Definition of an Unexpected Ligand Recognition Motif for
v
6 Integrin*
Sabine
Kraft
,
Beate
Diefenbach
,
Raj
Mehta§,
Alfred
Jonczyk¶,
G. Albrecht
Luckenbach
, and
Simon L.
Goodman
From the Departments of
Biomedical Research
Immunology/Oncology and ¶ Medicinal Chemistry, Merck KGaA,
Darmstadt 64271, Germany and § Merck London, Medical
Research Council Collaborative Centre, 1-3 Burtonhole Lane, Mill Hill,
London NW7 1AD, United Kingdom
 |
ABSTRACT |
Integrin interactions with extracellular matrix
proteins are mediated by brief oligopeptide recognition sequences, and
synthetic peptides containing such sequences can inhibit integrin
binding to the matrix. The RGD peptide motif is recognized by many
integrins including
v
6, a specific receptor for fibronectin
thought to support epithelial cell proliferation during wound healing
and carcinoma progression. We report here the discovery of an
unexpected non-RGD recognition motif for integrin
v
6. We compared
the recognition profiles of recombinant
v
6 and
v
3 integrins
by using phage display screening employing 7-mer and 12-mer peptide
libraries. As predicted, phages binding strongly to
v
3 contained
ubiquitous RGD sequences. However, on
v
6, in addition to RGD-
containing phages, one-quarter of the population from the 12-mer
library contained the distinctive consensus motif DLXXL. A
synthetic DLXXL peptide,
RTDLDSLRTYTL, selected from the phage sequences
(clone-1) was a selective inhibitor of RGD-dependent ligand
binding to
v
6 in isolated receptor assays (IC50 = 20 nM), and in cell adhesion assays (IC50 = 50 µM). DLXXL peptides were highly specific
inhibitors of
v
6-fibronectin interaction as synthetic scrambled
or reversed DLXXL peptides were inactive. NH2-
and COOH-terminal modifications of the flanking amino acids suggested
that the preceding two and a single trailing amino acid were also
involved in interaction with
v
6. The DLXXL sequence
is present in several matrix components and in the
chain of many
integrins. Although there is as yet no precise biological role known
for DLXXL, it is clearly a specific inhibitory sequence for
integrin
v
6 which has been unrecognized previously.
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INTRODUCTION |
Integrins are a family of heterodimeric class I transmembrane
receptors involved in numerous cell-matrix and cell-cell adhesion phenomena (1). They can be grouped roughly into three classes: the
1
series, which are ubiquitous receptors for extracellular matrix (2);
the
2 series, which are activatable on leukocytes and are triggered
during the inflammatory response (3); and the
v series, which bind
and mediate the cell response to provisional extracellular matrices
found during wound repair and other pathological processes (4).
The integrins
5
1 (5),
IIb
3 (6),
8
1 (7),
v
1 (8),
v
3 (9), and
v
6 (10) all bind the Arg-Gly-Asp- (RGD) peptide
sequence in fibronectin, where it is presented in a constrained loop
(11). Soluble RGD-containing peptides can inhibit the interaction of
each of these integrins with fibronectin. However, to analyze the
function of individual fibronectin receptors in a particular cellular
environment it is useful to have more specific inhibitors, and a
variety of inhibitory antibodies, modified peptides, and non-peptidic
substances has been developed. However, as yet no inhibitor has been
discovered specific for
v
6.
v
6 is a rare integrin, induced
during repair processes in epithelia (10, 12). Its only known
specificities are for fibronectin (10), where it can be the
dominant receptor mediating cell adhesion (13), and for tenascin (14).
v
6 is believed to be involved in supporting the proliferation of
epithelia during repair processes (15), and it can promote the
proliferation of carcinoma cells (16).
Phage display technology has proved useful for identifying novel
specific peptide sequences that act as ligand mimetics (17, 18).
Accordingly, both constrained cyclic and linear peptide libraries have
been used to discover novel peptides that interact with integrins
(19-23). But, with few exceptions, these peptides contain RGD
sequences, whereas the non-RGD sequences found have only bound weakly.
