Cell-Adhesive Activity and Receptor-Binding Specificity of the Laminin-Derived YIGSR Sequence Grafted onto Staphylococcal Protein A

Abstract

Laminin contains multiple oligopeptide motifs to promote cell adhesion and migration. One of these motifs is YIGSR within the Bl chain. We reconstituted the cell-adhesive activity of YIGSR motif by grafting it onto a truncated form of the Staphylococcal protein A (designated tSPA) via cassette mutagenesis. When coated on a polystyrene surface, the YIGSRgrafted tSPA (YIGSR-tSPA) promoted attachment and spreading of mouse melanoma and human rhabdomyosarcoma cells, but not of hamster fibroblasts. The cell-adhesive activity of YIGSR-tSPA was abolished by amino acid substitution or scrambling of the inserted YIGSR sequence. Divalent cations Mn²⁺ and Mg²⁺, but not Ca²⁺, promoted the cell adhesion to YIGSR-tSPA. Interestingly, the YIGSR-tSPA-mediated cell adhesion was barely inhibited by the linear peptide CDPGYIGSR-NH₂, , but was strongly inhibited by the cyclic peptide CDPGYIGSRC and another peptide PEILDVPST, which is a specific inhibitor for integrin α₄β₁. Among various anti-integrin antibodies, anti-α, and anti-β₁ antibodies specifically inhibited the cell adhesion to YIGSR-tSPA. In support of these observations, adhesion of rhabdomyosarcoma cells to intact laminin was also partially inhibited by synthetic PEILDVPST peptide and anti-α₄ antibody. These results, taken together, indicate that the YIGSR motif exerts its cell-adhesive activity through interaction with integrin α₄β₁.