Cell Type-specific Differences in Glycosaminoglycans Modulate the Biological Activity of a Heparin-binding Peptide (RKRLQVQLSIRT) from the G Domain of the Laminin alpha 1 Chain*

Matthew P. HoffmanDagger §, Jean A. EngbringDagger , Peter K. NielsenDagger , John VargasDagger , Zachary SteinbergDagger , Arezo J. KarmandDagger , Motoyoshi Nomizu, Yoshihiko YamadaDagger , and Hynda K. KleinmanDagger

From the Dagger  Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370 and the  Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan

Received for publication, January 26, 2001, and in revised form, March 28, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin alpha 1 chain, has diverse biological activities with different cell types. The heparan sulfate side chains of syndecan-1 on human salivary gland cells were previously identified as the cell surface ligand for AG73. We used homologous peptides from the other laminin alpha -chains (A2G73-A5G73) to determine whether the bioactivity of the AG73 sequence is conserved. Human salivary gland cells and a mouse melanoma cell line (B16F10) both bind to the peptides, but cell attachment was inhibited by glycosaminoglycans, modified heparin, and sized heparin fragments in a cell type-specific manner. In other assays, AG73, but not the homologous peptides, inhibited branching morphogenesis of salivary glands and B16F10 network formation on Matrigel. We identified residues critical for AG73 bioactivity using peptides with amino acid substitutions and truncations. Fewer residues were critical for inhibiting branching morphogenesis (XKXLXVXXXIRT) than those required to inhibit B16F10 network formation on Matrigel (N-terminal XXRLQVQLSIRT). In addition, surface plasmon resonance analysis identified the C-terminal IRT of the sequence to be important for heparin binding. Structure-based sequence alignment predicts AG73 in a beta -sheet with the N-terminal K (Lys2) and the C-terminal R (Arg10) on the surface of the G domain. In conclusion, we have determined that differences in cell surface glycosaminoglycans and differences in the amino acids in AG73 recognized by cells modulate the biological activity of the peptide and provide a mechanism to explain its cell-specific activities.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Heparan sulfate proteoglycans (HSPGs)1 are abundant in both the extracellular matrix and on cell surfaces. The diverse biological activities of HSPGs largely depend on interactions between specific oligosaccharide sequences of the glycosaminoglycan (GAG) side chains and protein sequences. Modifications of the GAG side chains provide structural heterogeneity and a basis for the exquisite specificity of its interactions (1-4). Heparin is used to study GAG interactions with cells and is the most negatively charged GAG, whereas heparan sulfate and chondroitin sulfates are the biological GAG ligands present on epithelial cell surfaces. The sulfation patterns of GAGs vary in different sites, cells, and tissues and at various times in development, allowing for protein- and cell-specific interactions (5-7). For example, extensive studies on the role of heparin and fibroblast growth factor receptor function have revealed important size and sulfation requirements of heparin for different tissue-specific FGF-mediated biological activities (8-10).

Laminins are a family of heparin-binding, biologically active glycoproteins found in basement membranes. Currently, there are five alpha , three beta , and three gamma  chains, which form 15 different heterotrimers (11, 12). Laminin isoforms are present at different times during development with tissue-specific expression patterns. Cell type-specific interactions between laminin isoforms and multiple integrins and heparan sulfate-containing receptors provide mechanisms for regulating the broad range of biological activities of laminins (13). Several heparin-binding sites have been identified on laminin chains and interactions with the heparan sulfate proteoglycans, including dystroglycan, syndecans, agrin, and perlecan, have been demonstrated (14, 15). The elastase fragment of the laminin alpha 1 chain, E3, which contains the C-terminal LG4 and LG5 modules, is an important heparin-binding site (16, 17). Recent studies have identified other heparin-binding sites in the homologous LG4-5 regions of laminin alpha 1 (14, 18), alpha 2 (14), alpha 3 (19), alpha 4 (20, 21), and alpha 5 (22, 23). Different heparin-binding sequences have been identified depending on the laminin isoform and the purified ligand, cell, or tissue type tested.

We have identified a biologically active sequence, AG73 (RKRLQVQLSIRT), from the laminin alpha 1 G4 module using a synthetic peptide approach (24). AG73 promotes attachment of multiple cell types (24, 25), induces salivary acinar cell differentiation (15), inhibits branching morphogenesis of embryonic salivary glands (26), stimulates neurite outgrowth (27), stimulates matrix metalloproteinase secretion by PC12 cells (28), and promotes liver metastasis by melanoma cells (29). AG73 inhibits human submandibular gland (HSG) and B16F10 melanoma cell (29) attachment to the E3 fragment of laminin-1. The receptor on HSG cells for AG73 was identified as syndecan-1, and the interaction occurs via the heparan sulfate side chains (15). Recent data from our laboratory with B16F10 cells suggest a similar AG73 receptor, with related but different GAG side chains.2

Here, we compare the activity of AG73 with the homologous sequences in the other laminin alpha -chains (A2G73-A5G73, Table I) to determine whether the active sequence is specific to the alpha 1 chain. We show that cell type-specific interactions with the peptides involve different GAGs. We identify the amino acids in AG73 that mediate its specific biological activity in different assays. Our data indicate that cell-specific activities of AG73 are mediated by interactions with cell surface GAGs and that specific amino acids in AG73 modulate different biological activities in a cell-specific manner.

