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INTRODUCTION |
Laminins are heterotrimeric basement membrane proteins that exert
multiple biological functions through interactions with other matrix
molecules and cell surface receptors. The regulation of these
interactions is critical to many biological processes, including cell
adhesion, migration, angiogenesis, tumor progression, and neurite
outgrowth (for review, see Ref. 1). The laminin family contains at
least 11 chains (five
, three
, and three
chains). The newly
discovered
5 chain is the most widely expressed member
of the laminin
chain family (2). Mutant mice that lack the
5 chain have defects in neural tube closure, digit
septation, placentation, and glomerulogenesis, suggesting that the
5 chain has multifunctional roles in tissue development
(3, 4). The laminin
5 chain associates with the
1 chain and either
1 or
2
chains to form laminin-10 and laminin-11, respectively (2). Laminin-10/11 is a ligand for several cell surface receptors, including
-dystroglycan (5),
3
1,
6
1, and
6
4
integrins (6, 7). Laminin-11 has been shown to inhibit neurite
outgrowth in vitro, while other laminin isoforms promote
neurite outgrowth, suggesting a unique role of the
5
chain in neural development (see Refs. 8 and 9, for review, see Ref.
10). Interestingly, SV2, a transmembrane keratan sulfate proteoglycan
found in synaptic vesicles, has recently been proposed to act as a
laminin
5 receptor, suggesting a role of the laminin
5 chain in nerve regeneration (11).
The N-terminal globular domain (domain VI, ~250-270 residues) is
specific for laminin and is the most conserved (~60% sequence identity) among the laminin domains (12). Studies with
function-blocking antibodies and cell binding assays have indicated
that domain VI of both the laminin
1 and
2 chains contains binding sites for the
1
1 and
2
1
integrins (13-15). Furthermore, domain VI is also capable of binding
heparin and heparan sulfate chains of perlecan (13-15). In addition to
binding functions for cell surface receptor and for matrix proteins,
domain VI is also essential for the self-assembly of laminins (13, 16,
18). Previous studies showed that the synthetic peptide RQVFQVAYIIIKA
(A-13), derived from domain VI of the laminin
1 chain,
binds
1 subunit-containing integrins (19). The active
core sequence (VAYI) of this peptide is conserved in the
5 chain, but the functional importance of this site
within domain VI has not yet been examined.
In the present work, we studied cell binding functions of mouse laminin
5 domain VI using site-directed mutagenesis and
synthetic peptides. We found that two sites, spaced by ~90 amino
acids, are required for cell binding. We also identified four residues within the two sites that are essential for the binding. In addition, we demonstrated that an arginine residue of one of the sites is critical for both heparin and cell binding. Our findings suggest that
the protein conformation surrounding these sites is important for cell
binding through integrin and heparan sulfate-containing cell surface receptors.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Vectors and Site-directed
Mutagenesis--
Mouse kidney cDNA was used as a template in
polymerase chain reaction to amplify sequences encoding domain VI and
the first four EGF1-like
repeats of domain V of the laminin
1 and
5 chain. Polymerase chain reaction was performed with
PfuTurbo DNA polymerase (Stratagene, La Jolla, CA)
using the following primers: 1, GAGAGAAAGCTTCGCACTCCCGGGGGCGATGGC; 2, GAGAGACTCGAGAGTGGCAGCTAGGCCCGTGGAATC; 3, GAGAGAAAGCTTCAGCAGAGAGGCTTGTTCCCTGC; 4, GAGAGACTCGAGAGGAGCAGCCCTCGGGGTTTCG. Primers 1 and 2 were used for
laminin
5 domain VI
(Arg1-His514), and primers 3 and 4 for the
laminin
1 domain VI
(Gln1-Ser484). For polymerase chain reaction
amplification of the EGF-like repeats of laminin
5
domain V (Arg273-His514), primers 2 and 5, GAGAGAAAGCTTCGCTGTGTCTGTCATGGCCACG, were used. In addition to the
coding sequences, these primers contained either a HindIII
or a XhoI restriction site. The polymerase chain reaction products were digested with HindIII and XhoI and
ligated into the expression vector pSecTag2/Hygro B (Invitrogen,
Carlsbad, CA). The resulting expression vectors encode the Ig
chain
leader sequence, a laminin domain, and a c-myc epitope
followed by a hexahistidine affinity tag sequence. Site-directed
mutagenesis was performed using the Quickchange kit method (Stratagene,
La Jolla, CA) and the N-terminal globular domain VI and the first four
EGF-like repeats of domain V of the mouse laminin
5
chain, which has been cloned into pBluescript II SK(+) (Stratagene). The following laminin
5 residues were individually
substituted with alanine: Tyr130, Arg225,
Lys229, Arg234, Arg236, and
Arg239. All expression constructions and mutations were
verified by DNA sequencing.
