Title: Cell Type-specific Differences in Glycosaminoglycans Modulate the Biological Activity of a Heparin-binding Peptide (RKRLQVQLSIRT) from the G Domain of the Laminin α1 Chain
Abstract: AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin α1 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 α-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 β-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. AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin α1 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 α-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 β-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. heparan sulfate proteoglycan glycosaminoglycan de-N-sulfated-N-acetylated heparin completely desulfated-N-acetylated heparin completely desulfated-N-sulfated heparin human submandibular gland Dulbecco's modified Eagle's medium bovine serum albumin phosphate-buffered saline embryonic day fibroblast growth factor degree of polymerization 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 (1Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (661) Google Scholar, 2Ornitz D.M. Bioessays. 2000; 22: 108-112Crossref PubMed Scopus (623) Google Scholar, 3Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 4Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (396) Google Scholar). 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 (5Gallagher J.T. Turnbull J.E. Lyon M. Int. J. Biochem. 1992; 24: 553-560Crossref PubMed Scopus (118) Google Scholar, 6Safaiyan F. Lindahl U. Salmivirta M. Biochemistry. 2000; 39: 10823-10830Crossref PubMed Scopus (37) Google Scholar, 7Tumova S. Woods A. Couchman J.R. Int. J. Biochem. Cell Biol. 2000; 32: 269-288Crossref PubMed Scopus (310) Google Scholar). 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 (8Pye D.A. Vives R.R. Turnbull J.E. Hyde P. Gallagher J.T. J. Biol. Chem. 1998; 273: 22936-22942Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 9Lundin L. Larsson H. Kreuger J. Kanda S. Lindahl U. Salmivirta M. 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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 (13Woods A. Couchman J.R. J. Biol. Chem. 2000; 275: 24233-24236Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). 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 (14Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. EMBO J. 1999; 18: 770-863Crossref Scopus (398) Google Scholar, 15Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The elastase fragment of the laminin α1 chain, E3, which contains the C-terminal LG4 and LG5 modules, is an important heparin-binding site (16Yurchenco P.D. Cheng Y.S. Schittny J.C. J. Biol. Chem. 1990; 265: 3981-3991Abstract Full Text PDF PubMed Google Scholar, 17Andac Z. Sasaki T. Mann K. Brancaccio A. Deutzmann R. Timpl R. J. Mol. Biol. 1999; 287: 253-264Crossref PubMed Scopus (95) Google Scholar). Recent studies have identified other heparin-binding sites in the homologous LG4–5 regions of laminin α1 (14Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. EMBO J. 1999; 18: 770-863Crossref Scopus (398) Google Scholar, 18Yoshida I. Tashiro K. Monji A. Nagata I. Hayashi Y. Mitsuyama Y. Tashiro N. J. Cell. Physiol. 1999; 179: 18-28Crossref PubMed Scopus (13) Google Scholar), α2 (14Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. EMBO J. 1999; 18: 770-863Crossref Scopus (398) Google Scholar), α3 (19Goldfinger L.E. Jiang L. Hopkinson S.B. Stack M.S. Jones J.C. J. Biol. Chem. 2000; 275: 34887-34893Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), α4 (20Yamaguchi H. Yamashita H. Mori H. Okazaki I. Nomizu M. Beck K. Kitagawa Y. J. Biol. Chem. 2000; 275: 29458-29465Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21Talts J.F. Sasaki T. Miosge N. Gohring W. Mann K. Mayne R. Timpl R. J. Biol. Chem. 2000; 275: 35192-35199Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and α5 (22Nielsen P.K. Gho Y.S. Hoffman M.P. Watanabe H. Makino M. Nomizu M. Yamada Y. J. Biol. Chem. 2000; 275: 14517-14523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 23Nielsen P.K. Yamada Y. J. Biol. Chem. 2001; 276: 10906-10912Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 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 α1 G4 module using a synthetic peptide approach (24Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). AG73 promotes attachment of multiple cell types (24Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 25Nomizu M. Kuratomi Y. Malinda K.M. Song S.Y. Miyoshi K. Otaka A. Powell S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), induces salivary acinar cell differentiation (15Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), inhibits branching morphogenesis of embryonic salivary glands (26Kadoya Y. Nomizu M. Sorokin L.M. Yamashina S. Yamada Y. Dev. Dyn. 1998; 212: 394-402Crossref PubMed Scopus (39) Google Scholar), stimulates neurite outgrowth (27Richard B.L. Nomizu M. Yamada Y. Kleinman H.K. Exp. Cell. Res. 1996; 228: 98-105Crossref PubMed Scopus (74) Google Scholar), stimulates matrix metalloproteinase secretion by PC12 cells (28Weeks B.S. Nomizu M. Ramchandran R.S. Yamada Y. Kleinman H.K. Exp. Cell. Res. 1998; 243: 375-382Crossref PubMed Scopus (37) Google Scholar), and promotes liver metastasis by melanoma cells (29Kim W.H. Nomizu M. Song S.Y. Tanaka K. Kuratomi Y. Kleinman H.K. Yamada Y. Int. J. Cancer. 1998; 77: 632-639Crossref PubMed Scopus (48) Google Scholar). AG73 inhibits human submandibular gland (HSG) and B16F10 melanoma cell (29Kim W.H. Nomizu M. Song S.Y. Tanaka K. Kuratomi Y. Kleinman H.K. Yamada Y. Int. J. Cancer. 1998; 77: 632-639Crossref PubMed Scopus (48) Google Scholar) 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 (15Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Recent data from our laboratory with B16F10 cells suggest a similar AG73 receptor, with related but different GAG side chains. 