Here we have used recombinant
v
6 expressed as a transmembrane
truncated soluble receptor to screen phage libraries displaying peptides of 7 or 12 amino acids. This revealed a strong and previously unpredicted recognition motif for
v
6 integrin which we describe here. Similar motifs are displayed in several extracellular matrix molecules.
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EXPERIMENTAL PROCEDURES |
Ligands--
The ligands fibronectin (24), vitronectin (25), and
fibrinogen (26) were purified from human blood and biotinylated as
described previously (27, 28).
Preparation of Integrins--
Human integrins were extracted
from native sources (
v
5,
IIb
3) or expressed as recombinant
soluble receptors using the baculovirus expression system (
v
3,
v
6).
IIb
3 was prepared from outdated human platelet concentrates (6)
as detailed previously (27) by affinity chromatography on
GRGDSPK-conjugated Sepharose CL-4B. The column was eluted with the
GRGDSPK, the peak containing
IIb
3 was concentrated ~5-fold, dialyzed, and stored at
80 °C.
v
5 was purified from human placenta (29). Term
placenta was minced in ~2 volumes of ice-cold solution A (0.05% w/v
digitonin, 2 mM CaCl2, 2 mM
Pefabloc (Merck), pH 7.4), then washed twice in solution A by
centrifugation (12,000 × gmax, 45 min, 4 °C) and resuspension. The final pellet was extracted by resuspension in
~4 volumes of ice-cold buffer B (100 mM octyl
-D-glucopyranoside, 1 mM CaCl2,
2 mM Pefabloc, in phosphate-buffered saline) and
centrifuged (12,000 gmax, 45 min, 4 °C). The
supernatant was recirculated over a P1F6 antibody column (16 h,
4 °C) (13). After washing with buffer C (0.1% Nonidet P-40 in
phosphate-buffered saline; ~10 bed volumes) and buffer D (0.1%
Nonidet P-40, 2 mM CaCl2, 10 mM
sodium acetate, pH 4.5; ~10 cv), bound material was eluted with
buffer E (buffer D adjusted to pH 3.1). The eluant was neutralized with
3 M Tris, pH 8.8, dialyzed against buffer C, and
concentrated ~10× using Aquacide II (Calbiochem). The purified
receptor was stored at
80 °C.
v
3 was purified in a soluble transmembrane truncated form from a
baculovirus expression system as detailed previously (28) with minor
modifications using 14D9.F8 antibody affinity chromatography (27).
Briefly, the extracellular domains of
v and
3 human integrin
chains were cloned into the pBacPAK expression system, the resulting
recombinant baculoviruses containing both chains were used to coinfect
High Five insect cells, and the soluble receptor was harvested from the
culture supernatant at 48-72 h of culture by passing the supernatant
over 14D9.F8 antibody affinity columns, washing, and eluting at pH 3.1. Peak fractions were neutralized, concentrated, and dialyzed before
shock freezing and storage at
80 C. The soluble human receptor
(
v
3-
TM) had ligand binding specificities indistinguishable
from the native receptor isolated from placenta (27).
v
6 was purified in a soluble transmembrane truncated form (13)
from a baculovirus expression system as detailed previously for
v
3 (28) using 14D9.F8 antibody affinity chromatography (27). The
6 cDNA clone pCDNAneo
6 was the generous gift of Dr. D. Sheppard (University of California, San Francisco). The procedure and
the cloning will be detailed
elsewhere.1 In brief, the
transfer vector pAcUW31 (CLONTECH Laboratories, Inc.) allowed simultaneous expression of two different target cDNAs
and was used to make recombinant baculovirus-expressing transmembrane
truncated
v
6. The preparation of truncated
v transfer vectors
was as described (28). Transmembrane truncated
v was excised from
v-
TM(pBac9) (28) using EcoRI and XbaI and
cloned into the EcoRI site of pAcUW31 downstream of the
polyhedrin promoter by blunt end ligation. Transmembrane truncated
6
cDNA was excised from pCDNAneo
6 (13) using EcoRI
and XbaI and cloned into the BamHI site of
pAcUW31 downstream of the polyhedrin promoter by blunt end ligation.