Expression and Purification of Recombinant Proteins--
The
expression vectors were transiently transfected into monkey kidney
COS-7 cells (CRL-1651, ATCC) using FuGENE 6 (Roche Molecular
Biochemicals, Indianapolis, IN). The cells were maintained at 37 °C
in a humid atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Generation of secreted
recombinant His-tagged laminin domains was confirmed by Western
blotting of serum-free conditioned medium using an
anti-His(C-terminal)-horseradish peroxidase monoclonal antibody
(Invitrogen). For recombinant production, conditioned medium
(Dulbecco's modified Eagle's medium containing 1% fetal calf serum)
was collected 7 days after transfection. The medium was cleared of
detached cells and debris through centrifugation, and 1 M
Tris-HCl, pH 8.0, and 5 M NaCl were added to final
concentrations of 50 mM and 0.5 M,
respectively. In addition, 0.2 M phenylmethanesulfonyl fluoride and 3 M imidazole, pH 6.0, were added to
concentrations of 1 and 10 mM, respectively. Nickel-charged
agarose resin (Probond, Invitrogen) was equilibrated with 50 mM Tris-HCl, 0.3 M NaCl, 10 mM
imidazole, pH 8.0, and incubated with the conditioned medium. After
incubation at 4 °C for 1 h, the resin was transferred to a
column and washed with 50 mM Tris-HCl, 0.3 M
NaCl, 20 mM imidazole, pH 8.0. The His-tagged proteins were
eluted with 50 mM Tris-HCl, 0.3 M NaCl, 250 mM imidazole, pH 8.0. Purified proteins were dialyzed against phosphate-buffered saline and quantified using a BCA protein assay kit with bovine serum albumin as a standard (Pierce, Rockford, IL). Purity was determined by SDS-polyacrylamide gel electrophoresis followed by colloidal Coomassie G-250 Blue (GelCode Blue, Pierce, Rockford, IL) staining and judged to >95%.
Single-site substitutions may result in a change of the overall fold of
domain VI. Gel filtration analysis of mutants, including Y130A and
R239A, were therefore performed in 0.05 M phosphate buffer
with 0.15 M NaCl, pH 7.0, using a Superose 12 HR 10/30 column on an ÄKTA EXPLORER design system (Amersham Pharmacia Biotech). The majority of the mutants and the wild-type domain VI
eluted at a volume corresponding to a molecular weight of ~65,000 (data not shown). The results indicate that these molecules are monomers without mis-folding.
Synthetic Peptides--
Peptide synthesis was performed on ABI
model 433A peptide synthesizers at the Facility for Biotechnology
Resources (U. S. Food and Drug Administration). All the peptides
were prepared with a C-terminal amide group. The peptides were purified
by reverse-phase high performance liquid chromatography and
characterized by mass spectrometry.
Real-time Heparin Binding Kinetics of Recombinant Proteins
Measured by Surface Plasmon Resonance--
Biotinylated heparin
(Celsus Laboratories, Inc., Cincinnati, OH) at 40 µg/ml in running
buffer (10 mM HEPES, pH 7.4, 150 mM NaCl,
including 0.005% surfactant P20) was immobilized on a
streptavidin-coated sensor chip (Sensor Chip SA, BIAcore, Inc.,
Piscataway, NJ) at 10 µl/min for 4 min to an immobilization level of
300 resonance units. In the affinity measurements, recombinant proteins
at different concentrations (50-200 nM) were injected on
the heparin-coated surface at 30 µl/min in the running buffer at
25 °C, and the binding and dissociation were registered (2 min each)
in a BIAcoreTM 1000 instrument (BIAcore, Inc.). The
streptavidin-heparin surface was regenerated at the end of each run by
two successive injections of 30 µl of 20 mM NaOH
containing 1 M NaCl. In control experiments with the same
concentrations of recombinant proteins, but with a blank streptavidin
sensor chip, no binding was seen. The sensorgrams obtained were
analyzed by nonlinear least square curve fitting using BIAevaluation
2.1 software assuming single-site association and dissociation models.