                              
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Table I
Summary table of results
Identical (medium shading) and conserved (light shading) residues in the AG73 homologs from the other laminin alpha -chains are highlighted. The other amino acid substitutions and truncations (light shading) are also highlighted.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Cell Culture-- The HSG cell line (30) was cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12), containing 5% fetal bovine serum (Biofluids, Rockville, MD), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). B16F10, a mouse melanoma cell line (31), was cultured in DMEM containing minimal essential medium with nonessential amino acids (Life Technologies, Inc.), 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum. Both cell types were maintained at 37 °C in a humidified, 5% CO2, 95% air atmosphere.

Preparation of Peptides and Beads-- All peptides were manually synthesized using the 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase strategy with a C-terminal amide form and purified by reverse phase high performance liquid chromatography as described previously (32). The peptide-resin beads were also synthesized as described previously (25).

Cell Attachment Assay with Peptide Beads-- Cell attachment to peptide beads was assayed in 48-well dishes. The dishes and peptide beads were blocked with 3% BSA in DMEM/F-12 for 1 h at 37 °C. After washing with 0.1% BSA in DMEM/F-12, 3.0 × 104 cells were added and incubated for 2 h at 37 °C. The beads were washed, stained with DiffQuik (Baxter, Miami, FL), and photographed.

Cell Attachment Assays-- Peptides were coated onto round-bottomed 96-well plates (Immulon 2B, Dynex Technologies, Chantilly, VA) by either drying overnight in 50 µl of distilled H2O or by incubating in 50 µl of PBS for 2 h at 37 °C. The wells were then blocked with 3% BSA in DMEM/F-12 for 1 h at 37 °C and then washed twice with 0.1% BSA in DMEM/F-12. 3.5 × 104 HSG cells in 100 µl of 0.1% BSA in DMEM/F-12 or 3.0 × 104 B16F10 cells in 50 µl of 0.1% BSA in DMEM were added per well for 30 min at 37 °C. The medium was gently removed from the wells, and the cells were stained with crystal violet for 10 min. After washing twice with water, the cells were lysed with 50 µl of 10% SDS and the optical density (600 nm) measured.

Biotinylated Heparin Binding Assay-- Peptide-coated plates, prepared as above, were blocked with 3% BSA in PBS for 30 min at 37 °C and washed with 0.1% BSA in PBS. Biotinylated, sized heparin (Heparin-BH with an average mass of 12.5 kDa, Celsus Laboratories, Inc., Cincinnati, OH), 20 ng/well in 0.1% BSA in PBS, was added to the wells and incubated for 30 min at 37 °C. The heparin was gently removed, the plate was washed twice with 0.1% BSA in PBS, and the bound biotinylated heparin was detected with streptavidin-alkaline phosphatase (Pierce). After incubation with the enzyme substrate, the optical density was measured at 405 nm. Initial dose-response experiments with biotinylated heparin binding to a fixed amount of peptide determined that 20 ng/well resulted in 50% of the maximal binding detected with streptavidin-alkaline phosphatase.

Inhibition of Cell Attachment-- GAGs, modified heparin, and heparin fragments were added to the cell attachment assay. GAGs (5 µg/ml) were incubated with the peptide-coated wells for 15 min, and then the cells were added. Heparin, heparan sulfate, chondroitin sulfates A, B, and C, keratan sulfate, hyaluronic acid (Sigma), de-N-sulfated-N-acetylated heparin (DNSNAc), completely desulfated-N-acetylated heparin (CDSNAc), and completely desulfated-N-sulfated heparin (CDSNS) (Seikagaku, Rockville, MD) were used. Sized heparin fragments (a gift from Dr. A. Marolewski, RepliGen Corp., Needham, MA) were prepared by alkaline depolymerization of heparin with an average mass of 5 kDa, (Enzyme Research Laboratories, South Bend, IN) that was fractionated on a Sephadex G50 column using ammonium bicarbonate buffer (33). Fractions were dried and weighed. Size was determined by gradient electrophoresis with comparison to known standards that had been prepared by capillary electrophoresis (34).

Inhibition of cell attachment by competition with peptides was used to determine IC50 values of competitor peptides. Decreasing amounts of substrate peptides AG73-A5G73 were used to determine the amount of peptide coating resulting in half-maximal cell attachment. The cells were preincubated in solution with different amounts of the competitor peptide for 10 min at 37 °C. Then the cells and competitor peptide were added to the wells, and cell attachment was quantitated as above. HSG and B16F10 cell attachment to AG73 was also competed with a series of peptides containing amino acid substitutions and truncations. The amount of peptide used to compete was the same as the amount of AG73 used to compete itself.

B16F10 Network Formation on Matrigel-- B16F10 cells (1.5 × 104 cells/well) were cultured in serum-free DMEM with 200 µg/ml peptides in 48-well flat-bottomed plates (Costar Corp., Cambridge, MA) coated with 133 µl of Matrigel (Becton Dickinson, Bedford, MA). After 18 h, the cells were fixed, stained with DiffQuik, and photographed.

Ex Vivo Salivary Gland Organ Culture-- Submandibular/sublingual salivary gland rudiments dissected from embryonic day 13 (E13) ICR mice were cultured on Whatman Nucleopore Track-etch filters (13 mm, 0.1-µm pore size, VWR, Buffalo Grove, IL) at the air/medium interface. The filters were floated on 240 µl of DMEM/F-12 in 50-mm glass-bottomed microwell dishes (MatTek, Ashland, MA). The medium was supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 150 µg/ml vitamin C, and 50 µg/ml transferrin. Six E13 gland rudiments were cultured on each filter at 37 °C in a humidified 5% CO2, 95% air atmosphere. Glands were photographed after ~1, 24, and 48 h, and the number of end buds was counted at each time point. Various peptide concentrations (26) were added to the medium at the beginning of the experiment.