The tandem vectors containing truncated
v and truncated
6 were
used to prepare recombinant baculovirus as described (28). The
recombinant baculoviruses were used to infect High Five insect cells,
and the soluble receptor was harvested from the culture supernatant at
48-72 h of culture by passing the supernatant over 14D9.F8 antibody
affinity columns, washing, and eluting at pH 3.1. All processes were
carried out at room temperature and in the absence of detergents. Peak
fractions were neutralized, concentrated, and dialyzed at 4 °C
before shock freezing and storage at
80 °C. The recombinant
soluble human receptor (
v
6-
TM) is biologically active and
retains ligand specificity (13).
The integrin
v
3-
TM,
v
5, and
v
6-
TM preparations
were ~95% pure as judged by anti-integrin
ELISA2 using
and
chain-specific monoclonal antibodies (data not shown) and by
SDS-polyacrylamide gel electrophoresis against molecular weight
standards (Bio-Rad).
Peptides--
Peptides were synthesized, purified, and analyzed
in-house as described (30) using an Fmoc
(N-(9-fluorenyl)methoxycarbonyl) strategy with acid-labile
side chain protection on acid-labile Wang resin using a commercially
available continuous flow peptide synthesizer (Milligen 9050 plus).
Purification was done by reversed phase HPLC and analysis by HPLC and
fast atom bombardment-mass spectrometry. The peptide EMD 66203, cyclic(Arg-Gly-Asp-(D-Phe)-Val), is abbreviated as c(RGDfV).
Antibodies--
17E6 and 14D9.F8 (anti-
v), LM609
(anti-
v
3), P4C10 (anti-
1), and P1F6 (anti-
v
5) all
inhibit cell adhesion mediated by their respective integrins and have
been described in detail elsewhere (27).
Selection of Integrin-binding Phages--
M13 phage display
libraries displaying linear peptides of 7 or 12 amino acids (PHD system
from New England Biolabs) were used to select integrin-binding phages.
Panning was performed as described in the product manual with the
following modifications. Integrins were diluted to 1.5 µg/ml in TBS++
(1 mM CaCl2, 1 mM
MgCl2, 0.01 mM MnCl2, 150 mM NaCl, 50 mM Tris, pH 7.4) and adsorbed onto
Petri dishes for 16 h at 4 °C. After blocking for 2 h with
0.5% BSA in TBS++, aliquots of the phage display libraries
(1011 plaque-forming units) were diluted in TBS++
containing 0.1% Tween 20, and phages were allowed to bind for 1 h
at 30 °C. Unbound phages were removed by washing with TBS++
containing 0.3% Tween 20, and bound phages were eluted with 0.1 M glycine, pH 2.2, and amplified in Escherichia
coli XL-1. Phages were prepared from the culture supernatant by
standard polyethylene glycol/NaCl precipitation and used for the next
round of panning. After the third round of panning randomly selected
phage plaques were amplified, and single-stranded DNA was sequenced
using the Amplitaq FS sequencing kit (Applied Biosystems) with the
primer 96gIII (New England Biolabs).
Phage Binding Assay--
Integrins were adsorbed onto 96-well
plates as described above for the Petri dishes. Phages amplified from
single plaques and purified by polyethylene glycol/NaCl precipitation
were serially diluted in TBS++ containing 0.1% Tween 20 and 0.1% BSA
and allowed to bind to integrins for 1 h. After washing, bound
phages were detected with an anti-M13 horseradish peroxidase-conjugated
antibody (Amersham Pharmacia Biotech) and 3,3',5,5'-
tetramethylbenzidine substrate.
Integrin Adsorption Control--
The adsorption of the integrins
to the plates was investigated using indirect ELISA. Integrins were
immobilized by adsorption on 96-well plates, and the plates were
blocked with BSA as described under "Selection of Integrin-binding
Phages" (above). 17E6 antibody, specific for the
v
chain of all
v integrins (27), was biotinylated (28), titrated in
phage diluent buffer (0.1% Tween 20, 0.1% BSA, TBS++), and allowed to
bind (1 h, 37 °C); after washing (0.3% Tween 20 and TBS++), bound
antibody was detected with anti-biotin alkaline phosphatase conjugate
(Bio-Rad) and p-nitrophenyl phosphate substrate.