Cell Binding Assays--
HT-1080 fibrosarcoma cells (CCL-121,
ATCC) were detached with 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in
PBS, washed with Dulbecco's modified Eagle's medium containing 0.1%
bovine serum albumin, resuspended to a concentration of 3 × 105 cells/ml, and incubated at 37 °C for 30 min. For
evaluation of the effects of synthetic peptides or monoclonal
antibodies against integrin subunits, cells were incubated with
peptides or antibodies at 37 °C for 30 min. Function-blocking
monoclonal antibodies against integrin
1 (FB12),
2 (P1E6),
3 (P1B5),
3
(ASC-1),
4 (P1H4),
5 (P1D6),
6 (NKI-GoH3),
V (P3G8), and
4 (ASC-3) were purchased from Chemicon International,
Inc., Temecula, CA. Anti-integrin
2 (A2-IIE10) was from
Upstate Biotechnology, Lake Placid, NY, and anti-integrin
1 (mAb13) was a gift from Dr. K. Yamada, National Institutes of Health. Assays were performed in 96-well round-bottom microtiter plates (Immulon-2HB, Dynex Technologies, Inc., Chantilly, VA). Wells were coated for 1 h at room temperature with 50 µl of
recombinant proteins or laminin-10/11 (Life Technologies, Inc., catalog
number 12163-010, Life Technologies) diluted with Dulbecco's PBS
(Dulbecco's PBS) and then blocked for 1 h at room temperature with 200 µl of 1% heat-denatured bovine serum albumin. After washing (Dulbecco's PBS), cells (100 µl) were added and incubated for 60 min
at 37 °C in a humidified atmosphere of 5% (v/v) CO2.
Wells were washed gently twice with Dulbecco's PBS and stained for 10 min with 0.2% (w/v) crystal violet (Sigma) in 20% (v/v) methanol. After washing with H2O, cells were dissolved in 10% SDS
(w/v), and the absorbance at 600 nm was measured. Each sample was
assayed in triplicate, and attachment to bovine serum albumin was
subtracted from all measurements.
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RESULTS |
Expression and Cell Binding Activity of Recombinant Laminin
5 N-terminal Globular Domain VI--
Constructs were
generated for expression of mouse laminin
5 domain VI
and the first four EGF-like repeats of domain V in mammalian cells
(Fig. 1). In addition, a similar
construct was also generated for laminin
1 domain VI,
which corresponds to that described previously (15). The secreted
recombinant proteins were purified by Ni-agarose chromatography from
the conditioned medium of transfected COS-7 cells with a yield of 1-2
µg/ml medium. The purity of the protein preparations was more than
95% as judged from Coomassie Blue-stained gels after
SDS-polyacrylamide gel electrophoresis (data not shown). Recombinant
fragments containing domains VI through IV of the laminin
1 and
2 chains have previously been shown
to promote binding of HT-1080 fibrosarcoma cells (13, 14). This cell
line was therefore used to analyze the adhesive properties of the
recombinant laminin
5 domain VI. HT-1080 cells showed
strong binding to domain VI, while the recombinant domain V, consisting
of the EGF-like repeats, showed no cell binding, demonstrating that the
cell binding activities reside in domain VI (Fig. 1).

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Fig. 1.
Expression of recombinant laminin
5 domain VI. Schematic
representation of the laminin 5 chain constructs used
for mammalian expression. The N-terminal globular domain VI and the
first four EFG-like repeats of domain V are indicated by VI
and E1-4, respectively. The positions of four sequences
(S1, S2, S6, and S7), two of which overlap, that inhibited HT-1080 cell
binding to laminin 5 domain VI are indicated. Cell
binding activity of recombinant laminin 5 domain VI
(A5VI) and V (A5V) is shown. HT-1080 cells were
added to wells coated with the laminin domains (10 µg/ml). Cell
attachment was quantified by crystal violet staining and is expressed
as percentage of domain VI (A5VI).
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Effect of Synthetic Peptides on Cell Binding to Laminin
5 Domain VI--
To localize potential cell-binding
sites on laminin
5 domain VI, we screened synthetic
peptides for their inhibitory effect on HT-1080 cell binding to domain
VI. We synthesized 15 peptides that corresponded to possible integrin
recognition sequences (for review, see Ref. 20) within laminin
5 domain VI and to active sequences previously
identified in laminin
1 domain VI (Table I). Four peptides (S1, S2, S6, and S7)
were found to inhibit HT-1080 cell binding to domain VI (Fig.
