Active sites of the laminin alpha1 chain LG4 module for syndecan binding and cell adhesion and spreading

Abstract

Laminins are a family of heteromeric glycoproteins in the basement membrane that exert multiple biological functions through interactions with other matrix molecules and cell surface receptors. Laminin α chains share 5 large subdomains (LG1-5) at their C-terminal region and have many biological activities. We previously identified active synthetic peptides AG73 and EF-1 from laminin α1 LG4 for binding to heparin/syndecan and integrin α2β1, respectively. In this study, we prepared recombinant LG4 and LG4-5 proteins, which contain site-specific mutations within AG73 and EF-1, and examined their activities for receptor binding and cell adhesion and spreading. We found that LG4 and LG4-5 bound heparin and syndecan-1, -2, and -4 but not glypican-1. When Ala was substituted for each of the three positively charged residues (Arg2719, Lys2720 and Arg2721) within the AG73 sequence of the recombinant proteins, binding activity was significantly reduced. LG4 was active for adhesion to fibroblasts, but mutant LG4 failed to adhere to fibroblasts. Cell spreading of fibroblasts to LG4 was inhibited by antibodies to integrin α2β1. A single substitution mutation (Asp2763 to Ala) within EF-1 of LG4 significantly decreased cell spreading, suggesting the involvement of the integrin for cell spreading. These results suggest that the AG73 site of LG4 bind cells through syndecan interactions and the EF-1 site is required for cell spreading through integrin α2β1.