Integrin-Ligand Competition Assays--
Integrin-ligand
competition assays were performed as described (30). Integrins were
immobilized as described under "Selection of
Integrin-binding Phages" (above). Serially
diluted peptides in TBS++ containing 0.1% BSA were added in parallel
with biotinylated fibronectin, vitronectin, or fibrinogen (to 1 µg/ml). After a 3-h incubation at 37 °C and washing with TBS++,
bound ligand was detected by incubation with an anti-biotin alkaline
phosphatase-conjugated antibody (Bio-Rad) followed by development with
a p-nitrophenyl phosphate substrate. The reaction was
stopped by the addition of NaOH and the color intensity read at 405 nm.
The optimal time course for the integrin-ligand interaction was
determined in preliminary experiments.
Inverted Integrin-Ligand Competition Assay--
v
6-
TM
was biotinylated as described previously for matrix proteins and
antibodies (28). Fibronectin was diluted (to 5 µg/ml) in TBS++ and
immobilized by adsorption to 96-well plates for 16 h at 4 °C as
described under "Selection of Integrin-binding Phages" (above).
After blocking for 2 h with 0.5% BSA in TBS++ (BTBS++) and
washing in TBS, biotinylated integrin (3 µg/ml in BTBS++) was added
in the presence of peptides serially diluted in the same buffer. After
incubation (3 h, 37 °C) and washing with TBS++, bound integrin was
detected by incubation with anti-biotin alkaline phosphatase-conjugated
antibody and color development as described above.
Cell Attachment Assays--
Cell culture of lines M21-L and
HT-29, and their use in cell attachment assays, has been described in
detail elsewhere (27, 31). M21-L is a human melanoma cell line selected
for null expression of
v integrins (32) which uses only
5
1 to
attach to fibronectin. HT-29 is a human carcinoma cell line that uses
only
v
6 integrin to attach to fibronectin (33). The integrin
profile of the cells was monitored routinely by fluorescence-activated
cell sorter and showed the expected lack of
v integrins on M21-L and
the lack of
5
1 and
v
3 on HT-29 (see also Fig. 6).
96-well plates were coated with fibronectin (12.5 µg/ml) and blocked
with BSA. Serially diluted peptides or antibodies were added followed
by cells (2.5 × 104 cells/well). After 1.5 h at
37 °C nonadherent cells were washed away and attached cells detected
using an assay measuring cellular hexosaminidase activity (31).
 |
RESULTS |
Recombinant soluble human integrins
v
3 and
v
6 and
native placental
v
5 and platelet
IIb
3 were purified by
antibody or ligand affinity chromatography. On SDS gels each
preparation showed the two major bands corresponding to the
and the
chains (Fig. 1A). The
chains migrated at the molecular mass predicted for the intact and
transmembrane truncated recombinant chains (
v full-length, 150 kDa;
v-
TM, 125 kDa;
IIb full-length, 145 kDa;
3 full-length, 105 kDa;
5 full-length, 100 kDa;
3-
TM, 85 kDa;
6-
TM, 90 kDa). Each integrin was biologically active and showed ligand binding
and divalent cation requirements predicted from the literature and also
as demonstrated independently by us (13, 27, 28, 30).

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Fig. 1.
Integrin preparation and adsorption.
Panel A, SDS-polyacrylamide gel electrophoresis (7.5% gel)
under nonreducing conditions of transmembrane truncated recombinant
v 3 (lane 1), placental v 5 (lane 2),
transmembrane truncated recombinant v 6 (lane 3), and
platelet IIb 3 (lane 4). The gel is stained with
Coomassie Blue. Arrowheads and numbers to the
right indicate the migration position of marker proteins and
their molecular mass in kDa. The band at ~65 kDa in lane 4 may be a serum albumin contaminant. Panel B, adsorption
control. Integrins v 3- TM (solid circles),
v 6- TM (open circles), and v 5 (solid
triangles down) were adsorbed to 96-well plates from 1.5-µg/ml
solutions and blocked as for phage display screens and ligand
competition assays. Biotinylated antibody 17E6 (27), serially diluted
as indicated, was added, and after incubation and washing bound
antibody was detected with anti-biotin-alkaline phosphatase conjugate.