2), whereas the other peptides showed no
or only small effects on the cell-domain interaction. A GRGDS peptide,
which is reported to block the function of various integrins, had no
effect on cell binding. The sequences of peptides S1 and S2 overlap
with four residues, but only S2 showed strong activity when tested in a
direct cell binding assay (data not shown). The S6 and S7 peptides
overlap by eight residues and showed no activity when tested for direct
cell binding (data not shown). None of the peptides inhibited cell
binding to laminin-10/11, suggesting that other cell-binding sites are
available on the intact molecule (data not shown). Taken together,
these results suggest that two sequences: S2,
123GQVFHVAYVLIKF135 and S6,
225RDFTKATNIRLRFLR239, separated by ~90
residues, directly interact with cell surface receptors within domain
VI.
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Table I
Synthetic peptides of the laminin 5 N-terminal domain VI
The underlined sequences compete with HT-1080 cell-domain VI
interactions.
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Fig. 2.
Effects of synthetic peptides on cell/domain
VI interactions. HT-1080 cells were incubated for 30 min at
37 °C with synthetic peptides (100 µg/ml) and added to wells
coated with laminin 5 domain VI (10 µg/ml). After
incubation for 1 h, cell binding was quantified. Values are
expressed as percentage of control without peptide (C) and
are the mean of three different experiments. Duplicate experiments gave
similar results.
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Cell Binding Activity of Laminin
5 Domain VI
Mutants--
We next examined the functional role of these two sites
within domain VI by alanine substitution mutagenesis. Our laboratory has previously identified several synthetic peptides active for cell
binding within domain VI of the mouse laminin
1 chain
(19). The S2 peptide corresponds to the highly active peptide A-13
(RQVFQVAYIIIKA) of laminin
1 domain VI. Deletion
analysis revealed that the VAYI sequence within A-13 is critical for
high cell binding activity (19). The active core sequence VAYI is
conserved in the laminin
5 chain with the exception of a
substitution of Ile with Val (Fig. 3). To
test the importance of the tyrosine in the VAYI site, we generated a
single substitution mutant by replacing tyrosine with alanine (Y130A).
This showed that Tyr130 plays a significant role for cell
binding, as the mutant domain VI with Y130A (A5VI-Y130A) reduced cell
binding to ~25% of that of wild-type domain VI (Fig.
4).

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Fig. 3.
Alignment of sequences of laminin domain VI
corresponding to synthetic peptides active for competition of
cell/domain VI interactions. Alignment of domain VI of laminin
1, 2, 3B, and
5 chains. All sequences are mouse. The sequences shown
correspond to the two regions active for cell binding within domain VI
defined by using synthetic peptides. The residues in bold
within the S2 and S6 sites of the laminin 5 chain were
replaced with Ala residues by site-directed mutagenesis of laminin
5 domain VI (A5VI) to generate A5VI-Y130A (S2 site),
A5VI-R225A (S6 site), A5VI-K229A (S6 site), A5VI-R234A (S6 site),
A5VI-R236A (S6 site), and A5VI-R239A (S6 site). Numbers
represent residues of the laminin 5 chain.
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Fig. 4.
Cell binding to laminin
5 domain VI mutants. Wells were
coated with laminin 5 domain VI mutants (10 µg/ml).
HT-1080 cells were added, and crystal violet staining was used to
assess the number of attached cells after 1 h. Data are given as
percentage of cell binding to the wild-type laminin 5
domain VI (A5VI). Each value represents the mean of three
separate determinations ± S.D. Duplicate experiments gave similar
results.
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The S6 peptide overlaps with two previously reported active synthetic
peptides (A-24 and A-25) derived from the laminin
1 chain (19). The S6 sequence is characterized by five basic residues (four Arg and one Lys). Notable, three of the four Arg residues are
conserved in domain VI of all the laminin
chains (Fig. 3). To
analyze the contribution of the individual Arg/Lys residues within the
S6 site to cell binding, we generated five single substitution mutants
by replacing Arg/Lys residues with Ala (R225A, K229A, R234A, R236A, and
R239A). All the mutants were expressed at a level similar to wild-type
domain VI, suggesting that the mutations did not cause unstable
synthesis of domain VI due to unfolding of the proteins (data not
shown). Cell binding to A5VI-K229A was very poor, while neither
A5VI-R234A nor A5VI-R236A had a significant effect on cell binding
(Fig. 4). R225A and R239A also showed a significant reduction in cell
binding, by ~60% of the wild type (Fig. 4). These data indicate that
four residues, Tyr130, Arg225,
Lys229, and Arg239, within these two regions
are crucial for cell binding.
