Hair-forming activity of human lymphocyte specific protein 1 requires cooperation between its caldesmon-like domains and the villin headpiece-like domains.

Abstract

LSP1 is an F-actin binding with multiple F-actin binding domains. Overexpression of LSP1 in NAD 47/89 patient's neutrophils created hair-like projections on the patient's neutrophil cell surfaces and inhibited neutrophil cell motility and transfection of LSP1 in serial cell lines recreate the NAD 47/89 phenotype and produce branching hair-like surface projections. Although LSP1 contains hair-forming ability and LSP1 F-actin binding domains have been defined, the LSP1 domains responsible for its hair-forming activity, the relationship to the F-actin binding domains, and the required domain interactions, if any, for hair formation are not well understood. To define the hair-forming domains of LSP1, the relationship to the known F-actin binding domains, and binding domain interactions, LSP1 truncates, which include or exclude the different F-actin binding domains, were created by PCR. LSP1 mutants were created by site-directed mutagenesis to define the amino acids important for hair formation. Sf9 cells were infected with recombinant baculovirus expressing the cDNA of LSP1 truncates and mutants, and the morphology of infected Sf9 cells was documented by DIC optics. Results show that (1) the hair-forming activity of LSP1 is localized to the basic C-terminal half of the molecule, which contains all of the F-actin binding domains; (2) both the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of LSP1; (3) basic amino acids in the villin headpiece regions are crucial for the hair-forming activity of LSP1 molecule. The results suggest cooperation between the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of human LSP1 in cells.