(Received for publication, January 31, 1995; and in revised form, June 1, 1995)
From the
The laminin 1 chain carboxyl-terminal globular domain has
been identified as a site of multiple biological activities. Using a
systematic screening for cell binding sites with 113 overlapping
synthetic peptide beads that covered this domain, we found 19 potential
active sequences. Corresponding synthetic peptides were evaluated for
direct cell attachment, spreading, and inhibition of cell spreading to
a laminin-1 substrate using several cell lines. Five peptides (AG-10,
AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with
cell-type specificities. Cell spreading on AG-10 was inhibited by
1 and
6 integrin antibodies and on AG-32 was inhibited by
1,
2, and
6 integrin antibodies. In contrast, cell
adhesion and spreading on peptide AG-73 were not inhibited by these
antibodies. The minimum active sequences of AG-10, AG-32, and AG-73
were determined to be SIYITRF, IAFQRN, and LQVQLSIR, respectively.
These sequences are highly conserved among the different species and
different laminin
chains, suggesting that they play a critical
role for biological function and for interaction with cell surface
receptors.
Laminin-1 is a major component and cell adhesion protein of the
basement membrane matrix(1, 2, 3) . There are
at least seven isoforms of laminin each consisting of three different
chains(4) . The most extensively characterized laminin,
laminin-1 (M = 900,000) from the mouse
Engelbreth-Holm-Swarm tumor consists of
1,
1, and
1
chains, which assemble into a triple-stranded coiled-coil structure at
the long arm to form a cross-like structure (4, 5) .
These chains have a similar domain structure except for the unique
COOH-terminal globular domain (G domain) on the
1 chain (6, 7, 8) . Laminin-1 has multiple biological
activities including promotion of cell adhesion, spreading, growth,
neurite outgrowth, tumor metastasis, and collagenase IV
secretion(1) . Several active sites of laminin-1 have been
identified using proteolytic fragments, recombinant proteins and
synthetic peptides (Fig. 1)(9, 10) . The YIGSR
sequence located on the
1 chain (positions 929-933) has been
shown to promote cell adhesion and migration and to inhibit
angiogenesis and tumor metastasis(11, 12) . The PDSGR
and F-9 (RYVVLPR) sequences located on the
1 chain were also found
to promote cell adhesion(13, 14) . An IKVAV sequence
located on the COOH-terminal end of the long arm of the
1 chain
was found to promote cell adhesion, neurite outgrowth, experimental
metastasis, collagenase IV secretion, angiogenesis, cell growth, and
tumor growth(15, 16, 17, 18) .
Figure 1: Structural model of laminin-1. The locations of cell binding sites are indicated by arrows. YIGSR was from Graf et al.(11) , PDSGR from Kleinman et al.(13) , IKVAV from Tashiro et al.(15) , F-9 (RYVVLPR) from Charonis et al.(57) and Skubitz et al.(14) , and RGD from Tashiro et al.(58) . E8 and E3 designate previously described proteolytic fragments of laminin-1(46) . The E8 and E3 fragments and the G domain are indicated by squarebrackets.
Several studies have focused on the biological activity of the G
domain of the laminin 1 chain. E8, a proteolytic fragment
containing the COOH-terminal long arm and the NH
-terminal
60% of the G domain, possesses a major cell binding activity that is
mediated through
6
1
integrin(19, 20, 21) . The site in E8 which
interacts with the
6
1 integrin has not been identified.
Recombinant and reconstitution experiments have suggested that this
activity is dependent on protein
conformation(22, 23) . Several synthetic peptides from
the G domain containing positively charged regions were found to
promote heparin binding, cell adhesion and neurite
outgrowth(24) . Moreover, the synthetic peptide (GD-6:
KQNCLSSRASFRGCVRNLRLLSR, corresponding to mouse laminin
1 chain
positions 3011-3032) was found to bind to the
3
1
integrin(25) . Recently, the SN peptide (a 20-mer synthetic
peptide from the mouse laminin
1 chain comprising residues
2179-2198) was found to inhibit lung alveolar formation and to
promote cell adhesion in vitro(26) .
In this paper,
we describe the systematic screening of biologically active sequences
in the mouse laminin 1 chain G domain (positions 2111-3060)
using a large set of overlapping peptides covalently bound to resin
beads. Using this assay system as the first stage of screening, we
examined the cell attachment activities of 113 different peptide beads.
Nineteen potential active sequences were identified from this peptide
bead screening. As a second screening, free synthetic peptides,
including these initially identified active sequences, were examined
for direct cell attachment activities on plastic and for inhibitory
effects on cell spreading to laminin-1. Five active peptides were
identified from the second screening. Several additional biological
activities were also evaluated for the five active synthetic peptides.