Kinetic Analysis of Interactions between Heparin and Laminin
5 Domain VI Mutants using Surface Plasmon
Resonance--
Since heparin binding activity has been localized to
domain VI of the laminin
1 and
2 chains
(13-15), cell binding to recombinant laminin
5 domain
VI was examined in the presence of heparin and other heparin-like
glycosaminoglycans. Heparin and heparan sulfate inhibited HT-1080 cell
binding to domain VI, whereas keratan sulfate was much less inhibitory
(Fig. 5). These results indicate that the
heparin-binding site overlaps with the cell-binding sites.

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Fig. 5.
Effects of heparin/heparan sulfate and EDTA
on cell binding to laminin 5
domain VI. HT-1080 binding assays were performed on control
without inhibitors (C), or in the presence of heparin
(HP), heparan sulfate (HS), keratan sulfate
(KS), and 5 mM EDTA. Each value represents the
mean of three separate determinations ± S.D. Duplicate
experiments gave similar results.
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To examine the relationships between the heparin and cell-binding site,
the binding kinetics between immobilized biotinylated heparin and the
laminin
5 domain VI mutants were measured directly by
real-time biomolecular interaction analysis using surface plasmon resonance on a BIAcoreTM system. Similar equilibrium
dissociation constants (Kd = 9-16 nM)
were obtained for the interactions of wild-type domain VI of the
laminin
5 chain and five of the mutants with heparin (Table II). Less than a 2-fold difference
was observed for the kinetic binding constants between wild-type domain
VI and five of the mutants including Y130A, R225A, K229A, R234A, and
R236A. Statistically significant differences in kinetic constants
derived from BIAcore experiments are generally considered to be at
least 5-10-fold. This demonstrates that the heparin binding activity was unchanged, indicating that the structural integrity of domain VI
was maintained in these mutants and that these positions were not part
of the heparin-binding site. In contrast, no binding of the R239A
mutant to heparin was observed even at a high protein concentration
(200 nM). Mutation of Arg239 was also found to
be critical for cell binding, reducing activity by ~60% compared
with wild-type domain VI (see above). Consistent with the cell binding
results, no heparin binding was observed for a recombinant protein
consisting of the first four EGF-like repeats of domain V. These
results demonstrate that the heparin-binding site is located within
domain VI. Interestingly, the binding affinity of domain VI of the
laminin
1 chain to heparin was about 4-fold weaker
(Kd = 45 nM), demonstrating differences
in affinity for heparin between the laminin domain VI isoforms. Taken
together, these results indicate that Arg239 within the S6
site is critical for both heparin and cell binding.
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Table II
Effect of mutagenesis on laminin 5 domain VI-heparin
interaction
Kinectic constants were obtained by surface plasmon resonance analysis
of real-time heparin-binding interactions of laminin 5
domain VI mutants, domain V, and laminin 1 domain VI.
Recombinant domains were analyzed at several concentrations and the
kinectic constants represent average values of 2-3 determinations.
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Effect of Integrin Monoclonal Antibodies on Cell Binding to Laminin
5 Domain VI--
Cell binding to laminin
5 and
1 domain VI and to laminins
containing either the
5 chain (laminin-10/11) or the
1 chain (laminin-1) was examined in the presence of
different anti-integrin antibodies to identify integrin receptors
involved in domain VI binding. The
1 integrin antibody
demonstrated partial inhibition of HT-1080 cell binding to
laminin-10/11 but strong inhibition of cell binding to laminin-1 (Fig.
6). The monoclonal antibody against
integrin
6 inhibited cell binding to laminin-1 but not laminin-10/11, while a monoclonal antibody against integrin
3 showed a weak inhibitory effect on cell binding to
laminin-10/11. Other antibodies against integrin subunits, including
4,
1,
2,
4,
5, and
V, had no effect on cell binding.
