©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Cell Binding Sites in the Laminin 1 Chain Carboxyl-terminal Globular Domain by Systematic Screening of Synthetic Peptides (*)

(Received for publication, January 31, 1995; and in revised form, June 1, 1995)

Motoyoshi Nomizu (1) Woo Ho Kim (1) Keizo Yamamura (1)(§) Atsushi Utani (1) Sang-Yong Song (1) Akira Otaka (2)(¶) Peter P. Roller (2)(¶) Hynda K. Kleinman (1) Yoshihiko Yamada (1)

From the  (1)Laboratory of Developmental Biology, NIDR, National Institutes of Health and the (2)Laboratory of Medicinal Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The laminin alpha1 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 beta1 and alpha6 integrin antibodies and on AG-32 was inhibited by beta1, alpha2, and alpha6 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 alpha chains, suggesting that they play a critical role for biological function and for interaction with cell surface receptors.


INTRODUCTION

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(r) = 900,000) from the mouse Engelbreth-Holm-Swarm tumor consists of alpha1, beta1, 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 alpha1 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 beta1 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 beta1 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 alpha1 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 alpha1 chain. E8, a proteolytic fragment containing the COOH-terminal long arm and the NH(2)-terminal 60% of the G domain, possesses a major cell binding activity that is mediated through alpha6beta1 integrin(19, 20, 21) . The site in E8 which interacts with the alpha6beta1 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 alpha1 chain positions 3011-3032) was found to bind to the alpha3beta1 integrin(25) . Recently, the SN peptide (a 20-mer synthetic peptide from the mouse laminin alpha1 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 alpha1 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.


MATERIALS AND METHODS

Synthetic Peptide Resins (Beads)

Peptide resins were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc)(^1)-based solid-phase method using simultaneous and multiple peptide synthesis strategy(29) . Starting with a polystyrene-polyoxyethylene support-based resin, Nova Syn TG resin (50 mg, substitution: 0.29 mmol/g, Novabiochem, La Jolla, CA), the respective amino acids were condensed manually in a stepwise manner. As a linker between peptide and resin, one glycine residue was added at the COOH terminus. For condensation, diisopropylcarbodiimide/N-hydroxybenzotriazole was employed, and for deprotection of N-Fmoc groups, 20% piperidine in dimethylformamide was employed. The following side chain protecting groups were used: Asn, Gln, and His, trityl; Asp, Glu, Ser, Thr, and Tyr, t-butyl; Arg, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; and Lys, t-butoxycarbonyl. Protected peptide resins were treated with trifluoroacetic acid-thioanisole-m-cresol-ethanedithiol-H(2)O (80:5:5:5:5, v/v) at 20 °C for 2 h, followed by washing with trifluoroacetic acid, and then this treatment was repeated one more time. The peptide-resin bond was stable under the condition, and the resulting deprotected peptide resins (here termed peptide beads) were washed with dimethylformamide, methanol, H(2)O, phosphate-buffered saline (PBS) and H(2)O (three times each), and then dried.

Synthetic Peptides and Laminin

All peptides were manually synthesized by the Fmoc strategy and prepared in the COOH-terminal amide form. Starting with a tris-(alkoxy)benzylamine resin(27) , the respective amino acids were condensed manually in a stepwise manner as described above. Resulting protected peptide resins were deprotected and cleaved from the resin using a two-step strategy of consecutive trifluoroacetic acid-thioanisole-m-cresol-ethanedithiol-H(2)O (80:5:5:5:5, v/v) at 20 °C for 2 h and 1 M trimethylsilyl bromide-thioanisole in trifluoroacetic acid in the presence of m-cresol and ethanedithiol at 20 °C for 1 h (30, 31) . Resulting crude peptides were purified by reverse-phase high performance liquid chromatography. Identity of the peptides was confirmed by amino acid analysis and fast atom bombardment mass spectral analysis. Amino acid analyses were performed at the Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. Mass spectra were measured in a glycerol matrix on a VG 7070E-HF double focusing mass spectrometer.

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).

