Identification of Biologically Active Sequences in the Laminin α4 Chain G Domain

Laminins are a family of trimeric extracellular matrix proteins consisting of α, β, and γ chains. So far five different laminin α chains have been identified. The laminin α4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin α4 chain. Here we have focused on the biological functions of the laminin α4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-α4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-α4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-α4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-α4G. These results suggest that the four active sites are involved in the biological functions of the laminin α4 chain G domain. Furthermore, rec-α4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.