dr. A. Sonnenberg, Central Laboratory
of the Netherlands Red Cross Blood Transfusion Service, Amsterdam,
Netherlands. Rabbit antisera to the C terminus of human
3B,
4,
5, and
v integrin subunits were kindly donated by Dr. E. Ruoslahti, La Jolla Cancer Research Institute, La Jolla, CA. Polyclonal
antibody against C terminus of the
7 subunit was prepared by
immunizing a synthetic peptide having sequence of cytoplasmic tail of
human
7B subunit (DAHPILAADWHPELG) in our laboratory. These
polyclonal antisera all recognized respective subunits from mouse
integrin because of the high interspecies conservation of the sequence
in the cytoplasmic region. Rat hybridoma cells secreting anti-mouse
4 integrin PS/2 were obtained from American Type Culture Collection
and ascites containing this antibody was produced. A recombinant
VCAM-1-mouse C
chain fusion protein was donated by Dr. D. Dottavio
(Sandoz Pharmaceuticals, East Hanover, NJ) and a CS1 peptide-rat serum albumin conjugate was a gift from Dr. E. Wayner (Fred Hutchinson Cancer
Research Center, Seattle, WA). Synthetic peptides GRGDSP and
DELPQLVTLPHPNLHGPEILDVPST (CS1) were purchased from Sigma. pp-vWF was
purified from bovine-washed platelets by immunoaffinity chromatography as described previously (23).
Wild-type human 1 integrin cDNA (24) was
subcloned into pBJ-1 vector and transfected into Chinese hamster ovary
(CHO) cells together with pFneo DNA containing the neomycin resistance
gene by electroporation as described previously (25). Stable clones were selected using 700 µg/ml G418 (Life Technologies, Inc.), and
cells expressing human
1 integrin most abundantly were selected by
single-cell sorting using an anti-
1 mAb. For obtaining CHO cells
expressing both human
4 and
1 integrins, wild-type human
4
cDNA (26) was transfected into
1 integrin-expressing clone together with CDhygro DNA containing the hygromycin-resistance gene and
maintained in the medium containing 400 µg/ml hygromycin. Stable CHO
cell line clones expressing high levels of human
4 were again
selected by single-cell sorting using an anti-
4 mAb. Expression
levels of each human integrin subunit were verified by
fluorescence-activated cell sorting analysis using FACScan (Becton
Dickinson, Mountain View, CA) according to the method described
previously (24).
A cDNA clone encoding the 8-kDa portion of human
pp-vWF was synthesized using reverse transcriptase-polymerase chain
reaction amplification of human placental mRNA. Briefly, total
mRNA was prepared using the guanidinium isothiocyanate/cesium
chloride method (27). First strand cDNA was then generated by
reverse transcription using a 3-oligonucleotide primer complementary to the C terminus of the 8-kDa fragment (Lys455) with a
terminal EcoRI restriction sequence
(5
-GAATTCTTTCAGGAGGGGGAGCTGGA-3
). The reverse transcribed cDNA
mixture was subjected to 30 amplification cycles of polymerase chain
reaction using a 5
primer with an EcoRI restriction
sequence (5
-GAATTCACTTCAAGAGCTTTGACAACAGATA-3
). The polymerase chain
reaction products were ligated into the pBluescript and transformed
into Escherichia coli JMl09. The cloned cDNA was identified by restriction analysis and sequenced using the dideoxy chain termination method. The insert was then subcloned into the EcoRI site of a modified version of the pGEX-2T expression
vector (Pharmacia, Milton Keynes, United Kingdom), which included
additional cloning sites in its polylinker, and were used to transform
JMl09. Recombinant clone (pGEX-r8k1A) was checked for its insert
orientation by restriction mapping. Using the same polymerase chain
reaction product as a starting material, two additional clones
containing an insert of the truncated version of the 8-kDa fragment
(r8k1B, corresponds to Ser373-Asp415, and
r8k2A, corresponds to Gln409-Lys455) were also
obtained. Primers used in the amplification of r8k1B were
antisense primer 5
-GAATTCTGCAGTCAGTCGCGGTCATCAGCACACT-3
and
sense primer 5
-GAATTCACTTCAAGAGCTTTGACAACAGATA-3
; primers for
amplification of r8k2A were antisense primer
5
-GAATTCTTTCAGGAGGGGGAGCTGGA-3
and sense primer
5
-GAATTCTGCAGAAACAGTGTGCTGATGACCGCGA-3
.