The non- v integrin control, IIb 3, gave background binding of
17E6 (A405 < 0.05) (data not shown).
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Recombinant
v
6 expressed as a transmembrane truncated soluble
receptor bound two distinct classes of phages in phage display panning
experiments from a linear 12-mer library as determined from the
sequences of more than 100 clones: those containing RGD sequences
(51%) and those containing an
XXDLXXLX motif (27%). Some phages
contained RGD and also continued with the motif as RGDLXXL
(9%), whereas others displayed the sequence RGDL (38%); the remaining
phages that were bound often contained DLXXL-related sequences. A selection of representative displayed sequences is shown
(Table I).
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Table I
Integrin binding clones from phage display library screens
Representative displayed peptide sequences from clones isolated by low
pH elution from recombinant v 6 and v 3. Sequences have been
selected to approximate the distribution of features found. RGD
sequences have been underlined and DLXXL sequences
highlighted bold and underlined.
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In the non-RGD sequences, the amino acid distribution at X
within
X1X2DLX3X4LX5
appeared nonrandom. Arg was favored at X1 and
X5, Thr/Ser/Asp/Gly at
X2, whereas at
X3X4 Ser/Thr
were often paired with a charged amino acid. These characteristics were
typified by a dominant clone with the sequence RTDLDSLRTYTL (clone-1).
Non-RGD, DLXXL-containing peptides were represented in only
5% of clones isolated from a 7-mer phage display library, suggesting
that sequences COOH-terminal to DLXXL are also involved in
integrin binding (Table I). Indeed, the sequence COOH-terminal to the
peptide insertion site in the phage pIII protein continues Gly-Gly-Gly
(i.e. the 7-mer library inserts would read
XXDLXXLGGG), and related sequences were not
isolated from the 12-mer library. Pro at
X3X4 was never found in a
DLXXL motif, and it was similarly excluded from the 4 amino
acids COOH-terminal of the DLXXL sequence, although not from
these positions in RGD-containing sequences. One sequence was found
where the motif was concatenated as GDLDLLKLRLTR. To investigate
whether the presence of DLXXL sequences was a library artifact, we also screened on
v
3. Here mainly RGD-containing phages were bound, the DLXXL sequence was absent, and RGDL
was present in fewer than 10% of clones. This distribution was
distinct from that of the
v
6 phage display library screen (Table
I) and similar to that reported for linear 15-mer library screens (23).
To eliminate the possibility that the differences in phage interactions
seen between
v
3 and
v
6 were artifacts caused by differential adsorption of integrins during the phage screen, we
estimated the amounts of each integrin adsorbed to the screening plates
under the conditions of the screen using an indirect ELISA technique
(Fig. 1B). The ELISA titration curves derived using the 17E6
antibody, which recognizes the
v chain, were similar in form, in
saturation level of antibody binding, and in the amount of antibody
needed to titrate 50% of the receptors. This indicated that similar
amounts of the
v integrins were adsorbed to the plates.
To test the specificity of interaction of phage clones we used a
quantitative ELISA to measure the binding of representative phages from
the
v
6 screen to immobilized purified integrins (Fig.
2). DLXXL-containing phages
bound only to
v
6, whereas RGD phages bound both
v
6 and
v
3 and weakly to
v
5 and to
IIb
3. This indicated that
the XXDLXXLX clones isolated in the screen were specific for
v
6.

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Fig. 2.
Binding to integrins of phages displaying
different peptides. Phage binding to immobilized integrins was
detected using an anti-M13 antibody. Phages displaying
RTDLDSLRTYTL (solid
circles),
GDLDLLKLRLTR (solid
squares), RGDAPSPNIFRL (triangles up),
QSAHRGDIPNVL (triangles down) binding to
v 6 (panel A), v 3 (panel B), v 5
(panel C), IIb 3 (panel D). Note that the
DLXXL-containing phages bind only v 6.
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RGD-containing peptides inhibit the interaction of RGD-containing
ligands with their integrin receptors. To test whether DLXXL peptides were able to inhibit integrin-ligand interaction, we synthesized the clone-1 peptide. We compared the effects of clone-1 peptide and RGD-containing peptides on ligands binding to integrins
v
3,
v
5, and
v
6 (Fig.
