Identification of Cell Binding Sites in the Laminin α5-Chain G Domain

Experimental Cell Research 277, 95–106 (2002) doi:10.1006/excr.2002.5540

Identification of Cell Binding Sites in the Laminin ␣5-Chain G Domain Masayoshi Makino,* Ikuko Okazaki,* Shingo Kasai,* Norio Nishi,* Maria Bougaeva,† Benjamin S. Weeks,† Akira Otaka,‡ Peter K. Nielsen,§ Yoshihiko Yamada,§ and Motoyoshi Nomizu* ,1 *Graduate School of Environmental Earth Science, Hokkaido University, Kita 10 Nishi 5, Kita-ku, Sapporo 060-0810, Japan; †Division of Mathematics and Sciences, Department of Biology, Adelphi University, Garden City, New York 11530; ‡Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan; and §Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4370

␤-chains, and three ␥-chains [2– 4]. Laminins have diverse biological activities including promotion of cell adhesion, cell migration, neurite outgrowth, tumor metastasis, and collagenase IV production [1]. The identification and characterization of several active sites for laminin-1 have been demonstrated previously using proteolytic fragments, recombinant proteins, peptidespecific antibodies, and synthetic peptides [5, 6]. Recently, we have screened for cell adhesion sequences on laminin-1 using a large set of overlapping synthetic peptides spanning the entire protein [7–10]. Most of the active peptides were derived from the globular domains, and several of them were suggested to play a critical role in binding to cell surface receptors [11]. Some of the peptides were also found to promote neurite outgrowth, angiogenesis, and tumor metastasis [12–20]. The amino acid sequence of the murine laminin ␣-chain has been deduced from cDNAs [21] and reveals relationships with the Drosophila ␣-chain [22]. Analysis of RNA expression shows that the murine laminin ␣5-chain is widely expressed and most abundant among the ␣-chains in the adult stages [21]. The ␣5chain is expressed at higher levels in adult tissues of the lung, heart, bone marrow, pancreas, and kidney, with low levels in the brain and skeletal muscle and even lower levels in the liver, gut, and skin [3, 23]. Laminin-10 and laminin-11 consist of three chains associated with ␣5, ␥1-, and either ␤1- or ␤2-chains [3]. Elimination of the laminin ␣5-chain has been shown to cause embryonic lethality in mice [24]. The ␣5-chain has been associated with diverse pathologies, such as defective glomerulogenesis, sickle red blood cell (RBC) adhesion, and diabetic retinopathy [25–27]. Laminin-10 and -11 were found to interact with ␣3␤1-integrin [28 –30]. Moreover, a recombinant LG4 module of the laminin ␣5-chain G domain interacts with a cell surface receptor containing heparan sulfate, and heparin-binding sites were identified using various synthetic peptides [31]. However, functional studies and

The laminins consist of at least 11 polypeptides (5 ␣-chains, 3 ␤-chains, and 3 ␥-chains) specific to basement membranes. Here we investigate the biological activity associated with the G domain of the newly identified laminin ␣5-chain using 113 overlapping synthetic peptides (positions 2679 –3635). Using HT-1080 cells, 21 peptides showed attachment activity either on peptide-coated tissue culture plates or to peptideconjugated Sepharose beads. Heparin inhibited cell attachment to 16 peptides, while ethylenediaminetetraacetic acid exhibited no inhibitory activity. Peptides A5G-27, A5G-65, and A5G-71 showed the strongest cell attachment, with the minimum active core sequences of the peptides being GIIFFL, HQNMGSVNVSV, and YLQFVG, respectively. Furthermore, these 16 peptides were tested for their ability to stimulate neurite outgrowth in the PC12 cells. A5G-3, A5G-33, A5G-71, A5G-73, A5G-81, and A5G-101 were the only peptides of the 16 that demonstrated the ability to promote neurite outgrowth. These results demonstrate that synthetic peptides with ␣5-chain G domain primary amino acid sequences possess some of the same biological activities attributable to the whole laminin and the ␣5-chain G domain. Therefore, these peptides may be useful in the investigation of laminin– receptor interactions and possibly mechanisms of laminin signal transduction. © 2002 Elsevier Science (USA)

INTRODUCTION

Laminins are heterotrimeric basement membrane proteins that have multiple biological functions through interactions with other matrix molecules and cell surface receptors [1]. Laminins consist of ␣-, ␤-, and ␥-chains, which are assembled into a triplestranded coiled-coil domain and form a crosslike structure [1]. To date, 15 laminin isoforms (laminins 1–15) have been identified with five distinct ␣-chains, three 1 To whom correspondence and reprint requests should be addressed. Fax: 81-11-706-2254. E-mail: [email protected].