Surface Plasmon Resonance-- Biotinylated, sized heparin (Heparin BH, Celsus Laboratories Inc., Cincinnati, OH) was prepared by oxidative cleavage with periodate, and the terminal aldehyde at the reducing end of the heparin was labeled with biotin hydrazide. Thus, the heparin would attach by its reducing end when immobilized to a streptavidin-coated sensor chip (Sensor Chip SA, BIAcore, Inc., Piscataway, NJ). Heparin attached to a sensor ship by the reducing end interacted more with protein than if attached at its midpoint (35). The heparin, at 40 µg/ml in 25 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.005% surfactant P20 was immobilized on the sensor chip at 10 µl/min for 4 min to an immobilization level of 300 resonance units. Peptides (20 µM in 25 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.005% surfactant P20) were injected on the heparin-coated surface at 30 µl/min in a BIA-coreTM 1000 instrument. The association rates (ka) and dissociation rates (kd) were registered (2 min each), and the KD was calculated from the equation KD= kd/ka. The streptavidin-heparin surface was regenerated between each run by two successive injections of 30 µl of 20 mM NaOH containing M NaCl. In control experiments, the peptides were run over a blank streptavidin chip. The sensorgrams were analyzed by non-linear least square curve fitting using BIAevaluation 2.1 software assuming single-site association and dissociation models.

Structural Alignment of the AG73 Peptide on the Crystal Structure of the alpha 2 LG5 Module-- AG73 was mapped on the crystal structure of the alpha 2 LG5 module (Protein Data Bank code 1qu0) using the RasMol program (36). The AG73 sequence aligns to Gly2955-Thr2966 of the alpha 2 sequence.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell Attachment to Laminin alpha -Chain Homologues of AG73-- Resin beads with peptides synthesized directly on the beads were initially used to compare HSG cell attachment to AG73 and to the homologous sequences from the other laminin alpha -chains. Cell attachment after 2 h to AG73, A2G73, and A3G73 was greater than to A4G73 and A5G73 (Fig. 1A). AG73T, a scrambled version of AG73, showed no cell attachment activity. Different amounts of peptides were then dried onto 96-well plates, and HSG and B16F10 cell attachment was compared. Both cell types bound to the AG73 homologues but not to AG73T. For both cell types, the relative amount of cell attachment activity of the peptides was similar: AG73 > A3G73 > A2G73 > A5G73 > A4G73 (Fig. 1, B and C). These data demonstrate that homologues of the laminin alpha 1 chain peptide AG73 are active for cell attachment, to varying degrees, when coated on culture plates.


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Fig. 1.   HSG and B16F10 cells adhere to AG73 and the homologous peptides from the laminin alpha -chains. A, cells were incubated with peptide beads for 2 h at 37 °C, and the beads were washed and stained with DiffQuik. There are similar levels of HSG cell attachment to AG73, A2G73, and A3G73 peptide beads and less cell attachment to beads with A4G73 and A5G73. Cells do not adhere to the scrambled peptide AG73T beads. B and C, cell adhesion assays are described under "Materials and Methods." Cells were incubated for 30 min at 37 °C in plates coated with increasing amounts of peptides. After washing, the cells were stained, lysed, and the A600 nm measured. HSG cells adhere to plates coated with AG73 > A3G73 > A2G73 > A4G73 > A5G73 > AG73T. B16F10 cell attachment to plates coated with increasing amounts of peptides. B16F10 cells adhere to plates coated with AG73 > A3G73 > A2G73 > A5G73 > A4G73 > AG73T. All experiments were repeated at least three times, graphs in B and C represent three wells/dose, and S.E. are indicated.

HSG and B16F10 Cell Attachment to AG73 Is Competed by AG73, A2G73, and A3G73-- The specificity of HSG and B16F10 cell attachment to AG73 was determined by competing attachment with the other homologous peptides. Increasing amounts of peptides in solution were added to both HSG and B16F10 cell attachment assays to AG73, and the IC50 for each peptide was determined (Table II). AG73 inhibited HSG and B16F10 cell attachment to AG73 with IC50 values of 15 and 27 µg/ml, respectively, and A2G73 inhibited HSG and B16F10 cell attachment to AG73 with IC50 values of 24 and 119 µg/ml, respectively. A3G73 inhibited HSG and B16F10 cell attachment with IC50 values of 13 and 277 µg/ml, respectively. However, A4G73 (IC50 > 400 µg/ml for both cell lines) could not inhibit HSG or B16F10 cell attachment to AG73, and A5G73 could not inhibit B16F10 cell attachment to AG73 but could compete HSG cell attachment with an IC50 of 241 µg/ml. These data show that HSG cell attachment to AG73 can be inhibited by AG73, A2G73, and A3G73 with similar IC50 values, suggesting they recognize a similar receptor. In contrast, B16F10 cell attachment to AG73 is more specific, in that A2G73, A3G73, A4G73, and A5G73 do not compete cell attachment with an IC50 similar to that for AG73 and may bind with lower affinity or recognize different cell surface receptors.