3). The clone-1 peptide specifically inhibited fibronectin binding to
v
6 with an IC50 of
20 nM but did not affect ligand binding to
v
3,
v
5, or
IIb
3 (IC50 > 50 µM). By
contrast, the peptide GRGDSPK inhibited ligand binding to all four
integrins with IC50
1 µM. A cyclic
peptide inhibitor c(RGDfV) showed specificity for
v
3
(IC50 = 10 nM) over
v
6 and
IIb
3
(IC50
1 µM).
v
3 also binds to
fibronectin. To test whether the effects of DLXXL peptides
were a result of the ligand used with
v
6, fibronectin, we also
examined the effect of clone-1 peptide on fibronectin binding to
v
3 (Fig. 4). The DLXXL
peptide also had no effect on fibronectin binding, whereas the GRGDSPK peptide inhibited (IC50 = 200 nM). Thus, the
results for vitronectin and fibronectin on
v
3 were similar.

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Fig. 3.
Effect of peptides on integrin-ligand
interaction. Biotinylated ligand binding to immobilized integrins
in the presence of peptides was detected using an anti-biotin antibody.
Values were converted to percentage of control (no peptide).
Solid circles,
RTDLDSLRTYTL; open
circles, GRGDSPK; open squares,
c(RGDfV); open diamonds, AGDV. Panel
A, fibronectin binding to v 6; panel B,
vitronectin binding to v 3; panel C, vitronectin
binding to v 5; panel D, fibrinogen binding to
IIb 3.
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Fig. 4.
Effect of peptides on fibronectin binding to
v 3. Fibronectin binding to v 3 was measured in the
presence of increasing amounts of
RTDLDSLRTYTL (solid
circles) or GRGDSPK (open circles). Values
were converted to percentage of control (no peptide).
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We also examined whether the assay geometry was producing an
artifactual binding of DLXXL to
v
6. In
vivo,
v
6 binds to insoluble fibronectin in the extracellular
matrix rather than to the soluble form used in the receptor assay here.
In addition, adsorption to plastic might change the conformation of the
v
6 integrin and so alter its specificity. We therefore tested the effect of DLXXL peptides on
v
6 binding to immobilized
fibronectin (Fig. 5). Once again, the
RTDLDSLRTYTL peptide strongly inhibited the
v
6-fibronectin
interaction (IC50 = 100 nM). Both c(RGDfV) and
GRGDSPK peptides were also effective in this assay configuration. Thus,
the effect of DLXXL peptide on
v
6 was probably not an artifact of integrin adsorption to plastic.

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Fig. 5.
Inverted integrin-ligand competition
assay. Biotinylated v 6- TM binding to fibronectin was
detected in the presence of the indicated concentrations of test
peptides. Bound integrin was detected with anti-biotin antibody.
Solid circles,
RTDLDSLRTYTL; open
circles, GRGDSPK; open squares,
c(RGDfV); open diamonds, AGDV.
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Clone-1 peptides were also tested in cell attachment assays. HT-29
carcinoma cell attachment to fibronectin was strongly inhibited by
clone-1 peptide and by GRGDSPK (Fig.
6A). This attachment was also
suppressed completely by the 17E6 (anti-
v) antibody, showing that it
was dependent on
v integrins. HT-29 attachment to fibronectin has
been shown to be mediated by
v
6. M21-L melanoma cells attached to
fibronectin, and this was inhibited by GRGDSPK but was little affected
by the clone-1 peptide (Fig. 6B). M21-L attachment was suppressed by either P4C10 (anti-
1) or P1D6 (anti-
5) antibodies, indicating that it was mediated by the
5
1 integrin. Together, these data indicated that the peptide displayed by clone-1 was an
active and specific inhibitor of
v
6 independent of the RGD sequence.

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Fig. 6.