Specific antibodies to integrins blocked adhesion to two of these
active peptides.
Mouse laminin-1 was prepared from the Engelbreth-Holm-Swarm tumor as described previously(32) . Human plasma fibronectin was a generous gift from Dr. S. K. Akiyama (National Institutes of Health, Bethesda, MD).
Inhibition of cell spreading was assayed in the 96-well plates, where each well was coated with 2 µg of laminin-1 or fibronectin. The cells were incubated in the presence of various concentrations of peptide in either the laminin-1- or fibronectin-coated wells. The percentage of spread cells was counted as described above.
Figure 2: Adhesion of HT-1080 cells on peptide beads. HT-1080 human fibrosarcoma cells were allowed to attach to peptide beads for 16 h. A, beads (Nova Syn TG resin); B, RGD beads (GRGDSG beads); C, IKVAV beads (SIKVAVSG beads); D, GKVAV beads (SGKVAVSG beads); E, YIGSR beads (YIGSRG beads); F, YIGSE beads (YIGSEG beads). RGD beads had numerous flattened cells attached on the surface, while IKVAV and YIGSR beads had scattered cell champs.
Figure 3:
Sequence and peptides from the laminin
1 chain G domain. Sequences were derived from the mouse laminin
1 chain(8) . Locations of peptide beads are indicated by arrows. Active peptide beads are shown by a bolddottedline. Cell attachment activities are
shown in brackets. (++), similar to the RGD
bead; (+), weaker than RGD bead but similar to the IKVAV
and YIGSR beads; (-), negative. GD-1, GD-2, GD-3, and
GD-4 reported by Skubitz et al.(24) , GD-6 reported by
Gehlsen et al.(25) , and SN peptide reported by Matter
and Laurie (26) are indicated by squarebrackets.
The cell attachment activities of the 113 peptide beads were tested using B16-F10 mouse melanoma cells (Fig. 3). Seven peptide beads (AG-10, 17, 32, 39, 64, 73, and 81) were found to have a strong cell attachment activity comparable to that of the RGD beads. Twelve peptide beads (AG-13, 22, 42, 53, 56, 68, 78, 80, 82, 86, 98, and 103) showed weak cell attachment activity similar to that of the YIGSR and IKVAV beads. The remaining 94 peptide beads did not show cell attachment activity. AG-42, which is a segment of the previously described active peptide GD-3 peptide(24) , and AG-80-AG-82, which are part of the previously described active peptide GD-4(24) , showed cell attachment activities in our peptide bead assay. Peptide beads comprising other previously reported active peptides including GD-1, GD-2, and GD-6 did not show cell attachment activity(24, 25) . AG-10, which is involved in the most recently described active SN peptide region, showed cell attachment activity(26) . For evaluating additional biological activities of the 19 sequences identified from the first screening, we prepared free synthetic peptides and tested them in more well established assays.
Figure 4:
Attachment of HT-1080 cells to synthetic
peptides. Peptides and laminin-1 were dissolved in HO,
added to 96-well tissue culture dishes, and dried overnight. HT-1080
human fibrosarcoma cells were added, and the number of attached cells
was assessed by crystal violet staining. Data are expressed as mean of
triplicate results.
The inhibitory effects of these peptides on cell spreading to a laminin-1 substrate were tested using RD human embryonal rhabdomyosarcoma cells (Fig. 5A). As a control, the RGD sequence containing fibronectin peptide segment (FIB-1: YAVTGRGDSPAS) was prepared and tested. AG-22 showed the strongest inhibition. AG-10 and AG-32 inhibited more than 50% of cell spreading to laminin-1 at concentrations of 250 or 500 µg peptide/ml, respectively. AG-73 showed a dose-dependent inhibitory effect on cell spreading at low concentrations (25-100 µg of peptide/ml). Since AG-10 and AG-73 showed toxicity to the RD cells at the peptide concentrations of 500 and 250 µg/ml, respectively (data not shown), these peptides were used at lower concentrations in this experiment. AG-56 and FIB-1 showed no effect on cell spreading. The 14 peptides, which showed cell attachment activity in the beads assay but were not active as peptides coated on plates, did not effect RD cell spreading to laminin-1 substrate at a final peptide concentration of 250 µg/ml (data not shown). Inhibitory effects of the peptides on RD cell spreading to a fibronectin substrate were also tested (Fig. 5B). In this assay, AG-22 showed the strongest inhibitory effect on cell spreading. FIB-1 showed a dose-dependent inhibitory effect as expected. The other peptides did not inhibit cell spreading on fibronectin.
