Identification of a major heparin and cell binding site in the LG4 module of the laminin alpha 5 chain

The G domain of the laminin alpha chains consists of five homologous G modu les (LG1-5) and has been implicated in various biological functions. In thi s study, we identified an active site for cell and heparin binding within t he laminin alpha 5 G domain using recombinant proteins and synthetic peptid es. Recombinant LG4, LG5, and LG4-5 modules were generated using a mammalia n expression system. The LG4 and LG4-5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inh ibited cell binding to the LG4-5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin beta(1) subunit. These result s suggest that the LG4-5 module interacts with a cell surface receptor cont aining heparan sulfate but not with integrins. Solid-phase assays and surfa ce plasmon resonance measurements demonstrated strong binding of the LG4 an d LG4-5 modules to heparin with K-D values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with gly cosidases demonstrated that N-linked carbohydrates on the LG5 module are co mplex-type oligosaccharides. The LG4-5 module, devoid of N-linked carbohydr ates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal of N-linked glycosylation. To localize ac tive sites on the LG4 module, various synthetic peptides were used to compe te with binding of the tandem module to heparin and cells. Peptide F4 (AGQW HRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to co mpete 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 bi nding within the laminin alpha 5 chain.