                              
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Table II
IC50 values of competitor peptides (µg/ml) during HSG and B16F10 cell adhesion to AG73

Biotinylated Heparin Binds to AG73, A2G73, and A3G73-- We used an enzyme-linked immunosorbent assay-type assay to investigate heparin binding to the peptides. Biotinylated, sized heparin (12.5 kDa) was incubated in peptide-coated 96-well plates and detected with streptavidin-horseradish peroxidase. The biotinylated heparin also inhibited HSG cell attachment to the peptides (data not shown). The biotinylated heparin bound in a saturable, dose-dependent manner to AG73, A2G73, and A3G73 with KD values (50% maximal binding) all in the nanogram range: 38 ng for A3G73, 107 ng for A2G73, and 121 ng for AG73 (Fig. 2). Biotinylated heparin bound to A4G73 and A5G73 with KD values in the microgram range, 4.4 µg for A4G73 and 21 µg for A5G73. AG73T showed some heparin binding when >30 µg of peptide/well were used, suggesting nonspecific trapping of heparin occurred at these doses. Thus, biotinylated heparin binding to the peptides follows a similar pattern of activity as the cell attachment to the peptides, in that AG73, A2G73, and A3G73 are more active than A4G73 and A5G73, although A3G73 binds to heparin with a higher affinity than AG73.


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Fig. 2.   Biotinylated heparin shows increased binding to wells coated with AG73, A2G73, and A3G73 compared with binding to wells coated with A4G73 and A5G73. Biotinylated heparin was incubated for 30 min at 37 °C in peptide-coated dishes and detected with streptavidin-horseradish peroxidase. Heparin binds to plates coated with A3G73 > A2G73 > AG73 > A4G73 > A5G73 > AG73T. The KD values (50% of maximal binding) for the peptides are listed. Triplicate wells were used for each condition, graphs are representative of at least three experiments, and S.E. are indicated.

Inhibition of Cell Attachment to the Peptides with GAGs and Modified Heparin-- Interestingly, different GAGs inhibited HSG and B16F10 cell attachment to the peptides, suggesting cell type-specific interactions with the peptides (Fig. 3). Various amounts of the GAGs were used to inhibit cell attachment to the peptides (data not shown). The concentration shown (5 µg/ml) highlights the differences (Fig. 3, A and B). HSG cell attachment to all five peptides was inhibited by heparin. Although cell attachment to A3G73 was only inhibited ~50% by the dose shown, it was completely inhibited at higher doses. HSG cell attachment to the peptides was inhibited by heparan sulfate, with the exception of attachment to A3G73, which was not inhibited even at higher doses. Chondroitin sulfate B inhibited HSG cell attachment to A4G73 and A5G73, and DNSNAc inhibited cell attachment to A5G73. Thus, cell attachment to A4G73 and A5G73 is inhibited by less sulfated GAGs. HSG cell attachment to the AG73 homologues was not inhibited by less sulfated GAGs, such as chondroitin sulfate-A and -C, keratan sulfate, CDSNAc, or CDSNS.


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Fig. 3.   Glycosaminoglycans and modified heparin inhibit HSG and B16F10 cell attachment to wells coated with the homologous peptides from the laminin alpha -chains in a cell type-specific manner. Cell adhesion assays are described under "Materials and Methods." Peptide-coated wells were incubated with GAGs (5 µg/ml) for 15 min, and then cells were added. A, inhibition of HSG cell attachment. B, inhibition of B16F10 cell attachment. Triplicate wells were used for each condition, graphs are representative of at least three experiments, and S.E. are indicated.

B16F10 cell attachment to the peptides was inhibited with different GAGs than HSG cells (Fig. 3B). Generally, B16F10 cell attachment was inhibited by less sulfated GAGs, including chondroitin sulfate B. Heparin and heparan sulfate inhibited B16F10 cell attachment to the AG73 homologues. Chondroitin sulfate B and DNSNAc heparin inhibited cell attachment to AG73, A4G73, and A5G73, but not to A2G73 and A3G73. Thus, a more sulfated region on the GAG mediates cell attachment to A2G73 and A3G73 and a less sulfated region not requiring an N-sulfate mediates cell attachment to AG73, A4G73, and A5G73. CDSNS heparin inhibited attachment to AG73, suggesting the interaction between B16F10 cells and AG73 requires only one sulfate/disaccharide. Chondroitin sulfates A and C and CDSNAc heparin did not inhibit cell attachment to any of the peptides. Taken together, these data indicate that different GAGs or similar GAGs with different patterns of sulfation potentially mediate the interactions between HSG and B16F10 cells with the peptides. A2G73 and A3G73 may bind to regions of higher sulfation than AG73, and A4G73 and A5G73 may bind to less sulfated regions or to chondroitin sulfate.

Inhibition of Cell Attachment with Sized Heparin Fragments-- The interaction of heparin with growth factors and their receptors is dependent on a critical size and sulfation pattern of the heparin chain (37). The inhibition of HSG cell attachment to the laminin peptides was also dependent on oligosaccharide size. Heparin oligosaccharides with a degree of polymerization of 10 saccharides (dp10) inhibited HSG cell attachment to AG73, A2G73, and A3G73 by ~50%, to A4G73 by ~90%, and to A5G73 by ~75% (Fig. 4A). Heparin oligosaccharides with dp12 inhibited HSG cell attachment to AG73, A2G73, and A3G73 by 60-70% and to A4G73 and A5G73 by 90%. HSG cell attachment to A4G73 was also inhibited ~85% by an oligosaccharide with dp8. The trend was similar for B16F10 cell attachment; heparin oligosaccharides with dp10 inhibited B16F10 cell attachment to AG73, A3G73, A4G73, and A5G73 by >50% but only inhibited cell attachment to A2G73 by ~25% (Fig. 4B). Interestingly, B16F10 cell attachment to A2G73 was not inhibited with dp20. Taken together with Fig. 3, the data further indicate that different types of cell surface GAGs may recognize the peptides with different sized areas of charge distribution or sulfation patterns.