Figure 5: Inhibition of RD human embryonal rhabdomyosarcoma cell spreading on laminin-1 and fibronectin by synthetic peptides. PanelA, peptide inhibition of RD cell spreading on laminin-1 substrate. PanelB, peptide inhibition of RD cell spreading on fibronectin. A 96-well tissue culture dish was coated with 2 µg of laminin-1 or fibronectin/well. Cells and peptides were added, and the percentage of spread cell was counted. Each value represents the mean of five separate determinations ± S.D. Duplicate experiments gave similar results.
Based on the second screening, we identified five biologically active sequences corresponding to the peptides AG-10, AG-22, AG-32, AG-56, and AG-73. We next focused on evaluating these five peptides to further define their biological activities.
Figure 6:
Effect of peptides and integrin antibodies
on rhabdomyosarcoma cell spreading. Cell spreading assays were
performed using RD human embryonal rhabdomyosarcoma cells as described
under ``Materials and Methods.'' PanelA,
dose-dependence curves on laminin-1 and on synthetic peptides. PanelB, inhibitory effect of anti-integrin
antibodies. Cell spreading assays were performed on untreated controls (a), or in the presence of anti-1 integrin monoclonal
antibody (mAb 13, 67 µg/ml) (b), anti-
2 integrin
monoclonal antibody ascites (P1E6, 1:33 dilution) (c),
anti-
3 integrin monoclonal antibody ascites (P1B5, 1:33 dilution) (d), anti-
6 integrin monoclonal antibody (GoH3, 2
µg/ml) (e), and mouse preimmune IgG (2 µg/ml) (f). The amounts of coated peptides were 2 µg/well for
laminin-1 and AG-73, and 10 µg/well for AG-10 and AG-32. Each value
represents the mean of three separate determinations ±
S.D.
Multiple peptide synthesis methodologies (41, 42) have been developed using the traditional
solid-phase method, tea-bag approach, multi-pin method, split synthesis
method, and spot synthesis(43, 44) . These
methodologies have been useful in many studies such as peptide
libraries for screening or identification of new or more effective
ligands that bind antibody, receptor, enzyme, or other host molecules.
Here we describe screening of active cell attachment peptides from the
carboxyl-terminal globular domain of the laminin 1 chain. We used
traditional multiple solid-phase synthesis methodology (29) for
preparing a large number of peptide resins followed by deprotection of
side chain protecting groups. The peptide beads were used directly in
the assays. Peptide beads, which contained covalently conjugated
synthetic peptides on Sepharose beads, were used previously to
determine cell adhesion and found to be a useful method for short
peptides(45) . The peptide bead assay system described here was
found to be a convenient method for a first screening of active cell
attachment sequences since preparation of and biological assays on
large numbers of peptide beads were relatively easy and quick. In our
first screening, 19 peptide beads of the 113 beads tested had cell
attachment activities. However, only five peptides showed cell adhesive
activities in the second and third screening assays using
peptide-coated and laminin-1-coated dishes.
Identifying active domains on proteins using short synthetic peptides has the potential for false positives as well as false negatives due to the possible different conformations that the peptide sequences can assume in the peptide form versus the actual protein. It is also possible that a peptide which is active in vitro may have no activity in the intact molecule and/or in vivo due to a cryptic location or inactivity. The question of the conformational structure of the peptides is an important one, which is difficult to address in vitro. Certainly the fact that synthetic peptides in general are much less active on a molar basis than the intact protein could be explained based on different active and inactive conformations the peptides can assume depending upon the assay. Here we have tested the peptides in three potentially different conformations including extended on the beads, dried (and packed) on the dishes, and in solution for the competition assays. By testing the peptides in three different possible conformational states, we anticipate that false positive activities are reduced. This likely explains why only 5 of the 19 active peptides identified in the bead assay were active in the other two assays. The most physiological of these would be the competition assay where the peptide is added in solution and used to block laminin-1 activity. Potential problems with this assay include the fact that the laminin-1 is coated on the dish and may not be conformationally relevant to its in vivo structure. It should also be noted that laminin-1 has many active sites and many cells have multiple receptors for these sites such that one peptide may not block activity due to the utilization of other sites and receptors. Despite these limitations, a number of active sequences on several adhesion proteins have been described and shown to be active in vivo(9, 10) . A logical course with these in vitro identified peptides would be to determine if the active sites are exposed in vivo either in normal tissues and/or in tissues undergoing development or remodeling. Certainly as a first step in trying to identify potential active domains that may have useful clinical applications, the peptide approach is very valuable.
