Identification and characterization of tissue specific active sites in the laminins

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Laminins are heterotrimeric basement membrane glycoproteins consisting of genetically distinct α, β, and γ chains. So far, five α, three β, and three γ chains have been identified, and at least fifteen isoforms (Laminin-1-15) are formed by various combinations of each subunit. Laminins have diverse biological activities including promotion of cell adhesion, growth, migration, neurite outgrowth, and differentiation. Our goal is to understand the biological role of the laminins in normal development and disease processes and to apply the active sequences for therapeutic application including tissue regeneration and engineering. Our approach has been to localize cell adhesion sites on the laminin molecules using a large set of synthetic peptides and recombinant proteins. We have identified about 20 active sequences from the laminin-1 using a systematic screening for cell binding sites with 673 overlapping synthetic peptides. First, we have focused on the biological functions of the lamini … More n αchain G domains and screened active sites using a recombinant protein and synthetic peptides. We prepared various recombinant proteins, comprising the G domains of laminin α1, α3 and α4 chains, and tested their cell attachment activity. Several recombinant proteins showed cell attachment and neurite outgrowth activities. We also synthesized more than 300 overlapping peptides covering the entire G domains and tested their cell attachment activity sing peptide-coated plates and peptide-conjugated Sepharose beads. More than 20 peptides promoted cell attachment and five peptides inhibited the biological activity mediated by the recombinant proteins. Some of the peptides were found to interact with integrins and syndecans. These results suggest that the active sites are involved in the biological functions of the laminin α chain G domains. These active peptides may be useful for defining of the molecular mechanism of laminin-receptor interactions and laminin-mediated cellular signaling pathways. Less