Antibodies

Rat monoclonal antibodies used were: mAb 13, an antibody to the beta1 integrin subunit (28) (a generous gift from Dr. S. K. Akiyama, National Institutes of Health) and GoH3, an anti-alpha6 integrin subunit antibody (purchased from AMAC, Westbrook, ME). Mouse monoclonal antibodies used were: P1E6, an anti-alpha2 integrin subunit antibody, and P1B5, an anti-alpha3 integrin subunit antibody. These antibodies and mouse preimmune IgG were purchased from Life Technologies, Inc.

Cells and Culture

The following cells were used for this study: B16-F10 mouse melanoma cells(33) , HT-1080 human fibrosarcoma cells(34) , SW480 human colon adenocarcinoma cells(35) , and RD human embryonal rhabdomyosarcoma cells (ATCC, CCL136). HT-1080 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). B16-F10 cells (a gift of Dr. I. J. Fidler, M. D. Anderson Hospital, Houston, TX) were maintained in Eagle's minimal essential medium (EMEM) supplemented with 5% FBS, and nonessential amino acids and vitamins. SW480 cells were cultured in RPMI 1640 (Life Technologies, Inc.) containing 10% FBS, antibiotics, and insulin (5 µg/ml). RD cells were maintained in EMEM supplemented with 10% FBS and twice the standard concentrations of amino acids and vitamins. Cell cultures were performed on tissue culture plates (Nunc, Inc., Naperville, IL). In some experiments, poly-hydroxyethyl methacrylate (HEMA)-coated plates (50 µl of 10 mg/ml poly-HEMA solution in each 96-well plate) were used to maintain cells in suspension(36) .

Cell Attachment Assay Using Peptide Beads

Cell attachment to peptide beads was assayed in 48-well dishes, which were blocked with 0.5 ml of DMEM/well containing 1% bovine serum albumin (BSA) for 1 h at 37 °C. Peptide beads (10 mg) were suspended in PBS (500 µl) and centrifuged, then the peptide beads were added to the 48-well dishes and washed with DMEM (500 µl 3 times). The B16-F10 or HT-1080 cells (5 10^4) were added to each well in a total volume of 500 µl of DMEM containing 0.1% BSA and incubated for 12 h at 37 °C in 5% CO(2), 95% air. The beads were stained by Diff Quik (Baxter, Miami, FL) and analyzed under the microscope.

Attachment Assay Using Laminin-1 and Synthetic Peptides

Cell attachment was assayed in round-bottomed 96-well plates (Nunc, Inc., Naperville, IL) coated with either various amounts of synthetic peptides or laminin-1. The peptides were dissolved in Milli-Q water, and 50 µl of the solution was added to each well, followed by drying overnight. The wells were blocked by addition of 100 µl of 3% BSA in DMEM (200 µl) at 37 °C for 1 h, then washed twice with DMEM containing 0.1% BSA. Cells, detached with 0.02% EDTA in PBS and resuspended in DMEM containing 0.1% BSA, were added (3 10^4/0.2 ml) to each well and incubated at 37 °C for 1 h in 5% CO(2). The attached cells were stained with 200 µl of 0.2% crystal violet aqueous solution in 20% methanol for 10 min. After washing, 200 µl of 1% SDS was used to dissolve the cells and the optical density at 560 nm was measured in a Titertek Multiscan. Peptide-coated wells without cells were processed simultaneously to subtract the background because some peptides at higher concentrations were stained with crystal violet.

Cell Spreading and Inhibition Assays

Wells of a 96-well plastic tissue culture plate (Costar, Cambridge, MA) were coated with various amounts of either laminin-1 or synthetic peptides, then incubated 2 h or dried over night. Wells were subsequently blocked with 3% BSA in DMEM for 1 h at room temperature. The cells were detached with trypsin and allowed to recover in the presence of 10% FBS for 30 min at 37 °C in the presence of CO(2). After washing twice with PBS, cells were placed in the coated wells at 5 10^3 cells/well. After 45 min of incubation at 37 °C, the cells were fixed with 3% formaldehyde, 3% glutaraldehyde, and the percentage of spreading RD cells was counted using phase contrast microscopy(37) .

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.