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0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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LAMININ ␣5-CHAIN G DOMAIN CELL BINDING SITES

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FIG. 2. Attachment of HT-1080 cells to peptide-coated plates. (A) Cell attachment was comparable to those on AG-73 (B). Ninety-six-well plates were coated with various amounts of synthetic peptides. The peptides were dissolved in H 2O and added to 96-well tissue culture dishes, and the number of attached cells was assessed by crystal violet staining. AG-73, derived from the laminin ␣1-chain G domain, was used as a positive control. Data are expressed as mean of triplicate results. Triplicate experiments gave similar results.

additional binding sites of the whole ␣5-chain G domain have not yet been carried out in detail. Here, we identified novel cell binding and neurite outgrowth promoting sequences on the C-terminal globular domain (G domain) of the ␣5-chain (position 2679–3635) by a systematic peptide screening using 113 overlapping synthetic peptides. One hundred thirteen overlapping synthetic peptides, which span the laminin ␣5-chain G domain, were tested for HT-1080 cell attachment activity on either peptide-coated tissue culture plates and/or peptide-conjugated Sepharose beads. The effect of heparin and ethylenediaminetetraacetic acid (EDTA) on HT-1080 cell attachment activity of the cell adhesive peptides was also studied. The results with the HT-1080 cells were compared to PC12 cell attachment activity on peptidecoated tissue-culture wells, and the biological activity of the peptides was further analyzed for the ability to promote neurite outgrowth in PC12 cells. MATERIALS AND METHODS Synthetic peptides, laminin-10/11, and recombinant protein. All synthetic peptides adapted for this study were manually synthesized by 9-fluorenylmethoxycarbonyl- (Fmoc) based solid-phase method and prepared with a C-terminal amide as described previously [7, 32]. The respective amino acids were condensed manually in a stepwise manner using 4-(2⬘,4⬘-dimethoxyphenyl-Fmoc-aminomethyl)-

phenoxy resin. For condensation, diisopropylcarbodiimide was employed, and for deprotection of N ␣-Fmoc groups, 20% piperidine in N,N-dimethylformamide was employed. The amino-acid side-chain protecting groups were 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. The resulting protected peptide resins were deprotected and cleaved from the resin with trifluoroacetic acid/ thioanisole/m-cresol/ethanedithiol/H 2O (80:5:5:5:5, v/v) at room temperature for 3 h. The resulting crude peptides were precipitated, washed with ethyl ether, and then purified by reverse-phase highperformance liquid chromatography (using Mightysil RP-18 GP column and a gradient of water/acetonitrile containing 0.1% trifluoroacetic acid). The purity of the peptides was confirmed by analytical high-performance liquid chromatography. The identity of the peptides was confirmed by a Sciex API IIIE triple quadruple ion spray mass spectrometer [33]. Six peptides were not dissolved in aqueous solution. These peptides were evaluated for their biological activity as peptides coupled to polystyrene beads [7]. Recombinant laminin ␣5-LG4-5 protein involving mouse laminin ␣5-chain (3237–3610) was prepared as described previously [31]. Human laminin-10/-11 was purchased from Life Technologies, Inc. (Gaithersburg, MD). Preparation of peptide-conjugated Sepharose beads. The synthetic peptides were coupled to cyanogen bromide- (CNBr) activated Sepharose 4B (Pharmacia Biotech AB, Uppsala, Sweden) as described previously [34]. The peptide solutions (0.2 ml, 1 mg/ml in Milli-Q H 2O) were mixed with 20 mg of the activated CNBr-activated Sepharose beads. A5G-42- and ethanolamine-coupled beads were prepared as a control. Amounts of coupled peptides were determined by amino acid analysis, (10 –20 ␮mol of peptides per 1 g of Sepharose beads) as described previously [34]. If the N-terminal amino acid was either glutamate or glutamic acid, one amino acid was extended at

FIG. 1. Sequences and peptides from the laminin ␣5-chain G domain. Sequences were derived from the mouse laminin ␣5-chain G domain (positions 2679 –3635) [21]. Location of peptides is indicated by arrows. A bold line shows active peptides in peptide-coated plate and/or peptide-conjugated bead assays. Cell attachment activity of insoluble peptides was determined on peptide-conjugated polystyrene beads as described previously [7].

