Identification of a Major Heparin and Cell Binding Site in the LG4 Module of the Laminin α5 Chain

• Published in: The Journal of biological chemistry 2000-05, Vol.275 (19), p.14517-14523
• Main Authors: Nielsen, Peter K., Gho, Yong Song, Hoffman, Matthew P., Watanabe, Hideto, Makino, Masayoshi, Nomizu, Motoyoshi, Yamada, Yoshihiko
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.19.14517

The G domain of the laminin α chains consists of five homologous G modules (LG1–5) and has been implicated in various biological functions. In this study, we identified an active site for cell and heparin binding within the laminin α5 G domain using recombinant proteins and synthetic peptides. Recombinant LG4, LG5, and LG4–5 modules were generated using a mammalian expression system. The LG4 and LG4–5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inhibited cell binding to the LG4–5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin β1 subunit. These results suggest that the LG4–5 module interacts with a cell surface receptor containing heparan sulfate but not with integrins. Solid-phase assays and surface plasmon resonance measurements demonstrated strong binding of the LG4 and LG4–5 modules to heparin with KD values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with glycosidases demonstrated thatN-linked carbohydrates on the LG5 module are complex-type oligosaccharides. The LG4–5 module, devoid of N-linked carbohydrates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal ofN-linked glycosylation. To localize active sites on the LG4 module, various synthetic peptides were used to compete with binding of the tandem module to heparin and cells. Peptide F4 (AGQWHRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to compete binding. Alanine replacements demonstrated that one arginine residue within F4 was important for cell and heparin binding. Our results suggest a critical role of the LG4 module for heparan sulfate-containing receptor binding within the laminin α5 chain.