Effect of peptides on cell attachment to
fibronectin. Cell attachment on fibronectin of HT-29 cells
(panel A) and M21-L cells (panel B) in the
presence of peptides and inhibitory antibodies is shown. Solid
circles,
RTDLDSLRTYTL; open
circles, GRGDSPK; open squares, 17E6
( v-inhibitory;) open triangles up, P4C10
( 1-inhibitory); open triangles down, P1D6
( 5-inhibitory). Values were converted to percentage of control
(control cell attachment in the absence of peptide for HT-29 and M21-L
was 54 and 47% of cells added, respectively). HT-29 attachment to
fibronectin is dependent on v integrins. M21-L attachment to
fibronectin is dependent on 5 1.
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We next investigated which elements of the
X1X2DLX3X4LX5
motif were important for its inhibitory activity. NH2- and
COOH-terminal truncated forms of the peptide were synthesized and
tested for their activity to block fibronectin binding to
v
6. The
data are summarized in Table II.
Truncation of the COOH terminus of DLXXL had little effect
on inhibitory activity until the group at X5 was
deleted, when activity diminished by 30-fold. Removal of the groups at
X1X2 also abolished the
activity, indicating that the core motif was the 8-amino acid sequence
X1X2DLX3X4LX5.
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Table II
Inhibition of fibronectin v 6 interaction by synthetic DLXXL
peptides
Biotinylated fibronectin (FN) binding to v 6 in the presence of
synthetic linear peptides was detected using an anti-biotin antibody.
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Although the specific selection of DLXXL sequences from a
highly degenerate (2 × 109 clones) display library
implies specificity of interaction, we examined this more directly by
investigating the effect of reversed (TRLSDLDTR) and scrambled
(LDTRTRLSD) peptides on
v
6-fibronectin interaction. These
peptides were > 4 orders of magnitude less active than the
corresponding DLXXL peptide (Fig.
7).

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Fig. 7.
Effect of peptides on fibronectin binding to
v 6. Fibronectin binding to v 6 was measured
in the presence of increasing amounts of
RTDLDSLRTY (solid
circles), RTDLYYLRTY
(solid squares),
RTDLYYLMDL (solid
triangles),
PVDLYYLMDL (solid
diamonds), GRGDSPK (open circles),
TRLSDLDTR (open triangles), or LDTRTRLSD (open
squares).
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The high specificity of the clone-1 sequence for
v
6 suggested
that it might represent a sequence in a native
v
6 ligand. Indeed,
a FASTA search of the GEMBL data bases revealed several extracellular
matrix components with related consensus sequences (Table
III) including fibrinogen
chain,
tenascin, laminin
1,
3, and
1,
2, and
3 chains. With the
exception of tenascin, which has been reported to bind in an
RGD-dependent way to
v
6 (14), none of these molecules
has been implicated previously as a ligand for
v
6, thus the
possible biological relevance of such homologies remains unknown.
Interestingly, the sequence DLYYLMDL is strongly conserved in human
integrin
chains and may interact directly with ligands, hinting
that the clone-1 sequence might function by disturbing the interaction
from the side of the receptor rather than as a ligand mimetic
(e.g. like an RGD peptide). To test this possibility, we
examined a synthetic DLXXL peptide sequence derived from the
6 chain, P132VDLYYLMDL141, for
its effect on fibronectin-
v
6 interaction (Fig. 7). Compared with
the corresponding 10-mer derived from clone-1, RTDLDSLRTY, the
6 peptide was > 4 orders of magnitude less active as an
inhibitor of this interaction. Once again, this supported the concept
that the sequences selected from the phage display library were highly specific. We examined the structural basis of the inhibition by exchanging the flanking sequences from the clone-1 10-mer with those of
the
6 DLXXL peptide. Exchange of the DLYYL core gave a
highly active inhibitor, as did the core and the trailing 3 amino
acids. Thus the 2 amino acids preceding the DLXXL sequence were also important for integrin inhibitory activity.
 |
DISCUSSION |
Here, we describe the discovery by phage display screening of an
unexpected binding sequence (DLXXL) for
v
6 integrin,
which has previously been thought to interact only with RGD sequences in its target ligands.
v
6 is a rare inducible integrin (12, 15),
and isolation from native tissue sources has not been reported.
v
6 has been prepared successfully by recombinant methods (13), and we have adapted this technology to produce biochemical amounts of
soluble receptor.1 As with
v
3 (28), we found that
soluble, recombinant
v
6 retains the biological activity,
specificity, and inhibitor profiles of the native receptor in
situ, thus confirming and extending the studies of Sheppard and
co-workers (13). Because the receptor is secreted into the serum-free
culture supernatant, affinity purification gives rise to a very highly
purified molecule with a minimal amount of other proteins1
and thus is an excellent target for a phage display screen.
