Abstract
AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin alpha1 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 (Lys(2)) and the C-terminal R (Arg(10)) 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.
Highlights
AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin ␣1 chain, has diverse biological activities with different cell types
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
The relative amount of cell attachment activity of the peptides was similar: AG73 Ͼ A3G73 Ͼ A2G73 Ͼ A5G73 Ͼ A4G73 (Fig. 1, B and C). These data demonstrate that homologues of the laminin ␣1 chain peptide AG73 are active for cell attachment, to varying degrees, when coated on culture plates
Summary
AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin ␣1 chain, has diverse biological activities with different cell types. 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]. Different heparin-binding sequences have been identified depending on the laminin isoform and the purified ligand, cell, or tissue type tested. 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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