Glutathione S-transferase (GST)-r8k1A fusion protein was
induced and isolated as follows. Briefly, 5 ml of overnight cultures of
JM109 transformed with recombinant plasmids were diluted 1:100 with
fresh LB medium containing 50 µg/ml ampicillin and cultured for about
4 h at 37 °C. Isopropyl--D-thiogalactoside was
then added to 0.5 mM and the culture continued for an
additional 2 h. Cells were then centrifuged, suspended in 1/30
volume of phosphate-buffered saline containing 1 mM
phenylmethylsulfonyl fluoride, and sonicated. As the fusion protein was
completely recovered in the insoluble fraction, extracts were
centrifuged and the pellet solubilized in 20 mM Tris-HCl,
pH 8.0, containing 8 M urea and 0.2 mM
-mercaptoethanol, separated on a gel filtration column of Sephacryl
S-200 equilibrated with the same buffer, and dialyzed against 20 mM Tris-HCl, pH 8.0, containing 1 M urea and
0.2 mM
-mercaptoethanol. The GST portion of the fusion
protein was cleaved by the addition of 5 units/ml bovine thrombin
(Sigma) for 14 h at room temperature. The cleavage mixture was
then applied to a gel filtration column of Sephacryl S-200, and the
cleaved 8-kDa fragment was further purified by reverse phase high
performance liquid chromatography on a RESOURCE RPC column (Pharmacia).
Fusion proteins of the truncated version of the 8-kDa fragment (r8k1B
and r8k2A) were recovered in the soluble fraction and purified by
glutathione-agarose affinity chromatography. Those proteins were pooled
and dialyzed against 150 mM NaCl, 10 mM
Tris-HCl, pH 7.4, and stored at
70 °C. Protein concentrations were
measured using the BCA assay (Pierce).
A series of 20-residue peptides (T1 to T5), which correspond to parts of the 8-kDa fragment, were synthesized by a multiple peptide synthesizer (Model 396, Advanced ChemTech Inc., Louisville, KY) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Shorter peptides (T2-15, T2-10, T1-8 and BP-5) were synthesized using an Applied Biosystems 430A peptide synthesizer with t-butoxycarbonyl chemistry. In both cases, peptides were purified by high performance liquid chromatography and verified by fast atom bombardment-mass spectrometry as described previously (28). Peptides (4 mg) were covalently coupled to 2 ml of packed beads of CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) according to the manufacturer's instruction.
Cell Adhesion AssayB16 murine melanoma, human monocytic
cell lines U937 and THP-1, human lymphoma cell lines MOLT-3 and Jurkat,
and human erythroleukemic cell line K562 were provided from the
Japanese Cancer Research Resources Bank (Tokyo, Japan) and cultured in
either Eagle's minimum essential medium containing 10% fetal calf
serum or RPMI 1640 medium containing 10% fetal calf serum and
non-essential amino acids. Adherent cells were grown to near confluency
and harvested by incubation with phosphate-buffered saline, pH 7.2, containing 2.5 mM EDTA and 2 mg/ml bovine serum albumin for
30 min at 37 °C. Detached cells as well as suspension cells were
washed three times and suspended in serum-free Eagle's minimum
essential medium or RPMI 1640 prior to the adhesion assay. In the case
of assay using human leukemia cells and CHO cells transfected with
human integrins, the cells were pretreated with 1:1500 dilution of
anti-human 1 activating mAb TS2/16 ascites to activate human
1
integrin. Cell adhesion assay was performed according to the method
described previously (8) with a slight modification. In brief, 6-mm
square chips cut from bacteriologic plastic dishes (Falcon 1029) were coated for 16 h at 4 °C with 50 µl of bovine pp-vWF (5 µg/ml), CS1-rat serum albumin (0.1 µg/ml), and bovine fibronectin
(10 µg/ml). Some peptides were coated by mounting peptide solution on
the chips and drying up at room temperature. It was confirmed by the
protein assay that more than 80% of ligands were absorbed on the
surface under these conditions. For VCAM-1, chips were first coated
with anti-mouse C
chain (2 µg/ml, Caltag Laboratories, South San
Francisco, CA) and then incubated with recombinant VCAM-1-mouse C
chain (2 nmol/chip). After blocking nonspecific protein-binding sites
by incubation with 10 mM Tris-HCl, 150 mM NaCl,
pH 7.4, containing 1% bovine serum albumin and 100 µg/ml mouse IgG
at room temperature for 1 h, the chips were placed at the bottom of 48-well tissue culture dishes (Costar 3548) and overlaid with 2-5 × 105 cells in 50 µl of serum-free medium.