RESULTS

Cell Attachment of RGD-, YIGSR-, and IKVAV-containing Peptide Beads

As controls for screening of active sites, several peptide resins containing well studied biologically active and inactive sequences were prepared and their cell attachment activity was tested. The RGD(38) , YIGSR(11) , and IKVAV (15) active sequences and the control YIGSE and GKVAV sequences were prepared on a polyethylene glycol-coated resin (Nova Syn TG resin) using the Fmoc-based solid-phase peptide synthesis methodology. Cell attachment activity of the peptide beads was tested using HT-1080 human fibrosarcoma cells (Fig. 2). The RGD-, YIGSR-, and IKVAV-containing peptide beads were found to have cell attachment activities, but many more cells were present and spread on the RGD beads (Fig. 2B). No attached cells were found on the resin alone or on either the YIGSE or GKVAV (control) sequence-containing peptide beads. These observations are comparable to the previous results using free synthetic peptides (11, 15, 38) . When we tested cell attachment activity of the above peptide beads using B16-F10 mouse melanoma cells, similar results were obtained (data not shown). These data suggested that this peptide bead assay system was applicable for screening of cell attachment active sites using short peptides. We therefore used this system as a first screening method to identify active sequences from the G domain of the alpha1 chain.


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.



First Screening: Cell Attachment Assay of Peptide Beads Involving G Domain of the alpha1 Chain

For screening cell attachment active sites of the laminin alpha1 chain carboxyl-terminal G domain, 113 different peptide beads comprising overlapping sequences of the whole G domain were prepared (Fig. 3). Peptides were generally 12 amino acids in length and overlapped with neighboring peptides by 4 amino acids. If the NH(2)-terminal amino acid was either glutamine or glutamic acid, one amino acid was extended at the NH(2) terminus to avoid pyroglutamine formation(39) . Cysteine residues were omitted.


Figure 3: Sequence and peptides from the laminin alpha1 chain G domain. Sequences were derived from the mouse laminin alpha1 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.

Cell Attachment Activities of Synthetic Peptides

Nineteen peptides corresponding to the sequences shown to have cell attachment activities in the first screening using the peptide bead assay and six peptides (AG-9, 20, 35, 85, 95, and 110), which did not show cell attachment activities in the first screening, were synthesized. As a positive control, the IKVAV sequence (15) containing 12-mer peptide (LAM-L: AASI- KVAVSADR; (40) ) was also prepared. Cell attachment activities of the synthetic peptides were tested using HT-1080 human fibrosarcoma cells. Five of the peptides from the G domain showed dose-dependent cell attachment activity (Fig. 4). AG-73 had the strongest cell attachment activity. AG-10 and AG-22 also showed stronger cell attachment activity than that of the IKVAV peptide. AG-32 and AG-56 showed similar activity to the IKVAV peptide. Cell attachment activities of the 25 different synthetic peptides were also tested using B16-F10 mouse melanoma and SW480 human colon adenocarcinoma cells (Table 1). AG-73 had the strongest cell attachment activity for all of the cell lines. AG-10 also showed strong cell attachment activity for all of the cells. AG-22, AG-32, and AG-56 showed similar activity to that of the IKVAV peptide, whereas the other peptides were not active.


Figure 4: Attachment of HT-1080 cells to synthetic peptides. Peptides and laminin-1 were dissolved in H(2)O, 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.

Effects of Integrin Antibodies on AG-10-, AG-32-, and AG-73-mediated Cell Spreading

Since HT-1080 cells attached to the peptide-coated plates but did not spread well (data not shown), we used RD human embryonal rhabdomyosarcoma cells to assess the integrins involved in attachment to the G domain peptides. RD cells attached and spread in a dose-dependent manner on AG-10, AG-32, and AG-73 peptide-coated plates (Fig. 6A). RD cells spread poorly on AG-22-coated plates and did not spread on AG-56-coated plates (data not shown). The effects of integrin antibodies on cell spreading mediated by AG-10, 32, and 73 were tested using anti-beta1, alpha2, alpha3, and alpha6 integrin antibodies (Fig. 6B, lanes b-e, respectively). The anti-beta1 integrin antibody inhibited laminin-1-mediated cell spreading. Cell spreading mediated by AG-10 was inhibited by both anti-beta1 and anti-alpha6 integrin antibodies. AG-32-mediated cell spreading was inhibited by anti-beta1, anti-alpha2, and anti-alpha6 integrin antibodies. Anti-beta1, alpha2, alpha3, and alpha6 integrin antibodies did not affect AG-73-mediated cell spreading. We conclude that AG-10 and AG-32 cell spreading was mediated by integrins, whereas the AG-73 peptide probably interacts with either a different integrin and/or a non-integrin receptor.