In this study, we identified five different cell binding sites from the G domain. AG-73 showed the strongest cell attachment activities with three different cells (HT-1080, B16-F10 and SW480 cells) and the other four peptides (AG-10, AG-22, AG-32 and AG-56) also showed variable binding activities with the cells. Furthermore, AG-10, AG-22, AG-32, and AG-73 reduced laminin-1-mediated cell spreading of RD cells. AG-22 did not show strong cell attachment activity relative to AG-73, but AG-22 showed a strong inhibitory effect on laminin-1-mediated RD cell spreading. AG-22 also inhibited fibronectin-mediated cell spreading. These results suggest that conformations of AG-22 in solution can readily bind surface receptors. Since the peptide showed different inhibitory effects on cell spreading to laminin-1 and different results with various cell types, these sequences likely show cell type specificities, which probably relate to the receptor amount, type, and affinity.
AG-10 and AG-32 located in the E8 fragment region (46) were active in cell spreading assays, and this activity
was blocked by integrin antibodies. Cell spreading of AG-10 was
mediated at least in parts through 6
1 integrin and AG-32
recognized
2,
6, and
1 integrin subunits. E8 fragments
was previously reported to interact with the
6
1
integrin(19, 20, 21) . A recombinant
1
chain consisting of the COOH-terminal portion of the long arm and the
entire G domain, however, was not active for
6
1
integrin-mediated cell adhesion. Reconstitution of the recombinant
1 and the
1-
1 dimer from the E8 fragment yielded a
component active for cell adhesion through
6
1 similar to the
E8 fragment(22) . Since the reconstituted molecule mimics a
structure similar to the E8 fragment in electron microscopy, this
activity is apparently dependent on conformation. Since AG-10 and AG-32
peptides are active for cell spreading and this activity was blocked by
6
1 integrin antibodies, these active sites in the recombinant
1 chain could be hidden by protein folding but the sites become
available and active due to a conformational change when the three
chains assemble.
Recently, the SN peptide (A chain residues
2179-2198) from the first loop of the carboxyl-terminal G domain
and the SINNNR sequence, which was a minimum active sequence of the SN
peptide, were shown to inhibit lung alveolar formation and promote cell
adhesion(26) . We also identified the same region (AG-10,
residues 2183-2194) as a cell attachment site, but we found its
active sequence to be a heptapeptide, SIYITRF, using systematical
NH- and COOH-terminal truncated peptides. In addition, the
AG-9 peptide and the peptide bead, which contained the SINNNR sequence,
did not show cell attachment activity. The reason for this difference
between our data and that already published is unclear. The previous
study used proteolytic fragments of the SN peptide for determination of
the minimum active sequence. It is possible that the proteolytic
fragments were not completely separated.
Previously, a series of
synthetic peptides (20 amino acids long) containing multiple
positively charged amino acids from the G domain were tested for
several biological activities(24, 25) . It was
reported that the GD-1, GD-2, GD-3, GD-4, and GD-6 peptides (location
of these peptides is shown in Fig. 3) had cell binding
activities, the GD-3 and GD-4 peptides interacted with
1 integrin
subunit, and the GD-6 peptide was a binding site for
3
1
integrin. In our study, the GD-3 and GD-4 regions showed cell
attachment activities in the peptide bead assay, but the peptides
corresponding to these regions did not show activity in direct adhesion
assays. The other three sequences, GD-1, GD-2, and GD-6, containing
peptide beads did not show cell attachment activities. The explanation
for the differences in the results are not clear at this time. Our
peptide beads and synthetic peptides were a little shorter in length
than those previously used, basically 12 amino acid residues, and did
not include cysteine residues.
Several biologically active sequences
of laminin-1 have been reported previously using synthetic peptides,
and all of these contained an arginine residue except for the IKVAV
sequence (Fig. 1). Moreover, the LRE sequence from the laminin
2 chain was also shown to have cell adhesive
activity(47, 48) . It thus appears that a positively
charged residue, mainly an arginine residue, seems to be critical for
the peptide ligand to interact with cell surface receptors. In the
present study, we identified three different minimum sequences of
AG-10, AG-32, and AG-73 for cell binding activities. These sequences
also contain one arginine residue each. The minimum sequences, SIYITRF
(AG-10h), IAFQRN (AG-32n), and LQVQLSIR (AG-73e), are highly conserved
on human laminin
1(49, 50) , human laminin
2
chain(51, 52, 53) , mouse laminin
2
chain(54) , and Drosophila laminin
chain(55, 56) . Thus, these corresponding regions of
AG-10, AG-32, and AG-73 in the chains could also have conserved cell
binding activities but that has not yet been demonstrated.