Cell binding to both laminin-1 and laminin-10/11 was dependent on
divalent cations, since it was abolished by 5 mM EDTA (Fig.
6), which supports the role of integrins or
-dystroglycan as
receptors. These results suggest that different integrin receptors
mediate HT-1080 cell binding to laminin-1 and laminin-10/11 and that
the
3
1 integrin binds laminin-10/11,
agreeing with previous reports (6, 7).

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Fig. 6.
Inhibition of cell binding by anti-integrin
antibodies and EDTA to laminin-10/11 and laminin-1. Cell binding
assays using HT-1080 cells were performed on controls without
inhibitors (control), or in the presence of rat preimmune
IgG (10 µg/ml) (IgG), function-blocking anti-integrin
monoclonal antibodies (10 µg/ml), and EDTA (5 mM). Wells
were coated with 2 µg/ml laminin-10/11 or laminin-1. The results are
expressed as percent binding of cells without inhibitors. Each value
represents the mean of three separate determinations ± S.D.
Duplicate experiments gave similar results.
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The monoclonal antibody against the
1 integrin subunit
inhibited HT-1080 binding to recombinant domain VI of both laminin
5 and
1 (Fig.
7, A and B). These
results show that
1 subunit containing integrins is
critical for cell binding to domain VI of both chains. Furthermore, 5 mM EDTA was also found to inhibit cell binding, supporting
cation and integrin dependence of the interaction (Fig. 5). We then
tested several monoclonal antibodies against integrin
subunits to
identify partners for the
1 integrins. Anti-integrin
3 antibodies strongly inhibited HT-1080 cell binding to
laminin
5 and
1 domain VI (Fig. 7,
A and B). Monoclonal antibodies against
1,
5, and
V had no or only
small effects on cell binding, indicating that these integrins were not
major mediators of cell binding to laminin
5 and
1 domain VI (Fig. 7, A and B).
HT-1080 cell binding to laminin
5 and
1
domain VI was reduced to about 30-50% by anti-integrin
2 and
4 antibodies. Integrin
6 antibody also weakly reduced cell binding to laminin
5 domain VI but not to laminin
1 domain
VI (Fig. 7B). Taken together, these results identify
3
1 integrin as a major receptor to
5 domain VI.

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Fig. 7.
Inhibition of cell binding by anti-integrin
antibodies to laminin 1 and
5 domain VI. Cell binding assays
were carried out without inhibitor (control) or in the presence of
function-blocking anti-integrin antibodies (10 µg/ml). Wells were
coated with 10 µg/ml recombinant domain VI of the laminin
1 (panel A) or 5 chain
(panel B). Each value represents the mean of three separate
determinations ± S.D. Duplicate experiments gave similar
results.
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DISCUSSION |
The newly discovered laminin
5 chain has been
implicated in various biological activities, such as angiogenesis and
nerve regeneration. Previously, we identified a cell-binding site
within the C-terminal G domain of the mouse laminin
5
chain using recombinant proteins and synthetic peptides (21). In the
present study, we identified cell-binding sites on the laminin
5 N-terminal globular domain VI.
Recombinant proteins of laminin
5 domain VI and the
first four EGF-like repeats of domain V were generated by mammalian
expression. The recombinant protein was highly active for HT-1080
fibrosarcoma cell binding, while a recombinant protein consisting of
only the EGF-like repeats showed no binding, indicating that domain VI contributed to the cell-binding site. This result is similar to previous reports from studies on recombinant fragments consisting of
domains VI through IV, of the laminin
1 and
2 chains (13-15). In addition, recombinant laminin
5 domain VI was also found to bind other cell lines
including mouse B16-F10 melanoma and mesangial cells, while weak
binding was observed for a human submandibular gland cell line,
indicating cell type-specific interaction of this domain (data not shown).
To localize cell-binding sites on laminin
5 domain VI,
we screened domain VI-derived synthetic peptides for their effect on
HT-1080 cell/domain VI interactions. Four peptides: S1,
115EVNVTLDLGQVFH127; S2,
123GQVFHVAYVLIKF135; S6,
225RDFTKATNIRLRFLR239; and S7,
232NIRLRFLRTNTL243, inhibited cell binding.