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the N-terminus to avoid pyroglutamine formation and to couple with the CNBr-activated Sepharose beads. Peptide-conjugated polystyrene beads were synthesized to evaluate cell attachment activity of the six insoluble peptides (A5G-46, -52, -55, -75, -83, and -104) on a polystyrene–polyoxyethylene-support-based resin, Nova Syn TG resin (Novabiochem, LaJolla, CA), by the Fmoc-based solid-phase methods as described previously [7]. For each peptide resin, 50 mg Nova Syn TG resin was used (substitution: 0.29 mmol/g), and the respective amino acids were condensed manually in a stepwise manner. The amino-acid side-chain protecting groups were the same as described above. The protected peptide resins were treated with trifluoroacetic acid/thioanisole/m-cresol/ ethanedithiol/H 2O (80:5:5:5:5, v/v) at room temperature for 3 h, followed by washing with trifluoroacetic acid, and then this treatment was repeated. The resulting deprotected peptide resins (peptide–polystyrene beads) were washed with dimethylformamide, methanol, H 2O, phosphate-buffered saline, and H 2O (three times each) and then dried. Cells and culture. HT-1080 human fibrosarcoma cells were used for this study [35]. HT-1080 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Inc.) containing 10% fetal bovine serum (FBS; Life Technologies, Inc.), 100 units/ml penicillin G sodium, and 100 ␮g/ml streptomycin (Life Technologies, Inc.). Cell cultures were maintained on 25- or 75-cm 2 tissue culture plates (Iwaki, Co. Ltd., Tokyo, Japan). The cells were maintained at 37°C in a humidified 5% CO 2 and 95% air atmosphere. Cells of the rat pheochromocytoma cell line, PC12, were cultured in DMEM containing 7.5% equine serum, 7.5% fetal bovine serum, 100 units/ml penicillin G sodium, and 100 ␮g/ml streptomycin. The cells were maintained on 25- or 75-cm 2 tissue culture plates in a humidified 5% CO 2 and 95% air atmosphere. Cell attachment assay using peptide-coated plastic plates. Cell attachment assays were performed in 96-well round bottom microtiter plates (HT-1080 cells) or 24-well tissue culture plates (PC12 cells) (Nunc, Inc., Naperville, IL) coated with a variety of synthetic peptides. Wells were coated with the various amounts of peptides in 50 ␮l of Milli-Q water and dried overnight at room temperature. The wells were blocked by addition of 100 ␮l of 3% bovine serum albumin (BSA) in DMEM for 1 h and then washed twice with DMEM containing 0.1% BSA. HT-1080 cells were detached with 0.02% trypsinEDTA and PC12 cells were detached by agitation and incubated at 37°C in 5% CO 2 for 20 min. The cells washed twice with DMEM containing 0.1% BSA, added to a concentration of 2 ⫻ 10 4 cells/0.1 ml to each well, and then incubated at 37°C for 1 h. The attached cells were stained with 0.2% crystal violet aqueous solution (200 ␮l) in 20% methanol for 10 min (HT-1080 cells) or with Diff Quik (PC12 cells) (Baxter Scientific Products, McGraw Park, IL). After removing the unattached cells by aspiration, for HT-1080 cells, 1% sodium dodecyl sulfate (200 ␮l) was used to dissolve the cells, and the optical density at 570 nm was measured in a microplate reader (Bio-Rad Module 550). PC12 cell attachment was determined by counting the number of the remaining cells in three random fields in each of three replicate wells. Peptide inhibition of cell attachment to laminin-10/-11 and recombinant protein. For peptide inhibition experiments, wells were coated either with laminin-10/-11 (0.1 ␮g/well) in 50 ␮l of Milli-Q water and incubated at 37°C for 2 h or with recombinant protein (1 ␮g/well) in 50 ␮l of Milli-Q water and incubated overnight at 4°C. HT-1080 cells were preincubated with 0.1 mg/ml peptides at 37°C for 10 min and then plated. After a 30-min incubation, the attached cells were measured as described above. All assays were carried out in triplicate, and each experiment was repeated at least three times. Cell attachment assay using peptide-conjugated Sepharose beads. Cell attachment assays to peptide-conjugated Sepharose beads were performed in 48-well plates (Nunc, Inc.). Peptide beads solution (200 ␮l) were added to the 48-well dishes and washed twice with DMEM containing 0.1% BSA. The HT-1080 cells were detached with 0.02%

FIG. 3. Neurite outgrowth promotion of PC12 cells on synthetic peptides. The ability of laminin-derived synthetic peptides to promote neurite outgrowth was tested. Peptides were plated in triplicate wells at the indicated concentrations as described under Materials and Methods. The percentage of PC12 cells extending neurites was determined for each well and the average ⫾ the standard error of the mean (SEM) was determined for each peptide concentration and presented in the graph above with the error bars representing the SEM.

trypsin-EDTA and recovered at 37°C in 5% CO 2 for 20 min. Next, the cells (5 ⫻ 10 4 cells/100 ␮l of DMEM containing 0.1% BSA) were incubated with 200 ␮l of peptide bead solution at 37°C for 1 h. The beads were stained for 10 min with 0.2% crystal violet aqueous solution (200 ␮l) in 20% methanol and analyzed under the microscope. Inhibition of cell attachment by heparin. Inhibition of cell attachment assays were performed in 96-well round-bottom microtiter plates (Nunc, Inc.). Various concentrations of peptide solutions were coated in each well. HT-1080 cells and wells were prepared the same as described for the peptide-coated plate assay [7]. Heparin solution was added to cell suspension at a concentration of 10 ␮g/ml and incubated at 37°C in 5% CO 2 for 15 min. The cell suspensions with DMEM containing 0.1% BSA were added to a concentration of 2 ⫻ 10 4 cells/100 ␮l in each well and incubated at 37°C for 30 min. The attached cells were stained and measured as described above. Inhibition of cell attachment by EDTA. In EDTA-inhibition experiments, various concentrations of peptide solution were coated to each well in the same way as heparin-inhibition experiments before plating the cells. EDTA solution was added to cell suspension at a concentration of 5 mM and incubated at 37°C in 5% CO 2 for 10 min. After that, the cells with DMEM containing 0.1% BSA were added to a concentration of 2 ⫻ 10 4 cells/100 ␮l in each well and incubated at 37°C for 30 min. The attached cells were stained and measured as described above. Neurite outgrowth assays. All neurite outgrowth assays were performed in triplicate wells of a 24-well plate. The wells were coated