After incubation at 37 °C for 90 min, the chips were picked up and
rinsed in cold phosphate-buffered saline to remove nonadherent cells,
and fixed with 1% glutaraldehyde in phosphate-buffered saline.
Adherent cells were either photographed or counted using a light
microscope with a calibrated grid marked on the ocular lens. An
adhesion assay using peptide-coupled Sepharose was conducted as
follows. Cell suspension was added to 96-well microtiter plate
containing monolayer of peptide-Sepharose beads and incubated at
37 °C for 90 min. The beads bearing adherent cells were fixed with
1% glutaraldehyde in phosphate-buffered saline and separated from
nonadherent cells by differential centrifugation, and stained with
Giemsa prior to observation.
Confluent B16 melanoma cells grown
in a 100-mm dish were detached as described above and iodinated by the
lactoperoxidase/glucose oxidase method (29). Cells were then lysed with
1 ml of 100 mM octyl--D-glucopyranoside in
10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl,
2.5 mM MgCl2, 1 mM
MnCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A at 4 °C for 15 min.
After removal of insoluble material by centrifugation, the extract was
mixed with an equal volume of peptide-Sepharose, gently shaken at room
temperature for 3 h, and packed into a column. After washing out
the unbound materials with 8 column volumes of a washing buffer (50 mM octyl-
-D-glucopyranoside, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2.5 mM MgCl2, 1 mM MnCl2)
followed by 1 column volume of a pre-elution buffer (same as washing
buffer without MgCl2 and MnCl2), the bound
materials were eluted with an elution buffer which contains 5 mM EDTA in the pre-elution buffer. Eluted materials were
collected at 1 ml/fraction and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5%
polyacrylamide gel under reducing conditions as described by Laemmli
(30) followed by autoradiography using Bioimaging Analyzer BAS 2000 system (Fuji Film Co., Tokyo, Japan).
Eluates from T2-15-Sepharose were subjected to immunoprecipitation assay using anti-integrin antibodies. Samples were incubated either with 3 µl of antisera or 1 µg of IgG. Protein G/Protein A-agarose (Oncogene Science, Inc.) was then added to the mixture and incubated at 4 °C for 4 h with shaking. The immunocomplexes were centrifuged and the beads were washed four times with washing buffer as described above. The samples were then boiled in reducing Laemmli sample buffer and analyzed by SDS-PAGE on a 7.5% polyacrylamide gel as described above.
In a previous study (8), we
have found that only a limited number of cell lines are capable of
adhering to pp-vWF, i.e. only two cell lines of melanoma
origin out of more than 15 cell lines of both normal and tumor tissue
origin adhered well to pp-vWF. Therefore, we thought at first that the
receptor for pp-vWF is rather specifically expressed on melanoma cells.
However, this was not the case because several cells of leukemic origin
adhered to pp-vWF (Table I). Two monocytic cell lines,
THP-1 and U937, as well as two lymphocyte-like cells, Jurkat and
MOLT-3, are capable of attaching to the pp-vWF substrate in the
presence of a human 1 integrin-activating monoclonal antibody (mAb)
TS2/16. In contrast, an erythroleukemic cell line K562, which also
expresses
1 integrin, did not adhere to pp-vWF even in the presence
of both TS2/16 and Mn2+ ion. It is therefore very likely
that the former four cell lines express integrin
subunit
responsible for adhesion to pp-vWF while K562 does not. When the
expression of various
subunit of integrin was analyzed by
fluorescence-activated cell sorting analysis, the adhesive property of
these cells corresponded well to the expression level of
4 integrin
(Table I). As it is known that several melanoma cells also express high
levels of
4
1 integrin, it is suggested that the
subunit of
the
1 integrin receptor for pp-vWF is
4. To confirm this, the
effect of anti-
4 integrin mAbs on the cell adhesion to pp-vWF was
assessed. As depicted in Table II, adhesion of human
leukemia cells to pp-vWF was completely inhibited by an anti-human
4
mAb HP2/1, as well as an anti-human
1 mAb 4B4, but not by an
anti-human
5 mAb BIIG2. None of the function-blocking antibodies
against
2,
3, and
6 subunit affected this adhesion (data not
shown). The adhesion of mouse melanoma B16 was also inhibited by an
anti-mouse
4 mAb PS/2, indicating that VLA-4 integrin is the
responsible adhesion receptor for pp-vWF on both melanoma and
hematopoietic cells.