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Fig. 4.   Sized heparin fragments inhibit HSG and B16F10 cell attachment in a cell type-specific manner. Cell adhesion assays are described under "Materials and Methods." Peptide-coated wells were incubated with sized heparin fragments (5 µg/ml) for 15 min, and then cells were added. A, inhibition of HSG cell attachment. B, inhibition of B16F10 cell attachment. Triplicate wells were used for each condition, graphs are representative of at least three experiments, and S.E. are indicated.

Branching Morphogenesis of Mouse Embryonic Day 13 (E13) Salivary Glands and B16F10 Network Formation on Matrigel Are Inhibited Only by AG73-- In cell attachment and heparin binding assays, AG73, A2G73, and A3G73 gave similar, although not identical results, suggesting they may bind a similar receptor. Therefore, we compared their biological activities in more complex assays. When E13 mouse embryonic salivary gland rudiments are cultured on filters, they undergo branching morphogenesis. When the homologous peptides from the other laminin alpha -chains were added to the assay, they had no inhibitory effect on branching (Fig. 5A). Therefore, AG73 activity is specific; even though the other peptides compete HSG cell attachment to AG73-coated wells in a more complex assay, they do not have the same activity as AG73.


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Fig. 5.   The biological activity of AG73 is specific compared with the other alpha -chain homologues. AG73 inhibits branching morphogenesis of embryonic day 13 submandibular gland culture and B16F10 melanoma cell network formation on Matrigel. A, salivary gland organ culture is described under "Materials and Methods." AG73 (200 µg/well) inhibits branching morphogenesis, whereas the homologous peptides were similar to the control, scrambled peptide, AG73T. Each assay contained at least five salivary glands, the assays were repeated at least three times, and a representative gland is shown. B, B16F10 melanoma cells cultured on Matrigel form networks within 18 h. When peptides are added to the media, only AG73 inhibits the network formation. Triplicate wells were used for each condition, the experiments were repeated at least three times, and a representative well is shown.

When B16F10 cells are cultured on basement membrane (Matrigel), they form networks within 18 h. These cell-cell and cell-matrix interactions are important in the invasive phenotype exhibited by malignant tumor cells on Matrigel (38). When the peptides were added to this assay, only AG73 inhibited network formation and the cells grew as a monolayer (Fig. 5B). Thus, AG73 was able to competitively inhibit specific cell-matrix interactions that the other peptides could not. Although the five homologous peptides are similar, based on the number of identical and conserved residues in their sequences (Table I), our data suggest that there are specific residues or motifs in AG73 that are responsible for its biological activity.

Amino Acid Substitutions and Truncations Identify Residues in AG73 Important for Its Biological Activity-- Peptides with a series of alanine substitutions, conserved and non-conserved substitutions in residues Ile10 and Arg11, and N-terminal truncations were synthesized and tested in various assays (Table I). Cell attachment assays to these peptides with both HSG and B16F10 cells showed similar results; no single amino acid substitution inhibited cell attachment. Truncations with less than 8 amino acids, AG73D and AG73G, however, did not support cell attachment.

The peptides with substitutions and truncations were tested in solution for their ability to compete HSG and B16F10 cell attachment to AG73. Alanine substitutions that resulted in the peptide with decreased ability to compete HSG cell attachment to AG73 were Arg3 right-arrow Ala, Leu4 right-arrow Ala, Val6 right-arrow Ala, Leu8 right-arrow Ala, Ile10 right-arrow Ala, and Thr12 right-arrow Ala. Truncated peptides that were unable to compete HSG cell attachment were AG73D and AG73G, which are both shorter than 8 amino acids, and AG73H, which was only missing the N-terminal Arg1 and the C-terminal Thr12.

Biotinylated heparin binding assays identified four peptides with alanine substitutions that resulted in decreased heparin binding: Leu4 right-arrow Ala, Val6 right-arrow Ala, Leu8 right-arrow Ala, and Ile10 right-arrow Ala (Table I). These substitutions resulted in lower heparin binding than AG73 but not as low as measured for A4G73 and A5G73. We compared the four residues in AG73 that are important for heparin binding to see if they are identical or conserved in A2G73, A3G73, A4G73, and A5G73 (Table I). In A2G73, which bound heparin similar to AG73 (Fig. 4), the two leucines (Leu4 and Leu8) are identical and the other two residues are conserved. Comparing A3G73, which had slightly better heparin binding (KD 38 ng), to AG73 (KD 121 ng), Leu4, Leu8, and Ile10 are identical and the other residue, Val6, is not conserved. However, when comparing these to A4G73, which had very low heparin binding, none of them are identical, two are conserved, and in A5G73, only one is identical and two conserved.

The series of peptides with substitutions and truncations were also tested for their ability to inhibit salivary gland branching morphogenesis to identify amino acids critical for AG73 function (Fig. 6, A and B). Alanine substitutions in AG73 at Leu4 right-arrow Ala, Val6 right-arrow Ala, and Ile10 right-arrow Ala showed no inhibition of branching. An alanine substitution at Thr12 right-arrow Ala, or a non-conserved substitution, Arg11 right-arrow Glu, resulted in peptides that only partly inhibited branching. Peptide truncations resulting in loss of AG73 activity were AG73B, a deletion of Lys2 from AG73A (AG73A still inhibited branching), and AG73H, which is a truncation of the C-terminal Thr12. These truncations were important to define the minimal sequence length that inhibits branching morphogenesis as AG73A (KRLQVQLSIRT). Taken together with the amino acid substitutions, the residues critical for function (i.e. inhibiting branching of salivary glands) are XKXLXVXXXIRT.