It is of interest that RGD-dependent extracellular matrix
binding to
v
6 is inhibited strongly by peptides containing
DLXXL sequences that bear no close similarity to sequences
in fibronectin, although they do resemble a sequence in tenascin.
DLXXL sequences have rarely been found in other phage
display studies on integrins; the only clone described in the
literature bearing an RGDLXXL sequence was a weak inhibitor
of
v
3 (23).
We were able to demonstrate directly that synthetic DLXXL
peptides, although strong inhibitors of
v
6, had minimal effects on the interaction of integrins
v
3,
v
5, and
IIb
3 with
their ligands. Although indirect, cell adhesion assays implied that
5
1 was also not affected. The DLXXL sequences,
therefore, represent the first specific inhibitory peptides for
v
6 integrin.
Analysis of the structures of phage-encoded
v
6-binding
XXDLXXLX peptides showed that although
RXDLXXL was a favored sequence, it was by no
means obligatory. Phages binding to
v
6 were found where displayed
arginine was replaced by hydroxylated, neutral, or basic residues or
was absent. The inhibitory effect of DLXXL peptides is
apparently not the result of nonspecific charge effects because neither
reversed nor scrambled versions of highly inhibitory DLXXL
peptides have significant effects on the
v
6-fibronectin interaction. Further structural constraints on the context of DLXXL were found in experiments to investigate the possible
biological significance of the
v
6- DLXXL interaction.
DLXXL sequences are found in seven integrin
chains
(Table III), in a region directly implicated in ligand binding (34). We
hypothesized that the DLXXL motif was involved in the
interaction of integrin
and
chains and that the
DLXXL peptide might act by interacting with a binding site
on the
chain, usually occupied by
chain sequences, so
disrupting integrin function. To test this, we examined a 10-mer peptide corresponding to a DLXXL sequence in the
6 chain.
It was inactive, indicating that the DLXXL peptides were not
acting by competing for
6 chain binding sites on the
v chain.
However, the study did reveal that the context of the DLXXL
sequence was crucial because appropriate NH2-terminal
flanking amino acids were critical for inhibitory activity, whereas
those in the COOH-terminal flanking region were less crucial.
At present we have no indication where, if at all, the
XXDLXXLX sequences play a biological
role with
v
6 integrin. As discussed previously for the CRRETAWAC
inhibitory peptide sequence for
5
1 (21),
XXDLXXLX may bind a site distinct from
the RGD site and act as an allosteric inhibitor of RGD-ligand binding.
But in the absence of kinetic data at present we believe that the
XXDLXXLX sequences are ligand
mimetics. Similar inhibitory mimetic binding sequences with no homology
to known ligands are found in the phage display literature (35, 36).
The unusual strong and specific inhibitory binding sequences that we
characterize here provide a unique pharmacological tool from which to
investigate
v
6 biological function further.
 |
ACKNOWLEDGEMENTS |
We thank E. Rosell (Merck LBI) for discussion
and introduction to the phage display technology, D. Sheppard (UCSF)
for discussion and access to
6 cDNA, and R. Dunker and I. Remitschka (Merck, kGaA) for biotechnological support.
 |
FOOTNOTES |
*
This work was funded in part by Bundesministerium für
Bildung und Forschung Project 0310767.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-6151-726-452; Fax: 49-6151-729-0452; E-mail:
goodman{at}merck.de.
The abbreviations used are:
ELISA, enzyme-linked
immunosorbent assay; HPLC, high performance liquid chromatography; BSA, bovine serum albumin.
1
B. Diefenbach, R. J. Mehta, A. Brown, E. Cullen,
J. Adams, D. Sheppard, R. Dunker, S. L. Goodman, and D. Güssow,
manuscript in preparation.
 |
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