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To
confirm that pp-vWF is a ligand for VLA-4 integrin, CHO cells were
transfected with cDNA coding for human integrin subunits and the
resultant stable transfectants were assessed for the ability to adhere
to pp-vWF. As shown in Fig. 1A, the
transfectant clones (1-CHO and
4
1-CHO) express high levels of
respective human integrin subunits. Both transfectants adhered well to
plasma fibronectin (Fig. 1B), probably by using intrinsic
5 subunit complexed with both intrinsic and transfected
1
subunit. On the other hand, only
4
1-CHO cells adhered to CS1 and
VCAM-1, the known ligands for VLA-4, indicating that they express
functional VLA-4 integrin. Furthermore,
4
1-CHO but not
1-CHO
adhered to pp-vWF. This adhesion of
4
1-CHO to pp-vWF was
completely inhibited by an anti-
4 mAb HP2/1 (data not shown). These
results strongly indicate that pp-vWF is a ligand for VLA-4
integrin.
Cell Adhesion Activity of Recombinant 8-kDa Fragment
As
pp-vWF does not contain any homologous sequence to the known VLA-4
ligand sequences (CS1 region in fibronectin and first and fourth Ig
domain in VCAM-1), we were interested in knowing what sequence in the
pp-vWF primary structure is involved in cell adhesion. In a previous
paper (8), we have already suggested that the cell adhesion site in the
pp-vWF molecule resides within the central region of about 8 kDa. We
have shown that an 8-kDa fragment generated by the lysylendopeptidase
digest of bovine pp-vWF promotes melanoma cell adhesion in a
dose-dependent manner. Furthermore, a mAb reactive with
this fragment blocked the adhesive activity of pp-vWF. To determine the
VLA-4 ligand sequence in the pp-vWF, we decided to construct a
recombinant protein corresponding to this region. As amino acid
sequence analysis of the isolated fragment suggests that it corresponds
to a fragment having Ser373 as the N terminus and extends
to Lys435 or Lys455, we prepared a recombinant
fragment containing sequence 373-455 in human pp-vWF (r8k1A, Fig.
2) using a conventional bacterial fusion protein
expression system. When coated on plastic surface, this recombinant
fragment did promote attachment and spreading of B16 melanoma (Fig.
3). When compared at molar basis, the fragment showed
similar coating concentration dependence as intact pp-vWF protein,
suggesting that this fragment contains the authentic cell attachment
site. To narrow down the location of the active site, we prepared other
clones expressing the N-terminal (r8k1B) and C-terminal (r8k2A) half of
the 8-kDa fragment (Fig. 2) and assessed the cell adhesion activity of
GST fusion protein of these peptides. As depicted in Fig.
4, B16 melanoma cells adhered to GST-r8k1B but not to
GST-r8k2A. Again the extent of cell adhesion to the recombinant
fragment (r8k1B) was comparable to that of intact pp-vWF (more than
80% of the input cells were adhered) indicating that the active site
was solely located in the N-terminal half 42-residue portion.
Cell Adhesion Activity of Synthetic Peptides Derived from the 8-kDa Fragment
To further narrow down the cell attachment site in the
8-kDa region, we synthesized a series of peptides corresponding to this
region. Five 20-residue peptides with overlapping sequence covering the
entire length of the 8-kDa fragment were synthesized (Table
III) and tested for their ability to support B16 mouse
melanoma cell adhesion. Among the 5 peptides, 3 were active in
supporting cell attachment (Fig. 5). However, the
adhesions to peptides designated T1 and T4 were completely reversed by
the addition of heparin. On the other hand, the adhesion to peptide T2
was not affected by heparin at all and, furthermore, was blocked by
treatment of cells with an anti-mouse 4 integrin mAb, which was also
observed with the intact pp-vWF. It can be concluded that the integrin recognition sequence is contained in this 20-residue portion of pp-vWF
(391-410). As adhesion to intact pp-vWF was not affected by heparin at
all, the cell attachment to T1 and T4 peptides could be a result of
nonspecific electrostatic interaction between the cell surface and
peptides, which was not exhibited when these sequences are included in
the intact protein.