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Fig. 6.   The amino acids in the AG73 sequence responsible for the inhibition of branching morphogenesis were identified using amino acid substitutions and deletions. A, salivary gland organ culture is described under "Materials and Methods." After 48 h of culture, the end buds were counted. AG73 (200 µg/well) inhibited branching morphogenesis, and the other peptides were added at a similar dose. Five glands were used for each condition, the graph is representative of at least three similar experiments, and S.E. are indicated. B, representative example of two glands after 48 h of culture with Val6 right-arrow Ala and Gln7 right-arrow Ala. Hematoxylin/eosin-stained sections of the glands highlight the ducts and the terminal lobules that form the branching structure of the gland. C, the peptides were also used to inhibit B16F10 network formation on Matrigel. The data are presented in Table I, and two representative examples are shown after 18 h of culture with Arg1 right-arrow Ala and Leu4 right-arrow Ala.

The peptides with amino acid substitutions and truncations were also used to define the sequence that inhibited B16F10 cell networks on Matrigel. Alanine substitution Arg1 right-arrow Ala and Lys2 right-arrow Ala inhibited and Arg3 right-arrow Ala partially inhibited network formation (Table I and Fig. 6C). AG73A (Arg1 truncation) inhibited and AG73B (Arg1/Lys2 truncation) partly inhibited B16F10 network formation. Taken together these results suggest the N-terminal RK- residues are not required for AG73 function in this assay, but any other truncation or substitution results in loss of AG73 activity. Thus, RLQVQLSIRT is the minimal sequence required for AG73 to inhibit B16F10 cell network formation. It also suggests that HSG and B16F10 cell surface GAGs recognize different amino acids in the AG73 sequence.

Surface Plasmon Resonance Analysis of Peptide Binding to Biotinylated Heparin-- Biotinylated, sized heparin was immobilized on a streptavidin sensor chip, and the association rates (ka), dissociation rates (kd), and the dissociation constants (KD) of a series of peptides with alanine substitutions in the AG73 sequence (Table I) were measured in solution (Table III). There was an absence of detectable binding of Thr12 right-arrow Ala and the scrambled peptide AG73T. Thr12 right-arrow Ala and AG73T both lack the biological activity of AG73; they do not inhibit branching morphogenesis of submandibular glands, do not inhibit B16F10 network formation on Matrigel, and are unable to compete attachment of HSG cells to AG73 (Table I). Many substitutions altered the ka by a few fold, which in this type of assay is not considered significant, but two substitutions, Ile10 right-arrow Leu and Arg11 right-arrow Lys, decreased the ka by greater than 5-fold. Ile10 right-arrow Leu partly inhibited HSG cell attachment to AG73, and Arg11 right-arrow Lys partly inhibited branching morphogenesis of submandibular glands; however, both peptides were unable to inhibit B16F10 network formation on Matrigel. Peptide substitutions only affected the kd by a few fold, except for Arg11 right-arrow Lys (kd = 1.5 × 10-3 M-1 × s-1), which resulted in a 5-fold decrease in kd compared with AG73 (kd = 8.2 × 10-3 M-1 × s-1). Interestingly, the KD values of the peptide substitutions were all in the low micromolar range. However, Ile10 right-arrow Leu (KD = 79 µM) had a 12-fold decrease in affinity compared with AG73 (KD = 6.4 µM). Therefore, surface plasmon resonance analysis suggests that the C-terminal Ile10, Arg11, and Thr12 are important for the interaction of AG73 in solution with heparin and mediate part of the biological activity of AG73. Interestingly, these three residues are not identical in A2G73-A5G73 and only A2G73 and A3G73 have two identical residues (Table I), which may explain their inability to inhibit branching morphogenesis of submandibular glands and to inhibit B16F10 network formation on Matrigel.

                              
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Table III
Surface plasmon resonance analysis of AG73 and peptides with amino acid substitutions; binding to heparin immobilized on the sensor chip
The association rate (ka), dissociation rate (kd), and the dissociation constant (KD) are shown.

Molecular Modeling Indicates That AG73 Is Located in a beta -Sheet in the G4 Module of the Laminin alpha 1 Chain-- The crystal structure of the laminin alpha 2 LG5 module and the alpha 2 LG4-LG5 module pair has been determined (39, 40). Using the structure of the laminin alpha 2 LG5 module and structure-based sequence alignment, the LG4 of laminin alpha 1 can be modeled and the location of AG73 predicted (Fig. 7). Based on this prediction, three of the residues (Lys2, Arg11, and Thr12) important for branching morphogenesis are predicted to be on the surface of the structure at either end of a beta -sheet. The other amino acids, Leu4, Val6, and Ile10, are internal in the structure. Five amino acids required for inhibition of B16F10 cell networks (Arg3, Val5, Lys7, Arg11, and Thr12) were surface-exposed on the predicted model.