2J. A. Engbring, unpublished data. Here, we compare the activity of AG73 with the homologous sequences in the other laminin α-chains (A2G73–A5G73, TableI) to determine whether the active sequence is specific to the α1 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.Table ISummary table of resultsIdentical (medium shading) and conserved (light shading) residues in the AG73 homologs from the other laminin α-chains are highlighted. The other amino acid substitutions and truncations (light shading) are also highlighted. Identical (medium shading) and conserved (light shading) residues in the AG73 homologs from the other laminin α-chains are highlighted. The other amino acid substitutions and truncations (light shading) are also highlighted. The HSG cell line (30Shirasuna K. Sato M. Miyazaki T. Cancer. 1981; 48: 745-752Crossref PubMed Scopus (284) Google Scholar) 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 (31Fidler I.J. Gersten D.M. Hart I.R. Adv. Cancer Res. 1978; 28: 149-250Crossref PubMed Scopus (612) Google Scholar), 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. 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 (32Nomizu M. Kuratomi Y. Song S.Y. Ponce M.L. Hoffman M.P. Powell S.K. Miyoshi K. Otaka A. Kleinman H.K. Yamada Y. J. Biol. Chem. 1997; 272: 32198-32205Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The peptide-resin beads were also synthesized as described previously (25Nomizu M. Kuratomi Y. Malinda K.M. Song S.Y. Miyoshi K. Otaka A. Powell S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). 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 × 104cells were added and incubated for 2 h at 37 °C. The beads were washed, stained with DiffQuik (Baxter, Miami, FL), and photographed. 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. 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. 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 (33Turnbull J.E. Gallagher J.T. Biochem. J. 1988; 251: 597-608Crossref PubMed Scopus (74) Google Scholar). Fractions were dried and weighed. Size was determined by gradient electrophoresis with comparison to known standards that had been prepared by capillary electrophoresis (34Desai U.R. Wang H. Ampofo S.A. Linhardt R.J. Anal. Biochem. 1993; 213: 120-127Crossref PubMed Scopus (92) Google Scholar). 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 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. 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 (26Kadoya Y. Nomizu M. Sorokin L.M. Yamashina S. Yamada Y. Dev. Dyn. 1998; 212: 394-402Crossref PubMed Scopus (39) Google Scholar) were added to the medium at the beginning of the experiment. 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 (35Caldwell E.E. Andreasen A.M. Blietz M.A. Serrahn J.N. VanderNoot V. Park Y., Yu, G. Linhardt R.J. Weiler J.M. Arch. Biochem. Biophys. 1999; 361: 215-222Crossref PubMed Scopus (41) Google Scholar). 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-core™ 1000 instrument. The association rates (ka ) and dissociation rates (kd ) were registered (2 min each), and theKD was calculated from the equationKD=kd /ka . The streptavidin-heparin surface was regenerated between each run by two successive injections of 30 μl of 20 mm NaOH containing 1m 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. AG73 was mapped on the crystal structure of the α2 LG5 module (Protein Data Bank code 1qu0) using the RasMol program (36Sayle R.A. Milner-White E.J. Trends Biochem. Sci. 1995; 20: 374Abstract Full Text PDF PubMed Scopus (2320) Google Scholar). The AG73 sequence aligns to Gly2955–Thr2966 of the α2 sequence. 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 α-chains. Cell attachment after 2 h to AG73, A2G73, and A3G73 was greater than to A4G73 and A5G73 (Fig. 1 A). 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, Band C). These data demonstrate that homologues of the laminin α1 chain peptide AG73 are active for cell attachment, to varying degrees, when coated on culture plates. 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 IC50for each peptide was determined (TableII). 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 IC50values 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.Table IIIC50 values of competitor peptides (μg/ml) during HSG and B16F10 cell adhesion to AG73Competitor peptideAG73A2G73A3G73A4G73A5G73HSG cells152413>400241B16F10 cells27119277>400>400 Open table in a new tab 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. 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. B16F10 cell attachment to the peptides was inhibited with different GAGs than HSG cells (Fig. 3 B). 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. The interaction of heparin with growth factors and their receptors is dependent on a critical size and sulfation pattern of the heparin chain (37Schlessinger J. Plotnikov A.N. Ibrahimi O.A. Eliseenkova A.V. Yeh B.K. Yayon A. Linhardt R.J. Mohammadi M. Mol. Cell. 2000; 6: 743-750Abstract Full Text Full Text PDF PubMed Scopus (965) Google Scholar). 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.4 A). 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.4 B). 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. 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 α-chains were added to the assay, they had no inhibitory effect on branching (Fig. 5 A). 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. 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 (38Kramer R.H. Bensch K.G. Wong J. Cancer Res. 1986; 46: 1980-1989PubMed Google Scholar). When the peptides were added to this assay, only AG73 inhibited network formation and the cells grew as a monolayer (Fig. 5 B). 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 (TableI), our data suggest that there are specific residues or motifs in AG73 that are responsible for its biological activity. Peptides with a series of alanine substitutions, conserved and non-conserved substitutions in res