LAMININ ␣5-CHAIN G DOMAIN CELL BINDING SITES with the peptides as described above for the attachment assay, except that wells were not treated with BSA. Cells used for neurite outgrowth assays were treated with 100 ng/ml of nerve growth factor for 24 h. Next the PC12 cells were harvested by agitation and then washed three times with serum-free DMEM (again, not containg BSA) and resuspended to 2 ⫻ 10 4 cells/ml in an assay medium of serum-free DMEM supplemented with 100 ␮g/ml transferrin, 5 ␮g/ml bovine insulin, 30 nM NaSeO 3, and 100 ␮g/ml gentamycin. To each peptide-coated well 1 ⫻ 10 4 cells were added and incubated in 5% CO 2 at 37°C for 24 h. After 24 h the cells were fixed and stained with Diff Quik. In each of the triplicate wells, a total of 100 cells were inspected for neurites at a final magnification of ⫻100. A cell was considered to be positive for neurite outgrowth if it had neurites at least two times the cell diameter or greater. The number of cells with neurites in each of the triplicate wells was averaged (300 cells counted for each treatment) and because 100 cells were viewed in each well, the average number of cells with neurites in each well represents the percentage of cells with neurites.

RESULTS

Design and Synthesis of the Laminin ␣5-Chain G Domain Peptides We prepared 113 peptides from the laminin ␣5-chain G domain for screening of cell binding sequences (Fig. 1). For the screening, peptides were generally 12 amino acids in length and overlapped with neighboring peptides by four amino acids. If the N-terminal amino acid was either glutamate or glutamic acid, one amino acid was extended at the N-terminus to avoid pyroglutamine formation [36]. Cysteine residues were omitted to avoid the influence of disulfide bonds. These peptides were dissolved in Milli-Q water except for A5G46, -52, -55, -75, -83, and -104, which were insoluble. Cell Attachment and Neurite Outgrowth Activities on Laminin ␣5-Chain Peptide-Coated Plastic Plates HT-1080 human fibrosarcoma cell attachment to the 107 soluble laminin ␣5-chain G domain peptides was evaluated using peptide-coated plastic plates (Fig. 1). AG-73 (RKRLQVQLSIRT), which is the strongest cell attachment peptide from the laminin ␣1-chain G domain [7], was used as a positive control. In the first stage of screening, 16 peptides showed cell attachment activity. A5G-27 (RLVSYNGIIFFLK), A5G-33 (ASKAIQVFLLAG), A5G-65 (HQNMGSVNVSVG), A5G-71 (GPLPSYLQFVGI), A5G-77 (LVLFLNHGHFVA), A5G82 (VRWGMQQIQLVV), and A5G-109 (APVNVTASVQIQ) showed strong cell attachment activity similar to that of AG-73. These seven peptides showed similar dose-dependent curves to that of AG-73 (Fig. 2A). Three peptides (A5G-3, -19, and -81) also showed moderate cell attachment activity in a dose-dependent manner, but their activity was weaker than that of AG-73 (Fig. 2B). Six peptides (A5G-26, -35, -64, -73, -99, and -101) showed weak cell attachment activity (Fig. 2B).

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TABLE 1 Synthetic Laminin ␣5-Chain G Domain Peptides and Their Biological Activities HT-1080 cell attachment a

PC12 Cell

Peptide

Sequence

Coated plate

Sepharose beads

Neurite outgrowth b

A5G-3 A5G-19 A5G-26 A5G-27 A5G-33 A5G-35 A5G-36 A5G-63 A5G-64 A5G-65 A5G-67 A5G-71 A5G-73 A5G-76 A5G-77 A5G-81 A5G-82 A5G-99 A5G-101 A5G-109 A5G-112 AG-73

GKNTGDHFVLYM VVSLYNFEQTFML RFDQELRLVSYN RLVSYNGIIFFLK ASKAIQVFLLAG VLVRVERATVFS TVFSVDQDNMLE RLRGPQRVFDLH FDLHQNMGSVN HQNMGSVNVSVG SRATAQKVSRRS GPLPSYLQFVGI RNRLHLSMLVRP APMSGRSPSLVLK LVLFLNHGHFVA AGQWHRVSVRWG VRWGMQQIQLVV VLLQANDGAGEF DGRWHRVAVIMG APVNVTASVQIQ KQGKALTQRHAK RKRLQVQLSIRT

⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫺ ⫺ ⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹ ⫺ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹

⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫺ ⫹ ⫹ ⫺ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫺ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫺ ⫹⫹ ⫹ ⫹⫹⫹

⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹

a For cell attachment assays, various amounts of peptides were coated on 96-well plate as described under Materials and Methods. In all cases, the biological activities of the peptides were quantitated and evaluated relative to those observed with AG-73 as shown in Fig. 2. Cell attachment was evaluated on the following subjective scale: ⫹⫹⫹, adhesion comparable to those on AG73; ⫹⫹, weak adhesion compared with that on AG73; ⫹, very weak adhesion compared with that on AG73; ⫺, no adhesion. Triplicate experiments gave similar results. b For neutite outgrowth assays, various amounts of peptides were coated on the wells as described under Materials and Methods. Cell attachment is represented as follows: ⫹, promote neurite outgrowth; ⫺, inactive.