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To obtain shorter peptides with the cell adhesion activity, we
synthesized truncated versions of T2 peptide. As it was expected that
shorter peptides might have problem in coating on the plastic surface
by ordinary protocol, we immobilized these peptides covalently on
Sepharose beads and performed a cell adhesion assay using the peptide-conjugated beads. As clearly depicted in Fig. 6,
both 15- and 10-residue truncated peptides (T2-15 and T2-10,
respectively) were active in supporting adhesion of B16 murine
melanoma. A peptide derived from the central portion of T1 (T1-8) did
not show any adhesive property under this condition. Although the
extent of adhesion (average number of adherent cells on beads) was
almost the same for T2-15 and T2-10, cell spreading on
T2-10-Sepharose was less obvious than T2-15, suggesting that the
15-residue extension is necessary for full activity.
Inhibition of the Cell Adhesion to pp-vWF by Soluble Peptides
When the effect of soluble peptides on the B16 cell
adhesion to pp-vWF was assessed, the CS1 peptide strongly inhibited the adhesion (Fig. 7). Complete inhibition was achieved at
30 µM. In contrast, a peptide containing RGD sequence did
not affect adhesion at all, even at concentrations as high as 1 mM. Both peptide T2-15 and T2-10 had an inhibitory effect
on the B16 adhesion to pp-vWF. When added at 300 µM,
T2-15 inhibited cell adhesion by 75%. T2-10 had lower inhibitory
activity compared with T2-15; the inhibition at 300 µM
was only 40%, suggesting that the affinity of T2-10 is significantly
lower than that of T2-15. Two control peptides, T1-8 and BP-5 (derived
from a distant region of pp-vWF sequence containing similar net charge
and hydrophilicity value), had essentially no inhibitory effect.
Similar inhibition by CS1, T2-15, and T2-10 was also observed with
the B16 cell adhesion to CS1-rat serum albumin but not with adhesions
to collagen and laminin (data not shown), suggesting that the effect of
these peptides is specific to VLA-4-mediated adhesion.
Binding of VLA-4 Integrin to T2-15 Peptide Immobilized on Sepharose Beads
The results presented above strongly indicate
that the T2-15 sequence (DCQDHSFSIVIETVQ) represents the VLA-4-binding
site in the pp-vWF molecule. To confirm this, binding of VLA-4 integrin to the T2-15 peptide was evaluated. As B16 murine melanoma cells adhered on T2-15-Sepharose (Fig. 6), it is obvious that the peptide retains active conformation to recognize its receptor even on the
Sepharose beads. A control peptide derived from the central portion of
T1 (T1-8) did not show any adhesive property under this condition.
These peptide-Sepharose conjugates were then used as affinity matrices
to isolate the cell surface receptor.
Octyl--D-glucopyranoside extract of surface-iodinated
B16 mouse melanoma was applied to an affinity column of
T2-15-Sepharose, unbound materials were removed by washing, and the
bound materials were eluted with EDTA. As shown in Fig.