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Fig. 7.   Mapping of the AG73 peptide on the crystal structure of the alpha 2 LG5 module shows it is located in a beta -sheet. View of peptide AG73 (shaded gray) mapped on the crystal structure of the alpha 2 LG5 module (Protein Data Bank code 1qu0) (39) using the RasMol program (36). The AG73 sequence aligns to Gly2955-Thr2966 of the alpha 2 sequence. The first three residues (RKR) of the AG73 sequence are located spatially close to a calcium-binding site (shaded black) that has been implicated in alpha -dystroglycan binding to the laminin alpha 2 chain.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Laminin-1 contains multiple heparin-binding sites, including AG73, a peptide from the G4 module that binds the heparan sulfate side chains of syndecan-1 (15). Here, we have defined the functional interactions of this peptide and the homologous sequences from the other laminin alpha -chains. Our data demonstrate that the cellular responses to this sequence vary and are dependent on the cell type and assays used as well as on the environment of the cells. When the homologous peptides from the other laminin alpha -chains are coated on dishes, their biological activities are similar with both salivary gland and melanoma cells in cell attachment assays and in enzyme-linked immunosorbent assay-type heparin-binding assays. The highest heparin-binding peptide did not correspond to the peptide with highest cell attachment activity, indicating that something other than charge is involved (i.e. something specific about the AG73 sequence or something specific to the heparan sulfate on the cell surface). Furthermore, the biological activity of AG73 is specific; in assays where the peptide is in solution (i.e. inhibiting branching morphogenesis of submandibular glands and inhibiting B16F10 cell network formation on Matrigel), the homologous peptides from the other laminin alpha -chains have no activity. Although it may seem surprising that such a small peptide has such specificity, there is precedent in the literature. RGD-containing peptides have been widely used to investigate integrin-dependent cell adhesion and have exquisite biological specificity (41, 42).

Peptides generally do not have well defined secondary structure but are flexible and so assume random conformation in solution. We see differences in the biological activity of a particular peptide depending on what assay it is used in and whether it is bound to a plate or in solution. When the 12 peptides with a single alanine substitution are bound to plates, they all support HSG cell adhesion, but only 6 are able to compete HSG cell adhesion to AG73 when they are added in solution (Table I). These data could suggest that peptides bound to plastic have a fixed or distorted conformation and thus less selective activity, whereas in solution the peptides assume a conformation with a more selective biological activity that may mimic the site on laminin alpha 1 in vivo. This is supported by our recent mutational analysis of the AG73 sequence in recombinant LG4-5 of laminin alpha 1, which indicates specific residues are critical for specific biological activities.3

HSG and B16F10 cell attachment to the AG73 homologues was inhibited to various extents by different GAGs and by heparins with modified sulfate groups, which provides important information about the potential receptors for the peptides on the cell surface. Typically, in vitro studies use heparin to analyze the putative heparin-binding sites on laminins; however, the cell surface receptors are heparan sulfates and, as our data suggest, potentially chondroitin sulfates. The specificity of interactions with GAGs is not determined by total sulfation but by unique sulfation patterns on the GAGs. Our data suggest that HSG and B16F10 cell attachment is dependent on specific sulfation patterns or different cell surface GAGs (Fig. 2) and a specific size of GAG (Fig. 4). The sulfation patterns on different GAGs are known to be clustered in regions of higher and lower sulfation (6, 43). The cell surface ligand for AG73 on HSG cells was identified as the heparan sulfate side chains of syndecan-1, whereas the cell surface ligand for B16F10 cells may be both the heparan and chondroitin sulfate side chains.

GAGs do not act nonspecifically, but have unique functions in signaling pathways during cell differentiation and morphogenesis (1). The interaction of heparin with growth factors and their receptors is dependent on a critical size and sulfation pattern of the heparin chain, resulting in both binding and activation of the cell surface receptor (9). Recently, heparin hexasaccharides were reported to have dual roles of FGFR binding and dimerization, and 6-O-sulfation played a pivotal role in both processes (37). Others showed that heparin dodeca- (dp12) and deca- (dp10) saccharide fractions were mitogenic, and a clear correlation between total sulfate content and receptor activation activity was demonstrated (8, 44). The subtle differences in the HSG and B16F10 cell GAG composition may enable different biological responses to the laminin-derived peptides in a cell-dependent manner.

When the different AG73 homologues were tested for their activity in more complex in vitro assays, AG73 was active but the homologues displayed little activity (Fig. 5). Branching morphogenesis of submandibular glands was only inhibited by AG73 and not by the other homologues. The developing mouse submandibular gland basement membrane contains the laminin alpha 1, alpha 2, alpha 3, and alpha 5 chains (26, 45, 46). Our results suggest that the AG73 sequence in the laminin alpha 1 chain has a specific role in the morphogenesis of the gland. Likewise, melanoma cells plated on basement membrane Matrigel form a complex network, which was only blocked by AG73 and not by the homologues. Matrigel contains only laminin-1; therefore, the interaction between B16F10 cells and the AG73 sequence on the laminin alpha 1 chain is specific and is not disrupted with the homologous peptides. In these more complex assays, the peptides were tested in solution, so changes in conformation could explain differences in activity. Both assays demonstrate a high degree of specificity for the AG73 sequence. Therefore, we determined which amino acids were required for the activity of AG73 in different assays. These data confirm that functional differences in AG73 activity depend on the region of the peptide recognized by the particular cell type and are in accordance with previous studies with PC12 cells. Although the amino acids required for PC12 cell attachment and neurite outgrowth were different, the amino acids required for neurite outgrowth and matrix metalloproteinase secretion were identical (28).