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-beta1 integrin monoclonal antibody (mAb 13, 67 µg/ml) (b), anti-alpha2 integrin monoclonal antibody ascites (P1E6, 1:33 dilution) (c), anti-alpha3 integrin monoclonal antibody ascites (P1B5, 1:33 dilution) (d), anti-alpha6 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.



Minimum Active Sequences of AG-10, AG-32, and AG-73

The cell attachment activities of AG-10, AG-32, and AG-73 were found to be stronger or comparable to those of the previously reported IKVAV-containing peptide ( Table 1and Fig. 4). Minimum sequences of AG-10, AG-32, and AG-73 for cell attachment activity were determined using systematically truncated NH(2)- and COOH-terminal peptides (Table 2). AG-10d (SIYITRFG), an NH(2)-terminal truncated peptide, still retained full activity, whereas a deletion of serine from AG-10d (AG-10e) resulted in complete loss of activity (Table 2, part A). The COOH-terminal deletion peptide, AG-10h (SIYITRF) also showed activity. Further deletion of phenylalanine (AG-10i, SIYITR) eliminated its cell binding activity. These results indicate that SIYITRF is a critical sequence for activity in AG-10. A structure-activity study of AG-32 was also performed using truncated peptides (Table 2, part B). An NH(2)-terminal truncated peptide AG-32d showed cell attachment activity, but the isoleucine deleted peptide (AG-32e) from AG-32d lost all activity. On the other hand, the COOH-terminal truncated peptide AG-32h showed good activities, while its asparagine-deleted peptide analog was found to be inactive. The minimum sequence of AG-32 for cell attachment activity was determined to be a hexapeptide IAFQRN (AG-32n). Similarly systematic NH(2)- and COOH-terminally truncated AG-73 peptides were evaluated for cell attachment activity (Table 2, part C). The minimum sequence of AG-73 for cell attachment activity was found to be an octapeptide, LQVQLSIR (AG-73e). Since the glutamine residue at the NH(2) terminus of the peptide is known to easily form a pyroglutamine, the NH(2)-terminal leucine residue of the octapeptide was not deleted(39) . The structure-activity studies of AG-10, AG-32, and AG-73 strongly suggest that the peptides interact with cell surface receptors or binding proteins involving short sequences of six to eight amino acids.




DISCUSSION

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 alpha1 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 alpha6beta1 integrin and AG-32 recognized alpha2, alpha6, and beta1 integrin subunits. E8 fragments was previously reported to interact with the alpha6beta1 integrin(19, 20, 21) . A recombinant alpha1 chain consisting of the COOH-terminal portion of the long arm and the entire G domain, however, was not active for alpha6beta1 integrin-mediated cell adhesion. Reconstitution of the recombinant alpha1 and the beta1-1 dimer from the E8 fragment yielded a component active for cell adhesion through alpha6beta1 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 alpha6beta1 integrin antibodies, these active sites in the recombinant alpha1 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(2)- 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 beta1 integrin subunit, and the GD-6 peptide was a binding site for alpha3beta1 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 beta2 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 alpha1(49, 50) , human laminin alpha2 chain(51, 52, 53) , mouse laminin alpha2 chain(54) , and Drosophila laminin alpha 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Dermatology, Kobe University School of Medicine, Kobe 650, Japan.

Present address: Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan.

(^1)
The abbreviations used are: Fmoc, 9-fluorenylmethoxycarbonyl; PBS, phosphate-buffered saline; EMEM, Eagle's minimal essential medium; FBS, fetal bovine serum; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HEMA, hydroxyethyl methacrylate; mAb, monoclonal antibody.


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