These peptides corresponded to sequences in the laminin
1 chain (A-12, A-13, A-24, and A-25) previously reported
to be highly active for cell binding (19). The S1 and S2 peptides
overlap, as do S6 and S7, thus the results indicate that at least two
sequences, 123GQVFHVAYVLIKF135 and
225RDFTKATNIRLRFLR239, spaced by ~90
residues, directly interact with cell surface receptors within domain
VI (Fig. 3). Alanine mutagenesis of recombinant domain VI identified
four positions within these two sequences as critically involved in
cell binding: Tyr130, Arg225,
Lys229, and Arg239. A comparison with the
sequences of domain VI of the laminin
1,
2, and
3B chains reveals that
Tyr130 and Arg239 are conserved, while
Arg225 and Lys229 are conserved between the
3 and
5 chains, indicating that the position of the cell-binding sites varies. These differences may be
important for regulation and specificity of receptor interactions within domain VI. The site-directed mutagenesis data also demonstrate that the two sites together contribute to a cell binding epitope and
imply that these cell-binding sites are highly dependent on the
conformation of domain VI. The importance of the three-dimensional structure of cell-binding sites has also been shown for other integrin
ligands, including fibronectin and vascular cell adhesion molecule-1,
where multiple contacts, involving several different ligand peptide
segments, are formed between ligand and receptor (22, 23).
Kinetic data obtained here by surface plasmon resonance analysis for
six single-site alanine substitution mutants of laminin
5 domain VI demonstrate that one position
(Arg239) within the cell-binding site is also critical for
heparin interactions. Cell binding to domain VI was sensitive to
inhibition by heparin/heparan sulfate, demonstrating overlap of cell
and heparin-binding sites. Interestingly, the binding constants for
binding to heparin show a 4-fold difference between domain VI of the
laminin
5 and
1 chain, with the
5 domain demonstrating highest affinity. Strong heparin
binding affinity suggests the potential to bind heparan sulfate
containing matrix molecules or cell surface receptors (21, 24, 25);
accordingly, these molecules may interact mainly with the basic
residues within the S6 site. Our results suggest that heparan
sulfate-containing cell surface receptor interaction is required for
efficient cell binding to laminin
5 domain VI. This is
in accordance with previous studies, which indicate that heparan
sulfate-containing cell surface receptors can function as co-receptors
for integrins and that these co-receptors are essential for cell
binding to some ligands, including the heparin III domain of
fibronectin and the angiogenic inducer Cyr61 (17, 26). The heparan
sulfate-containing cell surface receptors may include syndecan-1 or
-dystroglycan; the latter has been shown to bind laminin-10/11 and
domain VI of the laminin
1 chain (8, 24). The role of
these interactions may be important for cell type-specific binding or
signaling (17, 26). The heparin-binding site on laminin
5 domain VI may also function as a binding site for
other matrix molecules, since it has been reported that laminin
1 domain VI binds to heparan sulfate chains of perlecan
(24). Accordingly, binding through the heparin-binding site may be a
mechanism for the regulation of interactions with cells and matrix assembly.
Several integrins have previously been implicated as receptors
for laminin-10/11, including
3
1,
6
1, and
6
4
(7). In this study, we demonstrate that domain VI of the laminin
5 chain is a binding site for integrins
3
1,
2
1,
4
1, and
6
1.
HT-1080 cell binding was completely blocked by anti-integrin
3 or
1 antibodies, while
function-blocking antibodies against the
2,
4, and
6 integrins showed a weaker
effect, suggesting that the
3
1 integrin
is a major mediator of cell binding. Small or no effects were observed
with antibodies against
1,
5, and
V integrins. Comparison with a recombinant protein of
laminin
1 domain VI showed similar integrin specificity
except for
6, where no effect was observed for laminin
1 domain VI. The inhibition results are in agreement
with the reported integrin specificity of recombinant fragments of
domains VI through IV, of the laminin
1 and
2 chains (13, 14). The previous studies used the same
cell line as here, but only results using anti-integrin
1 and
2 antibodies were reported. Our
results using various monoclonal antibodies suggest that several
integrins bind domain VI of the laminin
5 and
1 chains and that these receptors bind similar
recognition sites within domain VI.
In conclusion, the present data represent the first mapping of sites
within the N-terminal globular domain VI of the mouse laminin
5 chain responsible for cell binding. Our results
suggest that heparan sulfate-containing receptors and integrins
recognize domain VI. We found that two sequences, spaced by ~90
residues within laminin
5 domain VI, are critical for
cell surface receptor binding and that at least four residues within
these two regions together form a binding site(s) critical for receptor binding.