8A, EDTA eluted two polypeptides of
approximate molecular sizes of 150 and 125 kDa in the early step of
elution from T2-15-Sepharose, followed by the elution of a 210-kDa
protein in a rather retarded fraction. This 210-kDa band was also seen in the eluted fraction from a control column, T1-8-Sepharose, suggesting that it is a nonspecifically bound protein. The apparent molecular mass of the two polypeptides shifted from 150/125 kDa to
145/115 kDa when analyzed under nonreducing conditions (data not
shown). These values are quite similar to that of the murine VLA-4
complex. To confirm that the bound material is VLA-4 integrin, its
reactivity toward available immunological probes for the various integrin
subunits was examined. The EDTA-eluted proteins were subjected to immunoprecipitation using antisera against the cytoplasmic tail of integrin subunits
3,
4,
5,
7,
v, and an
anti-human
6 rat mAb which can also recognize mouse
6 (GoH3). As
clearly shown in Fig. 8B, only anti-
4 antibody could
immunoprecipitate radiolabeled polypeptide from the material eluted
from T2-15-Sepharose. The relative molecular mass of the polypeptide
is about 150 kDa, suggesting that the 125-kDa band (
1 subunit) was
lost during the immunoprecipitation procedure. This is consistent with
a notion that
4
1 integrin complex tends to be dissociated during
immunoprecipitation experiments conducted in the presence of EDTA (14,
31). It is concluded that T2-15 peptide can directly bind to VLA-4
complex in the presence of divalent cation and contains a novel ligand sequence for the VLA-4 integrin.
We report herein that the receptor responsible for cell adhesion
to pp-vWF is VLA-4 (4
1 integrin). At first we tested more than 20 cell lines, which grow on culture dishes, for their ability to adhere
to pp-vWF and found that only the cultured tumor cell lines with
melanoma origin, including mouse melanoma B16, human melanoma G-361 and
MeWo, and hamster melanoma RPMI 1846, adhered on pp-vWF substrate (data
not shown). We then thought that the receptor for pp-vWF might be a
melanoma-specific molecule. When we tested cells of hematopoietic
origin, however, most of those floating cells attached well to pp-vWF.
It became clear that the receptor responsible for adhesion to pp-vWF
was rather widely distributed among hematopoietic cells but its
expression on adherent cells was limited to several melanoma cells. In
a previous paper (8), we reported that the cell adhesion to pp-vWF was
mediated by the
1 class of integrin using a function-blocking
anti-
1 mAb, but could not identify the corresponding
subunit.
Today 10
subunits (
1-9 and
v) are known to be associated
with
1 integrin. Among those,
4
1 integrin (VLA-4) is known as
a rather common antigen on hematopoietic cells (32) and it is also
known that several melanoma cells express high levels of VLA-4 (33). As
the expression of
4 but not other
subunits correlated with the
adhesive activity of hematopoietic cells to pp-vWF, we tested the
effect of anti-
4 mAbs on the cell adhesion to pp-vWF and found they
completely inhibited adhesion. This strongly indicates that VLA-4 is
the receptor for pp-vWF. Other mAbs with function blocking ability,
including anti-
2(6F1),
3(P1B5),
5(BIIG2),
6(GoH3), and
v(LM609), did not affect adhesion (data not shown), ruling out these
subunits as receptor candidates. Finally, transfection of
4 integrin
cDNA into CHO cells resulted in acquisition of adhesion activity
toward pp-vWF as well as other well known ligands; CS1 and VCAM-1. It
is concluded at this point that the cell adhesion receptor to pp-vWF is
4
1 integrin.
VLA-4 is an integrin complex that recognizes both CS1 region in
fibronectin and VCAM-1. Lymphocyte adhesion to endothelial cells is
primarily mediated by interaction of lymphocyte VLA-4 with VCAM-1
expressed on cytokine-activated endothelial cells and this pathway is
thought to be central to the lymphocyte recruitment to the site of
inflammation (34, 35). In addition, VLA-4/VCAM-1 interaction is
involved in lymphocyte homing to high endothelial venule in peripheral
lymph nodes where VCAM-1 is constitutively expressed (36). Leukocyte
extravasation at the site of inflammation as well as the vascular wall
injury is also thought to be mediated by leukocyte integrins, including
VLA-4 although other adhesion receptors such as selectins play some
part (10). These adhesions need to occur immediately at the right
place, which means the adhesive ligands must be distributed spatially
and timely. Fibronectin is a molecule abundantly found in almost all
tissues and its distribution is hard to be controlled by immediate
inflammatory response. VCAM-1 is an inducible cell surface molecule on
endothelial cells and is a desirable docking molecule for leukocyte
recruitment. However, VCAM-1 must be newly synthesized before
expression on the cells and it is only detected after 2 h
treatment of cultured endothelial cells with tumor necrosis factor-
(37). This means that VCAM-1 cannot mediate leukocyte adhesion in the
very early steps in the inflammation. VCAM-1/VLA-4 pathway also cannot
work when the endothelial cells are seriously injured or removed from
the vascular wall. If there is another molecule that can mediate
leukocyte adhesion in the very early step of inflammatory response, the
whole system would be more effective. Vonderheide and Springer (38)
have suggested that there is an unknown ligand for VLA-4 on endothelium because VLA-4-dependent adhesion of lymphoid cells to
cultured endothelial cells is not completely inhibited by antibodies
against VCAM-1 and fibronectin. Hahne et al. (39) have also
suggested that there is an unknown leukocyte adhesion mechanism on
mouse endothelioma cells that is not mediated by VCAM-1, ICAM-1,
E-selectin, or P-selectin. Our results that VLA-4 also recognizes
pp-vWF as an adhesion substrate may suggest that the adhesion to pp-vWF functions as a physiologically important pathway of leukocyte recruitment to the inflammatory/vascular injury sites. Biosynthesis of
vWF precursor is limited in megakaryocytes and endothelial cells (4).