With PC12 cells, A5G-71, A5G-77, and A5G-88 showed the strongest attachment activity (data not shown). A5G-3, A5G-26, A5G-27, A5G-33, and A5G-65 also showed strong PC12 cell attachment activity, while A5G-28, A5G-35, A5G-64, A5G-73, and A5G-82 showed moderate activity. A5G-75 and A5G-101 showed relatively weak activity. With regard to neurite outgrowth, A5G-3, A5G-33, A5G-65, A5G-71, A5G-73, A5G-81, and A5G-101 had the activity (Fig. 3). Cell Attachment Assay Using Peptide-Conjugated Sepharose Beads Next, we evaluated cell adhesion to covalently conjugated peptides on Sepharose beads. One hundred seven soluble peptides were coupled with CNBr-acti-

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FIG. 4. Adhesion of HT-1080 cells on peptide-Sepharose beads. HT-1080 cells were allowed to attach to peptide-Sepharose beads for 1 h and were stained with crystal violet. (A) A5G-27, (B) A5G-33, (C) A5G-65, (D) A5G-71, (E) AG-73, (F) A5G-42.

vated Sepharose beads. As a positive control, AG-73, which previously showed cell attachment and spreading on Sepharose beads [34], was used. Cell attachment activities on the peptide-Sepharose beads were tested using HT-1080 cells. Fourteen peptides showed cell attachment (Table 1). Three of the peptide-Sepharose beads (A5G-27, -65, and -71) showed strong cell attachment and spreading activity similar to that observed with the AG-73-conjugated Sepharose beads (Fig. 4). Four peptides (A5G-33, -77, -82, and -109) showed moderate cell attachment and spreading activity, but their activity was weaker than that of AG-73. Seven peptides (A5G-36, -63, -67, -76, -81, -99, and -112) were found to be weak in cell attachment and spreading activity. Cell attachment activity of six insoluble peptides (A5G-46, -52, -55, -75, -83, and -104) was tested using peptide-conjugated polystyrene beads

as described previously [7]. These six peptides were not active in the assay (data not shown). Effect of Peptides on Cell Attachment to Laminin-10/-11 and Recombinant Protein Eight peptides (A5G-27, -33, -65, -71, -77, -81, -82, and -109) showed strong cell attachment activity in the both plate and bead assays (Table 1). We evaluated the ability of these attachment positive peptides for their ability to inhibit cell attachment to laminin-10/-11 and recombinant ␣5LG4-5. A5G-81 and A5G-109 slightly inhibited HT-1080 cell attachment to laminin-10/-11, and only A5G-81 significantly inhibited HT-1080 cell attachment to the LG4-5 protein (Figs. 5A and 5B). These results suggest that an active site for A5G-81 is available on laminin ␣5-chain LG4-5 modules.

LAMININ ␣5-CHAIN G DOMAIN CELL BINDING SITES

FIG. 5. Effect of peptides on cell attachment to laminin-10/-11 and recombinant ␣5-LG4-5. Graph showing the percentage of HT1080 cells that attach to laminin-10/-11 (0.1 ␮g/well) (A) and recombinant ␣5LG4-5 (1 ␮g/well) (B) in the presence of 0.1 mg/ml peptides. Laminin-10/11 and recombinant ␣5LG4-5 attachment levels were normalized at 100%. Data are expressed as mean of triplicate results. Triplicate experiments gave similar results.

Effects of Heparin and EDTA on the Cell Attachment Next, the effects of heparin on HT-1080 cell attachment were evaluated by 16 active peptides which showed cell attachment activity in the plate assays. AG-73 was used as a positive control. Cell attachment to AG-73 was inhibited by heparin as shown previously [9]. Cell attachment to four peptides (A5G-19, -35, -77, and -101) was slightly inhibited by heparin, whereas attachment to the remaining 12 peptides were significantly inhibited (Fig. 6). The effects of EDTA on HT1080 cell attachment were also evaluated by the 16 active peptides. However, EDTA did not inhibit cell attachment to all 16 peptides (Fig. 6). Active Core Sequences of A5G-27, A5G-65, and A5G-71 We next determined the active core sequence of the three strongest active peptides, A5G-27, A5G-65, and A5G-71. The structural requirements for biological activity of these peptides were determined using systematically truncated N-terminal and C-terminal peptides