An important paradigm has emerged in the FGF field for the role of GAGs in regulating tissue-specific functions of FGFs (2). The amino acid residues in the heparan sulfate-binding region of the FGFs are not completely conserved, suggesting that the affinity or specificity of different FGFs for their receptors depend on specific heparan sulfate sequences. The comparison of heparan sulfates from various tissues and cell types reveals differences in sulfation patterns (6). Tissue-specific differences in the structure of heparan sulfate modify the activity of FGFs. For example, tissue-specific heparan sulfate fragments differentially activate FGFs 1, 2, and 4 (47). The sulfation patterns of the heparan sulfates also regulate FGF activity, with 2-O-sulfation being required for FGF binding to the FGF receptor and 6-O-sulfation required for receptor activation. Therefore, tissue-specific patterns of O-sulfation and the local concentration of heparan sulfate regulate the activity and specificity of FGFs (2). This model may provide a framework to understand the function of the multiple heparin-binding sites that have been described in laminins, particularly in their G domains. Sites that are identified in vitro as heparin-binding, in fact, bind to heparan sulfate and potentially chondroitin sulfates in vivo. Laminin expression is developmentally regulated in tissue- and site-specific locations. Variation in the amino acid sequences of heparin-binding sites would potentially modulate the affinity and specificity of the interaction, thus providing a mechanism for regulating different functions of a laminin isoform within a tissue or between different tissues. The heparan sulfate-containing receptors for laminins include dystroglycan, syndecans, agrin, and perlecan, and cell type-specific interactions involving these GAG-containing receptors and the multiple integrin receptors could be modified by differences in the amino acid sequences of the heparin-binding sites on laminin isoforms. Laminin isoforms bind to various integrins, but different laminin isoforms can bind to the same integrins yet have different functions (12). Similarly, the interaction with heparan sulfates could provide a mechanism to modulate the functions of laminin isoforms. Syndecans, a family of transmembrane proteoglycans, bind a wide range of components, including the AG73 peptide, through their heparan sulfate side chains. All cells and tissues except B-stem cells express syndecans in cell-, tissue-, and developmentally specific patterns (48, 49). Specific interactions between different laminin isoforms and syndecans have not been reported and could provide a mechanism for regulating cell-, tissue-, or developmentally specific interactions with laminin isoforms.

The biological activity of a peptide sequence within a molecule is affected by the conformation of the surrounding protein. Previously, we reported that AG73 inhibits HSG cell attachment to the E3 fragment, which contains the AG73 sequence and partially inhibits cell attachment to laminin-1, suggesting it is a functional sequence in the intact molecule (15). When we align the sequence of AG73 using structure-based sequence alignment, it forms beta  strand C in the crystal structure of the alpha 2 LG5 module. Our data here suggest that of the six surface-exposed residues, Lys2, Arg3, Val5, Lys7, Arg11, and Thr12, three are critical to inhibit branching morphogenesis, and five are necessary to inhibit B16F10 cell network formation on Matrigel. Mutation of these residues in a recombinant molecule would be necessary to confirm the sequence is active in vivo. It is possible that AG73 binds to heparan sulfate and blocks the interaction with other heparin-binding sites on laminin. Interestingly, the basic residues Arg1, Lys2, and Arg3 are solvent-exposed and located spatially close to a calcium-binding site and several basic residues that were previously implicated in alpha -dystroglycan and heparin binding of both the alpha 1 LG4 and alpha 2 LG5 modules (17, 39). Alanine mutagenesis of basic residues mapped heparin- and alpha -dystroglycan-binding residues to two sites in the alpha 1 LG4 module, with the sequences GKGRTK (K3 mutant) and EYIKRK (K1 mutant) (17). A minor site, HYARARN (R1 mutant), was also found to be critical for binding of alpha -dystroglycan but not for heparin. It is interesting to note that the three heparin- and/or alpha -dystroglycan-binding sites are identical to AG78 (HFMFDLGKGRTK), AG81 (AHVKTEYIKRK), and AG86 (LGGLPSHYRAR), three cell-binding sequences identified earlier by systematic screening of synthetic peptides (24).

In conclusion, multiple levels of regulation control the interactions between laminins and their cell surface and extracellular matrix receptors, as evidenced by the broad range of biological activities of laminins. We have investigated one biologically active heparin-binding sequence from the G domain of the laminin alpha 1 chain and determined that cell type-specific differences in cell surface GAGs modulate the biological activity of this peptide in both in vivo and in vitro experimental systems. It remains to be determined whether cell and tissue type-specific sulfation patterns and GAG composition will effect the interactions of other recently identified heparin-binding sites on the different laminin isoforms and thereby modulate their function.

    ACKNOWLEDGEMENTS

We thank Melinda Larsen and Harry Grant for critical reading of the manuscript.

    FOOTNOTES

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

§ To whom correspondence should be addressed: NIDCR, National Institutes of Health, 30/430, 30 Convent Dr., MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-1660; Fax: 301-402-0897; E-mail: mhoffman@mail.nih.gov.

Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M100774200

2 J. A. Engbring, unpublished data.

3 P. K. Nielsen, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: HSPG, heparan sulfate proteoglycan; GAG, glycosaminoglycan; DNSNAc, de-N-sulfated-N-acetylated heparin; CDSNAc, completely desulfated-N-acetylated heparin; CDSNS, completely desulfated-N-sulfated heparin; HSG, human submandibular gland; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; E, embryonic day; FGF, fibroblast growth factor; dp, degree of polymerization.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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