Only platelets and endothelial cells are places that pp-vWF protein
exists in the whole body and pp-vWF is immediately released from these
cells upon activation by agonists such as thrombin (40). As pp-vWF has
the ability to bind to both collagen and laminin (5, 7), it may be
deposited at the exposed subendothelial matrix, thus presenting an
adhesion site for VLA-4-bearing leukocytes. In preliminary experiments,
we have observed that pp-vWF rapidly accumulates at injured sites of
glomerular vasculature during the experimental glomerulonephritis
while mature vWF does not.2 It is possible
that pp-vWF functions as an emergency tag for leukocytes/lymphocytes targeting and mediates successful
recruitment and infiltration of these inflammatory cells together
with VCAM-1.
We found that an amino acid sequence, DCQDHSFSIVIETVQ, derived from the
midregion of human pp-vWF, was a novel ligand sequence of VLA-4. A
peptide with this sequence by itself could promote cell adhesion in an
4
1 integrin-dependent manner, and, moreover, direct
binding of the VLA-4 integrin complex to this peptide is observed.
Although the shorter version of the peptide, T2-10 (DCQDHSFSIV), also
had cell adhesion activity, the inhibitory activity of this peptide on
cell adhesion to pp-vWF was significantly lower than that of T2-15. It
suggests that the 10-residue portion is essential for the recognition
by VLA-4, but the C-terminal extension increases the affinity and/or
stabilizes the favorable conformation of the peptide. CS1 and T2-15
peptide both can inhibit the cell adhesion to pp-vWF as well as to
CS1-bovine serum albumin, although these peptides share no homologous
motif in their sequences. It is possible that both peptides can bind to
the ligand binding pocket of VLA-4 in a similar way, thus inhibiting
the interaction of each other. Although the tertiary structure for the
pp-vWF protein has not been resolved, a computer-aided prediction of
secondary structure of the region containing this sequence by using the
algorithm of Robson (41) suggests that the segment
Asp395-Phe401 form a
-turn structure (data
not shown). It is possible that this segment forms loop structures
facing outward and is thus available to interact with the cell surface.
As this sequence contains neither the essential VLA-4-recognition motif
identified in CS1 (EILDV) (42) nor that in VCAM-1 (QIDSPL) (43), it
seems that this sequence represents a novel integrin-binding motif. However, it contains a sequence, VIET, which is similar to the sequence
around the essential glutamate residue (Glu34) in ICAM-1
(GIET) (44). As these sequences all contain some oxygenated residues
(Asp, Glu, Thr, or Ser) as well as isoleucine residue, these different
but rather similar sequences might be utilized as general integrin
binding motifs in each molecule together with other structures that
define the specificity. Determination of minimum essential residues for
the binding to the integrin within the T2-15 sequence is underway.
In conclusion, our finding that pp-vWF serves as a ligand for VLA-4 integrin provides new insights on the physiological significance of this vWF gene product which was originally thought as a mere propeptide without any particular functions. Furthermore, elucidation of mechanisms of the cell adhesion to pp-vWF will help in our understanding of mechanisms underlying the melanoma metastasis as well as vascular inflammation.
We thank Drs. C. Morimoto, M. E. Hemler, B. S. Coller, C. Damsky, A. Sonnenberg, E. Ruoslahti, D. Dottavio, and E. Wayner for their valuable gifts.