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(Table 2). A5G-27d (YNGIIFFLK), an N-terminal truncated peptide, still retained full activity, whereas a deletion of tyrosine from A5G-27d (A5G-27e) resulted in decreased activity in the plate assays. A C-terminal deletion peptide A5G-27g (RLVSYNGIIFFL) showed cell attachment activity but weaker than that of A5G27. Further deletion of leucine from A5G-27h (RLVSYNGIIFF) eliminated its cell binding activity. These results indicate that the six-amino-acid sequence GIIFFL is critical for activity in A5G-27 in the plate assays. A5G-27d (YNGIIFFLK), an N-terminal truncated peptide, still retained full activity, whereas a deletion of tyrosine from A5G-27e (NGIIFFLK) eliminated its cell binding activity in the peptide-conjugated Sepharose bead assay. These results indicate that the eight-amino-acid sequence YNGIIFFL is critical for activity of A5G-27 in the Sepharose bead assay. Next, a deletion of histidine and glutamine from A5G-65a (NMGSVNVSVG) had no cell binding activity in either assay. A C-terminal deletion peptide A5G-65e (HQNMGSVNVSV) eliminated its cell binding activity in both assays. These results indicate that the 11amino-acid sequence HQNMGSVNVSV is critical for activity of A5G-65 in the Sepharose bead assay. A5G-71d (SYLQFVGI), an N-terminal truncated peptide, still retained full activity, whereas a deletion of serine from A5G-71e (YLQFVGI) resulted in decreased activity in both assays. Furthermore, a deletion of tyrosine from A5G-71f (LQFVGI) eliminated its cell binding activity in both assays. C-terminal deletion peptide A5G-71g (GPLPSYLQFVGI) showed weak cell attachment activity in the peptide-coated plate assay but showed no cell attachment activity in the Sepharose bead assay. A deletion of glycine from A5G-71h (GPLPSYLQFVG) eliminated its cell binding activity in the peptide-coated plate assay. These results indicate that the six-amino-acid sequence YLQFVG is critical for activity in the plated-coated assay and the seven-amino-acid sequence YLQFVGI is critical for activity in the Sepharose bead assay. DISCUSSION

Previously, we identified several biologically active sites on laminin-1 by screening the activities of laminin-derived synthetic peptides [7–10]. The solid-phase peptide synthesis methodologies have recently undergone remarkable development, with effective deprotecting and new coupling reagents suitable for shortened synthesis times [37, 38]. In earlier studies, our attempt to utilize these methodologies to identify cell binding sites provided several meaningful results. First, synthetic peptides can be used to develop synthetic peptide libraries with varying cell adhesion assays, such as peptide-coated plates and peptide-conjugated Sepharose beads. Second, synthetic peptides can

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FIG. 6. Inhibition of HT-1080 cell attachment to peptides by heparin and EDTA. HT-1080 cells were allowed to attach to peptide-coated plates in the absence (white bars) or presence (striped bars) of 5 mM EDTA or presence (black bars) of 10 ␮g/ml heparin. Round-bottom 96-well plates were coated with AG-73 (0.5 ␮g/well), A5G-27, A5G-33, A5G-65, A5G-71, A5G-82, and A5G-109 (1 ␮g/well), A5G-3, A5G-19, A5G-26, A5G-64, A5G-77, A5G-81, A5G-99, and A5G-101 (12.5 ␮g/well), or A5G-35 and A5G-73 (50 ␮g/well). For the EDTA inhibition assay, 5 mM EDTA was added to cell suspensions and then cells were plated. Heparin (10 ␮g/ml) was added to the plates before plating the cells. After a 30-min incubation, attached cells were assessed by crystal violet staining. Each value represents the mean of three separate determinations ⫾ SD. Duplicate experiments gave similar results.

be used to identify new small to moderately sized bioactive peptide sequences derived from the larger recombinant laminin molecule. Third, synthetic peptides can be used to identify new ligands and delineate minimum active core sequences with alanine substituted or N- and C-truncated peptides. Overall, synthetic peptides can be used to better understand the biological role and activities of whole molecules during normal development and in disease processes. Moreover, the use of synthetic peptides may lead to the development of new therapeutic reagents for treatment of various diseases and for tissue repair. In this study, we have employed systematic peptide screening to identify cell binding sequences and neurite outgrowth-promoting sequences from the laminin ␣5-chain G domain using 113 synthetic peptides. We identified several cell attachment laminin sequences using peptide-coated plates and peptide-conjugated Sepharose beads. In the first screening, 16 and 14 of the peptides showed cell attachment activities on the peptide-coated plates and the peptide-conjugated Sepharose beads, respectively. Nine of the peptides showed cell attachment activities on both assays. The

different results obtained from the two assays may be explained by inactivation of the peptides by aggregation on the tissue culture plate, while on the Sepharose beads the peptides are flexible and able to form active conformations [39]. The combination of both assays is quite useful for identification of biologically active peptides. In the inhibition assays, cell attachment to 12 of the 16 peptides was significantly inhibited by heparin, suggesting that these 12 peptides bind to heparin. Furthermore, EDTA did not inhibit cell attachment to any of the 16 peptides, suggesting that cell attachment of these peptides might not be mediated by integrins. Using this combination of adhesion assays, three peptides (A5G-27, A5G-65, and A5G-71) strongly promoted cell attachment. The active core sequence of A5G-27 was GIIFFL in the peptide-coated plastic plate assay and YNGIFFL in the peptide-conjugated Sepharose bead assay. The active core sequence of A5G-65 was HQNMGSYNVSV in both assays. The active core sequence of A5G-71 was YLQFVG in the peptide-coated plastic plate assay and YLQFVGI in the peptide-conjugated Sepharose bead assay. Recently, the crystal structure of the LG5 module of

LAMININ ␣5-CHAIN G DOMAIN CELL BINDING SITES

TABLE 2 Cell Attachment Activity of N- and C-Terminal Truncated Peptides of A5G-27, A5G-65, and A5G-71 HT-1080 cell attachment activity b

Peptides

Sequence a

Coated plate

Sepharose beads

A5G-27 A5G-27 A5G-27a A5G-27b A5G-27c A5G-27d A5G-27e A5G-27f A5G-27g A5G-27h A5G-27i A5G-27j

RLVSYNGIIFFLK LVSYNGIIFFLK VSYNGIIFFLK SYNGIIFFLK YNGIIFFLK NGIIFFLK GIIFFLK RLVSYNGIIFFL RLVSYNGIIFF RLVSYNGIIF RLVSYNGII

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺

A5G-65 A5G-65 A5G-65a A5G-65b A5G-65c A5G-65d A5G-65e A5G-65f A5G-65g A5G-65h

HQNMGSVNVSVG NMGSVNVSVG MGSVNVSVG GSVNVSVG SVNVSVG HQNMGSVNVSV HQNMGSVNVS HQNMGSVNV HQNMGSVN

⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺

⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫺

A5G-71 A5G-71 A5G-71a A5G-71b A5G-71c A5G-71d A5G-71e A5G-71f A5G-71g A5G-71h A5G-71i A5G-71j

GPLPSYLQFVGI PLPSYLQFVGI LPSYLQFVGI PSYLQFVGI SYLQFVGI YLQFVGI LQFVGI GPLPSYLQFVG GPLPSYLQFV GPLPSYLQF GPLPSYLQ

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺

a Sequences of the synthetic peptides are given in the single-letter code. b Activity was scored on the following subjective scale: ⫹⫹⫹, activity comparable to that of original peptides; ⫹⫹, activity apparent but weaker than that of original peptides; ⫹, activity apparent but much weaker than that of original peptides; ⫺, no activity. Active core sequences are written in bold. All peptides have C-terminal amides.

the laminin ␣2-chain revealed a 14-stranded ␤-sandwich [40]. The Lys and Arg residues were suggested to be essential for heparin binding in the LG4-5 modules of the laminin-␣1 chain [41]. Further, several heparin binding sites in the LG4 modules of the ␣3-, ␣4-, and ␣5-chains were identified using recombinant proteins and synthetic peptides [31, 42, 43]. Basic amino acids were found to be critical for heparin binding and/or cell

103

attachment [31, 42, 43], and the aromatic amino acids Tyr and His were also found to be important for biological activity [31, 42]. All the active peptides newly identified in this study contained either basic amino acids (Arg and Lys) and/or aromatic amino acids (Tyr, Phe, and His) except for A5G-109 (Table 1). The basic and aromatic amino acids may be involved in critical roles for the biological functions of the 20 peptides. Previously, we demonstrated that an active peptide (F4: AGQWHRVSVRWG, here A5G-81) in the LG4 module of laminin-␣5 chain was a major heparin binding site using recombinant proteins and synthetic peptides [31]. It was suggested that the LG modules of the laminin ␣5-chain do not bind to ␣-dystroglycan in the absence of calcium ions [40]. In recent investigations the laminin G domains have been shown to interact with syndecans, a transmembrane proteoglycan [11, 43, 44]. Our present study also suggests that ␣5-chain G domain active peptides may bind to syndecans. A comparison of the most active mouse sequences with the human ␣5-chain showed strong sequence identity and homology (Fig. 7), while, A5G-109 showed less homology, possessing nine nonconserved amino acids. Inhibition of cell attachment to a laminin ␣5LG4-5 substrate by A5G-81 suggests that this site is active and available on the intact molecule. These results confirm both the importance of tertiary protein conformation and linear active sites in cell-substrate interactions. Proteolytic fragments of some proteins have been found to possess activities that are not exhibited in the intact molecules. Fragments of laminin-5 generated by MMP-9 were found to induce cell migration and tumor metastases [45]. Also, the antiangiogenesis factors angiostatin and endostatin are fragments of plasminogen activator and collagen, respectively [46, 47]. The laminin-␣5 chain active peptides, insofar as they represent primary amino acid laminin sequence fragments, may exhibit activities associated with proteolytic laminin fragments but not the intact laminin molecule. With regard to neurite outgrowth, the ability for native laminin-5 to promote neurite outgrowth was demonstrated in SY5Y neuroblastoma cells [48] in central and peripheral chick embryonic neurons [49]. Laminin-11 has also been shown to promote neurite initiation in motor neurons [50], however, in these studies, laminin-11 was not as potent a stimulator of neurite initiation as laminin-1 and indeed previous studies have shown that laminin-11 serves as a stop signal for neurites extending on laminin-1 [51]. While the ability for laminin-5 and laminin-11 to both initiate and stop neurite outgrowth was not localized to a specific domain, it is clear that the initiation of neurite outgrowth using the synthetic peptide in our study here reflects the ability of laminin-5 and laminin-11 to initiate and modulate neurite outgrowth.

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FIG. 7. Homology between active peptides from the mouse laminin ␣5-chain and the human ␣5-chain. Amino acid sequences of the nine active peptides (A5G-27, -33, -65, -71, -77, -81, -82, and -109) from the mouse ␣5-chain were compared with corresponding sequences of human ␣5-chain [51].

In summary, we present data which show that several peptides had cell attachment activity and the ability to promote neurite outgrowth. Interestingly, when we searched for sequence homologies in the core sequence (YLQFVG) of A5G-71, homologies were found with zinc protease in Treponema pallidum and proteins in Caenorhabditis elegans. While the significance of these homologies is unclear, screening laminin peptides for biological activities may help focus investigation into the basic biological mechanisms of laminin activity during development and may also contribute to the search for therapeutic agents capable of reversing or healing the effects of degenerative diseases.

8.

Nomizu, M., Kuratomi, Y., Song, S. Y., Ponce, L. M., Hoffman, M. P., Powell, S. K., Miyoshi, K., Otaka, A., Kleinman, H. K., and Yamada, Y. (1997). Identification of cell binding sequences in mouse laminin ␥1 chain by a systematic peptide screening. J. Biol. Chem. 272, 32198 –32205.

9.

Nomizu, M., Kuratomi, Y., Malinda, K. M., Song, S. Y., Miyoshi, K., Otaka, A., Powel, S. K., Hoffman M. P., Kleinman, H. K., and Yamada, Y. (1998). Cell binding sequences in mouse laminin ␣1 chain. J. Biol. Chem. 273, 32491–32499.

10.

Nomizu, M., Kuratomi, Y., Ponce, M. L., Song, S. Y., Miyoshi, K., Otaka, A., Powel, S. K., Hoffman, M. P., Kleinman, H. K., and Yamada, Y. (2000). Cell binding sequences in mouse laminin ␤1 chain by a systematic peptide screening. Arch. Biochem. Biophys. 378, 311–320.

11.

Hoffman, M. P., Nomizu, M., Roque, E., Amichay, N., Lee, S., Yamada, Y., and Kleinman, H. K. (1998). Laminin-1 and laminin-2 G domain synthetic peptides bind syndecan-1 and promote acinar-like development of a human submandibular gland (HSG) cell line. J. Biol. Chem. 273, 28633–28641.

12.

Richard, B. L., Nomizu, M., Yamada, Y., and Kleinman, H. K. (1996). Identification of synthetic peptides derived from laminin alpha 1 and alpha 2 (Merosin) chains with cell type specificity for neurite outgrowth. Exp. Cell Res. 228, 98 –105.

13.

Song, S. Y., Nomizu, M., Yamada, Y., and Kleinman, H. K. (1997). Liver metastasis formation by laminin-1 peptide (LQVQLSIR)-selected B16-F10 melanoma cells. Int. J. Cancer 71, 436 – 441.

14.

Kim, W. H., Nomizu, M., Song, S. Y., Tanaka, K., Kuratomi, Y., Kleinman, H. K., and Yamada, Y. (1998). Laminin ␣1 chain sequence Leu-Gln-Val-Gln-Leu-Ser-Ile-Arg (LQVQLSIR) enhances murine melanoma cell metastasis. Int. J. Cancer 77, 632– 639.

15.

Weeks, B. S., Nomizu, M., Ramchandran, R. S., Yamada, Y., and Kleinman, H. K. (1998). Laminin and the RKRLQVQLSIRT laminin alpha globular domain stimulate matrix metalloproteinase secretion in PC12 cells. Exp. Cell Res. 243, 375– 382.

16.

Powell, S. K., Williams, C. C., Nomizu, M., Yamada, Y., and Kleinman, H. K. (1998). Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro. J. Neurosci. Res. 54, 233–247.

17.

Kuratomi, Y., Nomizu, M., Nielsen, P., Tanaka, K., Song, S. Y., Kleinman, H. K., and Yamada, Y. (1999). Identification of metastasis-promoting sequences in the mouse laminin ␥1 chain. Exp. Cell Res. 249, 386 –395.

18.

Malinda, K. M., Nomizu, M., Chung, M., Delgado, M., Kuratomi, Y., Yamada, Y., Kleinman, H. K., and Ponce, M. L. (1999). Identification of laminin alpha1 and beta1 chain pep-

This work was supported in part by the Grants-in-Aid for Scientific Research from Ministry of Education, Science, Sports, and Culture of Japan (Nos. 11470480, 11139201, and 12215002).

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